HPLC METHODS FOR
RECENTLY APPROVED
PHARMACEUTICALS
HPLC METHODS FOR
RECENTLY APPROVED
PHARMACEUTICALS
George Lunn
A JOHN WILEY & SONS, INC., PUBLICATION
Copyright 2005 by John Wiley & Sons, Inc. All rights reserved.
Published by John Wiley & Sons, Inc., Hoboken, New Jersey.
Published simultaneously in Canada.
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Library of Congress Cataloging-in-Publication Data:
Lunn, George.
HPLC methods for recently approved pharmaceuticals / George Lunn.
p. cm.
Includes index.
ISBN 0-471-66941-5 (cloth)
1. High performance liquid chromatography. 2. Drugs–Analysis. I. Title.
RS189.5.H54L773 2005
615′ .1–dc22
2004016046
Printed in the United States of America.
10 9 8 7 6 5 4 3 2 1
CONTENTS
Preface / xi
Acknowledgements / xiii
About This Book / xv
Abacavir / 1
Acarbose / 5
Acetyl sulfisoxazole / 6
Acrivastine / 7
Adapalene / 10
Adefovir dipivoxil / 11
Adrenocorticotropic hormone / 13
Afloqualone / 15
Alacepril / 16
Alclometasone 17,21-dipropionate / 18
Alitretinoin / 21
Allethrin / 24
Almotriptan / 27
Alosetron / 29
Amcinonide / 30
Aminolevulinic acid / 33
Amprenavir / 36
Anagrelide / 42
Anakinra / 43
Apraclonidine / 45
Aprepitant / 46
Aranidipine / 48
Arotinolol / 49
Arteether / 52
Articaine / 54
Asparaginase / 57
Atazanavir sulfate / 58
Atipamezole / 60
Atomoxetine hydrochloride / 62
Atorvastatin / 64
Atosiban / 66
Balofloxacin / 67
Bambermycins / 69
Befunolol / 70
Benzalkonium chloride / 71
Betaine / 72
Bethanechol chloride / 74
Bexarotene / 75
Biapenem / 77
Bimatoprost / 79
Bioresmethrin / 80
Bivalirudin / 81
Boldenone / 82
Bosentan / 83
β-Boswellic acid / 86
Brimonidine / 88
Bromfenac / 90
Brovincamine / 92
Bucillamine / 93
Budipine / 94
Bulaquine / 95
Butacaine / 97
Butamben / 99
Butoconazole / 100
Butyl flufenamate / 101
Cambendazole / 102
Candesartan cilexetil / 104
Capecitabine / 106
Casanthranol / 108
Caspofungin / 109
v
vi
Contents
Castor oil / 112
Cefbuperazone / 113
Cefditoren / 114
Cefoselis / 116
Cefozopran / 117
Cefuzonam / 118
Celecoxib / 119
Cerivastatin / 123
Cetrorelix / 125
Cetyl alcohol / 128
Cevimeline hydrochloride / 130
Chlorobutanol / 132
Chloroprocaine / 133
Chorionic gonadotropin / 134
Cilnidipine / 135
Cimetropium bromide / 136
Cisatracurium besylate / 137
Citric acid / 139
Clioquinol / 142
Clobetasol 17-propionate / 143
Clopidogrel / 147
Clopidol / 149
Cloricromen / 151
Clorsulon / 152
Colistin / 153
Cypermethrin / 155
Dalfopristin / 156
Dalteparin / 158
Daptomycin / 159
Deferiprone / 161
Deflazacort / 162
Desloratadine / 164
Desogestrel / 166
Desoximetasone / 167
Desoxycorticosterone / 169
Dexrazoxane / 172
Dextran / 174
Diacerein / 176
Dichloroacetic acid / 177
Dichlorophen / 178
Diclazuril / 179
Dihydrotachysterol / 181
Dimethyl sulfoxide / 183
Dinitolmide / 185
Dipivefrin / 186
Dithiazanine iodide / 187
Docarpamine / 188
Dofetilide / 189
Dolasetron / 191
Donepezil / 193
Doxefazepam / 195
Doxercalciferol / 196
Dropropizine / 198
Drospirenone / 199
Droxicam / 200
Droxidopa / 201
Ebrotidine / 202
Edaravone / 204
EDTA / 206
Efavirenz / 208
Efrotomycin / 212
Egualen / 213
Eletriptan / 214
Emtricitabine / 215
Enoxaparin sodium / 217
Entacapone / 218
Eperisone / 220
Eplerenone / 222
Epoprostenol / 224
Eprosartan / 225
Eptazocine / 227
Eptifibatide / 228
Erdosteine / 229
Ergotamine / 230
Ertapenem / 234
Ethopabate / 236
Ethyl icosapentate / 237
Etonogestrel / 238
Etoricoxib / 240
Etorphine / 242
Exemestane / 243
Ezetimibe / 245
Fadrozole / 247
Falecalcitriol / 248
Fenoxycarb / 250
Fenticonazole / 251
Fexofenadine / 253
Flomoxef / 257
Florfenicol / 258
Fludrocortisone / 260
Fluprostenol / 262
Flurandrenolide / 264
Flurithromycin / 267
Flurogestone acetate / 268
Fluticasone propionate / 270
Flutrimazole / 273
Fomepizole / 274
Fomivirsen / 276
Fondaparinux / 277
Formestane / 278
Contents
Formoterol / 279
Fosamprenavir calcium / 281
Fosinopril / 283
Fosphenytoin / 284
Frovatriptan / 286
Fumagillin / 288
Galantamine / 290
Ganirelix / 292
Gatifloxacin / 293
Gefitinib / 295
Gemcitabine / 296
Gemifloxacin / 298
Gestodene / 300
Gestrinone / 301
Glycerin / 302
Guanabenz / 304
Guanadrel / 305
Halobetasol propionate / 306
Halofuginone / 308
Hetastarch / 310
Hydroquinone / 311
Hygromycin B / 312
Iloprost / 313
Imatinib / 314
Imidocarb / 316
Iobenguane / 318
Iodixanol / 320
Iopanoic acid / 322
Iopromide / 324
Ioversol / 326
Ipratropium bromide / 327
Ipriflavone / 328
Isoflupredone / 329
Isopropamide iodide / 330
Itopride / 332
Kinetin / 333
Lafutidine / 334
Lamivudine / 335
Latanoprost / 339
Leflunomide / 341
Lercanidipine / 343
Letrozole / 345
Levetiracetam / 346
Levonordefrin / 348
Levosimendan / 349
Lidamidine / 351
Lincomycin / 352
Lindane / 354
Linezolid / 355
Liothyronine / 357
Lomerizine / 358
Lopinavir / 359
Loteprednol etabonate / 362
Marbofloxacin / 364
Masoprocol / 367
Maxacalcitol / 368
Medetomidine / 369
Meglutol / 371
Melatonin / 372
Melengestrol acetate / 375
Memantine / 378
Menthol / 380
Mepenzolate bromide / 381
Mepixanox / 383
Mequinol / 384
Methenamine / 385
Methoprene / 386
Methoxychlor / 387
Methyltestosterone / 392
Metrizamide / 393
Metyrosine / 394
Micafungin / 396
Milnacipran / 398
Mirtazapine / 400
Misoprostol / 405
Mizolastine / 406
Moexipril / 411
Mofezolac / 412
Mometasone furoate / 413
Monensin / 415
Morantel / 416
Mosapride / 417
Moxifloxacin / 420
Moxonidine / 423
Nadifloxacin / 424
Naftopidil / 425
Nandrolone / 427
Narasin / 429
Nartograstim / 430
Nateglinide / 431
Nebivolol / 433
Nelfinavir / 435
Nequinate / 440
Neridronic acid / 441
Nevirapine / 443
Nicarbazin / 447
Nilutamide / 448
Nipradilol / 449
Nitazoxanide / 450
Nitenpyram / 452
vii
viii
Contents
Nomegestrol / 453
Nonoxynol-9 / 454
Nystatin / 455
Octocrylene / 456
Oleic acid / 457
Olmesartan / 469
Olopatadine / 470
Orbifloxacin / 472
Orlistat / 475
Oseltamivir / 477
Oxaliplatin / 479
Oxiconazole / 481
Panipenem / 483
Parecoxib / 484
Paricalcitol / 485
Pazufloxacin / 487
Penciclovir / 488
Pentaerythritol tetranitrate / 490
Pentosan polysulfate / 491
Perflubron / 492
Perospirone / 493
Phenazopyridine hydrochloride / 494
Phentermine / 497
Phosphatidylcholine / 501
Phosphatidylglycerol / 504
Piketoprofen / 508
Pilsicainide / 509
Pioglitazone / 511
Pipercuronium bromide / 514
Pirlimycin / 515
Poloxalene / 518
Pramipexole / 519
Pranlukast / 521
Prednicarbate / 523
Propionylpromazine / 524
Propoxycaine hydrochloride / 527
Propylene glycol / 528
Propylhexedrine / 529
Protirelin / 530
Prulifloxacin / 532
Pyrethrins / 533
Quetiapine / 536
Quinfamide / 539
Quinupristin / 540
Rabeprazole / 542
Ractopamine / 544
Raloxifene / 547
Ramosetron / 549
Rapacuronium bromide / 551
Remifentanil / 553
Repaglinide / 555
Ricinoleic acid / 557
Rifaximin / 558
Rilmazafone / 559
Risedronate sodium / 560
Rizatriptan / 561
Rofecoxib / 562
Ropinirole / 565
Rosiglitazone / 566
Rosuvastatin calcium / 568
Sarafloxacin / 569
Selamectin / 571
Sermorelin / 572
Sibutramine / 574
Sildenafil / 576
Simethicone / 579
Sivelestat / 580
Sodium oxybate / 582
Somatropin / 583
Squalane / 584
Squalene / 585
Stanozolol / 587
Succimer / 588
Succinylcholine chloride / 589
Sulfabromomethazine / 591
Sulfachlorpyridazine / 592
Sulfaethoxypyridazine / 596
Sulfamerazine / 598
Sulfanitran / 600
Sultamicillin / 602
Tacalcitol / 604
Talipexole / 605
Taltirelin / 607
Technetium Tc 99m bicisate / 608
Tegaserod / 609
Telithromycin / 610
Telmesteine / 611
Telmisartan / 612
Temocapril / 614
Temozolomide / 615
Tenofovir disoproxil fumarate / 617
Teprenone / 620
Teriparatide / 621
Tetrachlorvinphos / 622
Tetrahydrozoline / 623
Thalidomide / 626
Thialbarbital / 628
Thyrotropin / 629
Tiagabine / 630
Tiletamine hydrochloride / 631
Contents
Tilmicosin / 633
Tiludronic acid / 634
Tirilazad / 635
Tirofiban / 637
Tiropramide / 639
Tizanidine / 641
Tolcapone / 643
Topiramate / 645
Topotecan / 647
Tosufloxacin / 649
Travoprost / 651
Trenbolone / 652
Treprostinil / 653
Trichlorfon / 655
Triethanolamine / 656
Trifluridine / 657
Cumulative Index / 685
Cross-Index to Other Substances / 703
Triptorelin / 658
Troleandomycin / 660
Troxipide / 661
Ubenimex / 662
Unoprostone isopropyl ester / 663
Valacyclovir / 664
Valdecoxib / 666
Valganciclovir / 668
Valrubicin / 670
Valsartan / 671
Zaleplon / 673
Zaltoprofen / 676
Zanamivir / 678
Zinostatin / 680
Zofenopril calcium / 681
Zolazepam hydrochloride / 683
ix
PREFACE
This book is a collection of procedures for the analysis of more than 390 pharmaceuticals using high-performance liquid chromatography (HPLC) and covers the
literature up to the end of 2003. The current volume is a continuation of HPLC
Methods for Pharmaceutical Analysis, published in four volumes from 1997 to 2000.
The previous volumes described methods published in the literature through the
middle of 1998.
The current work lists procedures for the analysis of drugs in three broad categories:
Drugs that have been approved since the previous volumes were published.
Drugs that were approved when the previous volumes were published but for
which analytical methods were not then available in the literature.
ž Drugs for which procedures allowing determination in a blood matrix have only
become available since the previous volumes were published.
ž
ž
Please note that mention of a drug does not necessarily mean that it is currently
approved for use in the United States or indeed in any country.
Despite the ready availability of computer-aided literature, searching this resource
is not exploited as much as it might be. One reason for this reluctance is, of course,
that a computer search merely produces a listing of possibly relevant references.
Tedious and time-consuming searches in the library are necessary to find the most
relevant reference that can be turned into a practical analytical procedure in the
searcher’s own laboratory. The reference finally chosen will, naturally, depend on
the individual circumstances, such as the matrix in which the drug is present,
availability of equipment, and so on. This book circumvents this lengthy process by
providing a number of abstracted and evaluated procedures for the analysis of each
drug. The analyst can rapidly identify a relevant procedure and put it into practice.
In addition to the analytical matrix, other factors may be important when choosing
an analytical procedure. Accordingly, we have noted such features of the analytical
procedures as sensitivity, mode of detection, other compounds that interfere with
the analysis, other drugs that may be determined at the same time, and so on.
Readers familiar with our previous publications, HPLC Methods for Pharmaceutical Analysis, Volumes 1–4 (George Lunn and Norman R. Schmuff, John Wiley,
New York, 1997–2000) and Handbook of Derivatization Reactions for HPLC (George
Lunn and Louise C. Hellwig, John Wiley, New York, 1998), will notice many similarities. The abstract structure is very similar, and the philosophy that the procedures
xi
xii
Preface
should be reproducible without reference to the original literature is unchanged.
A new feature is that the retention times (in minutes) of other drugs that may be
determined using the same system have been added in parentheses after the drug
name. Other data, such as the limit of detection (LOD), may also be added. The
retention time is the number without units. Unlike the previous volumes, this book
is not available on a CD in an electronic form.
At the end of the book a Cumulative Index and a Cross-Index to Other Substances
are provided. The Cumulative Index provides a comprehensive listing of the drugs
covered in this book and the previous volumes. The Cross-Index lists the other
compounds that may also be chromatographed under the conditions described in
the monographs in this book. Using the information in the monographs it may be
possible to develop chromatographic procedures for these compounds.
GEORGE LUNN
ACKNOWLEDGEMENTS
I am grateful for the use of the National Institutes of Health Library, the FDA
Medical Library, and the National Library of Medicine and I would like to express
my appreciation for the hard work of the staff of these libraries, particularly those
diligent workers who reshelve the journal volumes after one of my forays. Although
many people have helped with the preparation of this work the mistakes are my
own. I would appreciate hearing from anyone who has corrections, comments, or
suggestions. I can be reached at lunng@cder.fda.gov.
The content of this volume does not necessarily reflect the views or policies
of the Food and Drug Administration, nor does the mention of trade names,
commercial products, or organizations imply endorsement by the U.S. Government.
Also, mention of a drug does not necessarily mean that it is currently approved for
use in the United States or indeed in any country.
G.L.
xiii
ABOUT THIS BOOK
SCOPE
Newly approved drugs were identified from a variety of sources including the FDA’s
annual lists of drug approvals (available at www.fda.gov/cder) and Annual Reports
in Medicinal Chemistry published by Elsevier/Academic Press.
The journals routinely surveyed for relevant articles are:
American Journal of Health-System Pharmacy
Analyst
Analytica Chimica Acta
Analytical Chemistry
Analytical Letters
Analytical Sciences
Antimicrobial Agents and Chemotherapy
Arzneimittelforschung
Biological and Pharmaceutical Bulletin
Biomedical Chromatography
Biopharmaceutics and Drug Disposition
Chemical and Pharmaceutical Bulletin
Chromatographia
Clinical Chemistry
Clinical Pharmacology and Therapeutics
Drug Metabolism and Disposition
Farmaco
Food Additives and Contaminants
Journal of Analytical Toxicology
Journal of AOAC International
Journal of Chromatographic Science
Journal of Chromatography, Part A and Part B
Journal of Clinical Pharmacology
Journal of Forensic Sciences
xv
xvi
About This Book
Journal of Liquid Chromatography & Related Technology
Journal of Pharmaceutical and Biomedical Analysis
Journal of Pharmaceutical Sciences
Journal of Pharmacology and Experimental Therapeutics
Pharmaceutical Research
Pharmazie
Therapeutic Drug Monitoring
Xenobiotica
Other journals were consulted when relevant articles were identified by computer searches.
The literature was surveyed from 1998 through the end of 2003, although methods
from some older articles (and a few from 2004) are included.
NOMENCLATURE
Each chapter is headed by the name and structure of the target compound as well as
other useful data such as the CAS Registry Number, molecular formula, molecular
weight, and Merck Index number (from the 13th edition).1 More useful information
such as melting point, solubility, optical rotation, references to reviews, and so on
can be found in the Merck Index.
In general, the United States Adopted Name (USAN)2 is used throughout to
identify each drug. Names of derivatives, such as esters, which would have different chromatographic properties, are identified by placing the derivative name in
parentheses after the retention time.
Increasingly, drugs previously marketed as racemates are being marketed as a
single enantiomer with the name changed to reflect the enantiomer. For example,
levofloxacin is the levorotatory form of ofloxacin. For an achiral HPLC method, the
chromatography of a single enantiomer is no different from that of the racemate.
In general, in this work and the preceding works, we have listed HPLC procedures
under the name of the racemate rather than the single enantiomer. The interested
reader is referred to the USP Dictionary2 (page 1208) for the naming conventions
used. Generally:
Levo rotatory
Levo rotatory
Dextro rotatory
Dextro rotatory
S isomer
R isomer
R isomer
S isomer
Prefix lev/levoPrefix arPrefix dex/dextroPrefix es-
For racemates, the rac- prefix is used.
In some cases, the chiral prefix is used. Thus, the following list shows the prefixes
that are used in the different volumes:
Dexrazoxane in this volume
Dextromethorphan in Volume 2
Dextromoramide in Volume 2
Dextrothyroxine in Volume 2
About This Book
Levallorphan in Volume 3
Levamisole in Volume 3
Levobunolol in Volume 3
Levodopa in Volume 3
Levonordefrin in Volume 3 and this volume
Levorphanol in Volume 3
Levosimendan in this volume
Levothyroxine in Volumes 1 and 3.
More generally, the name of the racemic compound is used. Thus,
For
Consult
Volume
Arformoterol
Formoterol
3, this volume
Dexamisole
Dexamphetamine
Dexbrompheniramine
Dexbudesonide
Dexchlorpheniramine
Dexfenfluramine
Dexibuprofen
Dexketoprofen
Dexmedetomidine
Dexmethylphenidate
Dexpropranolol
Dexsotalol
Dextroamphetamine
Dextropropoxyphene
Dexverapamil
Levamisole
Amphetamine
Brompheniramine
Budesonide
Chlorpheniramine
Fenfluramine
Ibuprofen
Ketoprofen
Medetomidine
Methylphenidate
Propranolol
Sotalol
Amphetamine
Propoxyphene
Verapamil
3
2
2
2
2
3
1, 4
1, 4
This volume
1
4
4
2
1, 4
1, 4
Esatenolol
Escitalopram
Esflurbiprofen
Esketamine
Esomeprazole
Esoxybutynin chloride
Eszopiclone
Atenolol
Citalopram
Flurbiprofen
Ketamine
Omeprazole
Oxybutynin chloride
Zopiclone
1, 2
2
3
3
1, 3
3
4
Levalbuterol
Levamfetamine
Levamphetamine
Levcycloserine
Levdobutamine
Levmetamfetamine
Levobetaxolol
Levobupivacaine
Levocarnitine
Levocetirizine
Levodropropizine
Albuterol
Amphetamine
Amphetamine
Cycloserine
Dobutamine
Methamphetamine
Betaxolol
Bupivacaine
Carnitine
Cetirizine
Dropropizine
1, 2
2
2
2
2
3
2
2
2
2
2, this volume
xvii
xviii
About This Book
Levofenfluramine
Levofloxacin
Levofuraltadone
Levoleucovorin
Levomenthol
Levomethadone
Levomoprolol
Levonorgestrel
Levopropoxyphene
Levopropylhexedrine
Levosalbutamol
Levosulpiride
Fenfluramine
Ofloxacin
Furaltadone
Leucovorin
Menthol
Methadone
Moprolol
Norgestrel
Propoxyphene
Propylhexedrine
Albuterol
Sulpiride
3
1, 3
3
3
3, this volume
3
3
1
1, 4
4, this volume
1, 2
4
Racementhol
Racemethorphan
Racemetirosine
Racemorphan
Racephedrine
Racepinephrine
Menthol
Dextromethorphan
Metyrosine
Levorphanol
Ephedrine
Epinephrine
3, this volume
2
This volume
3
3
3
BIBLIOGRAPHIES
For reasons of space, it is not possible to abstract every relevant paper, and so at the
end of some chapters an Annotated Bibliography lists other relevant papers. After
the citation, a few features of the method that are not obvious from the title of the
paper may be briefly mentioned to help the reader decide if this paper may be of use.
For example, the limit of quantitation of the method may be cited. Unless otherwise
mentioned, it may be assumed that a method involves liquid–liquid extraction of a
biological fluid from a human and uses reversed-phase HPLC with UV detection.
Thus, if a method uses solid-phase extraction (SPE) or fluorescence detection, this
will be mentioned.
ABSTRACT STRUCTURE
The detailed procedures given normally contain the following sections. Of course,
not all papers give full details, so some sections may be missing.
Matrix
Sample preparation
Guard column
Column
Mobile phase
Flow rate
Injection volume
Retention time
Detector
About This Book
xix
Internal standard
Column temperature
Extracted
Simultaneous
Also
Noninterfering
Interfering
Limit of detection
Limit of quantitation
Key words
Reference
ABSTRACT CONVENTIONS
Also
Column
Column temperature
Derivatization
Detector
Extracted
Flow rates
Guard column
Injection volume
Interfering
Matrix
Mobile phase
Compounds that can be analyzed at the same time. It is not
specified whether they interfere, but they can be
extracted. See also Extracted, Simultaneous.
Dimensions are length (mm) × internal diameter (mm), and
the material is stainless steel unless otherwise indicated.
If other than ambient (all temperatures are in degrees C).
Pre-column unless otherwise mentioned (in Key Words).
Wavelengths in nanometers
Compounds that can be extracted from the matrix in
question and analyzed at the same time and do not
interfere. See also Also, Simultaneous.
In milliliters per minute.
Dimensions are length (mm) × internal diameter (mm).
In microliters (µL). Injection volume may be either the
volume actually injected or the volume of the injection
loop. If it is the volume actually injected, this value is
also given in the Sample preparation section. If the
actual injection volume is not given in the Sample
preparation section, the Injection volume given is that of
the injection loop.
Compounds that interfere with the analysis of the target
compound. Compounds that interfere with the
chromatography of the internal standard (IS) are listed
under simultaneous (another IS can always be selected or
an external standard procedure can be used).
A controlled vocabulary is used (see below)
Ratios are v/v and gradients are linear, unless otherwise
noted. Times given when describing gradient elution and
other procedures such as column switching are the times
for each step, e.g., ‘‘MeOH:water 15:85 for 4 min, to 50:50
over 2 min, maintain at 50:50 for 4 min.’’ If we were to
include the cumulative times (t) in the example above it
would read: ‘‘MeOH:water 15:85 for 4 min (t = 4), to 50:50
over 2 min (t = 6), maintain at 50:50 for 4 min (t = 10).’’
xx
About This Book
Noninterfering
Retention time
Simultaneous
SPE
Species
Compounds that do not interfere with the analysis for
various reasons, e.g., they are not extracted, they are not
detected.
In minutes. This is frequently estimated from a reproduced
chromatogram, and so the accuracy may not be great.
When available, retention times are given for the
analyte, the internal standard, and other compounds that
may be chromatographed under the same conditions. For
the internal standard and other compounds that may be
chromatographed under the same conditions, the
retention times are given in parentheses after the
compound name.
Compounds that can be analyzed at the same time and do
not interfere. Note that the compound cannot necessarily
be extracted from the matrix in question (although it may
be). See also Also, Extracted.
For the sake of consistency, conditioning procedures for
solid-phase extraction (SPE) cartridges are always
described at the beginning of the sample preparation
sections. Bear in mind, however, that the conditioning
procedure should be carried out just prior to use. Thus, if
sample preparation is a lengthy procedure, it may be
necessary to delay SPE cartridge conditioning until the
step requiring the cartridge.
If other than human, noun is used instead of adjective, e.g.,
cow not bovine. In some cases, human may be specified.
For example, if both human blood and rat blood are
analyzed, both human and rat will be indicated (in Key
Words).
MATRIX
To help with searching, a controlled vocabulary is used to limit the number of terms
in the matrix section. For example, the terms raw material, drug substance, or API
(active pharmaceutical ingredient) are not used; the term bulk is used instead. In
a number of cases, the matrix is associated with various key words, which can be
used to narrow the search. For example, the matrix term blood has the key words
plasma, serum, and whole blood associated with it. Thus, if you are interested in the
determination of the drug in blood in general, you should look in the matrix field for
blood. If, however, you are specifically interested in finding the drug in plasma, you
should look in the key words field for plasma.
Matrix
Bile
Blood
Bulk
CSF
Formulations
Microsomal incubations
Associated Key Words
Plasma, serum, whole blood
Capsules, creams, injections, ointment, tablets, etc.
About This Book
Milk
Perfusate
Reaction mixtures
Saliva
Tissue
Urine
xxi
Brain, heart, kidney, liver, muscle, spleen, etc.
ABBREVIATIONS
BHT
DMSO
E
em
EtOH
ex
F
GPC
h
HPLC
ID
IS
L
LOD
LOQ
M
MeCN
MeOH
min
mL
mM
MS
MSPD
MTBE
nM
psi
s
SEC
SFC
SFE
SPE
Temp
U
UV
vol
2,6-Di-tert-butyl-4-methylphenol, butylated hydroxytoluene
Dimethyl sulfoxide
Electrochemical detection
Emission wavelength
Ethanol
Excitation wavelength
Fluorescence detection
Gel permeation chromatography
Hour
High-performance liquid chromatography
Internal diameter
Internal standard
Liter
Limit of detection or some other description indicating that this is the
smallest concentration or quantity that can be detected or analyzed for
Lower limit of quantitation, either given as such in the paper or taken as
the lower limit of the linear quantitation range
Molar (i.e., moles/L)
Acetonitrile
Methanol
Minutes
Milliliter
Millimolar (i.e., millimoles/L)
Mass spectrometric detection
Matrix solid phase dispersion
Methyl tert-butyl ether
Nanomolar (i.e., nanomoles/L)
Pounds/sq. in. (1 psi = 6.89476 kPa)
Seconds
Size Exclusion Chromatography
Supercritical fluid chromatography
Supercritical fluid extraction
Solid phase extraction
Temperature
Units
Ultraviolet detection
Volume
xxii
About This Book
PIC REAGENTS
These reagents are offered by Waters as buffered solutions containing the following
compounds:
PIC A is tetrabutylammonium sulfate
PIC B5 is pentanesulfonic acid
PIC B7 is heptanesulfonic acid.
WORKING PRACTICES
In general, good working practice, for example, using high-grade materials is
assumed. Solutions should be protected from light, and silanized glassware should
be used unless you have good reason to believe that these precautions are not
necessary. Details of solution preparation are generally not given. It should be
remembered that the preparation of a dilute aqueous solution of a relatively waterinsoluble compound can frequently be made by dissolving the compound in a small
volume of a water-miscible organic solvent and diluting this solution with water.
A number of excellent texts3 – 9 discuss good working practices and procedures in
HPLC. Please note that all the temperatures are in degrees C.
It is also assumed that safe working practices are observed. Organic solvents
should only be evaporated in a properly functioning chemical fume hood, correct
protective equipment should be worn when dealing with potentially hazardous
biological materials, and waste solutions should be disposed of in accordance with
all applicable regulations.
A number of solvents are particularly hazardous. For example, benzene is a
human carcinogen;10 chloroform,11 dichloromethane,12 dioxane,13 and carbon tetrachloride 14 are carcinogenic in experimental animals; and DMF 15 and MTBE 16,17
may be carcinogenic. Organic solvents are, in general, flammable and toxic by
inhalation, ingestion, and skin absorption. Sodium azide is carcinogenic and toxic
and liberates explosive, volatile, toxic hydrazoic acid when mixed with acid. Sodium
azide can form explosive heavy metal azides, for example, with plumbing fixtures,
and so should not be discharged down the drain.18 Disposal procedures have been
described for a number of hazardous drugs and reagents,18 and a procedure for the
hydrolysis of acetonitrile in waste solvent to the much less toxic acetic acid and
ammonia19,20 has been described. n-Hexane is surprisingly toxic.21
REFERENCES
1. O’Neil, M.J., Ed., The Merck Index, 13th edition, Merck & Co. Inc, Whitehouse Station,
NJ, 2001.
2. United States Pharmacopeial Convention. USP Dictionary of USAN and International
Drug Names, United States Pharmacopeial Convention, Rockville, MD, 2004.
3. Snyder, L.R.; Kirkland, J.J. Introduction to Modern Liquid Chromatography, 2nd edition, John Wiley & Sons, New York, 1979.
4. Lawrence, J.F. Organic Trace Analysis by Liquid Chromatography, Academic Press,
New York, 1981.
5. Sadek, P.C. The HPLC Solvent Guide, John Wiley & Sons, New York, 1996.
About This Book
xxiii
6. Snyder, L.R.; Kirkland, J.J.; Glajch, J.L. Practical HPLC Method Development, 2nd
edition, John Wiley & Sons, New York, 1997.
7. Meyer, V.R. Pitfalls and Errors of HPLC in Pictures, Hüthig, Heidelberg, Germany,
1997.
8. Meyer, V.R. Practical High-Performance Liquid Chromatography, 3rd edition, John
Wiley & Sons, Chichester, UK, 2000.
9. Sadek, P.C. Troubleshooting HPLC Systems. A Bench Manual, John Wiley & Sons,
New York, 2000.
10. Lewis, R.J. Sr. Sax’s Dangerous Properties of Industrial Materials, 8th edition, Van
Nostrand Reinhold, New York, 1992, pp. 356–358.
11. Lewis, R.J. Sr. Sax’s Dangerous Properties of Industrial Materials, 8th edition, Van
Nostrand Reinhold, New York, 1992, pp. 815–816.
12. Lewis, R.J. Sr. Sax’s Dangerous Properties of Industrial Materials, 8th edition, Van
Nostrand Reinhold, New York, 1992, pp. 2311–2312.
13. Lewis, R.J. Sr. Sax’s Dangerous Properties of Industrial Materials, 8th edition, Van
Nostrand Reinhold, New York, 1992, pp. 1449–1450.
14. Lewis, R.J. Sr. Sax’s Dangerous Properties of Industrial Materials, 8th edition, Van
Nostrand Reinhold, New York, 1992, pp. 701–702.
15. Lewis, R.J. Sr. Sax’s Dangerous Properties of Industrial Materials, 8th edition, Van
Nostrand Reinhold, New York, 1992, p. 1378.
16. Belpoggi, F.; Soffritti, M.; Maltoni, C. Methyl-tertiary-butyl ether (MTBE) – a gasoline additive – causes testicular and lympho-haematopoietic cancers in rats, Toxicol.Ind.Health., 1995, 11, 119–149.
17. Mehlman, M.A. Dangerous and cancer-causing properties of products and chemicals in
the oil refining and petrochemical industry: Part XV. Health hazards and health risks
from oxygenated automobile fuels (MTBE): lessons not heeded, Int.J.Occup.Med.Toxicol.,
1995, 4, 219–236.
18. Lunn, G.; Sansone, E.B. Destruction of Hazardous Chemicals in the Laboratory, 2nd
edition, John Wiley & Sons, New York, 1994.
19. Gilomen, K.; Stauffer, H.P.; Meyer, V.R. Detoxification of acetonitrile – water wastes
from liquid chromatography, Chromatographia, 1995, 41, 488–491.
20. Gilomen, K.; Stauffer, H.P.; Meyer, V.R. Management and detoxification of acetonitrile
wastes from liquid chromatography, LC.GC, 1996, 14, 56–58.
21. Meyer, V. A safer solvent, Anal.Chem., 1997, 69, 18A.
Abacavir
H
N
N
Molecular formula: C14 H18 N6 O
Molecular weight: 286.33
CAS Registry No: 136470-78-5 (base), 188062-50-2 (sulfate)
Merck Index: 13,1
HO
N
N
N
NH2
SAMPLE
Matrix: blood
Sample preparation: Condition a 1 mL 100 mg Bond Elut-C SPE cartridge with 1 mL
MeOH and 1 mL 100 mM pH 7.0 ammonium acetate buffer. Heat plasma at 58◦ for 1 h
to inactivate HIV. Vortex 800 µL plasma with 300 µL 2 µg/mL hexobarbital in 25 mM
pH 7.0 ammonium acetate buffer for 30 s and centrifuge at 18 000 g for 5 min. Add 1 mL
of the supernatant to the SPE cartridge, wash with 1 mL 100 mM pH 7.0 ammonium
acetate buffer, suck dry for 1 min, elute with 800 µL MeOH. Evaporate the eluate to
dryness under a stream of nitrogen at 40◦ and reconstitute the residue with 100 µL
mobile phase. Vortex for 30 s, centrifuge at 18 000 g for 3 min, and inject an 80 µL
aliquot.
HPLC VARIABLES
Guard column: 20 × 3.9 5 µm Polarity dC18 (Waters)
Column: 150 × 3.9 5 µm Polarity dC18 (Waters)
Column temperature: 40
Mobile phase: Gradient. A was 10 mM pH 6.5 ammonium acetate buffer. B was 10 mM
pH 6.5 ammonium acetate buffer:MeCN:MeOH 20:50:30. A:B 96:4 for 15 min, to 36:64
over 15 min, maintain at 36:64 for 3 min, re-equilibrate at initial conditions for 7 min.
Flow rate: 1.1
Injection volume: 80
Detector: UV 269 for 11 min, UV 250 for 3 min, UV 271 for 10 min, UV 230 for 9 min
CHROMATOGRAM
Retention time: 25.1
Internal standard: hexobarbital (30.6)
Limit of quantitation: 10.0 ng/mL
OTHER SUBSTANCES
Extracted: didanosine (13.6), lamivudine (8.6), nevirapine (27.3), stavudine (15.7), zalcitabine (5.9), zidovudine (23.8)
Noninterfering: tenofovir
KEY WORDS
plasma; SPE
REFERENCE
Rezk, N.L.; Tidwell, R.R.; Kashuba, A.D.M. Simultaneous determination of six HIV nucleoside analogue
reverse transcriptase inhibitors and nevirapine by liquid chromatography with ultraviolet absorbance
detection, J.Chromatogr.B, 2003, 791, 137–147.
SAMPLE
Matrix: blood
Sample preparation: Condition a 100 mg Dual Zone C18 SPE cartridge (Diazem) with
2 mL MeOH and 2 mL water. Dilute 500 µL serum with 1 mL water, add to the SPE
cartridge, wash with 500 µL water, elute with 1 mL MeOH. Evaporate the eluate to
HPLC Methods for Recently Approved Pharmaceuticals. by George Lunn
Copyright 2005 John Wiley & Sons, Ltd. ISBN: 0-471-66941-5
1
2
Abacavir
dryness with vortexing under reduced pressure at 40◦ and reconstitute the residue with
300 µL MeOH, inject a 10 µL aliquot.
HPLC VARIABLES
Column: two 150 × 4.6 3 µm Luna C18 columns in series
Column temperature: 60
Mobile phase: Gradient. MeCN:water from 5:95 to 45:55 over 20 min.
Flow rate: 0.85
Injection volume: 10
Detector: UV 250
CHROMATOGRAM
Retention time: 17
Limit of detection: 75 ng/mL
OTHER SUBSTANCES
Extracted: didanosine (10.5, LOD 120 ng/mL), lamivudine (9.5, LOD 260 ng/mL), stavudine (11.5, LOD 40 ng/mL), zalcitabine (7.5, LOD 440 ng/mL), zidovudine (16, LOD
30 ng/mL)
KEY WORDS
SPE; serum
REFERENCE
Simon, V.A.; Thiam, M.D.; Lipford, L.C. Determination of serum levels of thirteen human immunodeficiency virus-suppressing drugs by high-performance liquid chromatography, J.Chromatogr.A, 2001,
913, 447–453.
SAMPLE
Matrix: blood
Sample preparation: Mix 300 µL plasma with 75 µL 20% perchloric acid for 30 s,
centrifuge at 1300 g for 15 min, inject a 100 µL aliquot.
HPLC VARIABLES
Guard column: 20 × 3.8 Symmetry C18 (Waters)
Column: 100 × 4.6 3.5 µm Symmetry C18 (Waters)
Column temperature: 41 ± 2
Mobile phase: MeCN:25 mM pH 7.0 phosphate buffer 15:85
Flow rate: 1
Injection volume: 100
Detector: UV 285
CHROMATOGRAM
Retention time: 4.8
Limit of quantitation: 20 ng/mL
OTHER SUBSTANCES
Simultaneous: didanosine, folic acid, ganciclovir, lamivudine, nevirapine, pyrazinamide,
ranitidine, rifampin, stavudine, sulfamethoxazole, trimethoprim, zidovudine
Noninterfering: adefovir, amprenavir, delavirdine, efavirenz, fluconazole, indinavir,
itraconazole, methadone, nelfinavir, oxazepam, pyrimethamine, rifampin, ritonavir,
saquinavir, zalcitabine
KEY WORDS
plasma
Abacavir
3
REFERENCE
Veldkamp, A.I.; Sparidans, R.W.; Hoetelmans, R.M.W.; Beijnen, J.H. Quantitative determination of abacavir (1592U89), a novel nucleoside reverse transcriptase inhibitor, in human plasma using isocratic
reversed-phase high-performance liquid chromatography with ultraviolet detection, J.Chromatogr.B,
1999, 736, 123–128.
SAMPLE
Matrix: blood
Sample preparation: Centrifuge plasma at 4000 g for 20 min using a Centrifree
micropartition device (Amicon), inject a 100 µL aliquot of the ultrafiltrate.
HPLC VARIABLES
Column: 250 × 4.6 Adsorbsphere C18
Mobile phase: Gradient. A was MeCN:water 80:20. B was 50 mM ammonium acetate
containing 0.1% triethylamine adjusted to pH 5.5. A:B from 0:100 to 50:50 over 30 min,
re-equilibrate at initial conditions for 10 min.
Flow rate: 1
Injection volume: 100
Detector: UV 260, UV 285
CHROMATOGRAM
Retention time: 23
OTHER SUBSTANCES
Extracted: carbovir (20)
KEY WORDS
rat; pharmacokinetics; plasma
REFERENCE
Daluge, S.M.; Good, S.S.; Faletto, M.B.; Miller, W.H.; St.Clair, M.H.; Boone, L.R.; Tisdale, M.; Parry,
N.R.; Reardon, J.E.; Dornsife, R.E.; Averett, D.R.; Krenitsky, T.A. 1592U89, a novel carbocyclic nucleoside analog with potent, selective anti-human immunodeficiency virus activity, Antimicrob.Agents
Chemother., 1997, 41, 1082–1093.
SAMPLE
Matrix: CSF, urine
Sample preparation: Centrifuge CSF or urine at 12 000 g for 5 min, dilute a 75 µL
aliquot to 750 µL with mobile phase, inject an aliquot.
HPLC VARIABLES
Column: 150 × 3.2 5 µm Kromasil C18 (Phenomenex)
Mobile phase: Gradient. MeOH:25 mM pH 4.0 ammonium acetate buffer from 5:95 to
50:50 over 30 min, re-equilibrate at initial conditions for 10 min.
Flow rate: 0.7
Detector: UV 295
CHROMATOGRAM
Retention time: 25.5
Limit of quantitation: 62 ng/mL (CSF), 629 ng/mL (urine)
OTHER SUBSTANCES
Extracted: metabolites, abacavir 5′ -glucuronide, abacavir 5′ -carboxylate
4
Abacavir
REFERENCE
Ravitch, J.R.; Moseley, C.G. High-performance liquid chromatographic assay for abacavir and its two
major metabolites in human urine and cerebrospinal fluid, J.Chromatogr., 2001, 762, 165–173.
ANNOTATED BIBLIOGRAPHY
Fung, E.N.; Cai, Z.; Burnette, T.C.; Sinhababu, A.K. Simultaneous determination of Ziagen and its
phosphorylated metabolites by ion-pairing high-performance liquid chromatography-tandem mass
spectrometry, J.Chromatogr.B, 2001, 754, 285–295. [LC-MS]
Sparidans, R.W.; Hoetelmans, R.M.W.; Beijnen, J.H. Liquid chromatographic assay for simultaneous
determination of abacavir and mycophenolic acid in human plasma using dual spectrophotometric
detection, J.Chromatogr.B, 2001, 750, 155–161.
Thomas, S.A.; Bye, E.; Segal, M.B. Transport characteristics of the anti-human immunodeficiency virus
nucleoside analog, abacavir, into brain and cerebrospinal fluid, J.Pharmacol.Exp.Ther., 2001, 298,
947–953.
Yuen, G.J.; Lou, Y.; Thompson, N.F.; Otto, V.R.; Allsup, T.L.; Mahony, W.B.; Hutman, H.W.
Abacavir/lamivudine/zidovudine as a combined formulation tablet: Bioequivalence compared with
each component administered concurrently and the effect of food on absorption, J.Clin.Pharmacol.,
2001, 41, 277–288.
Aymard, G.; Legrand, M.; Trichereau, N.; Diquet, B. Determination of twelve antiretroviral agents
in human plasma sample using reversed-phase high-performance liquid chromatography,
J.Chromatogr.B, 2000, 744, 227–240. [for amprenavir; efavirenz; indinavir; nelfinavir; ritonavir;
saquinavir; abacavir; didanosine; lamivudine; stavudine; nevirapine; zidovudine]
McDowell, J.A.; Lou, Y.; Symonds, W.S.; Stein, D.S. Multiple-dose pharmacokinetics and pharmacodynamics of abacavir alone and in combination with zidovudine in human immunodeficiency virusinfected adults, Antimicrob.Agents Chemother., 2000, 44, 2061–2067.
Kumar, P.N.; Sweet, D.E.; McDowell, J.A.; Symonds, W.; Lou, Y.; Hetherington, S.; LaFon, S. Safety
and pharmacokinetics of abacavir (1592U89) following oral administration of escalating single doses
in human immunodeficiency virus type 1-infected adults, Antimicrob.Agents Chemother., 1999, 43,
603–608.
McDowell, J.A.; Chittick, G.E.; Ravitch, J.R.; Polk, R.E.; Kerkering, T.M.; Stein, D.S. Pharmacokinetics
of [14 C]abacavir, a human immunodeficiency virus type 1 (HIV-1) reverse transcriptase inhibitor,
administered in a single oral dose to HIV-1-infected adults: a mass balance study, Antimicrob.Agents
Chemother., 1999, 43, 2855–2861.
Wang, L.H.; Chittick, G.E.; McDowell, J.A. Single-dose pharmacokinetics and safety of abacavir
(1592U89), zidovudine, and lamivudine administered alone and in combination in adults with human
immunodeficiency virus infection, Antimicrob.Agents Chemother., 1999, 43, 1708–1715.
Acarbose
Acarbose
Molecular formula: C25 H43 NO18
5
OH
HO
HO
Molecular weight: 645.60
CAS Registry No: 56180-94-0
Merck Index: 13, 18
OH
H
H3C
N
HO
O
OH
O
HO
OH
O
OH
O
HO
OH
O
OH
OH
SAMPLE
Matrix: formulations
Sample preparation: Powder tablet, extract 3 times with 5 mL aliquots of water with
sonication for 15 min with vortexing at 5 min intervals each time, centrifuge at 2750 g
for 5 min, combine supernatants, make up to 20 mL with water. Dilute a 50 µL aliquot
to 1 mL with MeOH, filter (0.2 µM), inject a 20 µL aliquot.
HPLC VARIABLES
Column: 250 × 4.6 5 µm Nucleosil-NH2
Mobile phase: MeOH:dichloromethane 65:35
Flow rate: 1
Injection volume: 20
Detector: ELSD, nebulizing gas air at 2.5 bar and 4 L/min, solvent evaporated at 40◦
CHROMATOGRAM
Retention time: 4.1
Limit of detection: 5 µg/mL
Limit of quantitation: 15 µg/mL
OTHER SUBSTANCES
Simultaneous: sucrose (3.5)
KEY WORDS
comparison with capillary electrophoresis; tablets
REFERENCE
Cherkaoui, S.; Daali, Y.; Christen, P.; Veuthey, J.-L. Development and validation of liquid chromatography and capillary electrophoresis methods for acarbose determination in pharmaceutical tablets,
J.Pharm.Biomed.Anal., 1998, 18, 729–735.
6
Acetyl sulfisoxazole
Acetyl sulfisoxazole
Molecular formula: C13 H15 N3 O4 S
Molecular weight: 309.35
CAS Registry No: 80-74-0
Merck Index: 13, 9041
O
O
S
H2N
O
O N
CH3
N
CH3
CH3
SAMPLE
Matrix: formulations
Sample preparation: Extract 1 mL suspension with three 15 mL aliquots of chloroform
(Caution! Chloroform is a carcinogen!), combine the organic layers and make up to 50 mL
with chloroform, filter (0.45 µm silver membrane, Selas Corp.). Evaporate a 2 mL aliquot
of the filtrate to dryness under a stream of nitrogen, reconstitute with 5 mL 330 µg/mL
benzanilide in MeCN, inject a 5 µL aliquot.
HPLC VARIABLES
Column: 300 × 4 10 µm µBondapak C18
Mobile phase: MeCN:water 40:60
Flow rate: 1.5
Injection volume: 5
Detector: UV 254
CHROMATOGRAM
Retention time: 7
Internal standard: benzanilide (11)
OTHER SUBSTANCES
Simultaneous: sulfanilamide (2.5), sulfisoxazole (3)
Noninterfering: erythromycin ethylsuccinate
KEY WORDS
oral suspensions
REFERENCE
Elrod, L. Jr.; Cox, R.D.; Plasz, A.C. Analysis of oral suspensions containing sulfonamides in combination
with erythromycin ethylsuccinate, J.Pharm.Sci., 1982, 71, 161–166.
ANNOTATED BIBLIOGRAPHY
Suber, R.L.; Edds, G.T. High performance liquid chromatographic determinations of sulfonamides by
ionic suppression, J.Liq.Chromatogr., 1980, 3, 257–268. [for sulfanilamide; sulfaguanidine; sulfamerazine; sulfamethazine; sulfapyridine; sulfisoxazole; N-acetylsulfisoxazole; sulfathiazole; in plasma]
Acrivastine
Acrivastine
N
N
Molecular formula: C22 H24 N2 O2
Molecular weight: 348.44
CAS Registry No: 87848-99-5
Merck Index: 13, 129
7
COOH
H3C
SAMPLE
Matrix: blood
Sample preparation: Mix 1 mL whole blood with 20 µL 1 µg/mL dibenzepin in
MeOH:water 50:50, add 300 µL pH 11 tris buffer, mix, add 500 µL butyl acetate,
vortex for 2 min, centrifuge. Preserve the aqueous layer (A). Remove the organic layer
and add it to 75 µL 10 mM ammonium acetate buffer containing 0.1% formic acid (pH
3.2), evaporate (?). Add 75 µL MeCN, sonicate for 5 min, centrifuge at 5000 rpm for
5 min, keep the extract as B. Add 20 µL 1 µg/mL enalapril in MeOH:water 50:50 and
120 mg NaCl to the aqueous layer (A), mix, add 500 µL pH 3 phosphate buffer, add
600 µL 8.5% phosphoric acid, add 5 mL dichloromethane:isopropanol 95:5, shake at
250 cycles/min in a bench-top shaker for 30 min, centrifuge at 5000 rpm for 5 min.
Remove the lower organic layer and evaporate it to dryness under a stream of air at
45◦ . Reconstitute the residue with 150 µL initial mobile phase, sonicate, centrifuge,
combine with extract B, inject a 30 µL aliquot. (Sample preparation from Gergov,M.;
Robson,J.N.; Ojanperä,I.; Heinonen,O.P.; Vuori,E. Simultaneous screening and quantitation of 18 antihistamine drugs in blood by liquid chromatography ionspray tandem
mass spectrometry. Forensic Sci.Inter. 2001, 121, 108–115.)
HPLC VARIABLES
Guard column: 40 mm long 4 µm Purospher RP-18 LiChro Cart 4-4
Column: 100 × 2.1 4 µm Genesis C18 (Jones Chromatography)
Column temperature: 35
Mobile phase: Gradient. MeCN:buffer from 20:80 to 100:0 over 10 min, maintain at
0:100 for 3 min, re-equilibrate at initial conditions for 5 min. (Buffer was 10 mM
ammonium acetate containing 0.1% formic acid (pH 3.2).
Flow rate: 0.2
Injection volume: 30
Detector: MS, PE Sciex API 365 triple stage quadrupole LC-MS-MS, PE Sciex Turbo Ion
Spray interface, positive ion mode, needle voltage 5.2 kV, nebulizer gas air at 60 psi,
curtain gas nitrogen at 40 psi, collision cell gas nitrogen at 40 psi, turbo ionspray heater
375◦ , heater gas flow 7 L/min
CHROMATOGRAM
Retention time: 5.7
Internal standard: dibenzepin, enalapril
Limit of detection: <20 ng/mL
OTHER SUBSTANCES
Extracted: acebutolol (3.8, LOD 0.1 µg/mL), acetaminophen (2.5, LOD <5 µg/mL),
alprazolam (6.1, LOD <0.02 µg/mL), alprenolol (5.4, LOD 0.01 µg/mL), amantadine
(3.4, LOD 0.1 µg/mL), amiloride (2.0, LOD 0.1 µg/mL), aminophenazone (2.8, LOD
<5 µg/mL), amiodarone (10.2, LOD 0.05 µg/mL), amitriptyline (6.6, LOD <0.02 µg/mL),
astemizole (5.8, LOD <0.02 µg/mL), atenolol (1.7, LOD 0.30 µg/mL), azacyclonol (5.1,
LOD 0.02 µg/mL), benzhexol (6.6, LOD <0.02 µg/mL), benzoylecgonine (3.3, LOD
0.01 µg/mL), betaxolol (5.5, LOD 0.01 µg/mL), biperidine (6.2, LOD <0.02 µg/mL),
bisoprolol (5.0, LOD <0.02 µg/mL), brompheniramine (5.3, LOD 0.002 µg/mL),
bupivacaine (5.1, LOD <0.02 µg/mL), buprenorphine (5.9, LOD 0.01 µg/mL),
buspirone (5.1, LOD 0.002 µg/mL), caffeine (2.8, LOD 1 µg/mL), carbamazepine
8
Acrivastine
(6.1, LOD <0.02 µg/mL), carbinoxamine (5.1, LOD 0.002 µg/mL), carisoprodol
(6.7, LOD <5 µg/mL), carvedilol (6.2, LOD <0.02 µg/mL), celiprolol (4.3, LOD
0.05 µg/mL), cetirizine (6.3, LOD 0.05 µg/mL), chlorcyclizine (6.6, LOD <0.02 µg/mL),
chlordiazepoxide (5.7, LOD <0.02 µg/mL), chlormezanone (5.8, LOD <5 µg/mL),
chloroquine (2.7, LOD 0.02 µg/mL), chlorpheniramine (5.1, LOD 0.002 µg/mL),
chlorpromazine (7.0, LOD 0.02 µg/mL), chlorpropamide (6.7, LOD <5 µg/mL),
chlorprothixene (7.0, LOD <0.02 µg/mL), cinnarizine (7.9, LOD <0.02 µg/mL),
citalopram (5.7, LOD <0.02 µg/mL), clemastine (7.7, LOD 0.02 µg/mL), clobazam
(7.3, LOD <0.02 µg/mL), clobutinol (5.3, LOD 0.02 µg/mL), clomethiazole (6.2,
LOD 0.5 µg/mL), clomipramine (7.1, LOD <0.02 µg/mL), clonazepam (6.6, LOD
<0.02 µg/mL), clonidine (2.8, LOD 0.1 µg/mL), clozapine (5.6, LOD <0.02 µg/mL),
cocaine (4.6, LOD <0.02 µg/mL), codeine (2.5, LOD 0.1 µg/mL), coumatetralyl
(8.4, LOD 0.05 µg/mL), cyclizine (5.8, LOD <0.02 µg/mL), dextropropoxyphene (6.6,
LOD 0.05 µg/mL), demoxepam (5.8, LOD 0.02 µg/mL), dextromethorphan (5.5, LOD
<0.02 µg/mL), diazepam (8.1, LOD 0.02 µg/mL), diltiazem (5.8, LOD <0.02 µg/mL),
diphenhydramine (5.7, LOD <0.02 µg/mL), dipyridamole (5.4, LOD 0.005 µg/mL),
disopyramine (4.4, LOD <0.02 µg/mL), dixyrazine (6.8, LOD 0.005 µg/mL), doxapram
(4.8, LOD <0.02 µg/mL), doxepin (5.9, LOD <0.02 µg/mL), dronabinol (12.3, LOD
0.05 µg/mL), ebastine (9.6, LOD 0.005 µg/mL), embutramide (6.7, LOD 0.005 µg/mL),
ergotamine (5.5, LOD 0.005 µg/mL), ethenzamide (5.0, LOD 0.05 µg/mL), ethylmorphine
(3.2, LOD 0.05 µg/mL), ethylparathion (9.7, LOD <5 µg/mL), etodroxizine (6.4, LOD
<0.02 µg/mL), felodipine (9.6, LOD 0.02 µg/mL), fenazepam (7.5, LOD <0.02 µg/mL),
fenfluramine (5.3, LOD <0.02 µg/mL), fenkamfamine (5.1, LOD <0.02 µg/mL),
fentanyl (5.5, LOD <0.02 µg/mL), fexofenadine (6.3, LOD <0.02 µg/mL), flecainide
(5.9, LOD <0.02 µg/mL), fluconazole (4.0, LOD 0.1 µg/mL), flumazenil (5.2, LOD
<0.02 µg/mL), flunitrazepam (7.1, LOD 0.002 µg/mL), fluoxetine (6.8, LOD 0.1 µg/mL),
flupentixol (7.5, LOD 0.18 µg/mL), fluvoxamine (6.3, LOD 0.02 µg/mL), glibenclamide
(8.5, LOD <0.02 µg/mL), glipizide (6.8, LOD <0.05 µg/mL), haloperidol (6.1,
LOD <0.02 µg/mL), histapyrrodine (6.3, LOD 0.02 µg/mL), hydrocodone (3.0, LOD
0.05 µg/mL), hydroxychloroquine (2.4, LOD <0.3 µg/mL), hydroxyzine (6.3, LOD
<0.02 µg/mL), imipramine (6.4, LOD 0.05 µg/mL), indomethacin (8.6, LOD 0.05 µg/mL),
isoniazid (2.2, LOD 3 µg/mL), isradipine (8.6, LOD 0.05 µg/mL), ketamine (3.6, LOD
<0.05 µg/mL), ketobemidone (3.3, LOD <0.05 µg/mL), ketoprofen (7.3, LOD 0.1 µg/mL),
ketorolac (6.2, LOD 0.05 µg/mL), labetalol (4.9, LOD 0.05 µg/mL), lamotrigine (4.0,
LOD 0.1 µg/mL), levocabastine (5.8, LOD 0.01 µg/mL), levomepromazine (6.5, LOD
0.02 µg/mL), lidocaine (3.7, LOD <0.05 µg/mL), loratadine (9.3, LOD 0.002 µg/mL),
lorazepam (6.6, LOD 0.02 µg/mL), lormetazepam (7.4, LOD <0.02 µg/mL), LSD
(4.7, LOD <0.02 µg/mL), malathion (8.9, LOD 10 µg/mL), maprotiline (6.4, LOD
<0.02 µg/mL), MDMA (3.3, LOD 0.02 µg/mL), meclozine (8.5, LOD <0.02 µg/mL),
medazepam (6.3, LOD <0.02 µg/mL), meloxicam (7.1, LOD 0.01 µg/mL), melperone
(5.0, LOD <0.02 µg/mL), meperidine (4.7, LOD <0.02 µg/mL), mepivacaine (3.7,
LOD <0.02 µg/mL), meprobamate (4.9, LOD 0.1 µg/mL), mesoridazine (5.4, LOD
<0.02 µg/mL), methamphetamine (3.3, LOD 0.05 µg/mL), methadone (6.7, LOD
<0.02 µg/mL), methylparathion (8.6, LOD 10 µg/mL), methylphenidate (4.2, LOD
<0.02 µg/mL), metoclopramide (3.8, LOD <0.02 µg/mL), metoprolol (4.1, LOD
0.02 µg/mL), metronidazole (2.6, LOD 1 µg/mL), mexiletine (4.4, LOD 0.05 µg/mL),
mianserin (5.7, LOD <0.02 µg/mL), midazolam (5.9, LOD <0.02 µg/mL), mirtazapine
(4.4, LOD <0.02 µg/mL), mizolastine (5.5, LOD 0.01 µg/mL), moclobemide (3.7,
LOD 0.05 µg/mL), molindone (4.0, LOD <0.02 µg/mL), monoacetylmorphine (2.7,
LOD 0.1 µg/mL), morphine (2.0, LOD 0.1 µg/mL), nicotine (2.2, LOD 0.05 µg/mL),
nifedipine (7.5, LOD 0.02 µg/mL), nikethamide (3.6, LOD <0.02 µg/mL), nitrazepam
(6.5, LOD <0.02 µg/mL), nizatidine (1.7, LOD 1 µg/mL), nomifensine (4.6, LOD
<0.02 µg/mL), nortriptyline (6.4, LOD <0.02 µg/mL), norverapamil (6.2, LOD 1 µg/mL),
noscapine (5.0, LOD <0.02 µg/mL), olanzapine (3.0, LOD 0.05 µg/mL), ondansetron
(4.6, LOD <0.02 µg/mL), orphenadrine (6.1, LOD <0.02 µg/mL), oxazepam (6.3,
LOD <0.02 µg/mL), oxcarbazepine (5.3, LOD 0.02 µg/mL), oxprenolol (4.7, LOD
0.02 µg/mL), oxycodone (2.8, LOD 0.05 µg/mL), papaverine (4.8, LOD <0.02 µg/mL),
paroxetine (6.2, LOD 0.02 µg/mL), pemoline (3.3, LOD 0.05 µg/mL), pentazocine
(5.0, LOD <0.02 µg/mL), pentifylline (7.3, LOD <5 µg/mL), pentoxyverine (6.6,
LOD <0.02 µg/mL), perphenazine (6.9, LOD 0.002 µg/mL), phenazone (3.9, LOD
Acrivastine
9
0.05 µg/mL), phencyclidine (5.3, LOD 0.05 µg/mL), pheniramine (4.1, LOD 0.02 µg/mL),
phenylbutazone (9.0, LOD <5 µg/mL), phenylpropanolamine (2.5, LOD 0.3 µg/mL),
phenytoin (6.1, LOD 0.05 µg/mL), pindolol (3.3, LOD 0.05 µg/mL), piroxicam (6.6, LOD
0.02 µg/mL), pitofenone (5.4, LOD <0.02 µg/mL), pizotifen (6.5, LOD <0.02 µg/mL),
practolol (1.8, LOD 0.1 µg/mL), prazosin (4.1, LOD 0.05 µg/mL), prilocaine (3.8, LOD
<0.02 µg/mL), primidone (4.0, LOD <5 µg/mL), procainamide (2.2, LOD 0.05 µg/mL),
prochlorperazine (7.5, LOD 0.02 µg/mL), promazine (6.2, LOD <0.02 µg/mL),
promethazine (6.0, LOD 0.05 µg/mL), propafenone (6.3, LOD <0.02 µg/mL), propranolol
(5.4, LOD 0.02 µg/mL), propyphenazone (6.6, LOD 0.50 µg/mL), pseudoephedrine (2.6,
LOD 1 µg/mL), quinine (4.2, LOD 0.02 µg/mL), ranitidine (1.8, LOD 0.1 µg/mL),
risperidone (4.9, LOD <0.02 µg/mL), rocurone (3.8, LOD 0.1 µg/mL), ropivacaine
(4.6, LOD <0.02 µg/mL), salicylamide (4.2, LOD <5 µg/mL), selegiline (4.1, LOD
0.05 µg/mL), sertindole (7.2, LOD <0.02 µg/mL), sertraline (6.8, LOD 0.02 µg/mL),
sulindac (6.5, LOD 0.02 µg/mL), simazine (6.0, LOD 0.1 µg/mL), sincocaine (6.5,
LOD <0.02 µg/mL), sisapride (5.9, LOD <0.02 µg/mL), sotalol (2.1, LOD 0.1 µg/mL),
strychnine (5.3, LOD 0.05 µg/mL), sulpiride (1.9, LOD 0.1 µg/mL), sulthiame (4.1, LOD
0.05 µg/mL), temazepam (7.2, LOD <0.02 µg/mL), terbutaline (2.3, LOD 0.1 µg/mL),
terfenadine (8.1, LOD 0.002 µg/mL), terodiline (6.7, LOD <0.02 µg/mL), tetracaine (5.7,
LOD <0.02 µg/mL), tetrahydrozoline (3.6, LOD 0.1 µg/mL), theobromine (2.3, LOD
<5 µg/mL), theophylline (2.4, LOD <5 µg/mL), thioridazine (7.5, LOD 0.02 µg/mL),
timolol (3.8, LOD 0.05 µg/mL), thiothixene (6.7, LOD 0.02 µg/mL), tolbutamide (7.1,
LOD <5 µg/mL), toremifene (8.7, LOD 0.02 µg/mL), tramadol (4.2, LOD 0.02 µg/mL),
trazodone (5.2, LOD <0.02 µg/mL), triamterene (3.2, LOD 0.1 µg/mL), triazolam (6.7,
LOD 0.002 µg/mL), trimeprazine (6.4, LOD <0.02 µg/mL), trimethoprim (3.1, LOD
0.05 µg/mL), trimipramine (6.7, LOD <0.02 µg/mL), venlafaxine (4.9, LOD 0.02 µg/mL),
verapamil (6.5, LOD <0.02 µg/mL), warfarin (7.9, LOD <0.02 µg/mL), yohimbine (4.5,
LOD <0.02 µg/mL), zolpidem (4.7, LOD <0.02 µg/mL), zopiclone (4.0, LOD 0.1 µg/mL)
KEY WORDS
whole blood
REFERENCE
Gergov, M.; Ojanperä, I.; Vuori, E. Simultaneous screening for 238 drugs in blood by
liquid chromatography-ionspray tandem mass spectrometry with multiple-reaction monitoring,
J.Chromatogr.B, 2003, 795, 41–53.
10
Adapalene
Adapalene
COOH
Molecular formula: C28 H28 O3
Molecular weight: 412.52
CAS Registry No: 106685-40-9
Merck Index: 13, 150
H3CO
SAMPLE
Matrix: formulations
Sample preparation: Inject an aliquot of a 0.1% gel.
HPLC VARIABLES
Column: 250 × 4 ODS-RP18 (Merck)
Mobile phase: MeCN:THF:water:trifluoroacetic acid 43:36:21:0.02
Flow rate: 1
Detector: UV 270
CHROMATOGRAM
Retention time: 6.1
OTHER SUBSTANCES
Noninterfering: tretinoin
KEY WORDS
gel
REFERENCE
Martin, B.; Meunier, C.; Montels, D.; Watts, O. Chemical stability of adapalene and tretinoin when
combined with benzoyl peroxide in presence and in absence of visible light and ultraviolet radiation,
Br.J.Dermatol., 1998, 139 (Suppl. 52), 8–11.
11
Adefovir dipivoxil
Adefovir dipivoxil
Molecular formula: C20 H32 N5 O8 P
Molecular weight: 501.47
CAS Registry No: 142340-99-6
Merck Index: 13, 151
NH2
CH3
CH3
N
N
O
N
CH3
O
N
O
P
O
O
O
O
O
CH3
CH3
CH3
SAMPLE
Matrix: blood
Sample preparation: Mix 100 µL plasma with 200 µL 0.1% trifluoroacetic acid in
MeCN. Evaporate the supernatant to dryness under reduced pressure at room temperature. Reconstitute with 0.34% chloroacetaldehyde in 100 mM pH 4.5 sodium acetate,
vortex, centrifuge. Heat the supernatant at 95◦ for 40 min, evaporate to dryness,
reconstitute with 100 µL 25 mM pH 6.0 potassium phosphate buffer containing 5 mM
tetrabutylammonium hydrogen phosphate, inject a 50 µL aliquot.
HPLC VARIABLES
Guard column: 15 × 3.2 Brownlee RP-18 Newguard
Column: 150 × 4.6 Zorbax RX-C18
Column temperature: 35
Mobile phase: Gradient. A was MeCN:25 mM pH 6.0 potassium phosphate buffer
containing 5 mM tetrabutylammonium hydrogen phosphate 2:98. B was MeCN:25 mM
pH 6.0 potassium phosphate buffer containing 5 mM tetrabutylammonium hydrogen
phosphate 65:35. A:B 100:0 for 2 min, to 0:100 over 13 min, re-equilibrate at initial
conditions for 10 min. (Only adefovir is detected in blood. However, the method is
reported to distinguish between adefovir and adefovir dipivoxil.)
Flow rate: 1.5
Injection volume: 50
Detector: F ex 236 em 420
KEY WORDS
derivatization; dog; pharmacokinetics; plasma
REFERENCE
Cundy, K.C.; Sue, I.-L.; Visor, G.C.; Marshburn, J.; Nakamura, C.; Lee, W.A.; Shaw, J.P. Oral formulations of adefovir dipivoxil: In vitro dissolution and in vivo bioavailability in dogs, J.Pharm.Sci., 1997,
86, 1334–1338.
SAMPLE
Matrix: blood
Sample preparation: Vortex 200 µL plasma with 50 µL 20% trichloroacetic acid in
water, centrifuge at 1300 g for 15 min. Remove 150 µL of the supernatant and mix it
with 50 µL 160 mM chloroacetaldehyde in water containing 2 M sodium acetate, vortex,
close the tube, heat at 98◦ for 30 min, cool to 2◦ , vortex, inject a 20 µL aliquot.
HPLC VARIABLES
Guard column: 10 × 3 R3 (Chrompack)
Column: 150 × 4.6 5 µm Chromspher C8
Column temperature: 40 ± 2
Mobile phase: MeCN:buffer 10:90 (Buffer was 10 mM pH 7.0 sodium phosphate buffer
containing 2 mM tetrabutylammonium hydrogen sulfate.)
Flow rate: 1.5
12
Adefovir dipivoxil
Injection volume: 20
Detector: F ex 254 em 425
CHROMATOGRAM
Retention time: 4.5
Limit of quantitation: 10 ng/mL
OTHER SUBSTANCES
Extracted: adefovir (4)
KEY WORDS
derivatization; plasma
REFERENCE
Sparidans, R.W.; Veldkamp, A.; Hoetelmans, R.M.W.; Beijnen, J.H. Improved and simplified liquid chromatographic assay for adefovir, a novel antiviral drug, in human plasma using derivatization with
chloroacetaldehyde, J.Chromatogr.B, 1999, 736, 115–121.
Adrenocorticotropic hormone
13
Adrenocorticotropic hormone
CAS Registry No: 9002-60-2
Merck Index: 13, 136
SAMPLE
Matrix: blood
Sample preparation: Condition a 1 mL Analytichem weak cation-exchange (carboxymethylhydrogen form, CBA) SPE cartridge with 1 mL 1% trifluoroacetic acid
in MeOH, 1 mL MeOH, and 2 mL water. Add 1 mL plasma to the SPE cartridge, rinse
the tube with 1 mL water, add the rinse to the SPE cartridge, wash with 1 mL 1%
trifluoroacetic acid in water, wash with 2 mL water, wash with 2 mL MeOH, elute with
2 mL 1% trifluoroacetic acid in MeOH. Evaporate the eluate to dryness under a stream
of nitrogen, reconstitute the residue in 100 µL MeOH:buffer 50:50, inject a 5–75 µL
aliquot. (Buffer was 5.7 g monochloroacetic acid, 2.0 g NaOH, and 0.2 g disodium EDTA
in 1 L water, pH 3.2.) (The procedure was not necessarily validated for this compound.)
HPLC VARIABLES
Column: 250 × 2 5 µm Ultrasphere octyl
Column temperature: 60
Mobile phase: Gradient. A was MeOH containing 10 mM sodium octanesulfonate. B
was buffer containing 10 mM sodium octanesulfonate. A:B from 45:55 to 70:30 over
30 min, maintain at 70:30 for 1 h. (The buffer was 5.7 g monochloroacetic acid, 2.0 g
NaOH, and 0.2 g disodium EDTA in 1 L water, pH 3.2.)
Flow rate: 0.3
Injection volume: 5–75
Detector: F ex 390 em 470 following post-column reaction. The column effluent
mixed with 400 mM NaOH pumped at 0.15 mL/min and 0.05% ninhydrin pumped
at 0.05 mL/min and the mixture flowed through a 12 m ×0.33 mm ID reaction coil at
70◦ to the detector.
CHROMATOGRAM
Retention time: 45
Limit of detection: 100 fmole
OTHER SUBSTANCES
Simultaneous: angiotensin I, angiotensin II, angiotensin III, atrial natriuretic peptide,
bombesin, bradykinin, gonadorelin (LHRH), somatoliberin, vasopressin
KEY WORDS
plasma; post-column reaction; SPE
REFERENCE
Rhodes, G.R.; Boppana, V.K. High-performance liquid chromatographic analysis of arginine-containing
peptides in biological fluids by means of a selective post-column reaction with fluorescence detection,
J.Chromatogr., 1988, 444, 123–131.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 300 × 3.9 10 µm µBondapak C18
Mobile phase: Gradient. A was 0.08% trifluoroacetic acid. B was MeCN:0.08% trifluoroacetic acid 70:30. A:B from 70:30 to 50:50 over 30 min.
14
Adrenocorticotropic hormone
Flow rate: 1
Detector: UV 206
CHROMATOGRAM
Retention time: 25
OTHER SUBSTANCES
Simultaneous: adrenocorticotropic hormone fragments, melanotropin
KEY WORDS
human
REFERENCE
McDermott, J.R.; Smith, A.I.; Biggins, J.A.; Al-Noaemi, M.C.; Edwardson, J.A. Characterization and
determination of neuropeptides by high-performance liquid chromatography and radioimmunoassay,
J.Chromatogr., 1981, 222, 371–379.
SAMPLE
Matrix: solutions
Sample preparation: Dissolve in 100 mM NaH2 PO4 adjusted to pH 2.1 with orthophosphoric acid, inject a 100 µL aliquot.
HPLC VARIABLES
Column: 250 × 4 Aquapore RP 300 (Kontron)
Mobile phase: Gradient. A was 100 mM NaH2 PO4 adjusted to pH 2.1 with orthophosphoric acid. B was MeOH. A:B from 90:10 to 35:65 over 180 min.
Flow rate: 1
Injection volume: 100
Detector: UV 225
CHROMATOGRAM
Retention time: 145
OTHER SUBSTANCES
Simultaneous: adrenocorticotropin hormone fragments, lipotropic hormone and fragments, melanotropin, endorphins, prolactin, somatropin, menotropins
KEY WORDS
pig
REFERENCE
Richter, W.O.; Schwandt, P. Separation of neuropeptides by HPLC: evaluation of different supports, with
analytical and preparative applications to human and porcine neurophysins, β-lipotropin, adrenocorticotropic hormone, and β-endorphin, J.Neurochem., 1985, 44, 1697–1703.
ANNOTATED BIBLIOGRAPHY
Capp, M.W.; Simonian, M.H. Separation of the major adrenal steroids by reversed-phase highperformance liquid chromatography, Anal.Biochem., 1985, 147, 374–381.
Janssen, P.S.; van Nispen, J.W.; Hamelinck, R.L.; Melgers, P.A.; Goverde, B.C. Application of reversedphase HPLC in some critical peptide separations, J.Chromatogr.Sci., 1984, 22, 234–238.
Smith, A.I.; McDermott, J.R. High-performance liquid chromatography of neuropeptides using radially
compressed polythene cartridges, J.Chromatogr., 1984, 306, 99–108.
15
Afloqualone
Afloqualone
Molecular formula: C16 H14 FN3 O
Molecular weight: 283.30
CAS Registry No: 56287-74-2
Merck Index: 13, 183
N
H2N
F
N
O
H3C
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: Chiralpak AS
Column temperature: 50
Mobile phase: Hexane:EtOH 95:5
Flow rate: 1.3
Detector: UV 254
CHROMATOGRAM
Retention time: 30, 35 (enantiomers)
KEY WORDS
chiral
REFERENCE
Application Guide for Chiral Column Selection, Second Edition; Chiral Technologies: Exton PA, 1995,
p. 43.
16
Alacepril
Alacepril
H
O
O
Molecular formula: C20 H26 N2 O5 S
Molecular weight: 406.50
CAS Registry No: 74258-86-9
Merck Index: 13, 200
N
O
COOH
H3C
S
N
CH3
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 × 4.6 10 µm Cosmosil 5C18-MS
Column temperature: 50
Mobile phase: Gradient. MeCN:10 mM pH 2.5 potassium phosphate buffer 0:100 for
2 min, to 75:25 over 7.5 min, maintain at 75:25 for 5.5 min. Isocratic. MeCN:buffer 40:60
Flow rate: 1.5
Detector: UV (wavelength not specified)
CHROMATOGRAM
Retention time: 10.9 (gradient) or 4.1 (isocratic)
OTHER SUBSTANCES
Simultaneous: acetaminophen (7.9), ampicillin (7.9), aspirin (10.0), caffeine (8.5), carbenicillin (9.5), cefotiam (7.2), chlorpromazine (10.8), cromolyn (8.9), enalapril (9.9),
loperamide (11.6), ofloxacin (8.3), procainamide (7.4), procaine (7.9), propranolol (9.6),
sultamicillin tosylate (8.3), tegafur (8.4), temocapril (12.3), theophylline (8.0), tulobuterol
(8.9) (gradient retention times; isocratic conditions may differ)
REFERENCE
Sugiyama, T.; Matsuyama, R.; Usui, S.; Katagiri, Y.; Hirano, K. Selection of mobile phases in highperformance liquid chromatographic determination for medicines, Biol.Pharm.Bull., 2000, 23,
274–278.
SAMPLE
Matrix: enzyme reactions
Sample preparation: Mix 40 µL enzyme reaction mixture with 200 µL MeCN, add
200 µL of a 20 µg/mL solution of n-propyl paraben, centrifuge, inject a 30 µL aliquot of
the supernatant.
HPLC VARIABLES
Column: 250 × 4.6 Cosmosil 5-C18 MS
Column temperature: 50
Mobile phase: MeCN:10 mM pH 2.5 potassium phosphate buffer 40:60
Flow rate: 1.5
Injection volume: 30
Detector: UV 220
CHROMATOGRAM
Internal standard: n-propyl paraben
Alacepril
17
OTHER SUBSTANCES
Extracted: deacetylalacepril
REFERENCE
Usui, S.; Kubota, M.; Iguchi, K.; Kiho, T.; Sugiyama, T.; Katagiri, Y.; Hirano, K. Sialic acid 9-Oacetylesterase catalyzes the hydrolyzing reaction from alacepril to deacetylalacepril, Pharm.Res.,
2003, 20, 1309–1316.
18
Alclometasone 17,21-dipropionate
Alclometasone 17,21-dipropionate
O
Molecular formula: C28 H37 ClO7
Molecular weight: 521.05
CAS Registry No: 66734-13-2
Merck Index: 13, 219
CH3
HO
CH3
H
O
H
CH3
O
O
O
O
CH3
CH3
H
Cl
SAMPLE
Matrix: formulations
Sample preparation: Condition a 3 mL 500 mg Megabond MF C18 SPE cartridge
(Varian) with 3 mL MeOH and 3 mL water. Sonicate 1 g cosmetic with 10 mL MeOH
or MeOH:dichloromethane 10:90 (depending on what appears visually to give best
solubility) at 40◦ for 10 min, centrifuge, collect the clear supernatant. Add 5 mL of the
supernatant to the SPE cartridge, wash with 4 mL acetone:water 20:80, wash with
1 mL n-hexane, elute with 4 mL diethyl ether. Evaporate the eluate to dryness under
reduced pressure, reconstitute the residue with 5 mL (or more) MeOH, inject a 10 µL
aliquot.
HPLC VARIABLES
Column: 250 × 4.6 5 µm endcapped Purospher RP-18
Column temperature: 25
Mobile phase: Isocratic. MeCN:water 60:40. Gradient. MeCN:water from 25:75 to 90:10
over 30 min, maintain at 90:10 for 10 min.
Flow rate: 1
Injection volume: 10
Detector: UV 239
CHROMATOGRAM
Retention time: k′ 2.55 (isocratic); 21.0 min (gradient)
Limit of detection: 300 ng/mL
OTHER SUBSTANCES
Simultaneous: amcinonide (isocratic k′ 3.18; gradient retention time (min) 22.6; LOD
0.1 µg/mL), betamethasone (isocratic k′ 0.18; gradient retention time (min) 11.8; LOD
0.1 µg/mL), betamethasone-17-acetate (isocratic k′ 0.73; gradient retention time (min)
15.4; LOD 0.3 µg/mL), betamethasone-17-benzoate (isocratic k′ 2.04; gradient retention
time (min) 20.6; LOD 0.3 µg/mL), betamethasone-17-propionate-21-stearate (isocratic k′
>13; gradient retention time (min) >35; LOD 0.5 µg/mL), betamethasone-17-propionate21-butyrate (isocratic k′ 5.91; gradient retention time (min) 26.1; LOD 0.4 µg/mL),
betamethasone-17-valerate-21-acetate (isocratic k′ 4.41; gradient retention time (min)
23.1; LOD 0.4 µg/mL), betamethasone-17-valerate (isocratic k′ 2.32; gradient retention
time (min) 21.4; LOD 0.3 µg/mL), betamethasone-17,21-dipropionate (isocratic k′ 4.00;
gradient retention time (min) 24.2; LOD 0.4 µg/mL), betamethasone-17,21-diacetate
(isocratic k′ 1.81; gradient retention time (min) 20.5; LOD 0.3 µg/mL), betamethasone17,21-divalerate (isocratic k′ 10.82; gradient retention time (min) 28.0; LOD 0.4 µg/mL),
betamethasone-21-acetate (isocratic k′ 0.77; gradient retention time (min) 15.6; LOD
0.3 µg/mL), betamethasone propionate (isocratic k′ 0.82; gradient retention time (min)
17.1; LOD 0.3 µg/mL), clobetasol propionate (isocratic k′ 3.41; gradient retention time
(min) 23.4; LOD 0.1 µg/mL), clobetasone butyrate (isocratic k′ 5.45; gradient retention
time (min) 26.3; LOD 0.1 µg/mL), cortisone (isocratic k′ 0.18; gradient retention time
Alclometasone 17,21-dipropionate
19
(min) 11.1; LOD 0.6 µg/mL), cortisone acetate (isocratic k′ 0.73; gradient retention time
(min) 15.2; LOD 0.6 µg/mL), dehydrocorticosterone (isocratic k′ 4.27; gradient retention
time (min) 22.3; LOD 0.5 µg/mL), deoxymethasone (isocratic k′ 0.64; gradient retention
time (min) 14.2; LOD 0.2 µg/mL), dexamethasone (isocratic k′ 0.27; gradient retention
time (min) 11.9; LOD 0.1 µg/mL), dexamethasone-21-acetate (isocratic k′ 0.91; gradient retention time (min) 16.1; LOD 0.2 µg/mL), dexamethasone isonicotinate (isocratic
k′ 1.05; gradient retention time (min) 17.7; LOD 0.4 µg/mL), dexamethasone pivalate
(isocratic k′ 3.45; gradient retention time (min) 24.1; LOD 0.3 µg/mL), dexamethasone
valerate (isocratic k′ 3.00; gradient retention time (min) 21.6; LOD 0.3 µg/mL), diflucortolone valerate (isocratic k′ 4.73; gradient retention time (min) 23.3; LOD 0.3 µg/mL),
fludrocortisone acetate (isocratic k′ 0.59; gradient retention time (min) 14.1; LOD
0.3 µg/mL), flumethasone pivalate (isocratic k′ 2.68; gradient retention time (min) 21.2;
LOD 0.3 µg/mL), fluocinolone acetonide (isocratic k′ 0.91; gradient retention time (min)
13.4; LOD 0.3 µg/mL), fluocinonide (isocratic k′ 1.45; gradient retention time (min)
20.5; LOD 0.1 µg/mL), fluocortin butyl ester (isocratic k′ 5.59; gradient retention time
(min) 24.6; LOD 0.3 µg/mL), fluocortolone caproate (isocratic k′ 6.59; gradient retention
time (min) 25.1; LOD 0.3 µg/mL), fluocortolone pivalate (isocratic k′ 4.50; gradient
retention time (min) 23.6; LOD 0.3 µg/mL), fluorometholone (isocratic k′ 0.59; gradient
retention time (min) 14.4; LOD 0.1 µg/mL), 9-α-fluoroprednisolone (isocratic k′ 0.18;
gradient retention time (min) 10.0; LOD 0.1 µg/mL), 9-α-fluoroprednisolone acetate
(isocratic k′ 0.50; gradient retention time (min) 13.9; LOD 0.2 µg/mL), flurandrenolide
(isocratic k′ 0.50; gradient retention time (min) 13.5; LOD 0.1 µg/mL), halcinonide
(isocratic k′ 1.64; gradient retention time (min) 20.6; LOD 0.1 µg/mL), hydrocortisone
(isocratic k′ 0.18; gradient retention time (min) 10.0; LOD 0.4 µg/mL), hydrocortisone17-butyrate (isocratic k′ 1.09; gradient retention time (min) 17.7; LOD 0.6 µg/mL),
hydrocortisone-21-acetate (isocratic k′ 0.77; gradient retention time (min) 15.3; LOD
0.6 µg/mL), hydrocortisone pivalate (isocratic k′ 2.27; gradient retention time (min)
20.4; LOD 0.8 µg/mL), methylprednisolone (isocratic k′ 0.55; gradient retention time
(min) 13.5; LOD 0.1 µg/mL), mometasone furoate (isocratic k′ 3.05; gradient retention
time (min) 22.0; LOD 0.2 µg/mL), prednisolone-21-acetate (isocratic k′ 0.60; gradient
retention time (min) 13.6; LOD 0.2 µg/mL), prednisolone acetonide (isocratic k′ 0.50;
gradient retention time (min) 13.0; LOD 0.3 µg/mL), prednisolone pivalate (isocratic
k′ 2.05; gradient retention time (min) 19.7; LOD 0.3 µg/mL), triamcinolone (isocratic
k′ 0.14; gradient retention time (min) 7.2; LOD 0.1 µg/mL), triamcinolone acetonide
(isocratic k′ 0.50; gradient retention time (min) 13.9; LOD 0.2 µg/mL), triamcinolone
diacetate (isocratic k′ 0.45; gradient retention time (min) 13.9; LOD 0.3 µg/mL)
KEY WORDS
cosmetics; SPE
REFERENCE
Gagliardi, L.; De Orsi, D.; Del Giudice, M.R.; Gatta, F.; Porrà, R.; Chimenti, P.; Tonelli, D. Development
of a tandem thin-layer chromatography-high-performance liquid chromatography method for the
identification and determination of corticosteroids in cosmetic products, Anal.Chim.Acta, 2002, 457,
187–198.
SAMPLE
Matrix: formulations
Sample preparation: Mix 1 g of a topical product, 5 mL 100 mM pH 4 citrate buffer
saturated with NaCl, and 10 mL ethyl acetate, shake vigorously by hand to ensure
that no large clumps stick to the tube, mix with the tube on its side on an oscillating
shaker for 15 min. Remove the upper ethyl acetate layer and wash with citrate buffer as
before. Dry the ethyl acetate over anhydrous sodium sulfate and evaporate to dryness.
Dissolve the residue in a mixture of 10 mL heptane and 5 mL MeCN:water 90:10.
Remove the upper heptane layer and extract it with 2 mL MeCN:water 90:10. Combine
the MeCN/water layers and evaporate them to dryness, dissolve the residue in 300 µL
MeOH, filter (0.45 µm nylon), inject a 5 µL aliquot.
20
Alclometasone 17,21-dipropionate
HPLC VARIABLES
Guard column: 15 × 3.2 7 µm Brownlee NewGuard C18
Column: 75 × 4.6 3.5 µm Symmetry C18 (Waters)
Mobile phase: Gradient. MeCN:water 18:82 for 2 min, to 82:18 over 12 min, maintain
at 82:18 for 3 min, re-equilibrate at initial conditions for 12 min.
Flow rate: 1
Injection volume: 5
Detector: UV 240
CHROMATOGRAM
Retention time: 10.93
Limit of detection: 0.001%
OTHER SUBSTANCES
Simultaneous: amcinonide
(10.90), beclomethasone 17,21-dipropionate (11.90),
betamethasone (6.52), betamethasone 21-acetate (8.47), betamethasone 17-benzoate
(10.28), betamethasone 17,21-dipropionate (11.42), betamethasone 17-valerate (10.25),
budesonide (8.55, 8.69 (epimers)), clobetasol 17-propionate (11.06), cortisone
(5.62), cortisone 21-acetate (8.07), dexamethasone (6.57), dexamethasone 21-acetate
(8.68), desonide (6.99), desoximetasone (7.60), desoxycorticosterone acetate (10.90),
desoxycorticosterone pivalate (14.45), diflorasone 17,21-diacetate (9.81), fluocinolone
acetonide (7.38), fluocinonide (9.79), flurandrenolide (7.36), fludrocortisone 21acetate (7.77), fluorometholone (7.67), flumethasone 21-pivalate (11.20), flunisolide
(7.14), fluprednisolone (5.46), fluticasone 17-propionate (11.19), halcinonide (10.72),
halobetasol propionate (10.98), hydrocortisone (5.50), hydrocortisone 21-acetate (7.65),
hydrocortisone 17-butyrate (8.66), hydrocortisone 21-cypionate (12.54), hydrocortisone
17-valerate (9.53), mometasone 17-furoate (11.24), methylprednisolone (6.31),
methylprednisolone 21-acetate (8.34), meprednisone (6.55), prednisolone (5.37),
prednisolone 21-acetate (7.47), prednisolone 21-tebuate (11.01), paramethasone
21-acetate (8.64), prednicarbate (10.72), prednisone (5.46), triamcinolone (4.15),
triamcinolone acetonide (7.04), triamcinolone 16,21-diacetate (7.49), triamcinolone
hexacetonide (13.12)
KEY WORDS
body wash, cream, gel, lotion, shampoo, spray
REFERENCE
Reepmeyer, J.C. Screening for corticosteroids in topical pharmaceuticals by HPLC with a scanning
ultraviolet detector, J.Liq.Chromatogr.Rel.Technol., 2001, 24, 693–709.
Alitretinoin
Alitretinoin
H3C
Molecular formula: C20 H28 O2
Molecular weight: 300.43
CAS Registry No: 5300-03-8
Merck Index: 13, 244
[9-cis-retinoic acid]
CH3
21
CH3
CH3
H3C
COOH
SAMPLE
Matrix: blood
Sample preparation: 1 mL plasma + 50 µL 500 µg/mL IS in MeOH:MeCN 50:50 +
1 mL 1 M pH 6.0 phosphate buffer, mix, add 6 mL MTBE, shake on a horizontal shaker
for 10 min, freeze the aqueous layer in a dry ice/acetone bath. Decant the organic layer
and evaporate it to dryness under nitrogen at 25◦ , reconstitute the residue with 200 µL
MeOH, add 100 µL 5 mM ammonium acetate, centrifuge at 13 000 g for 3 min, inject a
100 µL aliquot. (Use silanized glassware. Process under yellow light.)
HPLC VARIABLES
Guard column: 10 × 2 5 µm Hypersil BDS C18
Column: 100 × 4.6 3 µm Microsorb Short One C18 (Rainin)
Column temperature: 36
Mobile phase: Gradient. A was 5 mM pH 2.7 ammonium acetate/acetic acid buffer. B
was 1% acetic acid in MeOH. A:B 30:70 for 6.5 min, to 20:80 over 0.5 min, to 11:80 over
14.4 min, to 30:70 over 0.5 min, maintain at 30:70 for 10 min.
Flow rate: 1
Injection volume: 100
Detector: UV 348
CHROMATOGRAM
Retention time: 21
Internal standard: all-trans-3,7-dimethyl-9-(2,4,6-trimethylphenyl)-2,4,6,8-nonatetraenoic acid (Ro 11–5036) (19)
Limit of detection: 2.5 ng/mL
OTHER SUBSTANCES
Extracted: isotretinoin (19.5), tretinoin (21.5), vitamin A (20.5)
KEY WORDS
plasma
REFERENCE
Dzerk, A.M.; Carlson, A.; Loewen, G.R.; Shirley, M.A.; Lee, J.W. A HPLC method for the determination
of 9-cis retinoic acid (ALRT1057) and its 4-oxo metabolite in human plasma, J.Pharm.Biomed.Anal.,
1998, 16, 1013–1019.
SAMPLE
Matrix: blood, food, formulations, tissue
Sample preparation: Serum. Extract one volume (20–100 µL) serum with three volumes isopropanol:dichloromethane 2:1 containing about 6 nM IS and 1 mM butylated
hydroxytoluene (BHT, antioxidant), add glacial acetic acid (1 µL/20 µL serum). Vortex
for 30 s, centrifuge for 1 min, inject a 20–70 µL aliquot of the supernatant. Tissue, food.
Homogenize 100–200 mg human or rat liver, 200 mg–2 g other tissues, or 2–5 g pulp
of fruits and fresh vegetables with 3–5 mL isopropanol:dichloromethane 2:1, make up
to 10 mL with isopropanol:dichloromethane 2:1. Vortex for 1 min, keep under argon
22
Alitretinoin
at −20◦ overnight, vortex for 1 min, return to the freezer. On the third day, vortex
the mixture, centrifuge or filter. Evaporate the supernatant or filtrate to dryness in
a rotary evaporator. Dissolve the residue in 200 µL isopropanol:dichloromethane 2:1,
inject a 20–40 µL aliquot. Multivitamin tablets. Grind tablet to a powder, add 10 mL
isopropanol:dichloromethane 2:1. Vortex for 1 min, keep under argon at −20◦ overnight,
vortex for 1 min, return to the freezer. On the third day, vortex the mixture, centrifuge
about 500 µL solution, inject a 50 µL aliquot of the supernatant.
HPLC VARIABLES
Guard column: C18 (Upchurch)
Column: 100 × 4.6 3 µm Microsorb MV
Mobile phase: Gradient. A was MeOH:water 75:25 containing 10 mM ammonium
acetate. B was MeOH:dichloromethane 80:20. A:B from 100:0 to 0:100 over 15 min,
maintain at 0:100 for 15–20 min, to 100:0 over 5 min, re-equilibrate at initial conditions
for 10 min.
Flow rate: 0.8
Injection volume: 50
Detector: UV 340
CHROMATOGRAM
Retention time: 10.2
Internal standard: retinyl acetate (13.8)
OTHER SUBSTANCES
Extracted: β-carotene (27.1), isotretinoin (9.9), all-trans retinal (13.8), all-trans retinyl
palmitate (24.1), all-trans retinyl stearate (26.4), tretinoin (10.5), vitamin A (12.9),
vitamin E (18.7),
KEY WORDS
human, ketchup, liver, mango, multivitamin tablets, papaya, rat, serum, spinach, tomato
REFERENCE
Barua, A.B.; Olson, J.A. Reversed-phase gradient high-performance liquid chromatographic procedure
for simultaneous analysis of very polar to nonpolar retinoids, carotenoids and tocopherols in animal
and plant samples, J.Chromatogr.B, 1998, 707, 69–79.
SAMPLE
Matrix: formulations
Sample preparation: Capsules. Cut open 10 capsules, sonicate three times at 30◦
for 5 min with 40 mL portions of MeCN:EtOH:1% acetic acid 70:20:10, centrifuge at
3500 rpm for 6 min. Filter the supernatants, combine, make up to 250 mL. Dilute a
1 mL aliquot to 10 mL with mobile phase, filter (nylon 0.45 µm), inject an aliquot.
Gel. Sonicate a portion with 8 mL mobile phase for 1 min, centrifuge at 3500 rpm
for 10 min. Filter the supernatant and make up to 10 mL. Dilute a 1 mL aliquot to
5 mL with mobile phase, filter (nylon 0.45 µm), inject an aliquot. Cream. Sonicate an
aliquot twice for 5 min with 4 mL portions of MeCN:EtOH:1% acetic acid 70:20:10,
centrifuge at 3500 rpm for 6 min. Filter the supernatants, combine, make up to 10 mL
with MeCN:EtOH:1% acetic acid 70:20:10. Dilute a 2 mL aliquot to 5 mL with mobile
phase, filter (nylon 0.45 µm), inject an aliquot.
HPLC VARIABLES
Column: 250 × 3.2 Phenomenex Prodigy 5ODS
Column temperature: 32 ± 2
Mobile phase: MeCN:EtOH:1% acetic acid 68:8:24
Flow rate: 0.4
Injection volume: 20
Detector: F ex 350 em 520
Alitretinoin
23
CHROMATOGRAM
Retention time: 31
Limit of detection: 11.09 pmole (S/N = 3)
OTHER SUBSTANCES
Simultaneous: isotretinoin (28.5), tretinoin (33)
KEY WORDS
avoid exposure to light, use amber-colored glassware, capsules, cream, gel
REFERENCE
Gatti, R.; Gioia, M.G.; Cavrini, V. Analysis and stability study of retinoids in pharmaceuticals by LC
with fluorescence detection, J.Pharm.Biomed.Anal., 2000, 23, 147–159.
ANNOTATED BIBLIOGRAPHY
Disdier, B.; Bun, H.; Placidi, M.; Durand, A. Excretion of oral 9-cis-retinoic acid in the rat, Drug
Metab.Dispos., 1996, 24, 1279–1281.
Disdier, B.; Bun, H.; Catalin, J.; Durand, A. Simultaneous determination of all-trans-, 13-cis, 9-cisretinoic acid and their 4-oxometabolites in plasma by high-performance liquid chromatography,
J.Chromatogr.B, 1996, 683, 143–154.
Gatti, R.; Gioia, M.G.; Di Pietra, A.M.; Cini, M. Determination of retinoids in galenicals by column liquid
chromatography with fluorescence and diode-array detection, J.Chromatogr.A, 2001, 905, 345–350.
Marchetti, M.-N.; Sampol, E.; Bun, H.; Scoma, H.; Lacarelle, B.; Durand, A. In vitro metabolism of three
major isomers of retinoic acid in rats. Intersex and interstrain comparison, Drug Metab.Dispos., 1997,
25, 637–646.
Miyagi, M.; Yokoyama, H.; Shiraishi, H.; Matsumoto, M.; Ishii, H. Simultaneous quantification of retinol,
retinal, and retinoic acid isomers by high-performance liquid chromatography with a simple gradiation,
J.Chromatogr.B, 2001, 757, 365–368.
Rühl, R.; Schweigert, F.J. Automated solid-phase extraction and liquid chromatographic method for
retinoid determination in biological samples, J.Chromatogr.B, 2003, 798, 309–316.
Shih, T.-W.; Lin, T.-H.; Shealy, Y.F.; Hill, D.L. Nonenzymatic isomerization of 9-cis-retinoic acid catalyzed by sulfhydryl compounds, Drug Metab.Dispos., 1997, 25, 27–32.
Van Merris, V.; Meyer, E.; De Wasch, K.; Burvenich, C. Simple quantification of endogenous retinoids
in bovine serum by high-performance liquid chromatography – diode-array detection, Anal.Chim.Acta,
2002, 468, 237–244.
Wyss, R.; Bucheli, F. Determination of endogenous levels of 13-cis-retinoic acid (isotretinoin), all-transretinoic acid (tretinoin) and their 4-oxo metabolites in human and animal plasma by high-performance
liquid chromatography with automated column switching and ultraviolet detection, J.Chromatogr.B,
1997, 700, 31–47.
Yamakoshi, Y.; Fukasawa, H.; Yamauchi, T.; Waki, H.; Kadowaki, T.; Shudo, K.; Kagechika, H. Determination of endogenous levels of retinoic acid isomers in type II diabetes mellitus patients. Possible
correlation with HbA1c values, Biol.Pharm.Bull., 2002, 25, 1268–1271.
24
Allethrin
Allethrin
Molecular formula: C19 H26 O3
Molecular weight: 302.41
CAS Registry No: 584-79-2
Merck Index: 13, 256
H3C
CH3
H3C
H3C
O
O
O
H3C
CH2
SAMPLE
Matrix: fruit, vegetables
Sample preparation: Prepare a cleanup column by placing 4 g Florisil, 1 g activated
charcoal, and a 20 mm layer of anhydrous sodium sulfate in a 400 × 10 glass column, wash with 40 mL toluene, wash with 40 mL toluene:MeCN 99:1. Homogenize
25 g chopped fruit or vegetable with 70 mL MeOH at high speed for 3 min, filter,
homogenize solid with 30 mL MeOH, and filter. Combine the filtrates and add them
to 60 mL toluene and 300 mL 10% NaCl in water, shake well for 3 min, let layers separate. Dry the organic layer by passing it through 20 g anhydrous sodium
sulfate in a 20 mm diameter column, concentrate to about 5 mL under reduced
pressure at 80◦ , add to the cleanup column, elute with 40 mL toluene:MeCN 99:1.
Evaporate the eluate just to dryness under reduced pressure at 80◦ , reconstitute
with 1 mL MeOH, inject an aliquot. (Reflux activated charcoal (20–40 mesh) with
1 M HCl for 4 h, wash with water until the washings are neutral, dry at 95–100◦
(J.Assoc.Off.Anal.Chem. 1983, 66, 1013). Heat 60–100 mesh Florisil at 200◦ for 24 h,
cool, add 4% water, mix thoroughly, store in a sealed jar (J.Assoc.Off.Anal.Chem. 1983,
66, 1003).)
HPLC VARIABLES
Column: 300 × 3.9 10 µm µBondapak C18
Column temperature: 50
Mobile phase: Gradient. MeCN:water from 62:38 to 78:22 over 32 min (Waters curve 6).
Flow rate: 1.5
Detector: UV 206
CHROMATOGRAM
Retention time: 11.55
Limit of detection: 50 ng/g
OTHER SUBSTANCES
Simultaneous: cypermethrin (21.05–22.08), permethrin (24.60, 27.01), tetramethrin
(13.08)
KEY WORDS
apple, cabbage, cucumber; peach, pear, tomato, SPE
REFERENCE
Pang, G.-F.; Chao, Y.-Z.; Liu, X.-S.; Fan, C.-L. Multiresidue liquid chromatographic method for simultaneous determination of pyrethroid insecticides in fruits and vegetables, J.AOAC Int., 1995, 78,
1474–1480.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: Two 250 × 4 Phase 3019 columns in series (Phenomenex)
Mobile phase: Hexane:1,2-dichloroethane:EtOH 500:30:0.15
Allethrin
25
Flow rate: 0.8
Detector: UV 230
CHROMATOGRAM
Retention time: 34, 36, 37, 39, 40, 42, 44, 46 (isomers)
REFERENCE
Phenomenex Catalogue, Phenomenex: Torrance CA, 1994, p. 1035.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Guard column: 50 × 4 40 µm pellicular material
Column: 250 × 4.6 5 µm Ultrasphere octadecylsilica
Mobile phase: MeOH:water 80:20
Flow rate: 1
Injection volume: 10
Detector: UV 254
CHROMATOGRAM
Retention time: k′ 4.68 (cis), k′ 5.32 (trans)
OTHER SUBSTANCES
Also analyzed: cyfluthrin (baythroid) (k′ 7.41 (cis, S), k′ 7.77 (trans, R), k′ 8.01 (cis, S), k′
8.73 (trans, R)), permethrin (k′ 14.9 (trans), k′ 19.5 (cis)), resmethrin (k′ 13.5 (cis), k′ 15.0
(trans)), tetramethrin (k′ 4.05 (cis), k′ 4.68 (trans))
REFERENCE
Abidi, S.L. Column selectivity in high-performance liquid chromatography of substituted gem-dimethylcyclopropanes, J.Chromatogr., 1986, 368, 59–76.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 × 4.6 5 µm Cyclobond I cyclodextrin-modified silica (Astec)
Mobile phase: MeCN:water 22:78
Flow rate: 1
Detector: UV 220
CHROMATOGRAM
Retention time: 7 (cis isomers), 9.5 (1R,trans, αS), 10.5 (1S,trans, αR), 13 (1R,trans,
αR), 15 (1S,trans, αS)
KEY WORDS
comparison with GC
REFERENCE
Kutter, J.P.; Class, T.J. Diastereoselective and enantioselective chromatography of the pyrethroid insecticides allethrin and cypermethrin, Chromatographia, 1992, 33, 103–112.
SAMPLE
Matrix: solutions
Sample preparation: Inject an aliquot of a 0.1–1 mg/mL solution in hexane.
26
Allethrin
HPLC VARIABLES
Guard column: 5 µm Spherisorb NH2
Column: 250 × 4.6 Pirkle ionic type 1-A column (Technicol)
Mobile phase: Hexane:isopropanol 99.85:0.15
Flow rate: 0.8
Detector: UV 230
OTHER SUBSTANCES
Also analyzed: cypermethrin, fenpropathrin, fenvalerate, tetramethrin
KEY WORDS
chiral
REFERENCE
Lisseter, S.G.; Hambling, S.G. Chiral high-performance liquid chromatography of synthetic pyrethroid
insecticides, J.Chromatogr., 1991, 539, 207–210.
SAMPLE
Matrix: urine
Sample preparation: Add 4 g solid NaCl, 3.5 mL MeCN, and 5 mL saturated NaCl
solution to 5 mL MeCN, shake for 1 min. Remove the MeCN layer and extract the
aqueous layer with 1 mL MeCN. Combine the MeCN layers and adjust to a known
volume (0.5–1 mL), mix, filter (0.45 µm), inject a 40 µL aliquot.
HPLC VARIABLES
Column: 150 × 3 3 µm Luna C18(2) (Phenomenex)
Column temperature: 30
Mobile phase: Gradient. MeCN:water 10:90 for 1 min, to 90:10 over 30 min, maintain
at 90:10 for 4 min, to 100:0 over 1 min, maintain at 100:0 for 10 min, return to initial
conditions over 1 min.
Flow rate: 0.5
Injection volume: 40
Detector: UV 235
CHROMATOGRAM
Retention time: 31.8
Limit of detection: 5 ng/mL
OTHER SUBSTANCES
Extracted: bifenthrin (37, LOD 5 ng/mL), cyfluthrin (34.3, LOD 5 ng/mL), fenvalerate
(35.3, LOD 2 ng/mL), cis-permethrin (35.7, LOD 5 ng/mL), trans-permethrin (36.3, LOD
5 ng/mL), phenothrin (36.4, LOD 5 ng/mL), m-phenoxybenzyl alcohol (21, LOD 5 ng/mL),
pyrethrin I (29.6, LOD 4 ng/mL), pyrethrin II (33.7, LOD 40 ng/mL), resmethrin (35.2,
LOD 5 ng/mL), tetramethrin (31.4, LOD 5 ng/mL)
REFERENCE
Loper, B.L.; Anderson, K.A. Determination of pyrethrin and pyrethroid pesticides in urine and water
matrices by liquid chromatography with diode array detection, J.AOAC Int., 2003, 86, 1236–1240.
Almotriptan
Almotriptan
H
O
O
Molecular formula: C17 H25 N3 O2 S
Molecular weight: 335.47
CAS Registry No: 154323-57-6
Merck Index: 13, 301
27
N
S
N
N
SAMPLE
Matrix: blood, urine
Sample preparation: Condition a C2 SPE cartridge (Baker) with 2 mL MeCN and
2 mL water. Dilute 500 µL plasma or 100 µL urine with 1 mL water containing IS,
mix, add to the SPE cartridge, wash with 750 µL MeCN:water 30:70, wash with 250 µL
water, elute with mobile phase over 1 min (straight onto column (?)).
HPLC VARIABLES
Guard column: Guardpak µBondapak CN
Column: 150 × 4 5 µm Spherisorb ODS-2
Mobile phase: MeCN:50 mM pH 4.0 sodium phosphate buffer:triethylamine 20:80:0.2
Flow rate: 1
Detector: UV 227
CHROMATOGRAM
Retention time: 6.5
Internal standard: 4-[3-(2-aminoethyl)-1H-indol-5-ylmethylsulfonyl]piperazine-1-carboxylic acid ethyl ester (10)
Limit of quantitation: 1 ng/mL (plasma), 50 ng/mL urine
KEY WORDS
plasma, SPE
REFERENCE
Jansat, J.M.; Costa, J.; Salvà, P.; Fernandez, F.J.; Martinez-Tobed, A. Absolute bioavailability, pharmacokinetics, and urinary excretion of the novel antimigraine agent almotriptan in healthy male volunteers,
J.Clin.Pharmacol., 2002, 42, 1303–1310.
SAMPLE
Matrix: microsomal incubations
Sample preparation: Mix 500 µL microsomal incubation with 1 mL 200 mM pH 4
sodium acetate buffer, centrifuge, inject an aliquot.
HPLC VARIABLES
Guard column: GuardPak µBondapak CN
Column: 300 × 3.9 10 µm µBondapak
Mobile phase: Gradient. A:B from 80:20 to 40:60 over 30 min. A was buffer. B was
MeCN:buffer 80:20. Buffer was 10 mM orthophosphoric acid containing 0.1% triethylamine, adjusted to pH 6.5 with NaOH.
Flow rate: 1
Detector: UV 227
CHROMATOGRAM
Retention time: 25
OTHER SUBSTANCES
Extracted: metabolites
28
Almotriptan
KEY WORDS
human, liver
REFERENCE
Salva, M.; Jansat, J.M.; Martinez-Tobed, A.; Palacios, J.M. Identification of the human liver enzymes
involved in the metabolism of the antimigraine agent almotriptan, Drug Metab.Dispos., 2003, 31,
404–411.
29
Alosetron
Alosetron
H
Molecular weight: 294.35
CAS Registry No: 122852-42-0, 122852-69-1 (HCl)
Merck Index: 13, 305
N
O
Molecular formula: C17 H18 N4 O
N
N
H3C
N
CH3
SAMPLE
Matrix: blood
Sample preparation: Condition a 100 mg LRC Bond Elut ethyl (C2) SPE cartridge
with 1 mL isopropanol and 1 mL buffer. Mix 1.1 mL plasma or serum with 1 mL buffer
containing 10 ng/mL IS, vortex, add 2 mL to the SPE cartridge, wash with 2 mL buffer,
dry with nitrogen for 30 s, wash with 2 mL MeCN, elute with two 2 mL aliquots of
MeCN:buffer 90:10. Evaporate the eluate to dryness under a stream of nitrogen at 40◦ ,
reconstitute the residue with 300 µL mobile phase, vortex, inject a 200 µL aliquot. (The
buffer was 10 mM ammonium acetate adjusted to pH 4.0 with glacial acetic acid.)
HPLC VARIABLES
Guard column: 15 × 4.6 7 µm Spherisorb cyanopropyl
Column: 100 × 4.6 5 µm Spheri cyanopropyl (Brownlee)
Column temperature: 45
Mobile phase: MeOH:THF:10 mM pH 4.0 ammonium acetate buffer 24:6:70
Flow rate: 0.5
Injection volume: 200
Detector: F ex 295 em 370
CHROMATOGRAM
Retention time: 10.1
Internal standard: GR87442, 6-fluoroalosetron (Glaxo) (13.7)
Limit of quantitation: 0.1 ng/mL
OTHER SUBSTANCES
Noninterfering: amitriptyline, carbamazepine, carmustine, chlorpromazine, cimetidine, cisplatin, cyclophosphamide, dexamethasone, diazepam, digoxin, etoposide, furosemide, haloperidol, ibuprofen, imipramine, indomethacin, methotrexate, phenobarbital, phenytoin, propranolol, ranitidine, theophylline, triazolam, warfarin
KEY WORDS
plasma; serum; SPE
REFERENCE
Lloyd, T.L.; Gupta, S.K.; Gooding, A.E.; Alianti, J.R. Determination of alosetron in human plasma or
serum by high-performance liquid chromatography with robotic sample preparation, J.Chromatogr.B,
1996, 678, 261–267.
30
Amcinonide
Amcinonide
O
H3C
Molecular formula: C28 H35 FO7
O
Molecular weight: 502.57
CAS Registry No: 51022-69-6
Merck Index: 13, 387
O
CH3
HO
O
CH3
F
H
O
H
O
SAMPLE
Matrix: formulations
Sample preparation: Condition a 3 mL 500 mg Megabond MF C18 SPE cartridge
(Varian) with 3 mL MeOH and 3 mL water. Sonicate 1 g cosmetic with 10 mL MeOH
or MeOH:dichloromethane 10:90 (depending on what appears visually to give the best
solubility) at 40◦ for 10 min, centrifuge, collect the clear supernatant. Add 5 mL of the
supernatant to the SPE cartridge, wash with 4 mL acetone:water 20:80, wash with
1 mL n-hexane, elute with 4 mL diethyl ether. Evaporate the eluate to dryness under
reduced pressure, reconstitute the residue with 5 mL (or more) MeOH, inject a 10 µL
aliquot.
HPLC VARIABLES
Column: 250 × 4.6 5 µm endcapped Purospher RP-18
Column temperature: 25
Mobile phase: Isocratic.MeCN:water 60:40. Gradient. MeCN:water from 25:75 to 90:10
over 30 min, maintain at 90:10 for 10 min.
Flow rate: 1
Injection volume: 10
Detector: UV 239
CHROMATOGRAM
Retention time: k′ 3.18 (isocratic); 22.6 min (gradient)
Limit of detection: 100 ng/mL
OTHER SUBSTANCES
Simultaneous: alclometasone dipropionate (isocratic k′ 2.55; gradient retention time
(min) 21.0; LOD 0.3 µg/mL), betamethasone (isocratic k′ 0.18; gradient retention time
(min) 11.8; LOD 0.1 µg/mL), betamethasone-17-acetate (isocratic k′ 0.73; gradient retention time (min) 15.4; LOD 0.3 µg/mL), betamethasone-17-benzoate (isocratic k′ 2.04;
gradient retention time (min) 20.6; LOD 0.3 µg/mL), betamethasone-17-propionate21-stearate (isocratic k′ >13; gradient retention time (min) >35; LOD 0.5 µg/mL),
betamethasone-17-propionate-21-butyrate (isocratic k′ 5.91; gradient retention time
(min) 26.1; LOD 0.4 µg/mL), betamethasone-17-valerate-21-acetate (isocratic k′ 4.41;
gradient retention time (min) 23.1; LOD 0.4 µg/mL), betamethasone-17-valerate (isocratic k′ 2.32; gradient retention time (min) 21.4; LOD 0.3 µg/mL), betamethasone-17,21dipropionate (isocratic k′ 4.00; gradient retention time (min) 24.2; LOD 0.4 µg/mL),
betamethasone-17,21-diacetate (isocratic k′ 1.81; gradient retention time (min) 20.5;
LOD 0.3 µg/mL), betamethasone-17,21-divalerate (isocratic k′ 10.82; gradient retention
time (min) 28.0; LOD 0.4 µg/mL), betamethasone-21-acetate (isocratic k′ 0.77; gradient
retention time (min) 15.6; LOD 0.3 µg/mL), betamethasone propionate (isocratic k′ 0.82;
gradient retention time (min) 17.1; LOD 0.3 µg/mL), clobetasol propionate (isocratic k′
3.41; gradient retention time (min) 23.4; LOD 0.1 µg/mL), clobethasone butyrate (isocratic k′ 5.45; gradient retention time (min) 26.3; LOD 0.1 µg/mL), cortisone (isocratic
k′ 0.18; gradient retention time (min) 11.1; LOD 0.6 µg/mL), cortisone acetate (isocratic
k′ 0.73; gradient retention time (min) 15.2; LOD 0.6 µg/mL), dehydrocorticosterone
(isocratic k′ 4.27; gradient retention time (min) 22.3; LOD 0.5 µg/mL), deoxymethasone
(isocratic k′ 0.64; gradient retention time (min) 14.2; LOD 0.2 µg/mL), dexamethasone
Amcinonide
31
(isocratic k′ 0.27; gradient retention time (min) 11.9; LOD 0.1 µg/mL), dexamethasone21-acetate (isocratic k′ 0.91; gradient retention time (min) 16.1; LOD 0.2 µg/mL),
dexamethasone isonicotinate (isocratic k′ 1.05; gradient retention time (min) 17.7; LOD
0.4 µg/mL), dexamethasone pivalate (isocratic k′ 3.45; gradient retention time (min)
24.1; LOD 0.3 µg/mL), dexamethasone valerate (isocratic k′ 3.00; gradient retention
time (min) 21.6; LOD 0.3 µg/mL), diflucortolone valerate (isocratic k′ 4.73; gradient
retention time (min) 23.3; LOD 0.3 µg/mL), fludrocortisone acetate (isocratic k′ 0.59;
gradient retention time (min) 14.1; LOD 0.3 µg/mL), flumethasone pivalate (isocratic
k′ 2.68; gradient retention time (min) 21.2; LOD 0.3 µg/mL), fluocinolone acetonide
(isocratic k′ 0.91; gradient retention time (min) 13.4; LOD 0.3 µg/mL), fluocinonide (isocratic k′ 1.45; gradient retention time (min) 20.5; LOD 0.1 µg/mL), fluocortin butyl ester
(isocratic k′ 5.59; gradient retention time (min) 24.6; LOD 0.3 µg/mL), fluocortolone
caproate (isocratic k′ 6.59; gradient retention time (min) 25.1; LOD 0.3 µg/mL), fluocortolone pivalate (isocratic k′ 4.50; gradient retention time (min) 23.6; LOD 0.3 µg/mL),
fluorometholone (isocratic k′ 0.59; gradient retention time (min) 14.4; LOD 0.1 µg/mL),
9-α-fluoroprednisolone (isocratic k′ 0.18; gradient retention time (min) 10.0; LOD
0.1 µg/mL), 9-α-fluoroprednisolone acetate (isocratic k′ 0.50; gradient retention time
(min) 13.9; LOD 0.2 µg/mL), flurandrenolide (isocratic k′ 0.50; gradient retention time
(min) 13.5; LOD 0.1 µg/mL), halcinonide (isocratic k′ 1.64; gradient retention time (min)
20.6; LOD 0.1 µg/mL), hydrocortisone (isocratic k′ 0.18; gradient retention time (min)
10.0; LOD 0.4 µg/mL), hydrocortisone-17-butyrate (isocratic k′ 1.09; gradient retention
time (min) 17.7; LOD 0.6 µg/mL), hydrocortisone-21-acetate (isocratic k′ 0.77; gradient
retention time (min) 15.3; LOD 0.6 µg/mL), hydrocortisone pivalate (isocratic k′ 2.27;
gradient retention time (min) 20.4; LOD 0.8 µg/mL), methylprednisolone (isocratic k′
0.55; gradient retention time (min) 13.5; LOD 0.1 µg/mL), mometasone furoate (isocratic
k′ 3.05; gradient retention time (min) 22.0; LOD 0.2 µg/mL), prednisolone-21-acetate
(isocratic k′ 0.60; gradient retention time (min) 13.6; LOD 0.2 µg/mL), prednisolone
acetonide (isocratic k′ 0.50; gradient retention time (min) 13.0; LOD 0.3 µg/mL), prednisolone pivalate (isocratic k′ 2.05; gradient retention time (min) 19.7; LOD 0.3 µg/mL),
triamcinolone (isocratic k′ 0.14; gradient retention time (min) 7.2; LOD 0.1 µg/mL),
triamcinolone acetonide (isocratic k′ 0.50; gradient retention time (min) 13.9; LOD
0.2 µg/mL), triamcinolone diacetate (isocratic k′ 0.45; gradient retention time (min)
13.9; LOD 0.3 µg/mL).
KEY WORDS
cosmetics; SPE
REFERENCE
Gagliardi, L.; De Orsi, D.; Del Giudice, M.R.; Gatta, F.; Porrà, R.; Chimenti, P.; Tonelli, D. Development
of a tandem thin-layer chromatography-high-performance liquid chromatography method for the
identification and determination of corticosteroids in cosmetic products, Anal.Chim.Acta, 2002, 457,
187–198.
SAMPLE
Matrix: formulations
Sample preparation: Mix 1 g of a topical product, 5 mL 100 mM pH 4 citrate buffer
saturated with NaCl, and 10 mL ethyl acetate, shake vigorously by hand to ensure
that no large clumps stick to the tube, mix with the tube on its side on an oscillating
shaker for 15 min. Remove the upper ethyl acetate layer and wash with citrate buffer as
before. Dry the ethyl acetate over anhydrous sodium sulfate and evaporate to dryness.
Dissolve the residue in a mixture of 10 mL heptane and 5 mL MeCN:water 90:10.
Remove the upper heptane layer and extract it with 2 mL MeCN:water 90:10. Combine
the MeCN/water layers and evaporate them to dryness, dissolve the residue in 300 µL
MeOH, filter (0.45 µm nylon), inject a 5 µL aliquot.
HPLC VARIABLES
Guard column: 15 × 3.2 7 µm Brownlee NewGuard C18
Column: 75 × 4.6 3.5 µm Symmetry C18 (Waters)
32
Amcinonide
Mobile phase: Gradient. MeCN:water 18:82 for 2 min, to 82:18 over 12 min, maintain
at 82:18 for 3 min, re-equilibrate at initial conditions for 12 min.
Flow rate: 1
Injection volume: 5
Detector: UV 240
CHROMATOGRAM
Retention time: 10.90
Limit of detection: 0.001%
OTHER SUBSTANCES
Extracted:
Simultaneous: alclometasone 17,21-dipropionate (10.93), beclomethasone 17,21-dipropionate (11.90), betamethasone (6.52), betamethasone 21-acetate (8.47), betamethasone 17-benzoate (10.28), betamethasone 17,21-dipropionate (11.42), betamethasone
17-valerate (10.25), budesonide (8.55, 8.69 (epimers)), clobetasol 17-propionate (11.06),
cortisone (5.62), cortisone 21-acetate (8.07), dexamethasone (6.57), dexamethasone 21acetate (8.68), desonide (6.99), desoximetasone (7.60), desoxycorticosterone acetate
(10.90), desoxycorticosterone pivalate (14.45), diflorasone 17,21-diacetate (9.81), fluocinolone acetonide (7.38), fluocinonide (9.79), flurandrenolide (7.36), fludrocortisone
21-acetate (7.77), fluorometholone (7.67), flumethasone 21-pivalate (11.20), flunisolide
(7.14), fluprednisolone (5.46), fluticasone 17-propionate (11.19), halcinonide (10.72),
halobetasol propionate (10.98), hydrocortisone (5.50), hydrocortisone 21-acetate (7.65),
hydrocortisone 17-butyrate (8.66), hydrocortisone 21-cypionate (12.54), hydrocortisone
17-valerate (9.53), mometasone 17-furoate (11.24), methylprednisolone (6.31), methylprednisolone 21-acetate (8.34), meprednisone (6.55), prednisolone (5.37), prednisolone
21-acetate (7.47), prednisolone 21-tebuate (11.01), paramethasone 21-acetate (8.64),
prednicarbate (10.72), prednisone (5.46), triamcinolone (4.15), triamcinolone acetonide
(7.04), triamcinolone 16,21-diacetate (7.49), triamcinolone hexacetonide (13.12)
KEY WORDS
body wash, cream, gel, lotion, shampoo, spray
REFERENCE
Reepmeyer, J.C. Screening for corticosteroids in topical pharmaceuticals by HPLC with a scanning
ultraviolet detector, J.Liq.Chromatogr.Rel.Technol., 2001, 24, 693–709.
Aminolevulinic acid
Aminolevulinic acid
33
O
H2N
COOH
Molecular formula: C5 H9 NO3
Molecular weight: 131.13
CAS Registry No: 106-60-5
Merck Index: 13, 445
SAMPLE
Matrix: blood, tissue
Sample preparation: Deproteinize plasma by adding perchloric acid to a final concentration of 800 mM. Neutralize the supernatant by adding solid sodium bicarbonate
until a pH of ca. 7.6 is reached. Homogenize tissue with 3 volumes of 10 mM pH
7.2 HEPES buffer containing 250 mM sucrose and 500 mM EDTA, centrifuge at 800 g
for 5 min. Mix 10 µL sample with 5 µL reagent and 35 µL water, let stand at room
temperature for 1 min, inject a 20 µL aliquot. (Prepare the reagent by dissolving 27 mg
o-phthalaldehyde in 500 µL MeOH, add 5 mL 100 mM sodium tetraborate, add 20 µL
mercaptoethanol, mix.)
HPLC VARIABLES
Column: 150 × 3.9 4 µm C18 (Waters)
Mobile phase: MeCN:50 mM pH 7.0 phosphate buffer 10:90 containing 2.4 mM EDTA
Flow rate: 1
Injection volume: 20
Detector: E, Shimadzu LECD 6A, glassy carbon working electrode at +0.45 V, Ag/AgCl
reference electrode
CHROMATOGRAM
Retention time: 44.6
Limit of detection: 50 nM
Limit of quantitation: 100 nM
KEY WORDS
brain; derivatization; human; liver; plasma; rat
REFERENCE
Costa, C.A.; Trivelato, G.C.; Demasi, M.; Bechara, E.J.H. Determination of 5-aminolevulinic acid in blood
plasma, tissues and cell cultures by high-performance liquid chromatography with electrochemical
detection, J.Chromatogr.B, 1997, 695, 245–250.
SAMPLE
Matrix: blood, urine
Sample preparation: 50 µL Plasma or urine + 3.5 mL reagent + 450 µL 10% formaldehyde, vortex for 3 s, heat at 100◦ for 10 min, cool in an ice bath, filter (0.8 µm, plasma
samples only), inject a 10 (urine) or 20 (plasma) µL aliquot. (Prepare the reagent by
mixing 15 mL acetylacetone, 10 mL EtOH, and 75 mL water.)
HPLC VARIABLES
Column: 150 × 4.6 Shim-pack CLC-ODS (Shimadzu)
Column temperature: 40
Mobile phase: MeOH:water:acetic acid 50:50:1
Flow rate: 0.7
Injection volume: 10–20
Detector: F ex 370 em 460
34
Aminolevulinic acid
CHROMATOGRAM
Retention time: 6.1
Limit of detection: 3 ng/mL
KEY WORDS
derivatization; plasma; protect from light
REFERENCE
Oishi, H.; Nomiyama, H.; Nomiyama, K.; Tomokuni, K. Fluorometric HPLC determination of deltaaminolevulinic acid (ALA) in the plasma and urine of lead workers: biological indicators of lead
exposure, J.Anal.Toxicol., 1996, 20, 106–110.
SAMPLE
Matrix: urine
Sample preparation: Centrifuge urine at 1000 g and store at −20◦ . 20 µL Urine +
5 mL acetylacetone:EtOH:4 g/L NaCl in water 15:10:75 + 450 µL 9.3% formaldehyde
solution, mix, boil for 15 min, cool with water, store sample in the dark at 15◦ until
injection, inject a 50 µL aliquot.
HPLC VARIABLES
Column: 150 × 4.6 5 µm TSK-80 TM (Tosoh)
Column temperature: 40
Mobile phase: Gradient. A was MeCN:MeOH:water:acetic acid 10:35:54:1. B was MeCN.
A:B 100:0 for 7.5 min, to 50:50 over 1.5 min, return to initial conditions over 2 min,
re-equilibrate for 2 min.
Flow rate: 0.8
Injection volume: 50
Detector: F ex 246 em 458
CHROMATOGRAM
Retention time: 7.3
Limit of detection: 10 ng/mL
KEY WORDS
derivatization; protect from light; improved version of A. Okayama et al. Clin.Chem. 1990,
36, 1494.
REFERENCE
Endo, Y.; Okayama, A.; Endo, G.; Ueda, T.; Nakazono, N.; Horiguchi, S. Improvement of urinary
δ-aminolevulinic acid determination by HPLC and fluorescence detection using condensing reaction
with acetylacetone and formaldehyde, Jap.J.Ind.Health, 1994, 36, 49–56.
ANNOTATED BIBLIOGRAPHY
Dalton, J.T.; Meyer, M.C.; Golub, A.L. Pharmacokinetics of aminolevulinic acid after oral and intravenous
administration to dogs, Drug Metab.Dispos., 1999, 27, 432–435. [derivatization]
Ho, J.; Guthrie, R.; Tieckelmann, H. Detection of δ-aminolevulinic acid, porphobilinogen and porphyrins
related to heme biosynthesis by high-performance liquid chromatography, J.Chromatogr., 1986, 375,
57–63. [derivatization]
Ho, J.W. Micro assay for urinary δ-aminolevulinic acid and porphobilinogen by high-performance liquid
chromatography with pre-column derivatization, J.Chromatogr., 1990, 527, 134–139.
Kondo, M.; Kimura, H.; Maekubo, T.; Tomita, T.; Senda, M.; Urata, G.; Kajiwara, M. Direct injection
method for quantitation of δ-aminolevulinic acid in urine by high-performance liquid chromatography,
Chem.Pharm.Bull., 1992, 40, 1948–1950. [derivatization]
Lim, C.K.; Rideout, J.M.; Samson, D.M. Determination of 5-aminolaevulinic acid and porphobilinogen by
high-performance liquid chromatography, J.Chromatogr., 1979, 185, 605–611.
Aminolevulinic acid
35
Meisch, H.U.; Reinle, W.; Wolf, U. Determination of 5-aminolevulinic acid in biological samples by
high-performance liquid chromatography, Anal.Biochem., 1985, 149, 29–34.
Minder, E.I. Measurement of 5-aminolevulinic acid by reversed phase HPLC and fluorescence detection,
Clin.Chim.Acta, 1986, 161, 11–18. [derivatization]
Miyajima, K.; Hirata, M.; Yoshida, T.; Kosaka, H.; Okayama, A. Study on measurement of delta-aminolevulinic acid in plasma by high-performance liquid chromatography, J.Chromatogr.B, 1994, 654,
165–169.
Okayama, A. Fluorimetric determination of urinary δ-aminolevulinic acid by high-performance liquid
chromatography and post-column derivatization, J.Chromatogr., 1988, 426, 365–369.
Okayama, A.; Fujii, S.; Miura, R. Optimized fluorometric determination of urinary delta-aminolevulinic
acid by using pre-column derivatization, and identification of the derivative, Clin.Chem., 1990, 36,
1494–1497.
Tomokuni, K.; Ichiba, M.; Hirai, Y.; Hasegawa, T. Optimized liquid-chromatographic method for fluorometric determination of urinary delta-aminolevulinic acid in workers exposed to lead, Clin.Chem.,
1987, 33, 1665–1667.
36
Amprenavir
Amprenavir
NH2
O
Molecular formula: C25 H35 N3 O6 S
Molecular weight: 505.64
CAS Registry No: 161814-49-9
Merck Index: 13, 594
O
O
O
N
H
N
OH
S
O
CH3
CH3
SAMPLE
Matrix: blood
Sample preparation: Condition a C18 SPE cartridge (Baker) with 3 mL MeOH and
3 mL water. Do not allow to run dry. Add 1 mL plasma to the SPE cartridge, wash with
2 mL water, suck dry for 1 min, elute with 2.6 mL MeOH. Evaporate a 1 mL aliquot of
the eluate to dryness under a stream of nitrogen at 40◦ , reconstitute the residue with
200 µL mobile phase, inject a 100 µL aliquot.
HPLC VARIABLES
Guard column: GuardPak µBondapak C18
Column: 250 × 4.6 5 µm Symmetry C18
Column temperature: 37
Mobile phase: MeCN:40 mM disodium hydrogen phosphate containing 4% octanesulfonic acid 50:50. (At the end of each session, wash column with MeOH:water 50:50 and
MeCN:water 80:20.)
Flow rate: 1.3
Injection volume: 100
Detector: UV 261 for 9 min, UV 241 for 11 min, UV 254 for 12 min
CHROMATOGRAM
Retention time: 5.6
Limit of quantitation: 25 ng/mL
OTHER SUBSTANCES
Extracted: efavirenz (15.2, LOQ 50 ng/mL), indinavir (4.8, LOQ 50 ng/mL), nelfinavir
(19.2, LOQ 50 ng/mL), ritonavir (12.8, LOQ 50 ng/mL), saquinavir (16.8, LOQ 5 ng/mL)
acebutolol, acetaminophen, acetylcysteine, acyclovir,
albendazole, alimemazine, alizapride, amikacin, amiodarone, amphotericin B,
ampicillin, aspirin, bepridil, buprenorphine, butobarbital, caffeine, calcium folinate,
captopril, carbamazepine, carbutamide, chloroquine, ciprofloxacin, clindamycin,
clofazimine, clofibrate, clonazepam, clonidine, cloxacillin, clozapine, cocaine,
codeine, cyamemazine, dantrolene, dexamethasone, dextropropoxyphene, diazepam,
diclofenac, didanosine, digoxin, dihydroergotamine, diltiazem, doxycycline, ethambutol,
flecainide, fluconazole, fluoxetine, fluvoxamine, foscarnet, furosemide, ganciclovir,
gentamicin, glibenclamide, granisetron, halofantrine, haloperidol, hydrocortisone,
imipramine, indomethacin, interferon alfa, isoniazid, itraconazole, josamycin,
ketoconazole, lamivudine, levomepromazine, lidocaine, loperamide, loratadine,
losartan, mefloquine, meprobamate, methadone, methylprednisolone, metoclopramide,
metronidazole, mianserin, moclobemide, morphine, nevirapine, nifedipine, niflumic
acid, nitrofurantoin, omeprazole, paroxetine, pentamidine, phenobarbital, phenytoin,
piracetam, prazosin, prednisolone, prednisone, primidone, propranolol, quinidine,
quinine, ranitidine, ribavirin, rifabutin, rifampin, roxithromycin, salicylic acid,
simvastatin, stavudine, sulfadiazine, sulfamethoxazole, sulpiride, thalidomide,
theophylline, trimethoprim, valproic acid, venlafaxine, vigabatrin, viloxazine,
zidovudine, zolpidem, zopiclone
Interfering: delavirdine, flunitrazepam
Noninterfering: abacavir,
Amprenavir
37
KEY WORDS
plasma; SPE
REFERENCE
Aymard, G.; Legrand, M.; Trichereau, N.; Diquet, B. Determination of twelve antiretroviral agents in
human plasma sample using reversed-phase high-performance liquid chromatography, J.Chromatogr.
B, 2000, 744, 227–240.
SAMPLE
Matrix: blood
Sample preparation: Mix 250 µL plasma with 50 µL MeOH, add 100 µL 2 µg/mL IS
in MeOH, add 250 µL 1 M NaOH, add 3 mL hexane:ethyl acetate 50:50, shake at high
speed for 25 min, centrifuge at 3000 g for 15 min. Evaporate the organic layer to dryness
under a stream of air, reconstitute the residue with 1 mL initial mobile phase, inject a
20 µL aliquot.
HPLC VARIABLES
Guard column: 10 × 2.1 Symmetry Shield
Column: 30 × 2.1 3.5 µm Symmetry C18
Mobile phase: Gradient. MeCN:5 mM pH 3.25 acetate buffer from 25:75 to 80:20 over
4 min using a nonlinear gradient (not specified).
Flow rate: 0.35
Injection volume: 20
Detector: MS, PE Sciex API 3000, turbo ionspray source, column effluent split 1:1 before
entering source
CHROMATOGRAM
Retention time: 2.7
Internal standard: Abbott A-86093 (3.2)
Limit of detection: 380 pg/mL
Limit of quantitation: 16.3 ng/mL
OTHER SUBSTANCES
Extracted: indinavir (2.0, LOQ 16.3 ng/mL, LOD 1.5 ng/mL), lopinavir (3.1, LOQ
16.3 ng/mL, LOD 750 pg/mL), nelfinavir (2.5, LOQ 16.3 ng/mL, LOD 330 pg/mL), ritonavir (2.9, LOQ 51.2 ng/mL, LOD 650 pg/mL), saquinavir (2.4, LOQ 16.3 ng/mL, LOD
780 pg/mL)
KEY WORDS
plasma
REFERENCE
Frerichs, V.A.; DiFrancesco, R.; Morse, G.D. Determination of protease inhibitors using liquid chromatography-tandem mass spectrometry, J.Chromatogr.B, 2003, 787, 393–403.
SAMPLE
Matrix: blood
Sample preparation: Mix 1 mL plasma with 200 µL 10 µg/mL IS in water, add 200 µL
100 mM NaOH, mix, add 4 mL diethyl ether, shake for 5 min, centrifuge at 2500 rpm for
5 min. Evaporate the organic layer to dryness under a stream of nitrogen, reconstitute
the residue with 200 µL initial mobile phase, inject a 100 µL aliquot.
HPLC VARIABLES
Column: 250 × 4.6 Stability RP18 (CIL, France)
38
Amprenavir
Mobile phase: Gradient. MeCN:50 mM pH 5.65 phosphate buffer from 36:64 to 64:36
over 25 min, to 80:20 (step gradient), maintain at 80:20 for 10 min, re-equilibrate at
initial conditions for 5 min.
Flow rate: 1.5
Injection volume: 100
Detector: UV 240 for 5 min, UV 215 for 22 min, UV 260 for rest of the run
CHROMATOGRAM
Retention time: 11.2
Internal standard: JR051012 (Janssen Cilag) (28.2)
Limit of quantitation: 50 ng/mL
OTHER SUBSTANCES
Extracted: efavirenz (19.9), indinavir (8.5), lopinavir (18.9), nelfinavir (24.1), nevirapine
(3.3), ritonavir (17.6), saquinavir (16.7)
Noninterfering: acetaminophen, amineptine, amphotericin B, aspirin, bromazepam,
buspirone, citalopram, clobazam, diazepam, didanosine, fluconazole, flunitrazepam, fluvoxamine, hydroxyitraconazole, isoniazid, itraconazole, lamivudine, loprazolam, lorazepam, metronidazole, minalcipram, nordiazepam, omeprazole, paroxetine, pyrimethamine, rifampin, sertraline, stavudine, sulfadiazine, trimethoprim, venlafaxine, zalcitabine, zidovudine, zolpidem, zopiclone
KEY WORDS
plasma
REFERENCE
Titier, K.; Lagrange, F.; Péhourcq, F.; Edno-Mcheik, L.; Moore, N.; Molimard, M. High-performance liquid chromatographic method for the simultaneous determination of the six HIV-protease inhibitors
and two non-nucleoside reverse transcriptase inhibitors in human plasma, Ther.Drug Monit., 2002,
24, 417–424.
SAMPLE
Matrix: microsomal incubations
Sample preparation: Extract 100 µL incubation mixture twice with 5 mL MTBE.
Evaporate the organic layer to dryness, reconstitute the residue with 100 µL MeCN,
inject a 30 µL aliquot.
HPLC VARIABLES
Column: 150 × 4.6 Beckman ODS Ultrasphere
Column temperature: 45
Mobile phase: Gradient. A was 0.1% formic acid in water. B was 0.1% formic acid in
MeCN. A:B 100:0 for 1 min, to 30:70 over 3 min, to 5:95 over 3 min, maintain at 5:95 for
3 min, to 100:0 over 1 min.
Flow rate: 0.35
Injection volume: 30
Detector: MS, Hewlett-Packard 5989B, electrospray ionization, selected ion monitoring,
m/z 506.6
CHROMATOGRAM
Limit of quantitation: 25 ng/mL
OTHER SUBSTANCES
Also analyzed: astemizole, indinavir, ketoconazole, methadone, nelfinavir, rifabutin,
rifampin, ritonavir, saquinavir, terfenadine, trimethoprim
KEY WORDS
human; liver; rat
Amprenavir
39
REFERENCE
Decker, C.J.; Laitinen, L.M.; Bridson, G.W.; Raybuck, S.A.; Tung, R.D.; Chaturvedi, P.R. Metabolism of
amprenavir in liver microsomes: role of CYP3A4 inhibition for drug interactions, J.Pharm.Sci., 1998,
87, 803–807.
ANNOTATED BIBLIOGRAPHY
Brophy, D.F.; Israel, D.S.; Pastor, A.; Gillotin, C.; Chittick, G.E.; Symonds, W.T.; Lou, Y.; Sadler, B.M.;
Polk, R.E. Pharmacokinetic interaction between amprenavir and clarithromycin in healthy male
volunteers, Antimicrob.Agents Chemother., 2000, 44, 978–984. [no HPLC of clarithromycin]
Chi, J.; Jayewardene, A.L.; Stone, J.A.; Motoya, T.; Aweeka, F.T. Simultaneous determination of five
HIV protease inhibitors nelfinavir, indinavir, ritonavir, saquinavir and amprenavir in human plasma
by LC/MS/MS, J.Pharm.Biomed.Anal., 2002, 30, 675–684.
Cociglio, M.; Hillaire-Buys, D.; Peyrière, H.; Alric, R. Performance analysis of a rapid HPLC
determination with the solvent demixing extraction of HIV antiproteases and efavirenz in
plasma, J.Chromatogr.Sci., 2003, 41, 80–86. [efavirenz; indinavir; amprenavir; ritonavir; saquinavir;
nelfinavir]
Dailly, E.; Thomas, L.; Kergueris, M.F.; Jolliet, P.; Bourin, M. High-performance liquid chromatographic
assay to determine the plasma levels of HIV-protease inhibitors (amprenavir, indinavir, nelfinavir,
ritonavir and saquinavir) and the non-nucleoside reverse transcriptase inhibitor (nevirapine) after
liquid-liquid extraction, J.Chromatogr.B, 2001, 758, 129–135.
Droste, J.A.H.; Verweij-van Wissen, C.P.W.G.M.; Burger, D.M. Simultaneous determination of the
HIV drugs indinavir, amprenavir, saquinavir, ritonavir, lopinavir, nelfinavir, the nelfinavir
hydroxymetabolite M8, and nevirapine in human plasma by reversed-phase high-performance liquid
chromatography, Ther.Drug Monit., 2003, 25, 393–399.
Edwards, J.E.; Brouwer, K.R.; McNamara, P.J. GF120918, a P-glycoprotein modulator, increases the
concentration of unbound amprenavir in the central nervous system in rats, Antimicrob.Agents
Chemother., 2002, 46, 2284–2286.
Faux, J.; Venisse, N.; Le Moal, G.; Dupuis, A.; Bouquet, S. Simultaneous determination of six HIV
protease inhibitors, one metabolite, and two non-nucleoside reverse transcriptase inhibitors in
human plasma by isocratic reversed-phase liquid chromatography after solid-phase extraction,
Chromatographia 2003, 58, 421–426. [amprenavir; indinavir; lopinavir; nelfinavir; ritonavir;
saquinavir; efavirenz; nevirapine; prazepam]
Gao, W.; Kishida, T.; Kimura, K.; Kageyama, M.; Sumi, M.; Yoshikawa, Y.; Shibata, N.; Takada, K.
Sensitive and simultaneous determination of HIV protease inhibitors in rat biological samples by liquid
chromatography-mass spectrometry, Biomed.Chromatogr., 2002, 16, 267–273. [indinavir; amprenavir;
saquinavir; nelfinavir]
Goujard, C.; Vincent, I.; Meynard, J.-L.; Choudet, N.; Bollens, D.; Rousseau, C.; Demarles, D.;
Gillotin, C.; Bidault, R.; Taburet, A.-M. Steady-state pharmacokinetics of amprenavir coadministered
with ritonavir in human immunodeficiency virus type 1-infected patients, Antimicrob.Agents
Chemother., 2003, 47, 118–123.
Gunawan, S.; Griswold, M.P.; Kahn, D.G. Liquid chromatographic-tandem mass spectrometric
determination of amprenavir (agenerase) in serum/plasma of human immunodeficiency virus type-1
infected patients receiving combination antiretroviral therapy, J.Chromatogr.A, 2001, 914, 1–4.
Huang, L.; Wring, S.A.; Woolley, J.L.; Brouwer, K.R.; Serabjit-Singh, C.; Polli, J.W. Induction of Pglycoprotein and cytochrome P450 3A by HIV protease inhibitors, Drug Metab.Dispos., 2001, 29,
754–760. [fosamprenavir; amprenavir; nelfinavir]
Justesen, U.S.; Pedersen, C.; Klitgaard, N.A. Simultaneous quantitative determination of the HIV
protease inhibitors indinavir, amprenavir, ritonavir, lopinavir, saquinavir, nelfinavir and the nelfinavir
active metabolite M8 in plasma by liquid chromatography, J.Chromatogr.B, 2003, 783, 491–500.
Keil, K.; Frerichs, V.A.; DiFrancesco, R.; Morse, G. Reverse phase high-performance liquid
chromatography method for the analysis of amprenavir, efavirenz, indinavir, lopinavir, nelfinavir
and its active metabolite (M8), ritonavir, and saquinavir in heparinized human plasma, Ther.Drug
Monit., 2003, 25, 340–346.
Leibenguth, P.; Le Guellec, C.; Besnier, J.-M.; Bastides, F.; Macé, M.; Gaudet, M.-L.; Autret-Leca, E.;
Paintaud, G. Therapeutic drug monitoring of HIV protease inhibitors using high-performance liquid
chromatography with ultraviolet or photodiode array detection, Ther.Drug Monit., 2001, 23, 679–688.
[indinavir; saquinavir; lopinavir; ritonavir; nelfinavir; amprenavir; carbamazepine]
40
Amprenavir
Marzolini, C.; Telenti, A.; Buclin, T.; Biollaz, J.; Decosterd, L.A. Simultaneous determination of the HIV
protease inhibitors indinavir, amprenavir, saquinavir, ritonavir, nelfinavir and the non-nucleoside
reverse transcriptase inhibitor efavirenz by high-performance liquid chromatography after solid-phase
extraction, J.Chromatogr.B, 2000, 740, 43–58.
Marzolini, C.; Béguin, A.; Telenti, A.; Schreyer, A.; Buclin, T.; Biollaz, J.; Decosterd, L.A. Determination
of lopinavir and nevirapine by high-performance liquid chromatography after solid-phase extraction:
application for the assessment of their transplacental passage at delivery, J.Chromatogr.B, 2002, 774,
127–140. [lopinavir; nevirapine; amprenavir; efavirenz; clozapine]
Pereira, A.S.; Kenney, K.B.; Cohen, M.S.; Eron, J.J.; Tidwell, R.R.; Dunn, J.A. Determination of
amprenavir, a HIV-1 protease inhibitor, in human seminal plasma using high-performance liquid
chromatography-tandem mass spectrometry, J.Chromatogr.B, 2002, 766, 307–317.
Poirier, J.-M.; Radembino, N.; Robidou, P.; Jaillon, P. Simultaneous determination of the five HIVprotease inhibitors: Amprenavir, indinavir, nelfinavir, ritonavir, and saquinavir in human plasma by
solid-phase extraction and column liquid chromatography, Ther.Drug Monit., 2000, 22, 465–473.
Poirier, J.M.; Robidou, P.; Jaillon, P. Simultaneous determination of the six HIV protease inhibitors
(amprenavir, indinavir, lopinavir, nelfinavir, ritonavir, and saquinavir) plus M8 nelfinavir metabolite
and the nonnucleoside reverse transcription inhibitor efavirenz in human plasma by solid-phase
extraction and column liquid chromatography, Ther.Drug Monit., 2002, 24, 302–309.
Polk, R.E.; Brophy, D.F.; Israel, D.S.; Patron, R.; Sadler, B.M.; Chittick, G.E.; Symonds, W.T.; Lou, Y.;
Kristoff, D.; Stein, D.S. Pharmacokinetic interaction between amprenavir and rifabutin or rifampin in
healthy males, Antimicrob.Agents Chemother., 2001, 45, 502–508.
Proust, V.; Toth, K.; Hulin, A.; Taburet, A.-M.; Gimenez, F.; Singlas, E. Simultaneous high-performance
liquid chromatographic determination of the antiretroviral agents amprenavir, nelfinavir, ritonavir,
saquinavir, delavirdine and efavirenz in human plasma, J.Chromatogr.B, 2000, 742, 453–458.
Rentsch, K.M. Sensitive and specific determination of eight antiretroviral agents in plasma by highperformance liquid chromatography-mass spectrometry, J.Chromatogr.B, 2003, 788, 339–350. [SPE;
amprenavir; efavirenz; indinavir; lopinavir; nelfinavir; nevirapine; ritonavir; saquinavir]
Sadler, B.M.; Hanson, C.D.; Chittick, G.E.; Symonds, W.T.; Roskell, N.S. Safety and pharmacokinetics
of amprenavir (141 W94), a human immunodeficiency virus (HIV) type I protease inhibitor, following
oral administration of single doses to HIV-infected adults, Antimicrob.Agents Chemother., 1999, 43,
1686–1692.
Sadler, B.M.; Chittick, G.E.; Polk, R.E.; Slain, D.; Kerkering, T.M.; Studenberg, S.D.; Lou, Y.;
Moore, K.H.P.; Woolley, J.L.; Stein, D.S. Metabolic disposition and pharmacokinetics of [14C]amprenavir, a human immunodeficiency virus Type 1 (HIV-1) protease inhibitor, administered as
a single oral dose to healthy male subjects, J.Clin.Pharmacol., 2001, 41, 386–396.
Sarasa-Nacenta, M.; López-Púa, Y.; Mallolas, J.; Blanco, J.L.; Gatell, J.M.; Carné, X. Simultaneous
determination of the HIV-protease inhibitors indinavir, amprenavir, ritonavir, saquinavir
and nelfinavir in human plasma by reversed-phase high-performance liquid chromatography,
J.Chromatogr.B, 2001, 757, 325–332.
Sparidans, R.W.; Hoetelmans, R.M.W.; Beijnen, J.H. Sensitive liquid chromatographic assay for
amprenavir, a human immunodeficiency virus protease inhibitor, in human plasma, cerebrospinal
fluid and semen, J.Chromatogr.B, 2000, 742, 185–192.
Tran, J.Q.; Petersen, C.; Garrett, M.; Hee, B.; Kerr, B.M. Pharmacokinetic interaction between
amprenavir and delavirdine: evidence of induced clearance by amprenavir, Clin.Pharmacol.Ther.,
2002, 72, 615–626.
Tréluyer, J.M.; Bowers, G.; Cazali, N.; Sonnier, M.; Rey, E.; Pons, G.; Cresteil, T. Oxidative metabolism
of amprenavir in the human liver. Effect of the CYP3A maturation, Drug Metab.Dispos., 2003, 31,
275–281.
Tribut, O.; Arvieux, C.; Michelet, C.; Chapplain, J.-M.; Allain, H.; Bentué-Ferrer, D. Simultaneous
quantitative assay of six HIV protease inhibitors, one metabolite, and two non-nucleoside reverse
transcriptase inhibitors in human plasma by isocratic reversed-phase liquid chromatography,
Ther.Drug Monit., 2002, 24, 554–562. [nevirapine; efavirenz; indinavir; amprenavir; nelfinavir;
ritonavir; lopinavir; saquinavir]
Turner, M.L.; Reed-Walker, K.; King, J.R.; Acosta, E.P. Simultaneous determination of nine
antiretroviral compounds in human plasma using liquid chromatography, J.Chromatogr.B, 2003, 784,
331–341. [indinavir; nelfinavir; saquinavir; ritonavir; amprenavir; delavirdine; efavirenz; lopinavir]
van Heeswijk, R.P.G.; Hoetelmans, R.M.W.; Harms, R.; Meenhorst, P.L.; Mulder, J.W.; Lange, J.M.A.;
Beijnen, J.H. Simultaneous quantitative determination of the HIV protease inhibitors amprenavir,
indinavir, nelfinavir, ritonavir and saquinavir in human plasma by ion-pair high-performance liquid
chromatography with ultraviolet detection, J.Chromatogr.B, 1998, 719, 159–168.
Amprenavir
41
Veronese, L.; Rautaureau, J.; Sadler, B.M.; Gillotin, C.; Petite, J.-P.; Pillegand, B.; Delvaux, M.;
Masliah, C.; Fosse, S.; Lou, Y.; Stein, D.S. Single-dose pharmacokinetics of amprenavir, a human
immunodeficiency virus type 1 protease inhibitor, in subjects with normal or impaired hepatic function,
Antimicrob.Agents Chemother., 2000, 44, 821–826.
Villani, P.; Feroggio, M.; Gianelli, L.; Bartoli, A.; Montagna, M.; Maserati, R.; Regazzi, M.B.
Antiretrovirals: simultaneous determination of five protease inhibitors and three nonnucleoside
transcriptase inhibitors in human plasma by a rapid high-performance liquid chromatography-mass
spectrometry assay, Ther.Drug Monit., 2001, 23, 380–388. [saquinavir; indinavir; ritonavir; nelfinavir;
amprenavir; nevirapine; delavirdine; efavirenz]
Yamada, H.; Kotaki, H.; Nakamura, T.; Iwamoto, A. Simultaneous determination of the HIV protease
inhibitors indinavir, amprenavir, saquinavir, ritonavir and nelfinavir in human plasma by highperformance liquid chromatography, J.Chromatogr.B, 2001, 755, 85–89.
Yu, L.; Bridgers, A.; Polli, J.; Vikers, A.; Long, S.; Roy, A.; Winnike, R.; Coffin, M. Vitamin E-TPGS
increases absorption flux of an HIV protease inhibitor by enhancing its solubility and permeability,
Pharm.Res., 1999, 16, 1812–1817.
42
Anagrelide
Anagrelide
Molecular formula: C10 H7 Cl2 N3 O
Molecular weight: 256.09
CAS Registry No: 68475-42-3
Merck Index: 13, 629
H
N
N
N
Cl
O
Cl
SAMPLE
Matrix: blood, urine
Sample preparation: Mix 2 mL plasma or urine with 2 mL 200 mM pH 7.0 phosphate
buffer, extract twice with 10 mL portions of ethyl acetate. Evaporate the organic layer
to dryness under a stream of nitrogen, reconstitute the residue with 60 µL DMSO, mix,
sonicate, inject a 40 µL aliquot.
HPLC VARIABLES
Guard column: 40 mm long µBondapak phenyl corasil
Column: 300 × 3.9 µBondapak phenyl
Mobile phase: MeCN:10 mM pH 4 sodium acetate buffer 25:75 for 10 min, DMSO for
8 min, return to original mobile phase
Flow rate: 2.5 for 13 min, 1 for 5 min, 2.5 for rest of the run
Injection volume: 40
Detector: UV 254; Radioactivity (14 C)
CHROMATOGRAM
Retention time: 6–8
KEY WORDS
plasma; radiolabeled
REFERENCE
Gaver, R.C.; Deeb, G.; Pittman, K.A.; Smyth, R.D. Disposition of anagrelide, an inhibitor of platelet
aggregation, Clin.Pharmacol.Ther., 1981, 29, 381–386.
Anakinra
43
Anakinra
Molecular weight: 17 000
CAS Registry No: 143090-92-0
Merck Index: 13, 5022
SAMPLE
Matrix: blood, tissue
Sample preparation: Inject a 50 µL aliquot of plasma or tissue homogenate supernatant.
HPLC VARIABLES
Guard column: 40 × 6 Spherogel TSK PWHR (Beckman)
Column: 300 × 7.8 5 µm Progel-TSK G2000 SWXL (Supelco)
Mobile phase: 10 mM pH 6.5 citrate buffer containing 140 mM NaCl and 0.5 mM EDTA
Flow rate: 0.5
Injection volume: 50
Detector: UV; Radioactivity (35 S); ELISA
CHROMATOGRAM
Retention time: 20
KEY WORDS
brain; gut; heart; kidney; liver; lung; muscle; plasma; rat; spleen
REFERENCE
Kim, D.C.; Reitz, B.; Carmichael, D.F.; Bloedow, D.C. Kidney as a major clearance organ for recombinant
human interleukin-1 receptor antagonist, J.Pharm.Sci., 1995, 84, 575–580.
SAMPLE
Matrix: solutions
Sample preparation: Inject a 100 µL aliquot of a 2–5 mg/mL solution in 10 mM pH
6.5 citrate buffer containing 140 mM NaCl and 0.5 mM EDTA.
HPLC VARIABLES
Column: 75 × 7.5 Bio-Gel SP-5-PW (Bio-Rad)
Mobile phase: Gradient. A:B from 99:1 to 40:60 over 60 min. A was 20 mM pH
5.5 2-(N-morpholino)ethanesulfonic acid monohydrate. B was 20 mM pH 5.5 2-(Nmorpholino)ethanesulfonic acid monohydrate containing 1.0 M NaCl.
Flow rate: 0.5
Injection volume: 100
Detector: UV 280
REFERENCE
Nahata, M.C.; Morosco, R.S.; Sabados, B.K.; Weber, T.R. Stability and compatibility of anakinra with
intravenous cimetidine hydrochloride or famotidine in 0.9% sodium chloride injection, J.Clin.Pharm.
Ther., 1995, 20, 97–99.
ANNOTATED BIBLIOGRAPHY
Chang, B.S.; Beauvais, R.M.; Arakawa, T.; Narhi, L.O.; Dong, A.; Aparisio, D.I.; Carpenter, J.F. Formation of an active dimer during storage of interleukin-1 receptor antagonist in aqueous solution,
Biophys.J., 1996, 71, 3399–3406.
44
Anakinra
Chang, B.S.; Reeder, G.; Carpenter, J.F. Development of a stable freeze-dried formulation of recombinant
human interleukin-1 receptor antagonist, Pharm.Res., 1996, 13, 243–249.
Nahata, M.C.; Morosco, R.S.; Sabados, B.K.; Weber, T.R. Stability and compatibility of anakinra with
ceftriaxone sodium injection in 0.9% sodium chloride or 5% dextrose injection, J.Clin.Pharm.Ther.,
1997, 22, 167–169.
45
Apraclonidine
Apraclonidine
Molecular formula: C9 H10 Cl2 N4
Molecular weight: 245.11
CAS Registry No: 66711-21-5
Merck Index: 13, 756
Cl
H
N
N
NH
Cl
NH2
SAMPLE
Matrix: solutions
Sample preparation: Inject a 50 µL aliquot of a solution in glutathione bicarbonated
Ringer’s solution (pH 7.4).
HPLC VARIABLES
Column: 150 × 4 5 µm Ultrasphere ODS
Mobile phase: MeCN:water 20:80 to 60:40 (?) containing 5 mM sodium heptanesulfonic
acid at pH 3.5
Flow rate: 1–1.5
Injection volume: 50
Detector: UV 254
OTHER SUBSTANCES
Simultaneous: clonidine
REFERENCE
Chien, D.S.; Homsy, J.J.; Gluchowski, C.; Tang-Liu, D.D.-S. Corneal and conjunctival/scleral penetration
of p-aminoclonidine, AGN 190342, and clonidine in rabbit eyes, Current Eye Res., 1990, 9, 1051–1059.
46
Aprepitant
Aprepitant
O
O
Molecular formula: C23 H21 F7 N4 O3
Molecular weight: 534.43
CAS Registry No: 170729-80-3
N
N
H
H H CH3
CF3
O
N
H
N
CF3
H
F
SAMPLE
Matrix: blood, tissue
Sample preparation: Mix 200 µL plasma with 20 ng IS and 1.7 mL water, add 500 µL
MeCN, add to a 500 mg Bond Elut C18 SPE cartridge, wash with 6 mL water, elute with
3 mL MeOH. Evaporate the eluate to dryness under a stream of nitrogen, reconstitute
the residue with 300 µL mobile phase, inject an aliquot. Alternatively, mix 50 µL plasma
or brain homogenate with 5 ng IS and 100 µL MeCN, vortex, centrifuge at 3000 g for
10 min, inject a 5–25 µL aliquot of the supernatant.
HPLC VARIABLES
Column: 50 × 4.6 5 µm Spherisorb C8
Mobile phase: MeCN:10 mM ammonium acetate:formic acid 55:45:0.1
Flow rate: 1
Injection volume: 5–25
Detector: MS, Sciex API III+, heated nebulizer interface, dwell time 450 ms, m/z 535 to
179
CHROMATOGRAM
Retention time: 1.5
Internal standard: desfluoroaprepitant (m/z 535 to 161) (1.5)
KEY WORDS
brain; ferret; plasma; SPE
REFERENCE
Huskey, S.-E.W.; Dean, B.J.; Bakhtiar, R.; Sanchez, R.I.; Tattersall, F.D.; Rycroft, W.; Hargreaves, R.;
Watt, A.P.; Chicchi, G.G.; Keohane, C.; Hora, D.F.; Chiu, S.-H.L. Brain penetration of aprepitant, a
substance P receptor antagonist, in ferrets, Drug Metab.Dispos., 2003, 31, 785–791.
SAMPLE
Matrix: blood, tissue
Sample preparation: Mix 3 mL plasma with 6 mL MeCN, centrifuge at 3000 g, evaporate the supernatant to dryness under a stream of nitrogen, reconstitute the residue
with 1 mL MeOH:water 40:60, inject a 250–400 µL aliquot of the supernatant. Homogenize the brain with 3 volumes of water. Vortex 10 mL homogenate with 90 mL MeCN,
sonicate for 5 min, centrifuge at 3000 g for 10 min, re-extract the pellet with 10 mL
MeOH. Combine the organic layers and add to a Bond Elut C18 SPE cartridge equipped
with an Acrodisc glass filter, elute with 5 mL MeOH:MeCN:water 50:25:25. Collect all
the cartridge effluent and evaporate to dryness under a stream of nitrogen, reconstitute
the residue with 5 mL MeOH, vortex, sonicate, centrifuge. Evaporate the supernatant
to dryness under a stream of nitrogen, reconstitute the residue with 1 mL MeOH:water
40:60, inject a 400 µL aliquot of the supernatant.
HPLC VARIABLES
Column: 250 × 4.6 Zorbax RX-C8
Aprepitant
47
Mobile phase: Gradient. A:B 65:35 to 20:80 over 40 min. A was 10 mM ammonium
acetate. B was MeCN:MeOH 92.8:7.2 containing 7.2 mM ammonium acetate. (Alternatively, A 10 mM ammonium acetate in water containing 0.1% trifluoroacetic acid and B
MeCN:MeOH 92.8:7.2 containing 7.2 mM ammonium acetate and 0.1% trifluoroacetic
acid with the same gradient.)
Flow rate: 1
Injection volume: 250–400
Detector: Radioactivity (14 C)
CHROMATOGRAM
Retention time: 26
Internal standard: desfluoroaprepitant (m/z 535 to 161) (1.5)
OTHER SUBSTANCES
Extracted: metabolites
KEY WORDS
brain; ferret; plasma; SPE
REFERENCE
Huskey, S.E.W.; Dean, B.J.; Bakhtiar, R.; Sanchez, R.I.; Tattersall, F.D.; Rycroft, W.; Hargreaves, R.;
Watt, A.P.; Chicchi, G.G.; Keohane, C.; Hora, D.F.; Chiu, S.H.L. Brain penetration of aprepitant, a
substance P receptor antagonist, in ferrets, Drug Metab.Dispos., 2003, 31, 785–791.
SAMPLE
Matrix: solutions
Sample preparation: Inject a 1 µL aliquot of a solution in MeOH:water 10:90.
HPLC VARIABLES
Column: 20 × 2 5 µm DASH BetaBasic C8 (ThermoHypersil Keystone)
Mobile phase: Gradient. A was MeCN:water:formic acid 5:95:0.1. B was MeCN:water:
formic acid 95:5:0.1. A:B 100:0 for 0.2 min, to 0:100 over 1.5 min.
Flow rate: 1.5
Injection volume: 1
Detector: MS, PE Sciex API-3000, turbo ionspray, electrospray 4500 V, ring 290 V, orifice
60 V, drying gas 400◦ , 20% of column effluent entered the detector, m/z 535.3–277
CHROMATOGRAM
Retention time: 1.4
OTHER SUBSTANCES
Simultaneous: amitriptyline (m/z 278.3–233) (1.1), diclofenac (m/z 296.1–215) (1.35),
enoxacin (m/z 321.2–234) (0.7), fenofibrate (m/z 360.9–233) (1.6), finasteride (m/z
373.2–317) (1.2), indinavir (m/z 614.4–421) (0.93), pioglitazone (357.2–134) (0.87),
raloxifene (m/z 474.1–112) (0.97)
REFERENCE
Romanyshyn, L.A.; Tiller, P.R. Ultra-short columns and ballistic gradients: considerations for ultra-fast
chromatographic liquid chromatographic-tandem mass spectrometric analysis, J.Chromatogr.A, 2001,
928, 41–51.
48
Aranidipine
Aranidipine
H
O
Molecular formula: C19 H20 N2 O7
Molecular weight: 388.37
CAS Registry No: 86780-90-7
Merck Index: 13, 772
CH3
H3C
N
CH3
O
OCH3
O
O
NO2
SAMPLE
Matrix: blood
Sample preparation: Add 20 ng nifedipine and 500 µL 100 mM pH 9.0 borate buffer
to 1 mL plasma, vortex for 10 s, add 6 mL toluene, shake mechanically for 10 min,
centrifuge at 1000 g for 15 min. Evaporate the organic layer to dryness under a stream
of nitrogen at 40◦ , reconstitute the residue with 200 µL mobile phase, inject a 50 µL
aliquot. (Carry out all steps under yellow fluorescent lighting.)
HPLC VARIABLES
Column: 150 × 4.6 5 µm Inertsil ODS-2
Column temperature: 40
Mobile phase: MeOH:360 mM sodium perchlorate 45:55
Flow rate: 0.8
Injection volume: 50
Detector: E, BAS LC-4B/17AT, +0.92 V versus Ag/AgCl
CHROMATOGRAM
Retention time: 16
Internal standard: nifedipine (26)
Limit of quantitation: 2.5 ng/mL
OTHER SUBSTANCES
Extracted: metabolites
KEY WORDS
dog; pharmacokinetics; plasma
REFERENCE
Iida, Y.; Kinouchi, Y.; Takeichi, Y.; Imai, T.; Otagiri, M. Simultaneous determination of a new dihydropyridine calcium antagonist (MPC-1304) and its metabolite in dog plasma by high-performance liquid
chromatography with electrochemical detection, J.Chromatogr., 1991, 571, 277–282.
49
Arotinolol
Arotinolol
O
H2N
Molecular formula: C15 H21 N3 O2 S3
CH3
CH3
S
S
N
N
S
Molecular weight: 371.55
CAS Registry No: 68377-92-4, 68377-91-3 (HCl)
Merck Index: 13, 797
OH
CH3
H
SAMPLE
Matrix: blood
Sample preparation: Add 100 µL 10 mg/mL IS in water to 500 µL plasma, make up
to 1 mL with water, add 100 µL 3 M pH 9 ammonium acetate, vortex vigorously for
2 min, centrifuge at 3000 g for 10 min. Extract the aqueous layer three times with 1 mL
portions of ether, evaporate the extracts to dryness under reduced pressure, reconstitute
the residue with 100 µL 100 mM HCl, inject an aliquot.
HPLC VARIABLES
Column: 250 × 4.6 Chirobiotic T (Advanced Separation Technologies)
Mobile phase: MeOH:acetic acid:triethylamine 100:0.1:0.1
Flow rate: 0.8
Detector: UV 317
CHROMATOGRAM
Retention time: 17.25 (S-(+)), 20.06 (R-(–) )
Internal standard: labetalol hydrochloride (21.98, 23.43 (enantiomers))
Limit of detection: 50 ng/mL
Limit of quantitation: 100 ng/mL
KEY WORDS
chiral; plasma
REFERENCE
Aboul-Enein, H.Y.; Hefnawy, M.M. Enantioselective determination of arotinolol in human plasma by
HPLC using teicoplanin chiral stationary phase, Biomed.Chromatogr., 2003, 17, 453–457.
SAMPLE
Matrix: blood, urine
Sample preparation: Condition a 1 mL C18 Bakerbond SPE cartridge with MeOH.
Mix 1 mL plasma with 50 µL 5 µg/mL alpiropride in water. Mix 100 µL pure or diluted
urine with 250 µL blank plasma and 100 µL 5 µg/mL alpiropride in water. Add the
sample to the SPE cartridge, wash three times with 1 mL portions of water, wash three
times with 1 mL portions of n-hexane:diethyl ether 50:50, elute with two 1 mL portions
of chloroform:triethylamine 90:10 (Caution! Chloroform is a carcinogen!). Evaporate the
eluate to dryness under reduced pressure, reconstitute the residue with 150 µL mobile
phase, inject a 100 µL aliquot.
HPLC VARIABLES
Guard column: 15 × 4.6 7 µm C18
Column: 250 × 4.6 5 µm ODS Hypersil
Column temperature: 25
Mobile phase: MeCN:MeOH:buffer 12.5:12.5:75 (The buffer was 67 mM pH 5.6 phosphate buffer containing 0.6 mM tetrabutylammonium chloride.)
Flow rate: 1.2
Injection volume: 100
Detector: F ex 310 em 395; UV 310
50
Arotinolol
CHROMATOGRAM
Retention time: 10.0
Internal standard: alpiropride (4.7)
Limit of detection: 0.11 ng/mL (plasma), 11 ng/mL (urine)
OTHER SUBSTANCES
Extracted: metabolites
KEY WORDS
pharmacokinetics; plasma; SPE
REFERENCE
Moulin, A.; Maillet, E.; Truffer, D.; Dufour, A. High performance liquid chromatographic determination
of arotinolol and AC 623, its main metabolite in biological samples, J.Liq.Chromatogr., 1992, 15,
151–164.
SAMPLE
Matrix: blood, urine
Sample preparation: Condition a 1 mL C18 Bakerbond SPE cartridge with MeOH.
Mix 1 mL plasma with 50 µL 5 µg/mL alpiropride in water. Mix 100 µL pure or diluted
urine with 250 µL blank plasma and 100 µL 5 µg/mL alpiropride in water. Add the
sample to the SPE cartridge, wash three times with 1 mL portions of water, wash three
times with 1 mL portions of n-hexane:diethyl ether 50:50, elute with two 1 mL portions
of chloroform:triethylamine 90:10 (Caution! Chloroform is a carcinogen!). Evaporate the
eluate to dryness under reduced pressure, reconstitute the residue with 150 µL mobile
phase, inject a 100 µL aliquot.
HPLC VARIABLES
Guard column: 15 × 4.6 7 µm diol
Column: 200 × 4.6 5 µm Lichrosorb diol
Column temperature: 25
Mobile phase: Dichloromethane containing 10 mM Z-glycyl-L-proline:MeOH 100:1
Flow rate: 2
Injection volume: 100
Detector: F ex 320 em 425
CHROMATOGRAM
Retention time: 12 (R-(–) ), 15 (S-(+))
Internal standard: alpiropride (7.5)
Limit of detection: 2 ng/mL
OTHER SUBSTANCES
Extracted: metabolites
KEY WORDS
chiral; plasma; SPE
REFERENCE
Moulin, A.; Truffer, D.; Maillet, E.; Dufour, A. High performance liquid chromatographic determination of the optical isomers of arotinolol and AC 623, its main metabolite, in biological samples,
J.Liq.Chromatogr., 1992, 15, 165–181.
Arotinolol
51
ANNOTATED BIBLIOGRAPHY
Nakamura, K.; Fujima, H.; Kitagawa, H.; Wada, H.; Makino, K. Preparation and chromatographic characteristics of a chiral-recognizing perphenylated cyclodextrin column, J.Chromatogr.A, 1995, 694,
111–118. [ibuprofen; chlorpheniramine; acetylpheneturide; alprenolol; arotinolol; atenolol; benzoin;
biperiden; bunitrolol; chlormezanone; chlorphenesin; eperisone; flavanone; oxprenolol; phenylethyl
alcohol; phenylethylamine; pindolol; proglumide; propranolol; trihexyphenidyl]
52
Arteether
Arteether
Molecular formula: C17 H28 O5
Molecular weight: 312.40
CAS Registry No: 75887-54-6
Merck Index: 13, 822
H
H3C
CH3
O O
O
H
H
H
CH3
O
H
O
CH3
SAMPLE
Matrix: blood
Sample preparation: Add 5 µL 10 µg/mL artemisinin in MeOH to 200 µL serum,
vortex, add 2 mL hexane, vortex for 1 min, centrifuge at 1000 g for 5 min, freeze in
liquid nitrogen. Repeat the extraction. Combine the organic layers and evaporate to
dryness, reconstitute the residue with 40 µL MeOH, inject a 20 µL aliquot.
HPLC VARIABLES
Column: 300 × 4.6 5 µm Ultracarb 5 ODS 20 (Phenomenex)
Mobile phase: MeOH:100 mM sodium acetate 80:20
Flow rate: 1
Injection volume: 20
Detector: MS, Quattro II triple quadrupole, electrospray, nebulizing gas nitrogen 10 L/h,
curtain gas nitrogen 250 L/h, ESI capillary at 3.5 kV, cone voltage 52 V, positive mode,
m/z 335 [M + Na]+, one tenth of column effluent was allowed into MS
CHROMATOGRAM
Retention time: 1.73 (α), 2.81 (β)
Internal standard: artemisinin (m/z 305) (1.02)
Limit of detection: 5 ng/mL
Limit of quantitation: 20 ng/mL
KEY WORDS
rat; serum
REFERENCE
Rajanikanth, M.; Madhusudanan, K.P.; Gupta, R.C. Liquid chromatographic-mass spectrometric method
for the determination of α-, β-arteether in rat serum, J.Chromatogr.B, 2003, 783, 391–399.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 × 4.6 5 µm IBO-SIL C18 (Phenomenex)
Mobile phase: MeOH:water 80:20
Flow rate: 0.9
Detector: UV 260
REFERENCE
Illapakurthy, A.C.; Sabnis, Y.A.; Avery, B.A.; Avery, M.A.; Wyandt, C.M. Interaction of artemisinin
and its related compounds with hydroxypropyl-β-cyclodextrin in solution state: experimental and
molecular-modeling studies, J.Pharm.Sci., 2003, 92, 649–655.
Arteether
53
ANNOTATED BIBLIOGRAPHY
Baker, J.K.; Yarber, R.H.; Hufford, C.D.; Lee, I.-S.; ElSohly, H.N.; McChesney, J.D. Thermospray mass
spectroscopy/high performance liquid chromatographic identification of the metabolites formed from
arteether using a rat liver microsome preparation, Biomed.Environ.Mass Spectrom., 1989, 18, 337–351.
Baker, J.K.; McChesney, J.D.; Chi, H.T. Decomposition of arteether in simulated stomach acid yielding
compounds retaining antimalarial activity, Pharm.Res., 1993, 10, 662–666.
Benakis, A.; Schopfer, C.; Paris, M.; Plessas, C.; Karayannakos, P.E.; Dondas, I.; Kotsarelis, D.;
Plessas, S.T.; Skalkeas, G. Pharmacokinetics of arteether in dog, Eur.J.Drug Metab.Pharmacokinet.,
1991, 16, 325–328.
Chi, H.T.; Ramu, K.; Baker, J.K.; Hufford, C.D.; Lee, I.S.; Zeng, Y.-L.; McChesney, J.D. Identification
of the in vivo metabolites of the antimalarial arteether by thermospray high-performance liquid
chromatography/mass spectrometry, Biol.Mass Spectrom., 1991, 20, 609–628.
Idowu, O.R.; Edwards, G.; Ward, S.A.; Orme, M.L’E.; Breckenridge, A.M. Determination of arteether in
blood plasma by high-performance liquid chromatography with ultraviolet detection after hydrolysis
with acid, J.Chromatogr., 1989, 493, 125–136.
Leo, K.U.; Grace, J.M.; Li, Q.; Peggins, J.; Mitchell, A.L.; Aguilar, T.; Brewer, T.G. Effects of Plasmodium
berghei infection on arteether metabolism and disposition, Pharmacology, 1997, 54, 276–284.
Leskovac, V.; Theoharides, A.D. Hepatic metabolism of artemisinin drugs – I. Drug metabolism in rat
liver microsomes, Comp.Biochem.Physiol.C, 1991, 99, 383–390. [arteether; dihydroartemisinin]
Li, Q.G.; Brueckner, R.P.; Peggins, J.O.; Trotman, K.M.; Brewer, T.G. Arteether toxicokinetics
and pharmacokinetics in rats after 25 mg/kg/day single and multiple doses, Eur.J.Drug
Metab.Pharmacokinet., 1999, 24, 213–223.
Melendez, V.; Peggins, J.O.; Brewer, T.G.; Theoharides, A.D. Determination of the antimalarial
arteether and its deethylated metabolite dihydroartemisinin in plasma by high-performance liquid
chromatography with reductive electrochemical detection, J.Pharm.Sci., 1991, 80, 132–138.
Rajanikanth, M.; Madhusudanan, K.P.; Gupta, R.C. An HPLC-MS method for simultaneous estimation of
α,β-arteether and its metabolite dihydroartemisinin, in rat plasma for application to pharmacokinetic
study, Biomed.Chromatogr., 2003, 17, 440–446.
Ramu, K.; Baker, J.K. Identification of the glucuronides of the hydroxylated metabolites of
the antimalarial arteether in rat plasma and urine by thermospray high-performance liquid
chromatography/mass spectrometry, J.Pharm.Sci., 1997, 86, 915–920.
54
Articaine
Articaine
Molecular formula: C13 H20 N2 O3 S
Molecular weight: 284.38
CAS Registry No: 23964-58-1, 23964-57-0 (HCl)
Merck Index: 13, 1884
O
OCH3
O
H
S
H3C
N
H
N
CH3
CH3
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 50 µL 2 µg/mL etidocaine in water + 100 µL
1 M NaOH + 3 mL heptane:ethyl acetate 90:10, shake for 2 min, centrifuge at 1200 g
for 10 min. Remove the organic phase and add it to 50 µL 50 mM sulfuric acid, shake for
2 min, centrifuge at 1200 g for 5 min. Remove the aqueous phase and add it to 820 µg
sodium acetate, inject a 40 µL aliquot. (The sodium acetate was measured out by adding
50 µL 200 mM sodium acetate in MeOH to the tube and evaporating the MeOH.)
HPLC VARIABLES
Column: 250 × 4 10 µm µBondapak C18
Column temperature: 30
Mobile phase: MeCN:10 mM sodium dihydrogen phosphate 7:93, adjusted to pH 2.1
Flow rate: 1
Injection volume: 40
Detector: UV 205
CHROMATOGRAM
Retention time: 19
Internal standard: etidocaine (10)
Limit of detection: 5 ng/mL
OTHER SUBSTANCES
Extracted: mepivacaine (15)
KEY WORDS
plasma; rabbit
REFERENCE
Le Guévello, P.; Le Corre, P.; Chevanne, P.; Le Verge, R. High-performance liquid chromatographic
determination of bupivacaine in plasma samples for biopharmaceutical studies and application to
seven other local anaesthetics, J.Chromatogr., 1993, 622, 284–290.
SAMPLE
Matrix: blood
Sample preparation: Condition a 1 mL 4 mm SDB-RPS SPE disk cartridge (3M
Empore) with 500 µL MeOH, 500 µL air, 500 µL water, and 1 mL air. Mix 1 mL
serum with 50 µL perchloric acid, let stand for 10 min, mix, centrifuge at 16 000 g for
10 min. Add 800 µL to the cartridge followed by 2 mL air. Wash with 800 µL 0.5%
phosphoric acid in MeOH:water 20:80, push through 1.5 mL air, wash with 700 µL
water, push through 2 mL air, elute with 500 µL MeOH containing 1% ammonia, push
through 1.2 mL air. Evaporate the eluate to dryness under a stream of air at 70◦ and
reconstitute the residue with 50 µL mobile phase, inject a 40 µL aliquot.
HPLC VARIABLES
Column: 125 × 3 5 µm Nucleosil 50-5 endcapped RP-8
Articaine
55
Column temperature: 35
Mobile phase: MeCN:buffer 12:88 (Buffer was 880 mL 20 mM potassium dihydrogen
phosphate containing 500 µL phosphoric acid, pH 3.)
Flow rate: 1
Injection volume: 40
Detector: UV 274
CHROMATOGRAM
Retention time: 9.5
Limit of quantitation: 10 ng/mL
OTHER SUBSTANCES
Extracted: articainic acid (3.5)
KEY WORDS
serum; SPE
REFERENCE
Richter, K.; Oertel, R. Solid-phase extraction and high-performance liquid chromatographic determination of articaine and its metabolite articainic acid in human serum, J.Chromatogr.B, 1999, 724,
109–115.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 × 4.6 10 µm Chiralcel OD
Mobile phase: n-Hexane:isopropanol 80:20
Flow rate: 0.4
Injection volume: 5
Detector: UV 274
CHROMATOGRAM
Retention time: 12, 14 (enantiomers)
KEY WORDS
chiral
REFERENCE
Rustichelli, C.; Ferioli, V.; Gamberini, G.; Stancanelli, R. Enantiomeric separation of local anaesthetic
drug by HPLC on chiral stationary phases, Chromatographia, 2001, 54, 731–736.
ANNOTATED BIBLIOGRAPHY
Chankvetadze, B.; Chankvetadze, L.; Sidamonidze, S.; Yashima, E.; Okamoto, Y. High performance liquid chromatography enantioseparation of chiral pharmaceuticals using tris(chloro-methylphenylcarbamate)s of cellulose, J.Pharm.Biomed.Anal., 1996, 14, 1295–1303. [oxazepam; oxazolam; hexobarbital; phenobarbital; thiopental; lorazepam; lopirazepam; camazepam; cloxazolam; ketazolam; pindolol;
propranolol; sotalol; alprenolol; bupranolol; acebutolol; penbutolol; toliprolol; enilconazole; econazole; miconazole; bifonazole; ornidazole; bayleton; metomidate; nisoldipine; nimodipine; isradipine;
nicardipine; triadimefon; pheniramine; doxylamine; chlorphenoxamine; carbinoxamine; azelastine;
mequitazine; paramethadione; norgestrel; tesicam; mesuximide; metofoline; tramadol; clofedanol;
lofexidine; clenbuterol; piprozolin; etozolin; doxapram; chlormezanone; aminoglutethimide; articaine;
etidocaine; nefopam]
Oertel, R.; Richter, K.; Weile, K.; Gramatte, T.; Berndt, A.; Feller, K. A simple method for the determination of articaine and its metabolite articainic acid in dentistry: application to a comparison of articaine
and lidocaine concentrations in alveolus blood, Methods Find.Exp.Clin.Pharmacol., 1993, 15, 541–547.
56
Articaine
Oertel, R.; Richter, K.; Gramatté, T.; Kirch, W. Determination of drugs in biological fluids by highperformance liquid chromatography with on-line sample processing, J.Chromatogr.A, 1998, 797,
203–209. [metoprolol; talinolol; celiprolol; tiracizine; triamterene; ajmaline; articaine; lamotrigine]
Rop, P.P.; Grimaldi, F.; Bresson, M.; Fornaris, M.; Viala, A. Liquid chromatographic analysis of
cocaine, benzoylecgonine, local anaesthetic agents and some of their metabolites in biological
fluids, J.Liq.Chromatogr., 1993, 16, 2797–2811. [cocaine; benzoylecgonine; procaine; p-aminobenzoic
acid; butacaine; tetracaine; articaine; prilocaine; o-toluidine; lidocaine; monoethylglycine xylidide;
bupivacaine; pipecolylxylidene; etidocaine; dibucaine; caffeine; amphetamine; ephedrine; epinephrine;
morphine; monoacetylmorphine; diamorphine; ethylmorphine; codeine; acetylcodeine; fluorescence
detection; UV detection; SPE]
Vree, T.B.; Baars, A.M.; van Oss, G.E.; Booij, L.H. High-performance liquid chromatography and preliminary pharmacokinetics of articaine and its 2-carboxy metabolite in human serum and urine,
J.Chromatogr., 1988, 424, 440–444.
Asparaginase
57
Asparaginase
Molecular weight: ca. 136 000
CAS Registry No: 9015-68-3
Merck Index: 13, 841
SAMPLE
Matrix: reaction mixtures
Sample preparation: Adjust to pH 7 with 2 M NaOH, inject a 20 µL aliquot.
HPLC VARIABLES
Column: 250 × 4 10 µm HEMA-BIO 1000 (Tessek, Prague) (hydroxyethyl methacrylate–type column)
Mobile phase: 100 mM Potassium dihydrogen phosphate adjusted to pH 6.9 with 2 M
NaOH
Flow rate: 0.8
Injection volume: 20
Detector: UV 210
CHROMATOGRAM
Retention time: 2.1
Limit of detection: 0.51 U/mL
REFERENCE
Barek, J.; Cvacka, J.; Zima, J.; De Méo, M.; Laget, M.; Michelon, J.; Castegnaro, M. Chemical degradation of wastes of antineoplastic agents amsacrine, azathioprine, asparaginase and thiotepa,
Ann.Occup.Hyg., 1998, 42, 259–266.
58
Atazanavir sulfate
Atazanavir sulfate
N
Molecular formula: C38 H52 N6 O7 .H2 O4 S
H2SO4
Molecular weight: 802.93
CAS Registry No: 229975-97-7
H3C
O
H3CO
CH3
CH3
H
N
N
H
O
OH
O
N
N
H
H3C
H
N
OCH3
O
CH3
CH3
SAMPLE
Matrix: blood
Sample preparation: Condition each well of a 3M Empore C2-SD 96 well plate
with 250 µL MeOH and 500 µL 0.1% acetic acid, do not allow to go dry. Add 50 µL
200 ng/mL IS in MeOH:water 60:40 to 200 µL MeOH:water 60:40 containing 5 million
cells, sonicate for 10 min, centrifuge at 2600 g for 10 min. Evaporate the supernatant
to dryness, reconstitute with 50 µL MeOH, add 200 µL water, add 250 µL 0.1% acetic
acid, mix, add to a well on the SPE plate, allow to pass through under vacuum over
2 min, wash with 500 µL 0.1% acetic acid, dry under vacuum for 2 min, elute twice with
200 µL portions of MeCN:MeOH 50:50, pulling to dryness after each portion. Evaporate
the eluate to dryness under a stream of nitrogen at 60◦ over ca. 40 min, reconstitute the
residue with 200 µL mobile phase, vortex, inject a 20 µL aliquot.
HPLC VARIABLES
Guard column: 10 × 2 5 µm YMC Basic
Column: 50 × 2 5 µm YMC Basic
Mobile phase: MeCN:MeOH:water:88% formic acid 30:30:40:0.025
Flow rate: 0.25
Injection volume: 20
Detector: MS, Sciex API 3000 turbo ionspray, electrospray, positive mode at 400◦ , m/z
705 to 335, IonSpray 4600 V, declustering potential 56 V, entrance potential −10 V,
focusing potential 220 V, TurboIon gas nitrogen 8 L/min, collision energy 42 V, collision
cell exit potential 24 V, dwell time 500 ms, pause time 5 ms
CHROMATOGRAM
Retention time: <4
Internal standard: 13 C6 -atazanavir
Limit of quantitation: 5 fmole/million cells
KEY WORDS
peripheral blood mononuclear cells; SPE
REFERENCE
Jemal, M.; Rao, S.; Gatz, M.; Whigan, D. Liquid chromatography-tandem mass spectrometric quantitative determination of the HIV protease inhibitor atazanavir (BMS-232632) in human peripheral blood
mononuclear cells (PBMC): practical approaches to PBMC preparation and PBMC assay design for
high-throughput analysis, J.Chromatogr.B, 2003, 795, 273–289.
SAMPLE
Matrix: blood
Sample preparation: Condition each well of a10 mg Oasis HLB 96 well SPE plate with
1 mL MeOH and 1 mL 0.1% acetic acid. Add 40 µL 5 µg/mL IS in water and 300 µL 0.1%
Atazanavir sulfate
59
acetic acid to 250 µL plasma, mix, add to a well of the SPE plate, wash with 500 µL 0.1%
acetic acid, wash with 500 µL MeOH:water 20:80, elute with 300 µL MeOH. Evaporate
the eluate to dryness under a stream of nitrogen at 60◦ , reconstitute the residue with
500 µL MeCN:MeOH:10 mM pH 5.5 ammonium acetate 30:30:40, inject a 15 µL aliquot.
HPLC VARIABLES
Column: 33 × 4.6 3 µm Uptisphere HDO C18 (Interchim)
Mobile phase: Gradient. MeCN:5 mM ammonium acetate 50:50 for 0.5 min, to 60:40
over 0.1 min, maintain at 60:40 for 1.7 min, return to initial conditions over 0.1 min,
re-equilibrate for 2.1 min.
Flow rate: 0.8
Injection volume: 15
Detector: MS, Micromass Quattro Ultima, atmospheric pressure electrospray ionization,
column effluent split 1:20 before entering MS, positive ion mode, capillary sprayer voltage 3.2 kV, sample cone voltage 80 V, source temperature 100◦ , desolvation temperature
350◦ , nebulizing gas nitrogen, cone gas nitrogen at 37 L/h, desolvation gas nitrogen at
500 L/h, collision gas argon at 2.6 µbar, collision energy was set at 40 eV, resolution set
at 0.7 mass units at half height for the first and third quadrupoles.
CHROMATOGRAM
Retention time: 2.3
Internal standard: 13 C6 -atazanavir
Limit of quantitation: 1 ng/mL
KEY WORDS
plasma; SPE
REFERENCE
Schuster, A.; Burzawa, S.; Jemal, M.; Loizillon, E.; Couerbe, P.; Whigan, D. Quantitative determination
of the HIV protease inhibitor atazanavir (BMS-232632) in human plasma by liquid chromatographytandem mass spectrometry following automated solid-phase extraction, J.Chromatogr.B, 2003, 788,
377–386.
60
Atipamezole
Atipamezole
CH3
H
N
Molecular formula: C14 H16 N2
Molecular weight: 212.29
CAS Registry No: 104054-27-5
Merck Index: 13, 866
N
SAMPLE
Matrix: blood
Sample preparation: Condition a Sep-Pak C18 SPE cartridge with water, MeOH,
and 100 mM ammonium acetate. 5 mL Plasma + 250 ng detomidine, add to the SPE
cartridge, wash with 100 mM ammonium acetate, elute with MeOH:100 mM ammonium
acetate 75:25. Evaporate the eluate to dryness under reduced pressure, reconstitute the
residue in 200 µL mobile phase, inject a 50 µL aliquot.
HPLC VARIABLES
Column: 150 × 4.6 5 µm Hitachi gel #3056
Mobile phase: MeOH:100 mM ammonium acetate 65:35
Flow rate: 1
Injection volume: 50
Detector: MS, Hitachi M-1000, APCI interface, drift voltage 21 V, nebulizer 260◦ , vaporizer 399◦ , multiplier voltage 1500 VF, m/z 213
CHROMATOGRAM
Retention time: 8
Internal standard: detomidine (m/z 187) (6.5)
Limit of quantitation: 1–2 ng/mL
OTHER SUBSTANCES
Extracted: medetomidine (7.5, m/z 201), midazolam (10.5, m/z 326)
KEY WORDS
pharmacokinetics; pig; plasma; SPE
REFERENCE
Kanazawa, H.; Nishimura, R.; Sasaki, N.; Takeuchi, A.; Takai, N.; Nagata, Y.; Matsushima, Y. Determination of medetomidine, atipamazole and midazolam by liquid chromatography-mass spectrometry,
Biomed.Chromatogr., 1995, 9, 188–191.
SAMPLE
Matrix: blood
Sample preparation: Mix 200 µL 250 mM NaOH with 500 µL plasma, add 6 mL
dichloromethane, mix gently for 10 min, centrifuge at 1700 g for 10 min. Evaporate
4 mL of the lower organic layer to dryness under a stream of nitrogen, reconstitute the
residue with 100 µL 50 mM pH 3.2 phosphate buffer, vortex for 1.5 min, centrifuge at
1700 g for 10 min, inject a 50 µL aliquot.
HPLC VARIABLES
Guard column: 20 × 4.6 5 µm Supelguard LC-DP
Column: 250 × 4.6 5 µm Supelcosil LC-DP
Mobile phase: MeCN:50 mM phosphate buffer:triethylamine 27:73:0.05, adjusted to pH
3.2
Flow rate: 1
Injection volume: 50
Atipamezole
61
Detector: UV 215
CHROMATOGRAM
Retention time: 16.2
Limit of detection: 5 ng/mL
OTHER SUBSTANCES
Extracted: medetomidine (14.6)
KEY WORDS
pharmacokinetics; plasma; reindeer
REFERENCE
Ranheim, B.; Horsberg, T.E.; Nymoen, U.; Soli, N.E.; Tyler, N.J.; Arnemo, J.M. Reversal of
medetomidine-induced sedation in reindeer (Rangifer tarandus tarandus) with atipamezole increases
the medetomidine concentration in plasma, J.Vet.Pharmacol.Ther., 1997, 20, 350–354.
ANNOTATED BIBLIOGRAPHY
Kanazawa, H.; Nagata, Y.; Matsushima, Y.; Takai, N.; Uchiyama, H.; Nishimura, R.; Takeuchi, A. Liquid
chromatography-mass spectrometry for the determination of medetomidine and other anaesthetics in
plasma, J.Chromatogr., 1993, 631, 215–220.
62
Atomoxetine hydrochloride
Atomoxetine hydrochloride
Molecular formula: C17 H21 NO.HCl
Molecular weight: 291.82
CAS Registry No: 82248-59-7
H
H3C
N
H
O
CH3
HCl
SAMPLE
Matrix: blood
Sample preparation: Add 500 µL plasma to a Varian SDB-XC SPE cartridge, wash
with 1 mL MeOH:water 15:85, elute with 750 µL MeCN containing 0.1% trifluoroacetic
acid. Evaporate the eluate to dryness under a stream of nitrogen at 45◦ , reconstitute
the residue with 100 µL MeCN, mix with 25 µL water, inject an aliquot.
HPLC VARIABLES
Column: 100 × 4.6 5 µm Brownlee Spheri-5 ODS
Mobile phase: MeCN:water 85:15 containing 5 mM ammonium acetate, 0.2% formic
acid, and 0.03% trifluoroacetic acid
Flow rate: 1
Detector: MS, PE Sciex API III, MS/MS, positive atmospheric pressure chemical ionization, heated nebulizer interface, m/z 256 to 44
CHROMATOGRAM
Limit of quantitation: 0.25 ng/mL
OTHER SUBSTANCES
Extracted: metabolites
KEY WORDS
dog; plasma; rat; SPE
REFERENCE
Mattiuz, E.L.; Ponsler, G.D.; Barbuch, R.J.; Wood, P.G.; Mullen, J.H.; Shugert, R.L.; Li, Q.; Wheeler,
W.J.; Kuo, F.; Conrad, P.C.; Sauer, J.-M. Disposition and metabolic fate of atomoxetine hydrochloride: Pharmacokinetics, metabolism, and excretion in the Fischer 344 rat and beagle dog, Drug
Metab.Dispos., 2003, 31, 88–97.
SAMPLE
Matrix: blood, urine
Sample preparation: Plasma. Mix 3 mL MeCN with 1.5 mL plasma, centrifuge, evaporate the supernatant to dryness under a stream of nitrogen, reconstitute the residue
with 200 µL MeCN:water 10:90, inject an aliquot. Urine. Lyophilize urine, reconstitute
with MeCN:water 10:90 to one-tenth original volume, vortex, filter (0.45 µm), inject an
aliquot.
HPLC VARIABLES
Column: 150 × 4.6 5 µm Zorbax Eclipse XDB-C18
Column temperature: 30
Mobile phase: Gradient. MeCN:50 mM ammonium acetate from 10:90 to 60:40 over
30 min. (Use 25 mM ammonium acetate for MS detector.)
Flow rate: 1
Atomoxetine hydrochloride
63
Detector: Radioactivity (14 C); MS, Finnigan TSQ 700 or TSQ 7000, positive electrospray,
collision gas argon, 0.2 mL/min of column effluent entered MS
CHROMATOGRAM
Retention time: 20
OTHER SUBSTANCES
Extracted: metabolites
KEY WORDS
plasma
REFERENCE
Sauer, J.-M.; Ponsler, G.D.; Mattiuz, E.L.; Long, A.J.; Witcher, J.W.; Thomasson, H.R.; Desante, K.A.
Disposition and metabolic fate of atomoxetine hydrochloride: the role of CYP2D6 in human disposition
and metabolism, Drug Metab.Dispos., 2003, 31, 98–107.
64
Atorvastatin
Atorvastatin
H3C
O
Molecular formula: C33 H35 FN2 O5
N
Molecular weight: 558.64
CAS Registry No: 134523-00-5
Merck Index: 13, 868
H
CH3
OH
OH
COOH
N
F
SAMPLE
Matrix: blood
Sample preparation: Mix 500 µL serum with IS, acidify to pH 6 with sodium acetate
buffer, extract with MTBE, centrifuge. Remove the organic layer and evaporate it to
dryness, reconstitute the residue, inject an aliquot.
HPLC VARIABLES
Column: YMC Basic
Mobile phase: Gradient. A:B from 70:30 to 45:55 over 1 min, maintain at 45:55 for
0.5 min, return to initial conditions over 0.1 min, maintain at 70:30 for 1.9 min. A
was MeOH:water:88% formic acid 5:95:0.0043. B was MeCN:MeOH:88% formic acid
95:5:0.0043.
Detector: MS, Finnigan TSQ-7000, electrospray, m/z 559-440
CHROMATOGRAM
Internal standard: deuterated atorvastatin
Limit of quantitation: 500 pg/mL
OTHER SUBSTANCES
Extracted: metabolites
KEY WORDS
pharmacokinetics; serum
REFERENCE
Kantola, T.; Kivistö, K.T.; Neuvonen, P.J. Effect of itraconazole on the pharmacokinetics of atorvastatin,
Clin.Pharmacol.Ther., 1998, 64, 58–65.
SAMPLE
Matrix: formulations
Sample preparation: Shake 10 tablets with 50 mL MeCN:THF:50 mM pH 4 ammonium citrate buffer 27:20:53 at 450 rpm for 1 h, make up to 100 mL with the same
solution, filter, dilute a 2 mL aliquot to 10 mL, inject an aliquot.
HPLC VARIABLES
Guard column: 4 × 3 5 µm C18 Luna (Phenomenex)
Column: 250 × 4.6 5 µm C18 Luna (Phenomenex)
Mobile phase: Gradient. MeCN:THF:20 mM pH 4.0 ammonium acetate buffer from
25:5:70 to 70:5:25 over 50 min, maintain at 70:5:25 for 10 min.
Flow rate: 1
Injection volume: 100
Detector: UV 248
CHROMATOGRAM
Retention time: 30
Atorvastatin
65
Limit of detection: 13 ng/mL
Limit of quantitation: 130 ng/mL
OTHER SUBSTANCES
Simultaneous: impurities
KEY WORDS
tablets
REFERENCE
Ertürk, S.; Aktas, E.S.; Ersoy, L.; Fiçicioglu, S. An HPLC method for the determination of atorvastatin
and its impurities in bulk drug and tablets, J.Pharm.Biomed.Anal., 2003, 33, 1017–1023.
ANNOTATED BIBLIOGRAPHY
Al-Rawithi, S.; Hussein, R.F.; Alzahrani, A. Sensitive assay for the determination of fluvastatin in
plasma utilizing high-performance liquid chromatography with fluorescence detection, Ther.Drug
Monit., 2003, 25, 88–92. [atorvastatin is IS]
Jacobsen, W.; Kuhn, B.; Soldner, A.; Kirchner, G.; Sewing, K.-F.; Kollman, P.A.; Benet, L.Z.; Christians, U. Lactonization is the critical first step in the disposition of the 3-hydroxy-3-methylglutaryl-CoA
reductase inhibitor atorvastatin, Drug Metab.Dispos., 2000, 28, 1369–1378. [microsomal incubations]
Mazzu, A.L.; Lasseter, K.C.; Shamblen, E.C.; Agarwal, V.; Lettieri, J.; Sundaresen, P. Itraconazole alters
the pharmacokinetics of atorvastatin to a greater extent than either cerivastatin or pravastatin,
Clin.Pharmacol.Ther., 2000, 68, 391–400. [SPE]
Miao, X.-S.; Metcalfe, C.D. Determination of cholesterol-lowering statin drugs in aqueous samples using
liquid chromatography-electrospray ionization tandem mass spectrometry, J.Chromatogr.A, 2003, 998,
133–141. [atorvastatin; lovastatin; pravastatin; simvastatin]
Prueksaritanont, T.; Tang, C.; Qiu, Y.; Mu, L.; Subramanian, R.; Lin, J.H. Effects of fibrates on metabolism of statins in human hepatocytes, Drug Metab.Dispos., 2002, 30, 1280–1287. [cerivastatin;
simvastatin; atorvastatin; rosuvastatin; pravastatin]
66
Atosiban
Atosiban
O
O
Molecular formula: C43 H67 N11 O12 S2
Molecular weight: 994.20
CAS Registry No: 90779-69-4
Merck Index: 13, 869
CH3
S
N
H
Ile-Thr-Asn-Cys-Pro-Orn-GlyNH2
O
SAMPLE
Matrix: solutions
HPLC VARIABLES
Guard column: Kromasil C8 pre-column
Column: 100 × 2.1 KR 100-5 C8 1572 (Hichrom)
Mobile phase: MeCN:water:triethylammonium phosphate 27:72.9:0.1
Flow rate: 0.2
Detector: UV 190
REFERENCE
Lundin, S.; Svedman, P.; Höglund, P.; Jönsson, K.; Broeders, A.; Melin, P. Absorption of an oxytocin
antagonist (antocin) and a vasopressin analogue (dDAVP) through a standardized skin erosion in
volunteers, Pharm.Res., 1995, 12, 2024–2029.
SAMPLE
Matrix: solutions
Sample preparation: Inject an aliquot of a solution in mobile phase.
HPLC VARIABLES
Column: 250 × 4.6 5 µm Spherisorb NH2 (Before use flush column with isopropanol at
60◦ (to remove hexane-shipping solvent) and then with aqueous trifluoroacetic acid (pH
2.0) at 75◦ (to protonate amino groups).)
Column temperature: 40
Mobile phase: MeCN:water 92.35:7.65 containing 2.5 mM ammonium acetate and
250 mM sodium perchlorate
Flow rate: 0.5–1.2
Detector: UV 210
CHROMATOGRAM
Retention time: 230 (0.5 mL/min), 130 (1.2 mL/min)
OTHER SUBSTANCES
Simultaneous: impurities
REFERENCE
Oyler, A.R.; Armstrong, B.L.; Cha, J.Y.; Zhou, M.X.; Yang, Q.; Robinson, R.I.; Dunphy, R.; Burinsky, D.J.
Hydrophilic interaction chromatography on amino-silica phases complements reversed-phase highperformance liquid chromatography and capillary electrophoresis for peptide analysis, J.Chromatogr.A,
1996, 724, 378–383.
Balofloxacin
Molecular formula: C20 H24 FN3 O4
OCH3
H3C
N
H
Molecular weight: 389.42
CAS Registry No: 127294-70-6
Merck Index: 13, 946
N
N
F
COOH
O
SAMPLE
Matrix: bile, blood, urine
Sample preparation: Dilute urine and bile 100-fold with 100 mM pH 7.4 sodium
phosphate buffer. Mix 100 µL plasma or diluted urine or bile with 100 µL IS solution,
100 µL 100 mM pH 7.4 sodium phosphate buffer, and 5 mL dichloromethane, shake for
10 min, centrifuge at 3000 rpm for 3 min. Remove the organic layer and evaporate it to
dryness under a stream of nitrogen, reconstitute the residue with 100 µL mobile phase,
inject a 20 µL aliquot.
HPLC VARIABLES
Column: 250 × 4.6 Capcell Pak C18
Mobile phase: MeCN:10 mM pH 2.5 potassium phosphate buffer containing 5 mM PIC
B-5 22:78
Flow rate: 1
Injection volume: 20
Detector: F ex 295 em 500
CHROMATOGRAM
Internal standard: l-cyclopropyl-6,8-difluoro-l,4-dihydro-7-(3-methylaminopiperidin-lyl)-4-oxoquinoline-3-carboxylic acid
Limit of quantitation: 10 ng/mL
OTHER SUBSTANCES
Extracted: metabolites
KEY WORDS
plasma; rat
REFERENCE
Nakagawa, T.; Ishigai, M.; Hiramatsu, Y.; Kinoshita, H.; Ishitani, Y.; Ohkubo, K.; Okazaki, A. Determination of the new fluoroquinolone balofloxacin and its metabolites in biological fluids by high
performance liquid chromatography, Arzneimittelforschung, 1995, 45, 716–718.
SAMPLE
Matrix: tissue
Sample preparation: Homogenize 100 mg mouse ears with 100 mM pH 7.2 phosphate
buffered saline.
HPLC VARIABLES
Column: Capcell Pak C18
Mobile phase: MeCN:10 mM potassium dihydrogen phosphate 22:78 containing 5 mM
1-pentanesulfonic acid
Detector: F ex 295 em 500
CHROMATOGRAM
Retention time: 7
HPLC Methods for Recently Approved Pharmaceuticals. by George Lunn
Copyright 2005 John Wiley & Sons, Ltd. ISBN: 0-471-66941-5
67
68
Balofloxacin
KEY WORDS
ear; mouse
REFERENCE
Marutani, K.; Matsumoto, M.; Otabe, Y.; Nagamuta, M.; Tanaka, K.; Miyoshi, A.; Hasegawa, T.; Nagano,
H.; Matsubara, S.; Kamide, R.; Yokota, T.; Matsumoto, F.; Ueda, Y. Reduced phototoxicity of a fluoroquinolone antibacterial agent with a methoxy group at the 8 position in mice irradiated with
long-wavelength UV light, Antimicrob.Agents Chemother., 1993, 37, 2217–2223.
ANNOTATED BIBLIOGRAPHY
Kozawa, O.; Uematsu, T.; Matsuno, H.; Niwa, M.; Nagashima, S.; Kanamaru, M. Comparative study of
pharmacokinetics of two new fluoroquinolones, balofloxacin and grepafloxacin, in elderly subjects,
Antimicrob.Agents Chemother., 1996, 40, 2824–2828.
Uematsu, T.; Ohsawa, Y.; Mizuno, A.; Nakashima, M. Analysis of a new fluoroquinolone derivative (Q35) in human scalp hair as an index of drug exposure and as a time marker in hair, Int.J.Legal Med.,
1994, 106, 237–243.
Bambermycins
69
Bambermycins
CAS Registry No: 11015-37-5
Merck Index: 13, 954
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 × 4 Superspher-100 RP18 endcapped
Mobile phase: MeCN:MeOH:buffer 25:35:40 (The buffer was 10 mg potassium dihydrogen phosphate in 1 L water, adjusted to pH 7.8 with dipotassium hydrogen phosphate.)
Flow rate: 0.4
Detector: UV 258
CHROMATOGRAM
Retention time: 24.6 (moenomycins A and C3)
KEY WORDS
According to the paper, moenomycins A and C3 can be separated using 100 × 2 5 µm
Nucleosil C18 with a 10 min gradient from 0.09% trifluoroacetic acid in MeCN to 0.1%
trifluoroacetic acid (sic) at 0.2 mL/min using electrospray MS triple quadrupole detection
(8.2 and 8.5 min, respectively).
REFERENCE
Subramaniam-Niehaus, B.; Schneider, T.; Metzger, J.W.; Wohlleben, W. Isolation and analysis of moenomycin and its biosynthetic intermediates from Streptomyces ghanaensis (ATCC 14672) wildtype and
selected mutants, Z.Naturforsch.C, 1997, 52, 217–226.
70
Befunolol
Befunolol
CH3
H3C
Molecular formula: C16 H21 NO4
N
H
O
OH
Molecular weight: 291.34
CAS Registry No: 39552-01-7,
39543-79-8 (HCl), 66717-59-7 ((S)-(–)
HCl), 66685-79-8 ((R)-(+) HCl)
Merck Index: 13, 1023
O
O
CH3
SAMPLE
Matrix: blood
Sample preparation: Condition a Sep-Pak C18 SPE cartridge with 5 mL MeOH, 5 mL
water, and 5 mL MeOH:water 30:70. Mix 1 mL plasma with 450 µL 3 µg/mL IS in
MeOH, centrifuge at 1700 g for 10 min, add the supernatant to the SPE cartridge,
wash with 5 mL MeOH:water 30:70, wash with 50 µL MeOH, suck dry under reduced
pressure for 1 min, elute with 5 mL MeOH. Evaporate the eluate to dryness under a
stream of nitrogen at 50◦ , reconstitute the residue with 200 µL mobile phase, inject a
150 µL aliquot.
HPLC VARIABLES
Column: 250 × 4 LiChrosorb RP-Select B
Mobile phase: MeCN:20 mM potassium dihydrogen phosphate 20:80
Flow rate: 1
Injection volume: 150
Detector: UV 290
CHROMATOGRAM
Retention time: 11
Internal standard: levobunolol (13)
Limit of quantitation: 1 ng/mL
OTHER SUBSTANCES
Extracted: dihydrobefunolol (UV 245, LOQ 10 ng/mL) (10)
KEY WORDS
pharmacokinetics; plasma; rabbit
REFERENCE
Nozaki, Y.; Imai, T.; Goto, K.; Imamura, Y.; Underberg, W.J.M.; Otagiri, M. Simultaneous determination of befunolol and its major metabolite, dihydrobefunolol, in plasma by high performance liquid
chromatography, Anal.Sci., 1989, 5, 395–397.
71
Benzalkonium chloride
Benzalkonium chloride
CH3
Molecular formula: C21 H38 ClN (C12), C23 H42 ClN (C14)
CH3
Molecular weight: 339.99 (C12), 368.04 (C14)
CAS Registry No: 8001-54-5
Merck Index: 13, 1058
N+
Cl−
R
n = C8H17 to C18H37
SAMPLE
Matrix: blood
Sample preparation: Mix 2 mL plasma with 4 mL water, add to a 3 mL Baker C18
SPE cartridge, wash with three 3 mL portions of water, wash with two 3 mL portions
of MeOH, wash with two 3 mL portions of ethyl acetate, elute with 4 mL MeOH:ethyl
acetate 50:50 containing 0.01% ammonium chloride, and evaporate the eluate to dryness
under a stream of nitrogen. Reconstitute the residue with 1 mL ethyl acetate, 1 mL 10%
sodium carbonate, and 200 µL 0.1% bromophenol blue. Evaporate the organic layer to
dryness under a stream of nitrogen, reconstitute the residue with 50 µL mobile phase,
and inject an aliquot. (The ammonium chloride is eliminated in the aqueous layer and
the benzalkonium forms an ion pair with the bromophenol blue and goes into the organic
layer.)
HPLC VARIABLES
Column: Spherisorb CN
Mobile phase: MeCN:buffer 90:10 (The buffer was a 161 mM pH 5.4 propionate buffer
made by mixing 75 mL 10% sodium carbonate, 1.5 L water, and 12 mL propionic acid.
Make up to 2 L with water.)
Flow rate: 2
Detector: UV 214, UV 254
CHROMATOGRAM
Retention time: 5.8 (C14), 6.2 (C12)
Limit of detection: 5 ng/mL (UV 214)
KEY WORDS
dog; human; plasma; SPE
REFERENCE
Bleau, G.; Desaulniers, M. High-performance liquid chromatographic assay of benzalkonium in plasma,
J.Chromatogr., 1989, 487, 221–227.
72
Betaine
Betaine
Molecular formula: C5 H11 NO2
O
−O
CH3
N+
CH3
CH3
Molecular weight: 117.15
CAS Registry No: 107-43-7
Merck Index: 13, 1182
SAMPLE
Matrix: blood, urine
Sample preparation: Dilute urine 10-fold with water. Mix 50 µL plasma or diluted
urine with 50 µL 100 mM potassium dihydrogen phosphate, vortex, add 900 µL reagent,
vortex, heat at 80◦ for 1 h, cool to room temperature, vortex, centrifuge at 1000 g, inject
a 15 µL aliquot of the supernatant. (Prepare the reagent by dissolving 66 mg 18-crown-6
and 1.39 g 4-bromophenacyl bromide in 100 mL MeCN.)
HPLC VARIABLES
Column: 250 × 4.6 5 µm Supelcosil LC-SCX
Mobile phase: MeCN:water 90:10 containing 22 mM choline
Flow rate: 1.5
Injection volume: 15
Detector: UV 254
CHROMATOGRAM
Retention time: 14.8
Limit of detection: 5 µM
OTHER SUBSTANCES
Extracted: N, N-dimethylglycine (12.7)
KEY WORDS
derivatization; plasma
REFERENCE
Laryea, M.D.; Steinhagen, F.; Pawliczek, S.; Wendel, U. Simple method for the routine determination of
betaine and N, N-dimethylglycine in blood and urine, Clin.Chem., 1998, 44, 1937–1941.
SAMPLE
Matrix: blood, urine
Sample preparation: Vortex 100 µL plasma or urine with 1 mL MeCN:MeOH 90:10,
add 300–400 mg drying mixture, mix thoroughly with occasional inversion for 1 h, centrifuge at 1000 g for 2 min. Remove a 200 µL portion of the supernatant and mix with
20 µL of a 10% suspension of magnesium oxide in water, vortex, add 50 µL 100 mM
4-bromophenacyl triflate in MeCN, continue mixing for 5 min, centrifuge at 1000 g for
2 min (Lever,M. et al. Anal.Biochem. 1992, 205, 14–21), add 10 µL of a suspension
of Dowex 1 in the dichloroacetate form (to eliminate excess derivatization reagent),
inject a 20 µL aliquot. (Drying mixture was 90% anhydrous disodium hydrogen phosphate and 10% argentous oxide. Prepare 4′ -bromophenacyl trifluoromethanesulfonate
as follows. Add 8.8 g p-bromobenzoyl chloride in 40 mL dry ether over 20–30 min to
100 mmol diazomethane stirred in an ice bath, stir in an ice bath for 8–9 h, let stand at
room temperature for 3 h, evaporate the solvent under reduced pressure, recrystallize
4′ -bromo-2-diazoacetophenone from ether/hexane (mp 123.5–124◦ d) (J.Am.Chem.Soc.
1951, 73, 5301). Condense 50 mL anhydrous sulfur dioxide in a flask fitted with a
calcium sulfate drying tube, cool in a dry ice/acetone bath, add 2.25 g 4′ -bromo-2diazoacetophenone, stir for 5 min, add 900 µL anhydrous trifluoromethanesulfonic acid
Betaine
73
from a freshly opened bottle in one portion, stir for 15 min, remove the cooling bath, after
30 min use an ice/water bath to evaporate the solvent. Dissolve the residue in 100 mL
boiling dichloromethane, treat twice with 5 g portions of decolorizing carbon, filter,
evaporate the filtrate, recrystallize the residue from pentane:dichloromethane 80:20
to give 4′ -bromophenacyl trifluoromethanesulfonate as colorless plates (mp 137–8◦ )
(J.Chromatogr. 1984, 299, 365.)
HPLC VARIABLES
Column: 250 × 4 5 µm silica (Merck)
Mobile phase: MeCN:dichloromethane:water 80:10:10 containing 9 mM triethylamine
and 6 mM citric acid
Flow rate: 1
Injection volume: 20
KEY WORDS
normal phase; plasma
REFERENCE
Lever, M.; Sizeland, P.C.; Bason, L.M.; Hayman, C.M.; Chambers, S.T. Glycine betaine and proline
betaine in human blood and urine, Biochim.Biophys.Acta, 1994, 1200, 259–264.
74
Bethanechol chloride
Bethanechol chloride
Molecular formula: C7 H17 ClN2 O2
CH3
H2N
O
O
N+
CH3
Cl−
CH3
CH3
Molecular weight:: 196.68
CAS Registry No: 590-63-6
Merck Index: 13, 1189
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 × 4.6 5 µm Bakerbond phenylethyl
Mobile phase: MeCN:water 33:67
Flow rate: 0.7
Detector: UV 200
CHROMATOGRAM
Retention time: 3.0
OTHER SUBSTANCES
Simultaneous: degradants
KEY WORDS
stability-indicating
REFERENCE
Allen, L.V. Jr.; Erickson,M.A. III. Stability of bethanechol chloride, pyrazinamide, quinidine sulfate,
rifampin, and tetracycline hydrochloride in extemporaneously compounded oral liquids, Am.J.Health
Syst.Pharm., 1998, 55, 1804–1809.
Bexarotene
Bexarotene
H3C
CH3
H3C
CH3
Molecular formula: C24 H28 O2
Molecular weight: 348.48
CAS Registry No: 153559-49-0
Merck Index: 13, 1196
75
CH2
CH3
COOH
SAMPLE
Matrix: bile, blood, microsomal incubation
Sample preparation: Mix plasma with 5 vol of MeOH, cool to −20◦ , centrifuge at 4◦ ,
evaporate the supernatant to dryness under reduced pressure, reconstitute the residue
with MeCN:10 mM ammonium acetate 40:60 containing 1% acetic acid, inject an aliquot.
Mix 1 mL (?) microsomal incubation with 1.5 mL ice-cold EtOH, cool at <5◦ for 1 h,
centrifuge, evaporate the supernatant to dryness under reduced pressure, reconstitute
the residue with MeCN:10 mM ammonium acetate 40:60 containing 1% acetic acid,
inject an aliquot. Dilute bile twofold with 10 mM ammonium acetate, centrifuge, inject
an aliquot.
HPLC VARIABLES
Column: 250 × 4.6 5 µm Microsorb MV C18
Column temperature: 40
Mobile phase: Gradient. A:B from 20:80 to 80:20 over 20 min, maintain at 80:20 for
15 min. A was MeCN:acetic acid 100:1. B was 10 mM ammonium acetate:acetic acid
100:1.
Detector: UV 262
CHROMATOGRAM
Retention time: 32
OTHER SUBSTANCES
Extracted: metabolites
KEY WORDS
dog; plasma; rat
REFERENCE
Howell, S.R.; Shirley, M.A.; Grese, T.A.; Neel, D.A.; Wells, K.E.; Ulm, E.H. Bexarotene metabolism in
rat, dog, and human, synthesis of oxidative metabolites, and in vitro activity at retinoid receptors,
Drug Metab.Dispos., 2001, 29, 990–998.
SAMPLE
Matrix: blood
Sample preparation: Mix 1 mL plasma with 20 µL 2.5 µg/mL IS, vortex for 10 s, add
five 250 µL portions of MeCN and one 1 mL portion of 500 mM HCl with each addition
followed by vortexing for a short period. Add 5 mL n-hexane:isoamyl alcohol 98:2, rotate
at 45 rpm for 20 min, centrifuge at 3200 g for 10 min, freeze, remove the organic layer.
Evaporate the organic layer to dryness under a stream of nitrogen at 40◦ , reconstitute
the residue with 400 µL MeCN, vortex for 30 s, sonicate for 2 min, add 100 µL 10 mM
ammonium acetate, centrifuge at 13 000 g for 5 min, inject a 50 µL aliquot.
HPLC VARIABLES
Column: 150 × 4.6 5 µm Inertsil ODS-2
Column temperature: 35
Mobile phase: MeCN:10 mM ammonium acetate:acetic acid 80:20:0.8
76
Bexarotene
Injection volume: 50
Detector: F ex 260 em 430
CHROMATOGRAM
Retention time: 8.5
Internal standard: LG100130 (4-[1-(5,6,7,8-tetrahydro-3-(1-methyl)ethyl-5,5,8,8-tetramethyl-2-naphthalenyl)ethenyl]benzoic acid, Ligand Pharmaceuticals, San Diego) (11)
Limit of detection: 0.3 ng/mL
OTHER SUBSTANCES
Extracted: metabolites
KEY WORDS
pharmacokinetics; plasma
REFERENCE
Van de Merbel, N.C.; van Veen, J.H.; Wilkens, G.; Loewen, G. Validated liquid chromatographic method
for the determination of bexarotene in human plasma, J.Chromatogr.B, 2002, 775, 189–195.
77
Biapenem
Biapenem
OH
H H
H3C
Molecular formula: C15 H18 N4 O4 S
Molecular weight: 350.40
CAS Registry No: 120410-24-4
Merck Index: 13, 1200
N
O
CH3
S
N
N+
N
COO−
SAMPLE
Matrix: blood, urine
Sample preparation: Mix plasma or urine with an equal volume of 1 M pH 7.0 MOPS
(3-(N-morpholino)propanesulfonic acid) buffer. Dilute plasma with 30% ammonium
sulfate solution, centrifuge at 3000 rpm.
HPLC VARIABLES
Column: 150 × 4.6 TSK gel ODS 80TM (Tosoh)
Mobile phase: MeCN:100 mM acetate buffer 1.5:98.5 (plasma) or MeCN:MeOH:sodium
1-octanesulfonate solution:acetic acid 110:12:480:3 (urine)
Flow rate: 1.2 (plasma), 1.1 (urine)
Detector: UV 300 (plasma), UV 310 (urine)
CHROMATOGRAM
Internal standard: 5-hydroxyindole-3-acetic acid (plasma), o-nitroacetanilide (urine)
Limit of detection: 100 ng/mL (plasma), 1 µg/mL (urine)
KEY WORDS
plasma
REFERENCE
Kozawa, O.; Uematsu, T.; Matsuno, H.; Niwa, M.; Takiguchi, Y.; Matsumoto, S.; Minamoto, M.; Niida, Y.;
Yokokawa, M.; Nagashima, S.; Kanamaru, M. Pharmacokinetics and safety of a new parenteral carbapenem antibiotic, biapenem (L-627), in elderly subjects, Antimicrob.Agents Chemother., 1998, 42,
1433–1436.
SAMPLE
Matrix: solutions
Sample preparation: Mix an aliquot of a solution in 50 mM pH 7.0 MOPS (3-(Nmorpholino)propanesulfonic acid) buffer with 2 aliquots of MeCN, vortex. Add a volume
of chloroform equal to 50% of the total volume (Caution! Chloroform is a carcinogen!),
mix well, centrifuge at 5000 g for 10 min, inject a 20 µL aliquot of the supernatant.
HPLC VARIABLES
Guard column: 100 × 4.6 Inertsil ODS-2
Column: 150 × 4.6 Inertsil ODS-2
Mobile phase: MeCN:50 mM pH 5.5 acetate buffer 2:100
Flow rate: 0.9
Injection volume: 20
Detector: UV 295
78
Biapenem
CHROMATOGRAM
Retention time: 6.3
REFERENCE
Hikida, M.; Kawashima, K.; Nishiki, K.; Furukawa, Y.; Nishizawa, K.; Saito, I.; Kuwao, S. Renal dehydropeptidase-I stability of LJC 10,627, a new carbapenem antibiotic, Antimicrob.Agents Chemother.,
1992, 36, 481–483.
79
Bimatoprost
Bimatoprost
H
H OH H
N
Molecular formula: C25 H37 NO4
Molecular weight: 415.57
CAS Registry No: 155206-00-1
CH3
O
HO
H
H
H
OH
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 150 × 4.6 5 µm Ultrasphere IP
Mobile phase: Gradient. A:B 100:0 for 1 min, to 40:60 over 16 min (+ curved), maintain
at 40:60 for 4 min, return to initial conditions over 1 min, re-equilibrate at initial
conditions for 8 min. A was MeCN:20 mM pH 2.8 potassium phosphate buffer 20:80. B
was MeCN:20 mM pH 2.8 potassium phosphate buffer 50:50.
Flow rate: 1
Injection volume: 20–100
Detector: UV, Radioactivity (3 H)
CHROMATOGRAM
Retention time: 16
OTHER SUBSTANCES
Simultaneous: metabolites
REFERENCE
Woodward, D.F.; Krauss, A.H.-P.; Chen, J.; Liang, Y.; Li, C.; Protzman, C.E.; Bogardus, A.; Chen, R.;
Kedzie, K.M.; Krauss, H.A.; Gil, D.W.; Kharlamb, A.; Wheeler, L.A.; Babusis, D.; Welty, D.; TangLiu, D.D.-S.; Cherukury, M.; Andrews, S.W.; Burk, R.M.; Garst, M.E. Pharmacological characterization of a novel antiglaucoma agent, bimatoprost (AGN 192024), J.Pharmacol.Exp.Ther., 2003, 305,
772–785.
80
Bioresmethrin
Bioresmethrin
Molecular formula: C22 H26 O3
Molecular weight: 338.44
CAS Registry No: 28434-01-7
Merck Index: 13, 1230
H3C CH3 O
H3C
H
H
H3C
O
O
SAMPLE
Matrix: urine
Sample preparation: Add 4 g solid NaCl, 3.5 mL MeCN, and 5 mL saturated NaCl
solution to 5 mL MeCN, shake for 1 min. Remove the MeCN layer and extract the
aqueous layer with 1 mL MeCN. Combine the MeCN layers and adjust to a known
volume (0.5–1 mL), mix, filter (0.45 µm), inject a 40 µL aliquot.
HPLC VARIABLES
Column: 150 × 3 3 µm Luna C18(2) (Phenomenex)
Column temperature: 30
Mobile phase: Gradient. MeCN:water 10:90 for 1 min, to 90:10 over 30 min, maintain
at 90:10 for 4 min, to 100:0 over 1 min, maintain at 100:0 for 10 min, return to initial
conditions over 1 min.
Flow rate: 0.5
Injection volume: 40
Detector: UV 235
CHROMATOGRAM
Retention time: 35.2
Limit of detection: 5 ng/mL
OTHER SUBSTANCES
Extracted: allethrin (31.8, LOD 5 ng/mL), bifenthrin (37, LOD 5 ng/mL), cyfluthrin
(34.3, LOD 5 ng/mL), fenvalerate (35.3, LOD 2 ng/mL), cis-permethrin (35.7, LO
5 ng/mL), trans-permethrin (36.3, LOD 5 ng/mL), phenothrin (36.4, LOD 5 ng/mL),
m-phenoxybenzyl alcohol (21, LOD 5 ng/mL), pyrethrin I (29.6, LOD 4 ng/mL), pyrethrin
II (33.7, LOD 40 ng/mL), tetramethrin (31.4, LOD 5 ng/mL)
REFERENCE
Loper, B.L.; Anderson, K.A. Determination of pyrethrin and pyrethroid pesticides in urine and water
matrices by liquid chromatography with diode array detection, J.AOAC Int., 2003, 86, 1236–1240.
Bivalirudin
81
Bivalirudin
Molecular formula: C98 H138 N24 O33
Molecular weight: 2180.28
CAS Registry No: 128270-60-0
Merck Index: 13, 1306
SAMPLE
Matrix: blood, urine
Sample preparation: Condition a 1 mL 30 mg Oasis HLB SPE cartridge with 1 mL
MeCN:trifluoroacetic acid 98:2 and 1 mL water. Plasma. Mix 200 µL plasma with
100 µL of a solution of IS in water, make up to 1100 µL with water, add 1 mL to the
SPE cartridge, wash with 500 µL water, wash with 500 µL MeOH:water 30:70, elute
with 1 mL MeCN:trifluoroacetic acid 98:2, evaporate the eluate to dryness under a
stream of nitrogen at 40◦ , reconstitute the residue with 100 µL MeOH:water 50:50,
centrifuge at 10 000 rpm at 4◦ for 10 min, inject a 30 µL aliquot. Urine. Mix 50 µL urine
with 400 µL of a solution of IS in water, make up to 500 µL with water, inject a 10 µL
aliquot.
HPLC VARIABLES
Column: 30 × 4.6 5 µm Hypersil BDS C18
Mobile phase: Gradient. A:B from 30:70 to 100:0 over 0.5 min, maintain at 100:0 for
2 min, return to initial conditions over 0.2 min, re-equilibrate at initial conditions for
1.5 min. A was MeCN:0.1% formic acid 60:40. B was 0.1% formic acid.
Injection volume: 10–30
Detector: MS, Perkin Elmer, positive ion mode, TurboIonSpray heater 400◦ , m/z 1090.9
to 227.1, dwell time 400 ms, ionspray voltage 5.2 kV, ring voltage 230 V, nebulizer gas
nitrogen at 12 units, TurboIonSpray gas nitrogen at 7.0 L/min, collision gas nitrogen
at 3 units, curtain gas nitrogen at 10 units, deflector −100 V, electron multiplier
2400–2700 V, collision energy −83 V
CHROMATOGRAM
Internal standard: Gln-9-hirulog (Polypeptide Laboratories, Torrance CA) (m/z 1097.9
to 199.1)
KEY WORDS
pharmacokinetics; plasma; SPE
REFERENCE
Robson, R.; White, H.; Aylward, P.; Frampton, C. Bivalirudin pharmacokinetics and pharmacodynamics:
effect of renal function, dose, and gender, Clin.Pharmacol.Ther., 2002, 71, 433–439.
82
Boldenone
Boldenone
CH3 OH
CH3
H
Molecular formula: C19 H26 O2
Molecular weight:: 286.41
CAS Registry No: 846-48-0
Merck Index: 13, 1315
H
H
O
SAMPLE
Matrix: blood, urine
Sample preparation: Adjust the pH of 3 mL serum to 9.6, add to an Extrelut 3 SPE
column, extract with 15 mL diethyl ether, evaporate the ether to dryness, reconstitute
the residue with 50 µL mobile phase containing 10 ng IS, inject an aliquot. Adjust
the pH of 3 mL urine to 9.6 with sodium bicarbonate:sodium carbonate 2:1, add to
an Extrelut 3 SPE column, extract with 15 mL diethyl ether, evaporate the ether to
dryness, reconstitute the residue with 50 µL mobile phase containing 10 ng IS, inject
the whole amount of aliquot.
HPLC VARIABLES
Column: 250 × 4.6 5 µm Ultrasphere ODS
Mobile phase: MeCN:water 37:63 (A) or THF:water 30:70 (B)
Flow rate: 1
Detector: ELISA (collect 300 µL fractions)
CHROMATOGRAM
Retention time: 13 (A), 10 (B)
Internal standard: methandienone (17 (A), 12 (B))
OTHER SUBSTANCES
Extracted: testosterone (20 (A), 15 (B))
KEY WORDS
horse; serum; SPE
REFERENCE
Hagedorn, H.-W.; Schulz, R.; Jaeschke, G. Identification and verification of the anabolic steroid boldenone
in equine blood and urine by HPLC/ELISA, Biomed.Chromatogr., 1994, 8, 63–68.
Bosentan
Bosentan
O
O
S
Molecular formula: C27 H29 N5 O6 S
H3C
Molecular weight: 551.62
CAS Registry No: 147536-97-8,
150726-52-6 (Na salt)
Merck Index: 13, 1341
H3C
H
N
N
OCH3
O
N
CH3
83
N
N
O
OH
SAMPLE
Matrix: blood
Sample preparation: Mix 500 µL plasma with 10 µL 5 µg/mL IS in EtOH, add
750 µL MeCN, vortex, store at 5◦ for 10 min. Add the supernatant at pH 11 to 7 mL
dichloromethane, shake on a rotating shaker for 20 min, centrifuge for 5 min. Evaporate the organic layer to dryness under reduced pressure, reconstitute the residue with
1 mL MeCN, centrifuge for 5 min. Evaporate the supernatant to dryness under reduced
pressure, reconstitute the residue with 100 µL MeCN:5 mM ammonium acetate:acetic
acid 30:70:1, inject a 30–90 µL aliquot.
HPLC VARIABLES
Guard column: 10 × 2 4 µm Superspher RP-18
Column: 125 × 2 4 µm Superspher RP-18
Column temperature: 35
Mobile phase: MeCN:5 mM ammonium acetate:acetic acid 75:25:1 or 70:30:1
Injection volume: 30–90
Detector: UV 270; MS, Perkin-Elmer SCIEX API III plus triple quadrupole, ionspray,
collision gas argon, collision energy 50 eV
CHROMATOGRAM
Retention time: 3.2
Internal standard: tetradeutero bosentan; Ro 47–8761 (4-cyclopropyl-N-[6-(2-hydroxyethoxy)-5-(2-methoxyphenoxy)[2,2′ -bipyrimidin]-4-yl]benzenesulfonamide)
Limit of quantitation: 0.5 ng/mL (MS)
KEY WORDS
plasma
REFERENCE
Lausecker, B.; Hopfgartner, G. Determination of an endothelin receptor antagonist in human plasma by
narrow-bore liquid chromatography and ionspray tandem mass spectrometry, J.Chromatogr.A, 1995,
712, 75–83.
SAMPLE
Matrix: blood, tissue
Sample preparation: Homogenize (Polytron) liver with 2 vol of 100 mM phosphate
buffer at 8000 rpm for 10–15 s. Mix 250 µL plasma or liver homogenate with
50 µL 2 µg/mL IS in MeCN:5 mM ammonium acetate:acetic acid 10:90:1, add 1 mL
MeCN:EtOH 50:50, mix, centrifuge at 14 000 g for 10 min. Remove the supernatant
and add it to 1 mL pH 4.0 buffer (Merck Tetrisol citrate/HCl), mix, add 6 mL
n-chlorobutane:dichloromethane 80:20, rotate at 40 rpm for 20 min. Evaporate the
organic layer to dryness under reduced pressure, reconstitute the residue with 1 mL
MeCN:5 mM ammonium acetate:acetic acid 10:90:1, inject a 950 µL aliquot onto column
A and elute to waste with mobile phase A. After 2 min, elute the contents of column A
onto column B with mobile phase B. (After the elution of the analytes, flush column B
with MeCN:acetic acid 99:1 at 0.35 mL/min for 1 min and then re-equilibrate with initial
mobile phase B at 0.35 mL/min for 30 s. Wash column A in backflush and forward flush
84
Bosentan
mode with MeCN:MeOH:5 mM ammonium acetate:acetic acid 45:45:10:1 at 1 mL/min
for 3.5 min, then re-equilibrate with mobile phase A at 1 mL/min for 2.5 min.)
HPLC VARIABLES
Column: A 25 × 4 5 µm Superspher RP-Select B; B 10 × 2 Superspher RP−18 + 150 × 2.1
Symmetry RP-18
Mobile phase: A 5 mM ammonium acetate containing 1% acetic acid; B Gradient. C:D
from 50:50 to 10:90 over 4.5 min. C was MeCN:MeOH:5 mM ammonium acetate:acetic
acid 25:25:50:1. D was MeCN:MeOH:5 mM ammonium acetate:acetic acid 45:45:10:1.
Flow rate: A 1; B 0.25
Injection volume: 950
Detector: MS, Perkin-Elmer SCIEX API 300 triple quadrupole, ionspray, collision gas
argon, collision energy 50 eV, m/z 552 to 202
CHROMATOGRAM
Retention time: 6.8
Internal standard: tetradeutero bosentan
Limit of quantitation: 1 ng/mL
OTHER SUBSTANCES
Extracted: metabolites
KEY WORDS
column-switching; dog; human; liver; plasma; rat
REFERENCE
Lausecker, B.; Hess, B.; Fischer, G.; Mueller, M.; Hopfgartner, G. Simultaneous determination of bosentan and its three major metabolites in various biological matrices and species using narrow bore liquid
chromatography with ion spray tandem mass spectrometric detection, J.Chromatogr.B, 2000, 749,
67–83.
SAMPLE
Matrix: blood
Sample preparation: Mix 250 µL plasma with 50 µL 2 µg/mL IS in MeCN:5 mM
ammonium acetate:acetic acid 10:90:1, add 750 µL MeOH. Mix the supernatant with
2 mL 50 mM pH 10 ammonium acetate buffer, add to a 30 mg Oasis SPE cartridge,
wash with 1 mL water, wash with 2 mL 20 mM phosphoric acid, wash with 2.1 mL
MeOH:water 20:80, wash with 1 mL water, elute with MeCN:MeOH:triethylamine
30:70:2. Evaporate the eluate to dryness, reconstitute the residue with 150 µL
MeCN:5 mM ammonium acetate:acetic acid 10:90:1, inject an aliquot.
HPLC VARIABLES
Column: 125 × 2 Superspher
Mobile phase: MeCN:MeOH:5 mM ammonium acetate:acetic acid 37.5:37.5:25:1
Flow rate: 0.25
Detector: MS, Perkin-Elmer SCIEX API 300 triple quadrupole, ionspray, collision gas
argon, collision energy 50 eV, m/z 552 to 202
CHROMATOGRAM
Retention time: 3
Internal standard: tetradeutero bosentan
Limit of quantitation: 1 ng/mL
OTHER SUBSTANCES
Extracted: metabolites
Bosentan
85
KEY WORDS
human; plasma; rat; SPE
REFERENCE
Lausecker, B.; Hess, B.; Fischer, G.; Mueller, M.; Hopfgartner, G. Simultaneous determination of bosentan and its three major metabolites in various biological matrices and species using narrow bore liquid
chromatography with ion spray tandem mass spectrometric detection, J.Chromatogr.B, 2000, 749,
67–83.
ANNOTATED BIBLIOGRAPHY
Dell, D.; Lausecker, B.; Hopfgartner, G.; van Giersbergen, P.L.M.; Dingemanse, J. Evolving bioanalytical
methods for the cardiovascular drug bosentan, Chromatographia, 2002, 55, S115–S119. [review]
Ubeaud, G.; Schmitt, C.; Jaeck, D.; Lave, T.; Coassolo, P. Bosentan, a new endothelin receptor antagonist:
prediction of the systemic plasma clearance in man from combined in vivo and in vitro data, Xenobiotica,
1995, 25, 1381–1390. [gradient; microsomal incubations; rat; mouse; dog; rabbit; metabolites]
Weber, C.; Schmitt, R.; Birnboeck, H.; Hopfgartner, G.; van Marle, S.P.; Peeters, P.A.M.; Jonkman,
J.H.G.; Jones, C.-R. Pharmacokinetics and pharmacodynamics of the endothelin-receptor antagonist bosentan in healthy human subjects, Clin.Pharmacol.Ther., 1996, 60, 124–137. [LC-MS; UV
detection; urine; plasma; LOQ 50 ng/mL]
86
β-Boswellic acid
β-Boswellic acid
CH3
H3C
CH3
Molecular formula: C30 H48 O3
Molecular weight: 456.70
CAS Registry No: 631-69-6
Merck Index: 13, 1343
CH3
H
CH3
H
CH3
HO
H
HOOC CH3
SAMPLE
Matrix: blood
Sample preparation: Add 1 mL plasma to an Extrelut NT SPE cartridge, let stand
for 15 min, elute with THF:hexane:ethyl acetate:isopropanol 32:32:32:3. Evaporate the
eluate to dryness under a stream of nitrogen at 60◦ , reconstitute the residue with
80 µL DMSO, add this solution to a 4 mL 150 mg Carbograph SPE cartridge (Alltech, preconditioned with 6 mL MeOH). Rinse the vial twice with 1 mL portions of
MeOH:isopropanol 95:5 and add the rinses to the Carbograph SPE cartridge, wash
with 3 mL MeOH:isopropanol 95:5, apply a vacuum for 5 s, elute with 2.5 mL cyclohexane:acetone 50:50. Evaporate the eluate to dryness under a stream of nitrogen at 60◦ ,
reconstitute the residue with 80 µL DMSO, inject an aliquot.
HPLC VARIABLES
Column: 250 × 3 5 µm ReproSil-Pur 120 ODS-3 (Dr Maisch, Ammerbuch, Germany)
Column temperature: 28
Mobile phase: Gradient. A:B from 62:38 to 51:49 over 20 min, to 39:61 over 15 min, to
32:68 over 5 min, to 31:69 over 5 min, to 0:100 over 5 min, maintain at 0:100 for 10 min,
re-equilibrate at initial conditions for 6 min. A was MeOH:water:acetic acid 80:20:0.2.
B was MeOH:acetic acid 100:0.2.
Flow rate: 0.56 for 50 min, 0.9 for 10 min, 0.56 for 6 min
Detector: UV 210
CHROMATOGRAM
Retention time: 32
OTHER SUBSTANCES
Extracted: similar triterpenic acids
KEY WORDS
plasma; SPE
REFERENCE
Büchele, B.; Simmet, T. Analysis of 12 different pentacyclic triterpenic acids from frankincense in human
plasma by high-performance liquid chromatography and photodiode array detection, J.Chromatogr.B,
2003, 795, 355–362.
SAMPLE
Matrix: plant resin
Sample preparation: Sonicate 100–500 mg resin in 3 mL MeOH for 10 min, centrifuge,
repeat extraction twice more. Combine the supernatants and make up to 10 mL with
MeOH, filter (0.45 µm), inject a 10 µL aliquot.
HPLC VARIABLES
Column: 150 × 4.6 4 µm Synergi MAX-RP 80 A (Phenomenex)
Column temperature: 60
β-Boswellic acid
87
Mobile phase: Gradient. A:B 65:35 for 7 min, to 90:10 over 21 min, maintain at 90:10
for 5 min, to 100:0 (step gradient), maintain at 100:0 for 5 min, re-equilibrate at initial
conditions for 10 min. A was MeCN containing 0.05% phosphoric acid. B was water
containing 0.05% phosphoric acid.
Flow rate: 1
Injection volume: 10
Detector: UV 210
CHROMATOGRAM
Retention time: 25
Limit of detection: 270 ng/mL
OTHER SUBSTANCES
Simultaneous: similar triterpenic acids
REFERENCE
Ganzera, M.; Khan, I.A. A reversed phase high-performance liquid chromatography method for the
analysis of boswellic acids in Boswellia serrata, Planta Med., 2001, 67, 778–780.
88
Brimonidine
Brimonidine
Molecular formula: C11 H10 BrN5
Molecular weight: 292.14
CAS Registry No: 59803-98-4
Merck Index: 13, 1361
H
H
N
N
N
Br
N
N
SAMPLE
Matrix: aqueous humor, blood
Sample preparation: Mix 100–300 µL serum or aqueous humor with 2 vol buffer,
centrifuge at 11 000 g for 10 min,, add the supernatant to a Sep-Pak C18 SPE cartridge,
wash with 5 mL buffer, elute with 5 mL MeCN:buffer 50:50. Evaporate the eluate to
dryness under a stream of nitrogen, reconstitute the residue with 100 µL mobile phase,
inject a 10–20 µL aliquot. (The buffer was 10 mM triethylamine adjusted to pH 3.2 with
phosphoric acid.)
HPLC VARIABLES
Guard column: 20 × 4.6 RP-18 (Supelco)
Column: 250 × 4.6 5 µm Supelcosil LC-18
Mobile phase: MeCN:buffer 10:90 (The buffer was 10 mM triethylamine adjusted to pH
3.2 with phosphoric acid.)
Flow rate: 1
Injection volume: 10–20
Detector: UV 248
CHROMATOGRAM
Retention time: 7
Limit of detection: 30 pg/mL
OTHER SUBSTANCES
Noninterfering: benzalkonium chloride, cyclopentolate, phenylephrine, tropicamide
KEY WORDS
serum; SPE
REFERENCE
Karamanos, N.K.; Lamari, F.; Katsimpris, J.; Gartaganis, S. Development of an HPLC method for determining the alpha2-adrenergic receptor agonist brimonidine in blood serum and aqueous humor of the
eye, Biomed.Chromatogr., 1999, 13, 86–88.
SAMPLE
Matrix: tissue
Sample preparation: Vortex <3–25 mg ocular tissue with 200 µL MeOH for 3 min,
centrifuge at 3000 g for 10 min. Mix a 35 µL aliquot of the supernatant with 65 µL
0.01% trifluoroacetic acid in water, inject the whole amount.
HPLC VARIABLES
Column: 300 × 3.9 15 µm Delta PAK C18 (Waters)
Mobile phase: MeOH:water:trifluoroacetic acid 35:65:0.01
Flow rate: 1
Injection volume: 100
Detector: UV 360
Brimonidine
89
CHROMATOGRAM
Retention time: 3.5
Limit of detection: 20 ng/mL
OTHER SUBSTANCES
Extracted: mitomycin C (6, LOD 10 ng/mL), timolol (10, LOD 50 ng/mL)
KEY WORDS
eye
REFERENCE
Xiong, X.; Lim, B.A.; Lat-Luna, M.; Chew, P.; Tan, D. Quantitation of mitomycin C in human ocular
tissues by high-performance liquid chromatography-photo-diode array detection, J.Chromatogr.B,
2001, 755, 65–72.
ANNOTATED BIBLIOGRAPHY
Acheampong, A.A.; Shackleton, M.; Tang-Liu, D.D.-S. Comparative ocular pharmacokinetics of brimonidine after a single dose application to the eyes of albino and pigmented rabbits, Drug Metab.Dispos.,
1995, 23, 708–712. [UV detection; radioactivity detection]
Acheampong, A.A.; Chien, D.-S.; Lam, S.; Vekich, S.; Breau, A.; Usansky, J.; Harcourt, D.; Munk, S.A.;
Nguyen, H.; Garst, M.; Tang-Liu, D. Characterization of brimonidine metabolism with rat, rabbit,
dog, monkey and human liver fractions and rabbit liver aldehyde oxidase, Xenobiotica, 1996, 26,
1035–1055.
Acheampong, A.A.; Shackleton, M.; John, B.; Burke, J.; Wheeler, L.; Tang-Liu, D. Distribution of brimonidine into anterior and posterior tissues of monkey, rabbit, and rat eyes, Drug Metab.Dispos.,
2002, 30, 421–429.
90
Bromfenac
Bromfenac
Molecular formula: C15 H12 BrNO3
Molecular weight: 334.17
CAS Registry No: 91714-94-2
Merck Index: 13, 1374
O
NH2
COOH
Br
SAMPLE
Matrix: blood, urine
Sample preparation: Mix 1 mL plasma with 1 mL MeCN, filter (0.2 µm) by centrifuging at 800 g for 15 min, inject a 500 µL aliquot of the filtrate. Mix 1 mL urine with 1 mL
100 mM pH 5 ammonium acetate buffer and 100 µL Glusulase (40 000 units), heat at
37◦ for 16 h, centrifuge at 800 g for 15 min, inject a 500 µL aliquot of the supernatant.
HPLC VARIABLES
Column: 150 × 4.6 5 µm HiChrom (Regis) (for electrospray negative ion MS (FinniganMAT TSQ 700) detection use 250 × 2 C18 DB (Supelco) column with the gradient below
at 0.2 mL/min)
Mobile phase: Gradient. MeCN:100 mM pH 4.9 ammonium acetate buffer from 10:90
to 45:55 over 50 min, maintain at 45:55 for 10 min, re-equilibrate at initial conditions
for 15 min.
Flow rate: 1
Injection volume: 500
Detector: UV 270
CHROMATOGRAM
Retention time: 42
OTHER SUBSTANCES
Extracted: metabolites
KEY WORDS
plasma
REFERENCE
Osman, M.; Chandrasekaran, A.; Chan, K.; Scatina, J.; Ermer, J.; Cevallos, W.; Sisenwine, S.F. Metabolic
disposition of 14 C-bromfenac in healthy male volunteers, J.Clin.Pharmacol., 1998, 38, 744–752.
SAMPLE
Matrix: blood
Sample preparation: Mix 500 µL plasma with 200 µL 1 µg/mL IS in water, add 7 mL
hexane, add 500 µL 5% ammonium hydroxide, shake rapidly on a reciprocating shaker
for 10 min, centrifuge at 550 g for 5 min, discard the organic layer. Add 1 mL 2 M HCl
to the aqueous layer, mix briefly, let stand for 20 min. Add 7 mL hexane, shake rapidly
for 10 min, centrifuge at 550 g for 5 min. Evaporate the organic layer to dryness under
a stream of nitrogen at 60◦ , reconstitute the residue with 200 µL mobile phase, inject a
100 µL aliquot.
HPLC VARIABLES
Column: 300 × 3.9 10 µm µBondapak C18
Mobile phase: MeCN:THF:50 mM pH 6.5 sodium acetate buffer 39:6:55
Flow rate: 1.5
Injection volume: 100
Detector: UV 270
Bromfenac
91
CHROMATOGRAM
Retention time: 9.5
Internal standard: 2-amino-3-(4-chlorobenzoyl)benzeneacetic acid (chloro analogue)
(8.5)
Limit of quantitation: 30 ng/mL
KEY WORDS
pharmacokinetics; plasma; rat
REFERENCE
Osman, M.A.; Dunning, L.K.; Cheng, L.K.; Wright, G.J. Determination of bromfenac in plasma by highperformance liquid chromatography, J.Chromatogr., 1989, 489, 452–458.
92
Brovincamine
Brovincamine
Molecular formula: C21 H25 BrN2 O3
Molecular weight: 433.35
CAS Registry No: 57475-17-9
Merck Index: 13, 1436
H
Br
HO
CH3O
O
N
N
CH3
SAMPLE
Matrix: blood
Sample preparation: Mix 1 mL plasma with 40 µL 10 µg/mL IS in MeCN, add 500 µL
ammonia (0.88):water 1:10, mix, add 5 mL diethyl ether, rotate for 10 min, centrifuge
at 2000 g for 10 min. Evaporate the organic layer to dryness under a stream of nitrogen
at 37◦ , rinse the walls of the tube with ether, again evaporate to dryness, reconstitute
the residue with 50 µL mobile phase, centrifuge at 2000 g for 10 min, inject an aliquot.
HPLC VARIABLES
Guard column: 70 × 2 25–37 µm Co:Pell ODS (Whatman)
Column: 300 × 4 10 µm µBondapak C18
Mobile phase: MeCN:buffer 35:65 (The buffer was 0.1% sodium dihydrogen phosphate
adjusted to pH 3.5 with phosphoric acid.)
Flow rate: 2
Detector: UV 232
CHROMATOGRAM
Retention time: 6.8
Internal standard: vincamine (3.6)
Limit of detection: 10 ng/mL
KEY WORDS
plasma
REFERENCE
Brodie, R.R.; Chasseaud, L.F. Determination of 11-bromovincamine in human plasma by highperformance liquid chromatography, J.Chromatogr., 1982, 228, 413–417.
Bucillamine
Bucillamine
Molecular formula: C7 H13 NO3 S2
O
H3C
H3C
COOH
N
SH
93
SH
H
Molecular weight: 223.32
CAS Registry No: 65002-17-7
Merck Index: 13, 1445
SAMPLE
Matrix: blood
Sample preparation: Mix 50 µL blood with 50 µL buffer, immediately add 300 µL
0.167 mM N-(1-pyrenyl)maleimide in MeCN, vortex, let stand at room temperature for
15 min, add 5 µL 167 mM HCl, centrifuge at 12 000 rpm for 2 min, inject an aliquot of
the supernatant. (The buffer was 10 mM pH 8.0 Tris-HCl containing 1 mM EDTA.)
HPLC VARIABLES
Column: 150 × 4.6 5 µm C18 (Kanto, Japan)
Mobile phase: MeCN:EtOH:water:orthophosphoric acid:acetic acid 210:250:290:0.5:0.5
Flow rate: 0.5
Injection volume: 20
Detector: F ex 330 em 380
CHROMATOGRAM
Retention time: 10
Limit of detection: 2.5 nM (S/N 3)
Limit of quantitation: 3 nM
KEY WORDS
derivatization; whole blood
REFERENCE
Higashi, Y.; Yamashiro, M.; Yamamoto, R.; Fujii, Y. HPLC analysis of bucillamine by derivatization with
N-(1-pyrenyl)maleimide in human blood, J.Liq.Chromatogr.Rel.Technol., 2003, 26, 3265–3275.
94
Budipine
Budipine
Molecular formula: C21 H27 N
N
CH3
CH3
CH3
Molecular weight: 293.44
CAS Registry No: 57982-78-2
Merck Index: 13, 1455
SAMPLE
Matrix: urine
Sample preparation: Adjust pH of urine to 5.0 with acetic acid, add 1300 U/mL
glusulase and 50 U/mL sulfatase, heat at 37◦ for 24 h, adjust pH to 9, pass through a
column of Amberlite XAD-2 resin, wash with 3 vol of water, elute with MeOH. Evaporate
the eluate to dryness, reconstitute the residue with water, adjust to pH 8, extract with
ethyl acetate. Wash the ethyl acetate extract with one-tenth the volume of saturated
NaCl, dry over anhydrous sodium sulfate, reconstitute with MeOH, inject an aliquot.
Alternatively, adjust pH of urine to 5.0 with acetic acid, add glusulase/sulfatase, heat
at 37◦ , adjust pH to 9, extract with dichloromethane:isopropanol 90:10. Evaporate the
extract to dryness under reduced pressure, reconstitute, inject an aliquot. (Both the
procedures are given.)
HPLC VARIABLES
Column: 10 µm Viosfer C8
Mobile phase: MeCN:water:isopropylamine 79.96:20:0.04
Detector: UV 220
OTHER SUBSTANCES
Extracted: metabolites
KEY WORDS
rat; SPE
REFERENCE
Caputo, O.; Grosa, G.; Ceruti, M.; Rocco, F.; Biglino, G. The metabolic fate of the anti-parkinsonian drug
budipine in rats, Eur.J.Drug Metab.Pharmacokinet., 1991, 16, 113–118.
95
Bulaquine
Bulaquine
Molecular formula: C21 H27 N3 O3
Molecular weight: 369.46
CAS Registry No: 223661-25-4
O
O
H
H
N
CH3
H
N
CH3
N
H3CO
SAMPLE
Matrix: formulations
Sample preparation: Extract capsule fill containing 5 mg bulaquine three times with
3 mL MeOH:dimethyloctylamine 99:1. Combine the extracts and make up to 10 mL
with the same solvent, filter, dilute 500 µL of the filtrate to 25 mL with MeOH, inject
an aliquot.
HPLC VARIABLES
Column: 250 × 4 5 µm Lichrospher RP select-B C8
Mobile phase: MeCN:10 mM pH 5.6 sodium acetate buffer 55:45
Flow rate: 1
Injection volume: 20
Detector: UV 265
CHROMATOGRAM
Retention time: 17.26
Limit of detection: 400 ng/mL
Limit of quantitation: 2 µg/mL
OTHER SUBSTANCES
Simultaneous: chloroquine (4.22), primaquine (5.72)
KEY WORDS
capsules; comparison with HPTLC
REFERENCE
Dwivedi, A.K.; Saxena, D.; Singh, S. HPLC and HPTLC assays for the antimalarial agents chloroquine,
primaquine and bulaquine, J.Pharm.Biomed.Anal., 2003, 33, 851–858.
SAMPLE
Matrix: blood
Sample preparation: Mix 500 µL plasma with 50 µL 1 M KOH, add 25 µL 20 µg/mL
IS in MeCN, add 3 mL extraction solvent, vortex for 1 min, centrifuge at 1000 g for
10 min, freeze the aqueous layer, remove the organic layer, repeat the extraction.
Evaporate the combined organic layers to dryness under reduced pressure, reconstitute the residue with 200 µL MeCN:50 mM pH 7.0 ammonium acetate buffer
50:50, centrifuge, inject an aliquot of the supernatant. (Extraction solvent was nhexane:isopropanol:dimethyloctylamine 98:2:0.1.)
HPLC VARIABLES
Guard column: 30 × 4.6 5 µm Spheri-5 cyano
Column: 220 × 4.6 5 µm Spheri-5 cyano
Mobile phase: Gradient. A:B from 55:45 to 90:10 over 15 min, maintain at 90:10 for
2 min, return to initial conditions over 2 min. A was MeCN:50 mM pH 6.0 ammonium
acetate buffer 65:35. B was 50 mM pH 6.0 ammonium acetate buffer.
96
Bulaquine
Flow rate: 1
Injection volume: 50
Detector: UV 261
CHROMATOGRAM
Retention time: 8
Internal standard: 3-bromoprimaquine diphosphate (13.5)
Limit of detection: 10 ng/mL
Limit of quantitation: 20 ng/mL
OTHER SUBSTANCES
Extracted: primaquine (11)
KEY WORDS
pharmacokinetics; plasma; rabbit
REFERENCE
Lal, J.; Mehrotra, N.; Gupta, R.C. Analysis and pharmacokinetics of bulaquine and its major metabolite
primaquine in rabbits using an LC-UV method – a pilot study, J.Pharm.Biomed.Anal., 2003, 32,
141–150.
ANNOTATED BIBLIOGRAPHY
Nitin, M.; Rajanikanth, M.; Lal, J.; Madhusudanan, K.P.; Gupta, R.C. Liquid chromatography-tandem
mass spectrometric assay with a novel method of quantitation for the simultaneous determination of
bulaquine and its metabolite, primaquine, in monkey plasma, J.Chromatogr.B, 2003, 793, 253–263.
Butacaine
Butacaine
Molecular formula: C18 H30 N2 O2
Molecular weight: 306.44
CAS Registry No: 149-16-6
Merck Index: 13, 1495
97
O
O
H2N
N
CH3
CH3
SAMPLE
Matrix: blood
Sample preparation: Buffer plasma with 100 mM pH 7.0 phosphate buffer, inject a
300 µL aliquot onto column A and elute to waste with mobile phase A; after 11 min,
backflush the contents of column A onto column B with mobile phase B. (Re-equilibrate
column A with mobile phase A at 1.8 mL/min for 3.3 min.)
HPLC VARIABLES
Column: A 25 × 4 LiChrospher RP-18 ADS; B 4 × 4 5 µm LiChrospher 100 RP−18 +
125 × 3 5 µm LiChrospher 100 RP-18
Column temperature: 35
Mobile phase: A 100 mM pH 7.0 phosphate buffer; B MeCN:50 mM pH 7.0 phosphate
buffer 50:50
Flow rate: 0.6
Injection volume: 300
Detector: UV 210
KEY WORDS
column-switching; plasma
REFERENCE
Baeyens, W.R.G.; Van Der Weken, G.; Haustraete, J.; Smet, E. Application of the ADS precolumnswitching technique to the separation of some drugs from plasma, Biomed.Chromatogr., 2000, 14,
61–63.
SAMPLE
Matrix: blood, urine
Sample preparation: 2 mL Whole blood, plasma, or urine + 1 mL saturated sodium
carbonate + 20 µL 100 µg/mL tetracaine, add to a 3 mL Extrelut SPE cartridge, elute
with 15 mL dichloromethane. Evaporate eluate to dryness under a stream of nitrogen
at 40◦ , reconstitute in 100 µL mobile phase, inject a 40 µL aliquot.
HPLC VARIABLES
Guard column: 5 × 6 µBondapak Guard Pak
Column: 300 × 3.9 10 µm µBondapak C18
Mobile phase: MeCN:100 mM ammonium acetate 50:50
Flow rate: 1.5
Injection volume: 40
Detector: UV 280; F ex 280 em 350
CHROMATOGRAM
Retention time: 10
Internal standard: tetracaine (14)
Limit of detection: 20 ng/mL
OTHER SUBSTANCES
Extracted: p-aminobenzoic acid (4), procaine (5)
98
Butacaine
Also analyzed: articaine, prilocaine, o-toluidine, lidocaine, bupivacaine, etidocaine,
dibucaine, caffeine, amphetamine, ephedrine, epinephrine, morphine, monoacetylmorphine, diamorphine, ethylmorphine, codeine, acetylcodeine
KEY WORDS
plasma; SPE; whole blood
REFERENCE
Rop, P.P.; Grimaldi, F.; Bresson, M.; Fornaris, M.; Viala, A. Liquid chromatographic analysis of cocaine,
benzoylecgonine, local anaesthetic agents and some of their metabolites in biological fluids, J.Liq.Chromatogr., 1993, 16, 2797–2811.
Butamben
Butamben
Molecular formula: C11 H15 NO2
Molecular weight: 193.24
CAS Registry No: 94-25-7
Merck Index: 13, 1502
99
O
O
CH3
H2N
SAMPLE
Matrix: blood
Sample preparation: Mix 500 µL plasma with 600 µL 100 mM KOH, add 1 mL 8.0 M
urea, vortex gently, add 4 mL ethyl acetate, vortex for 1 min, centrifuge at 3000 g for
10 min. Evaporate the organic layer to dryness under a stream of nitrogen, reconstitute
the residue with 200 µL MeOH, inject a 20 µL aliquot. (Perform the analyses under
yellow light.)
HPLC VARIABLES
Guard column: Corasil
Column: 250 × 4.6 5 µm Spherisorb ODS 2
Mobile phase: MeOH:10 mM pH 6.1 ammonium acetate buffer 62:38
Flow rate: 1
Injection volume: 20
Detector: UV 247
CHROMATOGRAM
Retention time: 8.45
OTHER SUBSTANCES
Extracted: nifedipine (7.14)
KEY WORDS
butamben is IS; plasma
REFERENCE
Thongnopnua, P.; Viwatwongsa, K. Quantitative analysis of nifedipine in plasma by high-performance
liquid chromatography, J.Pharm.Biomed.Anal., 1994, 12, 119–125.
100
Butoconazole
Butoconazole
N
Molecular formula: C19 H17 Cl3 N2 S
Molecular weight: 411.78
CAS Registry No: 64872-76-0; 64872-77-1 (nitrate)
Merck Index: 13, 1525
N
Cl
S
Cl
Cl
SAMPLE
Matrix: solutions
Sample preparation: Extract swab with MeOH or MeOH:THF 95:5, add IS, filter,
inject an aliquot.
HPLC VARIABLES
Column: 250 × 4.6 Partisil 10 SCX (Whatman)
Mobile phase: MeOH:135 mM pH 4.35 potassium acetate buffer 61:39
Flow rate: 1
Detector: UV 230
CHROMATOGRAM
Internal standard: 1-benzylimidazole
REFERENCE
Weinstein, L.; Henzel, M.R.; Tsina, I.W. Vaginal retention of 2% butoconazole nitrate cream: comparison
of a standard and a sustained-release preparation, Clin.Ther., 1994, 16, 930–934.
Butyl flufenamate
Butyl flufenamate
CH3
O
O
101
H
N
CF3
Molecular formula: C18 H18 F3 NO2
Molecular weight: 337.34
CAS Registry No: 67330-25-0
Merck Index: 13, 4158
SAMPLE
Matrix: formulations
Sample preparation: Add 100–300 mg gel ointment to 3 mL MeOH, mix vigorously,
filter (0.2 µm), inject a 10 µL aliquot.
HPLC VARIABLES
Column: 300 × 3.9 10 µm µBondapak C18
Mobile phase: MeOH
Flow rate: 1
Injection volume: 10
Detector: UV 280
CHROMATOGRAM
Retention time: 4.3
KEY WORDS
ointment
REFERENCE
Yamamura, K.; Yamada, J.-I.; Yotsuyanagi, T. High-performance liquid chromatographic assay of antiinflammatory drugs incorporated in gel ointments. Separation and stability testing, J.Chromatogr.,
1985, 331, 383–388.
Cambendazole
Molecular formula: C14 H14 N4 O2 S
Molecular weight: 302.36
H
CH3
H3C
O
N
O
N
N
S
N
H
CAS Registry No: 26097-80-3
Merck Index: 13, 1733
SAMPLE
Matrix: abomasal fluid, blood, duodenal fluid, rumen fluid
Sample preparation: Shake 4 mL plasma, rumen fluid, abomasal fluid, or duodenal
fluid with 4 mL pH 7.4 phosphate buffer and 20 mL ether on a rotary mixer for 10 min,
remove 16 mL of the ether layer, add 20 mL ether, shake on a rotary mixer for 10 min,
remove 20 mL of the ether layer. Combine the ether layers and evaporate them under
a stream of nitrogen at 60◦ to dryness, reconstitute in 50 µL MeOH, sonicate, inject a
5 µL aliquot.
HPLC VARIABLES
Column: 100 × 8 ODS Hypersil
Mobile phase: MeOH:50 mM ammonium carbonate 65:35
Flow rate: 1.5
Injection volume: 5
Detector: UV 292
CHROMATOGRAM
Retention time: 4.3
Limit of detection: 20 ng/mL
OTHER SUBSTANCES
Extracted: albendazole (7.5), fenbendazole (10), mebendazole (5), oxfendazole (3), oxibendazole (5.2), parbendazole (11), thiabendazole (3.7)
KEY WORDS
plasma; sheep
REFERENCE
Bogan, J.A.; Marriner, S. Analysis of benzimidazoles in body fluids by high-performance liquid chromatography, J.Pharm.Sci., 1980, 69, 422–423.
SAMPLE
Matrix: egg, tissue
Sample preparation: Condition an SDB (styrol-divinyl-benzene) SPE cartridge (Baker)
with 3 mL MeOH and 3 mL water. Vortex 3 g muscle or liver with ? µL 10 µg/mL IS in
MeOH, 1.5 g sodium sulfate, 500 µL 4 M potassium carbonate, and 5 mL ethyl acetate.
Centrifuge at 2500 g for 5 min and remove the organic layer. Repeat the extraction.
Combine the organic layers and evaporate them to dryness under a stream of nitrogen
at 50◦ or under reduced pressure. Add 5 mL n-hexane to the residue, shake to get a
good mixture, add 1 mL EtOH/HCl, vortex, centrifuge at 1000 g for 2 min, discard the
upper layer. Evaporate the lower layer to dryness under reduced pressure, reconstitute
the residue with 500 µL 10 mM pH 5.5 ammonium acetate and 500 µL MeOH, add to
the SPE cartridge, wash with 3 mL water, wash with two 3 mL portions of MeOH:water
50:50, dry under vacuum, elute with 3 mL MeOH:ethyl acetate 20:80. Evaporate the
eluate to dryness under reduced pressure, reconstitute the residue with 250 µL mobile
phase, inject a 4 (ion spray) or 15 (turbo ion spray) µL aliquot. Preparation of egg
samples is similar except that the mixture is vortexed for 10 s and sonicated for 15 min
102
HPLC Methods for Recently Approved Pharmaceuticals. by George Lunn
Copyright 2005 John Wiley & Sons, Ltd. ISBN: 0-471-66941-5
Cambendazole
103
after the addition of the ethyl acetate. (EtOH/HCl was made by mixing 66 mL EtOH
with 33 mL 200 mM HCl.)
HPLC VARIABLES
Column: 150 × 2.1 5 µm Zorbax RX
Mobile phase: MeCN:10 mM ammonium acetate containing 0.5% acetic acid 60:40
Flow rate: 0.04 (ion spray), 0.2 (turbo ion spray)
Injection volume: 4–15
Detector: MS, Perkin-Elmer SCIEX API 365 tandem, ion spray or turbo ion spray, m/z
303–261
CHROMATOGRAM
Retention time: 1.9
Internal standard: 2-n-butylmercaptobenzimidazole (2.7) (m/z 207–151)
Limit of detection: 4 ng/g
Limit of quantitation: 6 ng/g
OTHER SUBSTANCES
Extracted: albendazole (2.5, m/z 266–233, LOD 19 ng/g, LOQ 305 ng/g), febantel (4, m/z
447–382, LOD 10 ng/g, LOQ 15 ng/g), fenbendazole (2.9, m/z 300–268, LOD 5 ng/g,
LOQ 7 ng/g), flubendazole (2.1, m/z 314–281, LOD 3 ng/g, LOQ 5 ng/g), mebendazole
(2, m/z 296–264, LOD 3 ng/g, LOQ 5 ng/g), oxfendazole (1.7, m/z 316–159, LOD 5 ng/g,
LOQ 8 ng/g), oxibendazole (2.2, m/z 250–176, LOD 4 ng/g, LOQ 6 ng/g), thiabendazole
(2, m/z 202–175, LOD 3 ng/g, LOQ 5 ng/g), triclabendazole (5.5, m/z 359–343, LOD
6 ng/g, LOQ 9 ng/g)
KEY WORDS
cow; egg; liver; muscle; pig; sheep; SPE
REFERENCE
Balizs, G. Determination of benzimidazole residues using liquid chromatography and tandem mass
spectrometry, J.Chromatogr.B, 1999, 727, 167–177.
ANNOTATED BIBLIOGRAPHY
Danaher, M.; O’Keeffe, M.; Glennon, J.D. Development and optimisation of a method for the extraction of benzimidazoles from animal liver using supercritical carbon dioxide, Anal.Chim.Acta, 2003,
483, 313–324. [albendazole; fenbendazole; mebendazole; thiabendazole; oxibendazole; flubendazole;
oxfendazole; netobimin; triclabendazole; cambendazole]
104
Candesartan cilexetil
Candesartan cilexetil
N
N
Molecular formula: C33 H34 N6 O6
Molecular weight: 610.66
CAS Registry No: 145040-37-5, 139481-59-7
(candesartan only)
Merck Index: 13, 1747
CH3
O
N N
N
O
O
O
O
N
H
CH3
O
SAMPLE
Matrix: blood, formulations
Sample preparation: Mix 1 mL plasma with 2 mL MeCN, vortex briefly, let stand at
room temperature for 5 min, centrifuge at 4000 g for 20 min, inject a 20 µL aliquot
of the supernatant. Mechanically shake a pulverized tablet with 100 mL MeOH for
10 min, centrifuge an aliquot at 4000 rpm for 5 min, filter (0.45 µm), dilute, inject a
20 µL aliquot.
HPLC VARIABLES
Column: 150 × 4.6 5 µm Supelcosil C18
Mobile phase: MeCN:MeOH:10 mM potassium dihydrogen phosphate 18:80:2
Flow rate: 1
Injection volume: 20
Detector: UV 260
CHROMATOGRAM
Retention time: 3.5
Limit of detection: 2 ng/mL
Limit of quantitation: 11 ng/mL
OTHER SUBSTANCES
Extracted: hydrochlorothiazide (6.5, LOD 3.58 ng/mL, LOQ 6.75 ng/mL)
Simultaneous: benazepril (4.5), cilazapril (2.5), hydroflumethiazide (11), lisinopril (7.3)
Noninterfering: amiloride, losartan, valsartan
KEY WORDS
plasma; tablets
REFERENCE
Erk, N. Simultaneous analysis of candesartan cilexetil and hydrochlorothiazide in human plasma and
dosage forms using HPLC with a photodiode array detector, J.Liq.Chromatogr.Rel.Technol., 2003, 26,
2581–2591.
SAMPLE
Matrix: blood
Sample preparation: Condition a 1 mL 100 mg Bond Elut C8 SPE cartridge with
2 mL MeOH and 1 mL 100 mM pH 2 phosphate buffer. Mix 250 µL plasma with IS,
add 250 µL 1 M phosphoric acid, shake, centrifuge at 10000 g at 4◦ for 5 min, add the
supernatant to the SPE cartridge, wash with 500 µL MeOH:100 mM pH 2 phosphate
buffer 50:50, dry at full vacuum for 20 min, elute with 500 µL MeOH. Add 100 µL
MeOH:ethylene glycol 90:10 to the eluate (to prevent adsorption of the drug), vortex,
evaporate to dryness under a stream of nitrogen at 40◦ , reconstitute the residue with
250 µL of the initial mobile phase, inject a 20 µL aliquot.
Candesartan cilexetil
105
HPLC VARIABLES
Guard column: 20 × 3.9 4 µm Novapak C18 (Waters)
Column: 300 × 3.9 10 µm µBondapak C18
Mobile phase: Gradient. MeCN:5 mM pH 4 acetate buffer from 30:70 to 60:40 over 15
min, to 95:5 over 6 min, return to initial conditions over 3 min, re-equilibrate at initial
conditions for 1 min.
Flow rate: From 1 to 1.2 over 15 min, maintain at 1.2 for 6 min, to 1 over 3 min, maintain
at 1 for 1 min
Injection volume: 20
Detector: F ex 250 em 375
CHROMATOGRAM
Retention time: 22.6
Internal standard: bumetanide (13.5)
Limit of quantitation: 3 ng/mL
OTHER SUBSTANCES
Extracted: metabolites, irbesartan (12.6, LOQ 50 ng/mL), losartan (11.5, LOQ
16 ng/mL), valsartan (14.4, LOQ 50 ng/mL)
KEY WORDS
plasma; SPE
REFERENCE
González, L.; López, J.A.; Alonso, R.M.; Jiménez, R.M. Fast screening method for the determination of
angiotensin II receptor antagonists in human plasma by high-performance liquid chromatography
with fluorimetric detection, J.Chromatogr.A, 2002, 949, 49–60.
ANNOTATED BIBLIOGRAPHY
González, L.; Alonso, R.M.; Jiménez, R.M. A high-performance liquid chromatographic method for screening angiotensin II receptor antagonists in human urine, Chromatographia, 2000, 52, 735–740.
[losartan; irbesartan; valsartan; candesartan; SPE]
Stenhoff, H.; Lagerström, P.O.; Andersen, C. Determination of candesartan cilexetil, candesartan and
a metabolite in human plasma and urine by liquid chromatography and fluorometric detection,
J.Chromatogr.B, 1999, 731, 411–417.
106
Capecitabine
Capecitabine
O
H
N
Molecular formula: C15 H22 FN3 O6
Molecular weight: 359.35
CAS Registry No: 154361-50-9
Merck Index: 13, 1759
O
CH3
F
N
H3C
O
N
O
OH OH
SAMPLE
Matrix: blood
Sample preparation: Add 50 µL 50 µg/mL IS in MeOH to a tube, evaporate to dryness
under reduced pressure at 30◦ for 20 min, add 500 µL plasma, add 500 µL 5 mM pH 6.8
ammonium acetate, vortex, let stand on ice for 15 min, centrifuge at 3000 g at 41◦ (sic)
for 10 min. Inject a 20 µL aliquot of the supernatant onto column A and elute to waste
with the mobile phase; after 0.8 min, divert the effluent from column A onto column B;
after another 19.1 min, elute only column A to waste.
HPLC VARIABLES
Column: A 20 × 2.1 Oasis HLB (Waters); B SecurityGuard C18 (Phenomenex) + 150 × 2
5 µm YMC ODS-AQ
Column temperature: 30 (column B only)
Mobile phase: Gradient. MeCN:5 mM pH 6.8 ammonium acetate 0:100 for 2 min, to
10:90 over 0.2 min, to 30:70 over 7.8 min, to 70:30 over 2 min, maintain at 70:30
for 3 min, to 0:100 over 0.2 min, maintain at 0:100 for 4.8 min, to 95:5 over 0.2 min,
maintain at 95:5 for 1 min, return to initial conditions over 0.2 min, re-equilibrate for
2.6 min.
Flow rate: 3 for 0.8 min, 0.2 for 19.2 min, then 3
Injection volume: 20
Detector: MS, electrospray, m/z 360, cone voltage 20 V, 33% of column effluent went to
MS
CHROMATOGRAM
Retention time: 18
Internal standard: 5-chloro-2′ -deoxyuridine (11.5, m/z 261, cone voltage 35 V)
Limit of quantitation: 1.4 ng/mL
OTHER SUBSTANCES
Extracted: metabolites
KEY WORDS
column-switching; pharmacokinetics; plasma
REFERENCE
Xu, Y.; Grem, J.L. Liquid chromatography-mass spectrometry method for the analysis of the anti-cancer
agent capecitabine and its nucleoside metabolites in human plasma, J.Chromatogr.B, 2003, 783,
273–285.
SAMPLE
Matrix: blood
Sample preparation: Mix plasma and IS with MeCN, centrifuge, add the supernatant
to a Bond Elut C18 SPE cartridge, elute with MeOH.
Capecitabine
107
HPLC VARIABLES
Column: 150 × 6 5 µm YMC-Pack C8-AM
Mobile phase: MeCN:buffer 40:60 (The buffer was 50 mL pH 4.0 citrate buffer (Titrisol,
Merck) diluted to 1200 mL with water.)
Flow rate: 1
Detector: UV 310
CHROMATOGRAM
Retention time: 6.2
Internal standard: Ro 09–1977 (8.8)
Limit of quantitation: 50 ng/mL
KEY WORDS
pharmacokinetics; plasma; SPE
REFERENCE
Reigner, B.; Verweij, J.; Dirix, L.; Cassidy, J.; Twelves, C.; Allman, D.; Weidekamm, E.; Roos, B.;
Banken, L.; Utoh, M.; Osterwalder, B. Effect of food on the pharmacokinetics of capecitabine and
its metabolites following oral administration in cancer patients, Clin.Cancer Res., 1998, 4, 941–948.
108
Casanthranol
Casanthranol
CAS Registry No: 8024-48-4
Merck Index: 13, 1893
SAMPLE
Matrix: solutions
Sample preparation: Inject a 20 µL aliquot of a solution in MeOH.
HPLC VARIABLES
Guard column: 4 × 4 5 µm LiChrospher 100 RP18
Column: 125 × 4 5 µm LiChrospher 100 RP18
Column temperature: 40
Mobile phase: MeCN:MeOH:water:acetic acid 5:55:39:1
Flow rate: 1
Injection volume: 20
Detector: UV 254
CHROMATOGRAM
Retention time: 6.1 (aloe-emodin), 20.8 (emodin)
OTHER SUBSTANCES
Extracted: rhein (9.1), sennidin A (16.4), sennidin B (24.8)
REFERENCE
Grimminger, W.; Witthohn, K. Analytics of senna drugs with regard to the toxicological discussion of
anthranoids, Pharmacology, 1993, 47(Suppl. 1), 98–109.
ANNOTATED BIBLIOGRAPHY
Matthees, D. Determination of emodin in feeds, J.Agric.Food Chem., 1983, 31, 453–454.
Koch, A. Metabolism of aloin – the influence of nutrition, J.Pharm.Biomed.Anal., 1996, 14, 1335–1338.
109
Caspofungin
Caspofungin
H2N
H
N
Molecular formula: C52 H88 N10 O15
Molecular weight: 1093.31
HO
H2N
O
N
H
CAS Registry No: 162808-62-0
Merck Index: 13, 1899
OH
O
N
H
O
O H N
HO
H H
O
H N
O
H N
H
HO
N
H O
OH
N
H
OH H C
3
CH3
CH3
CH3
OH
HO
SAMPLE
Matrix: blood
Sample preparation: Condition a 3 mL diol SPE cartridge (Baker) with 3 mL MeOH
and 3 mL water. Mix 1 mL plasma with 10 µL 50 µg/mL IS in MeCN:0.1% trifluoroacetic acid adjusted to pH 3.0 with triethylamine 35:65 and 250 µL 1 M pH 4.9
potassium acetate buffer, add to SPE cartridge, wash with 3 mL water, wash with 3 mL
MeOH, elute with 1 mL MeOH containing 250 mM ammonium hydroxide and 0.1%
trifluoroacetic acid. Evaporate the eluate to dryness under reduced pressure at 50◦ ,
reconstitute the residue with 100 µL mobile phase, inject a 50 µL aliquot. (See also
Schwartz,M. et al. Anal.Chim.Acta 1997, 352, 299–307.)
HPLC VARIABLES
Column: 150 × 4.6 5 µm Zorbax SB C80
Mobile phase: MeCN:buffer 35:65. After 5.5 min, wash column with MeCN:buffer 90:10
for 0.9 min, re-equilibrate at initial conditions for 5 min. (The buffer was 675 µL
trifluoroacetic acid in 1 L water, adjusted to pH 3.0 with ammonium hydroxide.)
Flow rate: 1.2
Injection volume: 50
Detector: MS, PE Sciex API III plus triple quadrupole, ion spray, 5% of column effluent
entered MS, m/z 1093.7
CHROMATOGRAM
Retention time: 5.5
Internal standard: isostere (oxygen replaces nitrogen at 5 position, m/z 1094.7) (6.8)
Limit of quantitation: 10 ng/mL (LOQ was 2.5 ng/mL using a 75 µL injection and
turbo ion spray, but precision and accuracy was less good)
KEY WORDS
plasma; SPE
REFERENCE
Chavez-Eng, C.M.; Schwartz, M.; Constanzer, M.L.; Matuszewski, B.K. Determination of a cyclic hexapeptide, a novel antifungal agent, in human plasma by high-performance liquid chromatography with ion
spray and turbo ion spray tandem mass spectrometric detection, J.Chromatogr.B, 1999, 721, 229–238.
SAMPLE
Matrix: blood
110
Caspofungin
Sample preparation: Mix plasma with MeCN/MeOH containing 0.1% trifluoroacetic
acid, vortex, centrifuge. Evaporate the supernatant to dryness under reduced pressure,
reconstitute the residue with 0.1% trifluoroacetic acid, vortex, centrifuge, inject an
aliquot.
HPLC VARIABLES
Column: 250 × 4.6 5 µm Zorbax RX C8
Mobile phase: Gradient. MeCN:0.1% trifluoroacetic acid 18:82 for 4 min, to 50:50 over
36 min.
Detector: Radioactivity (3 H)
CHROMATOGRAM
Retention time: 32
OTHER SUBSTANCES
Extracted: metabolites
KEY WORDS
plasma
REFERENCE
Balani, S.K.; Xu, X.; Arison, B.H.; Silva, M.V.; Gries, A.; deLuna, F.A.; Cui, D.; Kari, P.H.; Ly, T.;
Hop, C.E.C.A.; Singh, R.; Wallace, M.A.; Dean, D.C.; Lin, J.H.; Pearson, P.G.; Baillie, T.A. Metabolites
of caspofungin acetate, a potent antifungal agent, in human plasma and urine, Drug Metab.Dispos.,
2000, 28, 1274–1278.
SAMPLE
Matrix: urine
Sample preparation: Evaporate to dryness under reduced pressure, reconstitute, inject
an aliquot.
HPLC VARIABLES
Column: 250 × 4.6 5 µm Luna C18 (Phenomenex) + 250 × 4.6 5 µm Zorbax SAX in series
Mobile phase: Gradient. MeCN:0.1% trifluoroacetic acid 0:100 for 15 min, to 80:20 over
25 min
Detector: Radioactivity (3 H)
CHROMATOGRAM
Retention time: 35
OTHER SUBSTANCES
Extracted: metabolites
REFERENCE
Balani, S.K.; Xu, X.; Arison, B.H.; Silva, M.V.; Gries, A.; deLuna, F.A.; Cui, D.; Kari, P.H.; Ly, T.;
Hop, C.E.C.A.; Singh, R.; Wallace, M.A.; Dean, D.C.; Lin, J.H.; Pearson, P.G.; Baillie, T.A. Metabolites
of caspofungin acetate, a potent antifungal agent, in human plasma and urine, Drug Metab.Dispos.,
2000, 28, 1274–1278.
ANNOTATED BIBLIOGRAPHY
Groll, A.H.; Gullick, B.M.; Petraitiene, R.; Petraitis, V.; Candelario, M.; Piscitelli, S.C.; Walsh, T.J. Compartmental pharmacokinetics of the antifungal echinocandin caspofungin (MK-0991) in rabbits,
Antimicrob.Agents Chemother., 2001, 45, 596–600.
Caspofungin
111
Petraitiene, R.; Petraitis, V.; Groll, A.H.; Sein, T.; Schaufele, R.L.; Francesconi, A.; Bacher, J.;
Avila, N.A.; Walsh, T.J. Antifungal efficacy of caspofungin (MK-0991) in experimental pulmonary
aspergillosis in persistently neutropenic rabbits: pharmacokinetics, drug disposition, and relationship
to galactomannan antigenemia, Antimicrob.Agents Chemother., 2002, 46, 12–23.
Schwartz, M.; Kline, W.; Matuszewski, B. Determination of a cyclic hexapeptide (L-743 872), a novel
pneumocandin antifungal agent in human plasma and urine by high-performance liquid chromatography with fluorescence detection, Anal.Chim.Acta, 1997, 352, 299–307.
112
Castor oil
Castor oil
CAS Registry No: 8001-79-4
Merck Index: 13, 1908
SAMPLE
Matrix: cosmetics
Sample preparation: Inject a 5–50 µL aliquot of a solution of the lipstick in MeCN.
HPLC VARIABLES
Column: 300 × 3.9 Bondapak C18
Mobile phase: Gradient. MeCN:water 50:50 for 5 min, to 100:0 (step gradient)
Flow rate: 2
Injection volume: 5–50
Detector: UV 254
CHROMATOGRAM
Retention time: 8, 20, 27 (multiple peaks)
OTHER SUBSTANCES
Simultaneous: lanolin (13, 14), propyl paraben (4)
KEY WORDS
lipstick
REFERENCE
Reuland, D.J.; Trinler, W.A. A comparison of lipstick smears by high performance liquid chromatography,
J.Forensic Sci.Soc., 1980, 20, 111–120.
113
Cefbuperazone
Cefbuperazone
Molecular formula: C22 H29 N9 O9 S2
Molecular weight: 627.66
H3C
H
N
N
O
O
O
HO
OCH3
H
O
N
H
N
H
CH3 O
S
N
CAS Registry No: 76610-84-9
Merck Index: 13, 1930
CH3
S
COOH
N
N
N N
SAMPLE
Matrix: blood
Sample preparation: Condition a 3 mL 200 mg Bond Elut C18 SPE cartridge with
two 3 mL portions of MeOH and two 3 mL portions of 5% acetic acid. Mix 500 µL
serum with 500 µL 10% acetic acid, add to the SPE cartridge, wash with 3 mL water,
elute with four 500 µL portions of MeOH:water 60:40. Evaporate the combined eluates
to dryness under a stream of nitrogen, reconstitute the residue with 250 µL water,
filter (0.22 µm). Inject a 100 µL aliquot onto column A and elute to waste with mobile
phase A. After an unspecified time, backflush the contents of column A onto column
B using mobile phase B. Monitor the effluent from column B. (Sato,K.; Kobayashi,K.;
Moore,C.M.; Mizuno,Y.; Katsumata,Y. Semi-quantitative analysis of cefaclor in human
serum by capillary high performance liquid chromatography/fast atom bombardment
mass spectrometry. Forensic Sci.Int. 1993, 59, 71–77.)
HPLC VARIABLES
Column: A 30 × 0.5 10 µm Develosil PhA (phenethyl); B 150 × 0.5 5 µm Develosil PhA
(phenethyl)
Mobile phase: A 10 mM ammonium acetate:glycerol 99.5:0.5, adjusted to pH 5 with
acetic acid; B MeOH:water:acetic acid:glycerol 40:59:0.5:0.5 (pH ca. 3)
Flow rate: A 0.05; B 0.004
Injection volume: 100
Detector: MS, JMS DX303 double focusing, xenon FAB ion source, gun current 10 mA,
voltage 3 kV, positive ion mode
CHROMATOGRAM
Retention time: 20.8
Limit of detection: 50–200 ng
OTHER SUBSTANCES
Extracted: cefaclor (14.4), cefamandole (31.0), cefazolin (19.7), cefixime (19.2), cefmenoxime (18.8), cefmetazole (21.4), cefoperazone (24.2), cefotaxime (16.1), cefotetan
(16.8), cefotiam (11.7), cefpiramide (20.9), cefsulodin (12.2), ceftazidime (12.0), ceftizoxime (15.6), ceftriaxone (16.7), cefuroxime (16.9), cefuzonam (32.0), cephalexin (14.8),
cephaloglycine (15.7), cephaloridine (15.6), cephalothin (34.7), flomoxef (18.3), latamoxef
(15.1)
KEY WORDS
column-switching; serum; SPE
REFERENCE
Kobayashi, K.; Sato, K.; Mizuno, Y.; Katsumata, Y. Capillary high-performance liquid chromatographyfast atom bombardment mass spectrometry of 24 cephem antibiotics, J.Chromatogr.B, 1996, 677,
275–290.
114
Cefditoren
Cefditoren
S
O
H H
H2N
N
Molecular formula: C19 H18 N6 O5 S3
Molecular weight: 506.59
CAS Registry No: 104145-95-1,
117467-28-4 (pivoxil)
Merck Index: 13, 1934
N
N
H
O
OCH3
N
S
S
CH3
N
COOH
SAMPLE
Matrix: blood
Sample preparation: Mix 200 µL serum or urine with 200 µL 5 µg/mL IS in MeCN,
centrifuge at 3500 rpm for 10 min, inject a 20 µL aliquot.
HPLC VARIABLES
Column: 150 × 4.6 ODS C18
Column temperature: 30
Mobile phase: MeOH:5% amine acetate (sic) 30:70
Flow rate: 1
Injection volume: 20
Detector: UV 295
CHROMATOGRAM
Retention time: 11.5
Internal standard: not specified (15.8)
KEY WORDS
pharmacokinetics; serum
REFERENCE
Li, J.T.; Hou, F.; Lu, H.; Li, T.Y.; Li, H. Phase I clinical trial of cefditoren pivoxil (ME 1207): pharmacokinetics in healthy volunteers, Drugs Exp.Clin.Res., 1997, 23, 145–150.
SAMPLE
Matrix: blood
Sample preparation: Mix 500 µL MeCN with ? µL plasma and ? µL 2 µg/mL IS
in MeCN:water 50:50, vortex at high speed for 30 s, centrifuge at 1180 g for 5 min.
Evaporate the supernatant to dryness, reconstitute the residue with 100–200 µL mobile
phase (?), inject a 10–25 µL aliquot.
HPLC VARIABLES
Column: 100 × 2 4 µm YMC J’sphere M80
Mobile phase: MeOH:10 mM pH 4.5 ammonium acetate 40:60
Flow rate: 0.2
Injection volume: 10–25
Detector: MS, PE-Sciex API-365 triple quadrupole, turbo ion spray source 450◦ , nitrogen
7 L/min, m/z 506.9–240.8
CHROMATOGRAM
Retention time: 6.2
Internal standard: cefotaxime (m/z 455.9–324) (2.2)
Limit of quantitation: 10 ng/mL
KEY WORDS
plasma
Cefditoren
115
REFERENCE
Zhu, T.; Cheung, B.W.Y.; Cartier, L.L.; Giebink, G.S.; Sawchuk, R.J. Simultaneous intravenous and
intramiddle-ear dosing to determine cefditoren influx and efflux clearances in middle ear fluid in freely
moving chinchillas, J.Pharm.Sci., 2003, 92, 1947–1956.
SAMPLE
Matrix: dialysate
Sample preparation: Inject a 10 µL aliquot of artificial middle ear fluid (pH 7.45
phosphate-buffered saline-containing 15 mM phosphate and 150 mM sodium) containing 200 ng/mL IS.
HPLC VARIABLES
Column: 100 × 4.6 YMC ODS-A S-5 C18
Mobile phase: MeOH:50 mM pH 4.0 ammonium acetate buffer 35:65
Flow rate: 0.5
Injection volume: 10
Detector: UV 295
CHROMATOGRAM
Retention time: 9.6
Internal standard: (+)-(6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-methoxyiminoacetamido]-3-[(Z)-2-(4-methylthiazol-5-yl)ethenyl]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-3-ene-2carboxylic acid (Meiji Seika Kaisha, Tokyo) (8.2)
Limit of quantitation: 5 ng/mL
OTHER SUBSTANCES
Extracted: metabolites
REFERENCE
Zhu, T.; Cheung, B.W.Y.; Cartier, L.L.; Giebink, G.S.; Sawchuk, R.J. Simultaneous intravenous and
intramiddle-ear dosing to determine cefditoren influx and efflux clearances in middle ear fluid in freely
moving chinchillas, J.Pharm.Sci., 2003, 92, 1947–1956.
116
Cefoselis
Cefoselis
Molecular formula: C19 H22 N8 O6 S2
Molecular weight: 522.57
CAS Registry No: 122841-10-5,
122841-12-7 (sulfate)
Merck Index: 13, 1945
S
O
H H
H2N
N
N
N
S
+
N
H
N
O
OCH3
COO−
N
NH2
OH
SAMPLE
Matrix: blood, CSF, tissue
Sample preparation: Mix 100 µL serum with 10 µL 100 mg/mL (sic) IS in 100 mM
pH 7.0 phosphate buffer and 100 µL MeCN, vortex for 10 s, centrifuge at 13000 rpm
for 2 min. Remove 100 µL of the supernatant and mix it with 400 µL 20 mM pH 2.5
phosphate buffer, vortex for 10 s, inject a 5 µL aliquot. Mix 30 µL CSF with 1 mg/mL IS
in 100 mM pH 7.0 phosphate buffer (?), inject a 50 µL aliquot. Homogenize brain tissue
with 5 vol of saline. Mix 200 µL homogenate with 20 µL 1 mg/mL IS in 100 mM pH 7.0
phosphate buffer and 200 µL MeCN, vortex for 10 s, inject a 50 µL aliquot.
HPLC VARIABLES
Column: 150 × 4.6 5 µm TSKgel ODS-80TM
Mobile phase: MeCN:20 mM pH 2.5 phosphate buffer 0.065:99.935
Flow rate: 1
Injection volume: 5–50
Detector: UV 254
CHROMATOGRAM
Internal standard: cefpirome sulfate
KEY WORDS
brain; rat; serum
REFERENCE
Nagata, M.; Yasuhara, M. Effect of experimental renal failure on the pharmacodynamics of cefoselisinduced seizures in rats, Biol.Pharm.Bull., 2001, 24, 1049–1052.
117
Cefozopran
Cefozopran
Molecular formula: C19 H17 N9 O5 S2
Molecular weight: 515.53
H2N
O
N
H H
S
N
N
N
CAS Registry No: 113359-04-9
Merck Index: 13, 1950
S
+
N
H
N
N
N
O
OCH3
COO−
SAMPLE
Matrix: blood, urine
Sample preparation: Serum. Mix 300 µL serum with 300 µL 5 mM pH 3.2 sodium
phosphate buffer and 800 µL MeCN, shake mechanically for 2 min, centrifuge at 13000 g
for 5 min. Remove 1.4 mL of the supernatant and add it to 2 mL dichloromethane, agitate
for 1 min, centrifuge at 2000 g for 5 min. Remove a 300 µL aliquot of the supernatant
and add it to 600 µL mobile phase, inject a 20 µL aliquot. Urine. Mix 100 µL urine with
900 µL of the aqueous portion of the mobile phase, inject a 5 µL aliquot.
HPLC VARIABLES
Guard column: 30 × 4 30–40 µm Perisorb RP18
Column: 125 × 4 5 µm Nucleosil 5C18
Mobile phase: MeCN:buffer 4.5:95.5 (Make mobile phase by adding 45 mL MeCN and
3 bottles of PIC B7 solution (15 M heptanesulfonic acid, pH 3.2 (Waters)) to water and
making up to 1 L with water.)
Flow rate: 1.5
Injection volume: 5 (urine), 20 (serum)
Detector: UV 235
CHROMATOGRAM
Retention time: 5.3
Limit of detection: 600 ng/mL (serum), 3.5 µg/mL (urine)
Limit of quantitation: 1 µg/mL (serum), 5 µg/mL (urine)
KEY WORDS
serum
REFERENCE
Borner, K.; Borner, E.; Lode, H. Determination of a new cephalosporin, SCE-2787, in serum and urine
by high-performance liquid chromatography, J.Chromatogr., 1993, 615, 174–179.
118
Cefuzonam
Cefuzonam
Molecular formula: C16 H15 N7 O5 S4
Molecular weight: 513.60
CAS Registry No: 82219-78-1
Merck Index: 13, 1967
S
O
H H
H2N
N
N
N
H
O
OCH3
S
N
S
S
N
COOH
N
SAMPLE
Matrix: blood
Sample preparation: Condition a 3 mL 200 mg Bond Elut C18 SPE cartridge with
two 3 mL portions of MeOH and two 3 mL portions of 5% acetic acid. Mix 500 µL
serum with 500 µL 10% acetic acid, add to the SPE cartridge, wash with 3 mL water,
elute with four 500 µL portions of MeOH:water 60:40. Evaporate the combined eluates
to dryness under a stream of nitrogen, reconstitute the residue with 250 µL water,
filter (0.22 µm). Inject a 100 µL aliquot onto column A and elute to waste with mobile
phase A. After an unspecified time, backflush the contents of column A onto column
B using mobile phase B. Monitor the effluent from column B. (Sato,K.; Kobayashi,K.;
Moore,C.M.; Mizuno,Y.; Katsumata,Y. Semi-quantitative analysis of cefaclor in human
serum by capillary high performance liquid chromatography/fast atom bombardment
mass spectrometry. Forensic Sci.Int. 1993, 59, 71–77.)
HPLC VARIABLES
Column: A 30 × 0.5 10 µm Develosil PhA (phenethyl); B 150 × 0.5 5 µm Develosil PhA
(phenethyl)
Mobile phase: A 10 mM ammonium acetate:glycerol 99.5:0.5, adjusted to pH 5 with
acetic acid; B MeOH:water:acetic acid:glycerol 40:59:0.5:0.5 (pH ca .3)
Flow rate: A 0.05; B 0.004
Injection volume: 100
Detector: MS, JMS DX303 double focusing, xenon FAB ion source, gun current 10 mA,
voltage 3 kV, positive ion mode
CHROMATOGRAM
Retention time: 32.0
Limit of detection: 200–1000 ng
OTHER SUBSTANCES
Extracted: cefaclor (14.4), cefamandole (31.0), cefazolin (19.7), cefbuperazone (20.8),
cefixime (19.2), cefmenoxime (18.8), cefmetazole (21.4), cefoperazone (24.2), cefotaxime
(16.1), cefotetan (16.8), cefotiam (11.7), cefpiramide (20.9), cefsulodin (12.2), ceftazidime (12.0), ceftizoxime (15.6), ceftriaxone (16.7), cefuroxime (16.9), cephalexin
(14.8), cephaloglycine (15.7), cephaloridine (15.6), cephalothin (34.7), flomoxef (18.3),
latamoxef (15.1)
KEY WORDS
column-switching; serum; SPE
REFERENCE
Kobayashi, K.; Sato, K.; Mizuno, Y.; Katsumata, Y. Capillary high-performance liquid chromatographyfast atom bombardment mass spectrometry of 24 cephem antibiotics, J.Chromatogr.B, 1996, 677,
275–290.
119
Celecoxib
Celecoxib
O
O
S
H2N
Molecular formula: C17 H14 F3 N3 O2 S
Molecular weight: 381.38
N
N
CF3
CAS Registry No: 169590-42-5
Merck Index: 13, 1968
H3C
SAMPLE
Matrix: blood
Sample preparation: Condition a 1 mL 100 mg Chromabond C18 SPE cartridge with
two 1 mL portions of MeCN and two 1 mL portions of water. Mix 200 µL plasma with
200 µL 100 mM pH 4.0 phosphate buffer and 25 µL 450 ng/mL IS in MeCN:water 50:50,
add to the SPE cartridge, wash with two 1 mL portions of water, dry under vacuum for
at least 5 min, elute with two 1 mL portions of dichloromethane. Evaporate the eluate to
dryness under a stream of nitrogen at 40◦ , reconstitute the residue with 200 µL mobile
phase, inject a 10 µL aliquot.
HPLC VARIABLES
Column: 30 × 2 5 µm Nucleosil C18
Mobile phase: MeCN:water:25% ammonium hydroxide 85:15:0.1
Flow rate: 0.2
Injection volume: 10
Detector: MS, PE Sciex API 3000 triple quadrupole, turbo ion spray interface, neg-
ative ion mode at -3700 V and 400◦ , auxiliary gas nitrogen 4.5 L/min, nebulizer gas
nitrogen 1.23 L/min, curtain gas nitrogen 1.08 L/min, collision gas nitrogen 2.92 × 1015
molecules/cm2 , m/z 380–316 (–32 eV)
CHROMATOGRAM
Retention time: 0.8
Internal standard: 4-[5-phenyl-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide (The IS is celecoxib lacking the methyl group. Preparation is as follows. Reflux
200 mL EtOH containing 16 mmol 4,4,4-trifluoro-1-phenyl-1,3-butadiene and 17.6 mmol
4-sulfonamidophenylhydrazine hydrochloride with stirring for 22 h, evaporate under
reduced pressure. Take up the residue in ethyl acetate, wash with water, wash with
saline, evaporate the organic solvent, recrystallize from n-hexane:ethyl acetate 50:50 to
obtain the IS as white needles.) (m/z 366–302 (–30 eV)) (0.8)
Limit of quantitation: 0.25 ng/mL
KEY WORDS
pharmacokinetics; plasma; SPE
REFERENCE
Bräutigam, L.; Vetter, G.; Tegeder, I.; Heinkele, G.; Geisslinger, G. Determination of celecoxib in
human plasma and rat microdialysis samples by liquid chromatography tandem mass spectrometry,
J.Chromatogr.B, 2001, 761, 203–212.
SAMPLE
Matrix: blood
Sample preparation: Mix 500 µL serum with 25 µL 30 µg/mL IS in MeCN, add 500 µL
saturated NaCl, add 1 mL MeCN, mix, add 8 mL chloroform (Caution! Chloroform is a
carcinogen!), extract for 15 min, centrifuge at 1500 g for 15 min. Evaporate the organic
120
Celecoxib
layer to dryness under a stream of nitrogen, reconstitute the residue with 100 µL mobile
phase, sonicate for 5 min, vortex, centrifuge for 15 min, inject a 10 µL aliquot.
HPLC VARIABLES
Column: 150 × 3 3 µm Prontosil C18 AQ
Column temperature: 15
Mobile phase: MeCN:water 60:40
Flow rate: 0.35
Injection volume: 10
Detector: F ex 240 em 380
CHROMATOGRAM
Retention time: 9.1
Internal standard: 4-[5-phenyl-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide (The IS is celecoxib lacking the methyl group. Preparation is as follows. Reflux
200 mL EtOH containing 16 mmol 4,4,4-trifluoro-1-phenyl-1,3-butadiene and 17.6 mmol
4-sulfonamidophenylhydrazine hydrochloride with stirring for 22 h, evaporate under
reduced pressure. Take up the residue in ethyl acetate, wash with water, wash with
saline, evaporate the organic solvent, recrystallize from n-hexane:ethyl acetate 50:50 to
obtain the IS as white needles.) (11.7)
Limit of quantitation: 12.5 ng/mL
KEY WORDS
pharmacokinetics; serum
REFERENCE
Schönberger, F.; Heinkele, G.; Mürdter, T.E.; Brenner, S.; Klotz, U.; Hofmann, U. Simple and sensitive
method for the determination of celecoxib in human serum by high-performance liquid chromatography
with fluorescence detection, J.Chromatogr.B, 2002, 768, 255–260.
SAMPLE
Matrix: blood
Sample preparation: Condition a 1 mL Oasis HLB SPE cartridge with 1 mL MeOH
and 1 mL water. Mix 500 µL plasma with 15 µL 10 µg/mL IS in MeOH and 300 µL
200 mM pH 5.0 sodium acetate buffer, add 1.8 mL MeCN, vortex, centrifuge at 15000 g
for 5 min. Evaporate the organic part of the supernatant under a stream of nitrogen
at 50◦ , dilute the remaining aqueous phase with 2 mL water, add to the SPE cartridge
at 0.07 mL/min, wash with two 1.5 mL portions of MeOH:water 5:95, elute with 2 mL
MeOH. Evaporate the eluate to dryness under a stream of nitrogen at 50◦ , reconstitute
the residue with 75 µL MeOH, inject a 50 µL aliquot.
HPLC VARIABLES
Column: 150 × 4.6 5 µm Luna C18 (Phenomenex)
Column temperature: 60
Mobile phase: Gradient. A:B 90:10 for 10 min, to 62:38 over 37 min, to 27:73 over
16 min, maintain at 27:73 for 5 min, return to initial conditions over 0.1 min, reequilibrate for 6.9 min. A was MeCN:10 mM pH 5.4 sodium phosphate buffer 10:90. B
was MeCN:10 mM pH 5.4 sodium phosphate buffer 80:20.
Flow rate: 1.5
Injection volume: 50
Detector: UV 254
CHROMATOGRAM
Retention time: 61
Internal standard: phenacetin (12)
Limit of quantitation: 10 ng/mL
Celecoxib
121
OTHER SUBSTANCES
Extracted: metabolites
KEY WORDS
pharmacokinetics; plasma; SPE
REFERENCE
Störmer, E.; Bauer, S.; Kirchheiner, J.; Brockmöller, J.; Roots, I. Simultaneous determination of celecoxib, hydroxycelecoxib, and carboxycelecoxib in human plasma using gradient reversed-phase liquid
chromatography with ultraviolet absorbance detection, J.Chromatogr.B, 2003, 783, 207–212.
SAMPLE
Matrix: formulations
Sample preparation: Weigh out capsule contents containing 5 mg celecoxib, dissolve
in 25 mL mobile phase, filter (Whatman No 1 paper), dilute with mobile phase, inject
an aliquot.
HPLC VARIABLES
Column: 250 × 4.6 5 µm Inertsil C8
Mobile phase: MeCN:water 65:35
Flow rate: 1.25
Injection volume: 20
Detector: UV 230
CHROMATOGRAM
Retention time: 8.05
Limit of detection: 25 ng/mL
Limit of quantitation: 75 ng/mL
KEY WORDS
capsules
REFERENCE
Saha, R.N.; Sajeev, C.; Jadhav, P.R.; Patil, S.P.; Srinivasan, N. Determination of celecoxib in pharmaceutical formulations using UV spectrophotometry and liquid chromatography, J.Pharm.Biomed.Anal.,
2002, 28, 741–751.
ANNOTATED BIBLIOGRAPHY
Abdel-Hamid, M.; Novotny, L.; Hamza, H. Liquid chromatographic-mass spectrometric determination
of celecoxib in plasma using single-ion monitoring and its use in clinical pharmacokinetics,
J.Chromatogr.B, 2001, 753, 401–408.
Abdel-Hamid, M.E. LC-MS analysis of selected sulfur-containing non-steroid antiinflammatory agents:
Applications to pharmaceutical products, J.Liq.Chromatogr.Rel.Technol., 2000, 23, 3095–3107.
[rofecoxib; sulindac; celecoxib; piroxicam; tenoxicam]
Chow, H.-H.S.; Anavy, N.; Salazar, D.; Frank, D.H.; Alberts, D.S. Determination of celecoxib in
human plasma using solid-phase extraction and high-performance liquid chromatography,
J.Pharm.Biomed.Anal., 2004, 34, 167–174.
Jayasagar, G.; Kumar, M.K.; Chandrasekhar, K.; SivaPrasad, P.; Madhusudan Rao, Y. Validated HPLC
method for the determination of celecoxib in human serum and its application in a clinical
pharmacokinetic study, Pharmazie, 2002, 57, 619–621. [tolbutamide is internal standard]
Mamidi, R.N.V.S.; Mullangi, R.; Kota, J.; Bhamidipati, R.; Khan, A.A.; Katneni, K.; Datla, S.;
Singh, S.K.; Rao, K.Y.; Rao, C.S.; Srinivas, N.R.; Rajagopalan, R. Pharmacological and
pharmacokinetic evaluation of celecoxib prodrugs in rats, Biopharm.Drug Dispos., 2002, 23, 273–282.
Mamidi, R.N.V.S.; Benjamin, B.; Ramesh, M.; Srinivas, N.R. Simple method for the determination of
rosiglitazone in human plasma using a commercially available internal standard, Biomed.Chromatogr.,
2003, 17, 417–420.
122
Celecoxib
Paulson, S.K.; Engel, L.; Reitz, B.; Bolten, S.; Burton, E.G.; Maziasz, T.J.; Yan, B.; Schoenhard, G.L.
Evidence for polymorphism in the canine metabolism of the cyclooxygenase 2 inhibitor, celecoxib, Drug
Metab.Dispos., 1999, 27, 1133–1142.
Paulson, S.K.; Kaprak, T.A.; Gresk, C.J.; Fast, D.M.; Baratta, M.T.; Burton, E.G.; Breau, A.P.; Karim, A.
Plasma protein binding of celecoxib in mice, rat, rabbit, dog and human, Biopharm.Drug Dispos., 1999,
20, 293–299.
Paulson, S.K.; Hribar, J.D.; Liu, N.W.K.; Hajdu, E.; Bible, R.H. Jr.; Piergies, A.; Karim, A. Metabolism
and excretion of [14 C]celecoxib in healthy male volunteers, Drug Metab.Dispos., 2000, 28, 308–314.
[SPE]
Paulson, S.K.; Zhang, J.Y.; Breau, A.P.; Hribar, J.D.; Liu, N.W.K.; Jessen, S.M.; Lawal, Y.M.; Cogburn, J.N.; Gresk, C.J.; Markos, C.S.; Maziasz, T.J.; Schoenhard, G.L.; Burton, E.G. Pharmacokinetics,
tissue distribution, metabolism, and excretion of celecoxib in rats, Drug Metab.Dispos., 2000, 28,
514–521.
Rao, D.S.; Srinivasu, M.K.; Narayana, C.L.; Reddy, G.O. LC separation of ortho and meta isomers of
celecoxib in bulk and formulations using a chiral column, J.Pharm.Biomed.Anal., 2001, 25, 21–30.
Rose, M.J.; Woolf, E.J.; Matuszewski, B.K. Determination of celecoxib in human plasma by normal-phase
high-performance liquid chromatography with column switching and ultraviolet absorbance detection,
J.Chromatogr.B, 2000, 738, 377–385.
Srinivasu, M.K.; Narayana, C.L.; Rao, D.S.; Reddy, G.O. A validated LC method for the quantitative
determination of celecoxib in pharmaceutical dosage forms and purity evaluation in bulk drugs,
J.Pharm.Biomed.Anal., 2000, 22, 949–956.
Stempak, D.; Gammon, J.; Klein, J.; Koren, G.; Baruchel, S. Single-dose and steady-state
pharmacokinetics of celecoxib in children, Clin.Pharmacol.Ther., 2002, 72, 490–497.
Tang, C.; Shou, M.; Mei, Q.; Rushmore, T.H.; Rodrigues, A.D. Major role of human liver microsomal
cytochrome P450 2C9 (CYP2C9) in the oxidative metabolism of celecoxib, a novel cyclooxygenase-II
inhibitor, J.Pharmacol.Exp.Ther., 2000, 293, 453–459.
Tang, C.; Shou, M.; Rodrigues, A.D. Substrate-dependent effect of acetonitrile on human liver microsomal
cytochrome P450 2C9 (CYP2C9) activity, Drug Metab.Dispos., 2000, 28, 567–572. [diclofenac;
phenytoin; celecoxib; tolbutamide; flurbiprofen; chlorpropamide]
Werner, U.; Werner, D.; Mundkowski, R.; Gillich, M.; Brune, K. Selective and rapid liquid
chromatography-mass spectrometry method for the quantification of rofecoxib in pharmacokinetic
studies with humans, J.Chromatogr.B, 2001, 760, 83–90.
Werner, U.; Werner, D.; Pahl, A.; Mundkowski, R.; Gillich, M.; Brune, K. Investigation of the
pharmacokinetics of celecoxib by liquid chromatography-mass spectrometry, Biomed.Chromatogr.,
2002, 16, 56–60. [rofecoxib is internal standard]
Yener, G.; Gonullu, U.; Uner, M.; Degim, T.; Araman, A. Effect of vehicles and penetration enhancers on
the in vitro percutaneous absorption of celecoxib through human skin, Pharmazie, 2003, 58, 330–333.
Cerivastatin
Cerivastatin
F
Molecular formula: C26 H34 FNO5
Molecular weight: 459.55
CAS Registry No: 145599-86-6
Merck Index: 13, 2004
123
OH
OH
COOH
H3CO
H3C
N
CH3
CH3
CH3
SAMPLE
Matrix: blood
Sample preparation: Add 50 µL serum to 500 µL chilled (4◦ ) 100 mM pH 5.0 sodium
acetate buffer, mix, add 50 µL IS in MeCN:water 50:50, vortex, add 5 mL MTBE,
shake mechanically for 15 min, centrifuge for 5 min, freeze in dry ice/MeOH. Remove
the organic layer, evaporate to dryness under reduced pressure with nitrogen at 35◦ ,
reconstitute the residue with 50 µL MeCN:10 mM pH 4.0 ammonium formate buffer
50:50, centrifuge, inject a 25 µL aliquot.
HPLC VARIABLES
Guard column: 10 × 2 YMC Basic
Column: 50 × 2 5 µm YMC Basic
Mobile phase: Gradient. MeCN:10 mM pH 4.0 ammonium formate buffer from 60:40
to 100:0 over 0.3 min, maintain at 100:0 for 0.7 min, return to initial conditions over
0.1 min, re-equilibrate for 2.4 min.
Flow rate: 0.3
Injection volume: 25
Detector: MS, Sciex API 365, positive turbo ion spray at 400◦ , ion spray +5.5 kV, orifice
voltage 45 V, nebulizer gas nitrogen 5.5 bar, turbo ion spray gas at 7 L/min, dwell time
200 ms, collision cell energy 45 eV, m/z 460.4-356.0 (acid), m/z 442.2–354.0 (lactone)
CHROMATOGRAM
Retention time: 1.52 (acid), 1.75 (lactone)
Internal standard: d3 -cerivastatin acid (1.49), d3 -cerivastatin lactone (1.72)
Limit of quantitation: 0.01 ng/mL
OTHER SUBSTANCES
Extracted: metabolites
KEY WORDS
serum
REFERENCE
Jemal, M.; Rao, S.; Salahudeen, I.; Chen, B.-C.; Kates, R. Quantitation of cerivastatin and its seven
acid and lactone biotransformation products in human serum by liquid chromatography-electrospray
tandem mass spectrometry, J.Chromatogr.B, 1999, 736, 19–41.
SAMPLE
Matrix: blood, formulations
Sample preparation: Vortex 2 mL serum and 500 µL MeCN for 2 min, centrifuge at
4000 g for 10 min, inject a 50 µL aliquot of the supernatant. Crush tablets and weigh
out an amount corresponding to 10 mg cerivastatin, sonicate with MeOH for 10 min,
make up to 10 mL with MeOH, filter, add IS to the filtrate, dilute with mobile phase,
inject a 50 µL aliquot.
124
Cerivastatin
HPLC VARIABLES
Column: 250 × 4.6 5 µm LC18 (Waters)
Mobile phase: MeCN:buffer 35:65 (Buffer was 10 mM potassium dihydrogen phosphate
adjusted to pH 3.1 with phosphoric acid.)
Flow rate: 1
Injection volume: 50
Detector: UV 232
CHROMATOGRAM
Retention time: 6.90
Internal standard: losartan (8.78)
Limit of detection: 0.62 ng/mL
Limit of quantitation: 207 ng/mL
KEY WORDS
serum; tablets
REFERENCE
Ozkan, S.A.; Ozkan, Y.; Aboul-Enein, H.Y. Quality control and drug dissolution studies of pharmaceutical
preparations containing cerivastatin sodium by means of RP-HPLC, J.Liq.Chromatogr.Rel.Technol.,
2002, 25, 251–262.
ANNOTATED BIBLIOGRAPHY
Boberg, M.; Angerbauer, R.; Fey, P.; Kanhai, W.K.; Karl, W.; Kern, A.; Ploschke, J.; Radtke, M.
Metabolism of cerivastatin by human liver microsomes in vitro. Characterization of primary metabolic
pathways and of cytochrome P450 isozymes involved, Drug Metab.Dispos., 1997, 25, 321–331. [LC-MS]
Krol, G.J.; Beck, G.W.; Ritter, W.; Lettieri, J.T. LC separation and induced fluorometric detection of
rivastatin in blood plasma, J.Pharm.Biomed.Anal., 1993, 11, 1269–1275.
Mazzu, A.L.; Lasseter, K.C.; Shamblen, E.C.; Agarwal, V.; Lettieri, J.; Sundaresen, P. Itraconazole alters
the pharmacokinetics of atorvastatin to a greater extent than either cerivastatin or pravastatin,
Clin.Pharmacol.Ther., 2000, 68, 391–400. [SPE]
Prueksaritanont, T.; Tang, C.; Qiu, Y.; Mu, L.; Subramanian, R.; Lin, J.H. Effects of fibrates on
metabolism of statins in human hepatocytes, Drug Metab.Dispos., 2002, 30, 1280–1287. [cerivastatin;
simvastatin; atorvastatin; rosuvastatin; pravastatin]
Cetrorelix
Cetrorelix
Cl
Molecular
formula: C70 H92 ClN17 O14
Molecular weight: 1431.06
CAS Registry
No: 120287-85-6
Merck Index: 13, 2036
125
H
O
N
N
H
N
H
O
O
O
Ser-Tyr-D-Cit-Leu-Arg-Pro-D-AlaNH 2
N
H3C
SAMPLE
Matrix: bile, feces, urine
Sample preparation: Bile. Dilute bile with 2 vol of 30% acetic acid, inject a 50–100 µL
aliquot. After each run, regenerate column with a water injection. Feces. Homogenize
feces with 1.5 vol of water. Extract 5 g homogenized feces three times with 5 mL
aliquots of MeOH:ethyl acetate 2:1 using an ultra turrax (TP 18–10; IKA, Staufen,
Germany) at about 10000 rpm with three 30 s applications, centrifuge at 10000 g for
15 min. Combine the supernatants, evaporate to dryness under reduced pressure at 40◦ ,
reconstitute the residue with 2 mL 30% acetic acid in water, filter (minisart GF and
minisart NML, 0.8 µm, Sartorius), inject a 50–100 µL aliquot. Urine. Directly inject a
50–100 µL aliquot of urine.
HPLC VARIABLES
Guard column: 30 × 3 Merck
Column: 250 × 3 5 µm LiChrospher WP 300 RP-18
Mobile phase: Gradient. A:B from 95:5 to 20:80 over 180 min, to 0:100 over 2 min, maintain at 0:100 for 10 min, return to initial conditions over 3 min. A was 0.1% trifluoroacetic
acid in water, adjusted to pH 2.0 with 1 M NaOH. B was MeCN:water:trifluoroacetic
acid 90:10:0.1, adjusted to pH 2.0 with 1 M NaOH.
Flow rate: 0.5
Injection volume: 50–100
Detector: Radioactivity (14 C); UV 226
CHROMATOGRAM
Retention time: 82
OTHER SUBSTANCES
Extracted: metabolites
KEY WORDS
dog; rat
REFERENCE
Schwahn, M.; Schupke, H.; Gasparic, A.; Krone, D.; Peter, G.; Hempel, R.; Kronbach, T.; Locher, M.;
Jahn, W.; Engel, J. Disposition and metabolism of cetrorelix, a potent luteinizing hormone-releasing
hormone antagonist, in rats and dogs, Drug Metab.Dispos., 2000, 28, 10–20.
SAMPLE
Matrix: blood, urine
Sample preparation: Plasma. Condition a 500 mg Sep-Pak Vac C8 with 20 mL
MeOH and 20 mL water. Vortex 1 mL plasma with 10 µL 20 µg/mL IS in mobile
phase, add 10 mL MeOH:water 10:90, mix, add to the SPE cartridge, wash with
10 mL MeOH:water 10:90, wash with 10 mL MeOH:water 50:50, elute with 10 mL
MeOH:water:trifluoroacetic acid 90:10:0.1. Evaporate the eluate to dryness under
126
Cetrorelix
reduced pressure, reconstitute the residue with 100 µL mobile phase, vortex, add
to an Ultrafree-MC/LCR filter (0.2 µm, area 0.2 cm2 ), filter while centrifuging at 3500 g
at 4◦ for 20 min, inject a 40 µL aliquot of the filtrate. Urine. Condition a 500 mg SepPak Vac C8 with 20 mL MeOH and 20 mL water. Mix 2 mL urine with 200 µL blank
human plasma, add 10 µL 20 µg/mL IS in mobile phase, mix, add 8 mL MeOH:water
10:70, mix, add to the SPE cartridge, wash with 10 mL MeOH:water 10:90, wash with
10 mL MeOH:water 50:50, elute with 10 mL MeOH:water:trifluoroacetic acid 90:10:0.1.
Evaporate the eluate to dryness under reduced pressure, reconstitute the residue with
100 µL mobile phase, vortex, add to an Ultrafree-MC/LCR filter (0.2 µm, area 0.2 cm2 ),
filter while centrifuging at 3500 g at 4◦ for 20 min, inject a 40 µL aliquot of the filtrate.
HPLC VARIABLES
Column: 150 × 2.1 5 µm µ-Bondasphere C18
Mobile phase: MeCN:water:trifluoroacetic acid 35:65:0.1
Flow rate: 0.2
Injection volume: 40
Detector: MS, Micromass Platform single-stage quadrupole, electrospray, negative ion
detection, nebulizer gas nitrogen at 30 L/h, drying gas nitrogen at 300 L/h, source
temperature 120◦ , capillary voltage 3 kV, m/z 1429, 1543, 1657
CHROMATOGRAM
Retention time: 6
Internal standard: brominated cetrorelix (dissolve 10 mg cetrorelix in 1 mL glacial
acetic acid, slowly add 15 µL 10% bromine in acetic acid while slowly stirring at room
temperature, stir for 1 h, evaporate to dryness under a stream of nitrogen, reconstitute
the residue with glacial acetic acid, store at 4◦ .) (m/z 1586, 1700, 1813) (8)
Limit of quantitation: 1 ng/mL
KEY WORDS
pharmacokinetics; plasma; SPE
REFERENCE
Niwa, M.; Enomoto, K.; Yamashita, K. Measurement of the novel decapeptide cetrorelix in human plasma
and urine by liquid chromatography-electrospray ionization mass spectrometry, J.Chromatogr.B, 1999,
729, 245–253.
SAMPLE
Matrix: blood
Sample preparation: Mix 4 mL plasma with 200 µL 1 M HCl, add 100 µL 1 µg/mL IS
in 10 mM acetic acid, add 9 mL ethyl acetate:1-butanol 90:10, extract on a Reax mixer
for 15 min, centrifuge at 1000 g for 3 min, remove 5 mL of the organic layer, add 5 mL
ethyl acetate:1-butanol 90:10, extract for 15 min, centrifuge at 1000 g for 3 min, remove
5 mL of the organic layer. Combine the organic layers and extract with 150 µL 10 mM
HCl, centrifuge at 1000 g for 3 min, inject a 150 µL aliquot of the aqueous layer.
HPLC VARIABLES
Column: 250 × 4 5 µm LiChrospher 60 RP-Select B
Mobile phase: MeCN:MeOH:50 mM pH 4 ammonium acetate buffer 25.5:25.5:49
Flow rate: 0.4
Injection volume: 150
Detector: F ex 227 em 340
CHROMATOGRAM
Retention time: 25
Internal standard: D-21740 (Ac-D-Nal1 -(p-Cl)-D-Phe2 -D-Pal3 -Ser4 -Tyr5 -D-Cit6 -Leu7 Arg8 -Ala9 -D-Ala10 -NH2 trifluoroacetate) (23)
Limit of quantitation: 2 ng/mL
Cetrorelix
127
KEY WORDS
plasma
REFERENCE
Raffel, H.H.; Locher, M.; Borbe, H.O. High-performance liquid chromatographic assay for the determination of the decapeptide cetrorelix, a novel luteinizing hormone-releasing hormone antagonist, in
human plasma, J.Chromatogr.B, 1994, 653, 102–105.
128
Cetyl alcohol
Cetyl alcohol
Molecular formula: C16 H34 O
Molecular weight: 242.44
CH3(CH2)14CH2OH
CAS Registry No: 36653-82-4
Merck Index: 13, 2037
SAMPLE
Matrix: solutions
Sample preparation: Prepare a solution of the alcohol by dissolving 10 mg alcohol
in 200 µL pyridine and diluting to 10 mL in MeCN. Mix 1 mL solution with 100 µL
reagent and 100 µL 2% 1-isopropyl-3-(3-dimethylaminopropyl)carbodiimide perchlorate
in MeCN, heat at 80◦ for 20 min, cool to room temperature, add 1 mL water, add 2 mL
isopropanol:water 50:50, add to a Sep-Pak ODS SPE cartridge, rinse the tube with
3 mL isopropanol:water 50:50, add the rinse to the SPE cartridge, wash with 3 mL
isopropanol:water 50:50, elute with 2 mL isopropanol, inject a 20 µL aliquot of the eluate. (The reagent was 10 mg 2-(4-carboxyphenyl)-5,6-dimethylbenzimidazole in 1 mL
pyridine, add 700 mg 4-piperidinopyridine, dilute to 10 mL with MeCN. Prepare 2-(4carboxyphenyl)-5,6-dimethylbenzimidazole as follows. Add 13 g 4-carboxybenzaldehyde
(terephthalaldehydic acid) in 400 mL EtOH dropwise to 4,5-dimethyl-1,2-phenylenediamine in 400 mL EtOH in an ice bath, after 1 h reflux for 8 h, cool to room temperature,
collect the precipitate, recrystallize three times from MeOH:water 50:50 to give 2-(4carboxyphenyl)-5,6-dimethylbenzimidazole as a white amorphous product (mp >300◦ ).
4-Piperidinopyridine is not commercially available but 4-dimethylaminopyridine or
4-pyrrolidinopyridine can be used instead although interferences are greater. Alternatively, 4-piperidinopyridine can be synthesized as follows. Add 200 mmol piperidine
dropwise with stirring to 15 g phosphorus pentoxide and 9.51 g 4-hydroxypyridine,
heat at 250◦ for 7 h, cautiously pour onto 200 g ice, add 400 mL 1 M NaOH, add
200 mL ether. Remove the ether layer and extract the aqueous layer three times with
100 mL portions of ether. Combine the organic layers and dry them over anhydrous
potassium carbonate, evaporate, distill the residue, recrystallize from petroleum ether
(bp 80–100◦ ) to give 4-piperidinopyridine (bp 167–170◦ /11 mm Hg; mp 79–80◦ ) (Synthesis 1978, 844). Alternatively, add 1.94 g 4-bromopyridine hydrochloride to 5 mL
50% NaOH, add 5 mL piperidine, add 2.72 g benzyltriethylammonium bromide, heat
at 100◦ for 5 h, remove excess piperidine by distillation, add 25 mL water, extract
four times with 25 mL portions of benzene. Combine the organic layers and dry
them over anhydrous sodium sulfate, boil the residue with petroleum ether to give
4-piperidinopyridine (mp 80◦ ) (Syn.Commun. 1979, 9, 251). Prepare 1-isopropyl-3-(3dimethylaminopropyl)carbodiimide perchlorate as follows. Stir 1.41 mol isopropylisocyanate in 750 mL dichloromethane at 5◦ , add 144 g 3-dimethylaminopropylamine
(N, N-dimethyl-1,3-propanediamine) in 250 mL dichloromethane at such a rate that the
temperature does not exceed 10◦ , add 500 mL triethylamine, add 300 g p-toluenesulfonyl
chloride in 300 mL dichloromethane at such a rate that the temperature does not exceed
10◦ , reflux for 3 h, add 400 g anhydrous sodium carbonate, add 3.5 L ice water, stir
vigorously for 30 min, remove the organic phase. Extract the aqueous phase three
times with 500 mL portions of dichloromethane. Combine the organic layers and dry
them over anhydrous sodium sulfate, evaporate under reduced pressure, distil the
residue to give 1-isopropyl-3-(3-dimethylaminopropyl)carbodiimide (bp 91–92◦ /10 mm
Hg (Ber. 1941, 74B, 1285)) (cf. Org.Syn. 1973, Coll Vol. V, 555). Prepare pyridine perchlorate from pyridine and 20% perchloric acid, crystallize from EtOH (Ber. 1926,
59, 446). Add 18 g pyridine perchlorate in portions to 100 mmol 1-isopropyl-3-(3dimethylaminopropyl)carbodiimide stirred in 200 mL dichloromethane at 0◦ , let stand
for 30 min, filter, add 200 mL anhydrous diethyl ether to the filtrate. Filter off the
precipitate and recrystallize it from dichloromethane/diethyl ether to give 1-isopropyl-
Cetyl alcohol
129
3-(3-dimethylaminopropyl)carbodiimide perchlorate (mp 88–90◦ ) (Chem.Pharm.Bull.
1985, 33, 5375).)
HPLC VARIABLES
Guard column: 50 × 4.6 7 µm Zorbax ODS
Column: 250 × 4.6 7 µm Zorbax ODS
Mobile phase: MeOH:isopropanol 85:15
Flow rate: 1
Injection volume: 20
Detector: F ex 338 em 428
CHROMATOGRAM
Retention time: 9.4
Limit of detection: 10–20 pg/mL
OTHER SUBSTANCES
Simultaneous: dodecyl alcohol (6.2), tetradecyl alcohol (7.2), stearyl alcohol (12.5),
eicosyl alcohol (16.5)
KEY WORDS
derivatization; SPE
REFERENCE
Katayama, M.; Masuda, Y.; Taniguchi, H. Determination of alcohols by high-performance liquid chromatography after pre-column derivatization with 2-(4-carboxyphenyl)-5,6-dimethylbenzimidazole,
J.Chromatogr., 1991, 585, 219–224.
130
Cevimeline hydrochloride
Cevimeline hydrochloride
N
O
Molecular formula: C10 H17 NOS.HCl.1/2H2 O
Molecular weight: 244.78
CAS Registry No: 153504-70-2, 107233-08-9 (free base)
S
HCl
CH3
H
1/2 H2O
SAMPLE
Matrix: blood
Sample preparation: Condition a Bond Elut Certify SPE cartridge with 3 mL MeOH
and 3 mL 100 mM pH 6.0 phosphate buffer. Mix 1 mL plasma with 100 µL water,
100 µL 1 µg/mL IS, and 2 mL 100 mM pH 6.0 phosphate buffer, add to the SPE
cartridge, wash with 4 mL 10 µM acetic acid, wash with 5 mL MeOH, elute with 6 mL
dichloromethane:isopropanol 80:20 containing 2% ammonia water. Concentrate the
eluate under a stream of nitrogen at 40◦ for 10 min, add 200 µL 0.02% DL-tartaric
acid in MeOH, evaporate to dryness under a stream of nitrogen at 40◦ , reconstitute the
residue with 200 µL MeOH, evaporate to dryness again, reconstitute the residue with
50 µL MeOH:water 20:80, inject an aliquot.
HPLC VARIABLES
Column: 250 × 4.6 5 µm Capcell pak C18 UG120
Mobile phase: Gradient. MeCN:0.075% heptafluorobutyric acid in water from 5:95 to
25:75 over 25 min.
Detector: MS, JEOL AX-505 W, 1% glycerol in MeOH is added post-column at 0.3 mL/min,
chamber temperature 60◦ , emission current 5 mA, collision gas xenon, FAB positive ion
CHROMATOGRAM
Limit of quantitation: 5 ng/mL
KEY WORDS
dog; pharmacokinetics; plasma; rat
REFERENCE
Washio, T.; Kohsaka, K.; Arisawa, H.; Masunaga, H. Pharmacokinetics and metabolism of the novel
muscarinic receptor agonist SNI-2011 in rats and dogs, Arzneimittelforschung, 2003, 53, 26–33.
SAMPLE
Matrix: urine
Sample preparation: Freeze-dry urine and reconstitute with 0.05% trifluoroacetic
acid, inject an aliquot.
HPLC VARIABLES
Column: 250 × 4.6 Inertsil ODS-2
Mobile phase: Gradient. MeCN:0.05% trifluoroacetic acid from 0:100 to 20:80 over
40 min.
Flow rate: 1
Detector: Radioactivity (14 C); Refractive Index
CHROMATOGRAM
Retention time: 29
OTHER SUBSTANCES
Extracted: metabolites
Cevimeline hydrochloride
131
REFERENCE
Washio, T.; Kohsaka, K.; Arisawa, H.; Masunaga, H.; Nagatsuka, S.-i.; Satoh, Y. Pharmacokinetics and
metabolism of radiolabelled SNI-2011, a novel muscarinic receptor agonist, in healthy volunteers,
Arzneimittelforschung, 2003, 53, 80–86.
132
Chlorobutanol
Chlorobutanol
Molecular formula: C4 H7 Cl3 O
Molecular weight: 177.46
CH3
H3C
HO
CCl3
CAS Registry No: 57-15-8
Merck Index: 13, 2148
SAMPLE
Matrix: formulations
Sample preparation: Dissolve ophthalmic ointment containing 20 mg chlorobutanol
in 50 mL hexane, extract 3 times with 15 mL portions of MeOH:water 75:25. Combine
the extracts, make up to 50 mL with MeOH:water 75:25, inject a 100 µL aliquot.
HPLC VARIABLES
Guard column: 10 × 2 47 µm Bondapak C18/Corasil
Column: 250 × 4 10 µm ODS-10 (Bio-Rad)
Mobile phase: MeOH:water 50:50
Flow rate: 1.8
Injection volume: 100
Detector: UV 210
CHROMATOGRAM
Retention time: 6
KEY WORDS
ophthalmic ointment
REFERENCE
Dunn, D.L.; Jones, W.J.; Dorsey, E.D. Analysis of chlorobutanol in ophthalmic ointments and aqueous
solutions by reverse-phase high-performance liquid chromatography, J.Pharm.Sci., 1983, 72, 277–280.
133
Chloroprocaine
Chloroprocaine
Molecular formula: C13 H19 ClN2 O2
Molecular weight: 270.76
CH3
O
O
H2N
N
CH3
Cl
CAS Registry No: 3858-89-7 (HCl)
Merck Index: 13, 2177
SAMPLE
Matrix: blood
Sample preparation: Mix 1 mL plasma with 50 µL 50 µg/mL lidocaine in water and
200 µL 2 M NaOH, extract with diethyl ether. Evaporate the organic layer to dryness
under a stream of nitrogen, reconstitute with 150 µL mobile phase, inject a 50 µL
aliquot.
HPLC VARIABLES
Column: 150 × 3.9 µBondapak C18
Mobile phase: MeCN:50 mM pH 5.8 sodium phosphate buffer 30:70
Flow rate: 1
Injection volume: 50
Detector: UV 210
CHROMATOGRAM
Retention time: 4
Internal standard: lidocaine (5)
Limit of detection: 100 ng/mL
OTHER SUBSTANCES
Extracted: metabolites
KEY WORDS
dog; pharmacokinetics; plasma
REFERENCE
Janicki, P.K.; Johnson, R.; Kambam, J.R. Rapid determination of chloroprocaine and its major metabolite,
2-chloroaminobenzoic acid, in plasma by high-performance liquid chromatography, J.Chromatogr.B,
1996, 675, 336–341.
134
Chorionic gonadotropin
Chorionic gonadotropin
CAS Registry No: 9002-61-3 (human), 9002-70-4 (horse)
Merck Index: 13, 2237
SAMPLE
Matrix: solutions
Sample preparation: Inject a 20 µL aliquot of a 1 mg/mL solution.
HPLC VARIABLES
Column: 250 × 4.6 10 µm Vydac 214TP
Mobile phase: Gradient. A:B from 30:70 to 0:100 over 40 min. A was 100 mM pH 6.8
sodium phosphate buffer. B was MeCN:100 mM pH 6.8 sodium phosphate buffer 50:50.
Flow rate: 2
Injection volume: 20
Detector: UV 220
CHROMATOGRAM
Retention time: 19
OTHER SUBSTANCES
Simultaneous: luteinizing hormone (20), thyroid-stimulating hormone (16)
REFERENCE
Chlenov, M.A.; Kandyba, E.I.; Nagornaya, L.V.; Orlova, I.L.; Volgin, Y.V. High-performance liquid chromatography of human glycoprotein hormones, J.Chromatogr., 1993, 631, 261–267.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Guard column: 40 × 4.1 SynChropak RSC (SynChrom)
Column: 250 × 4.1 SynChropak RP-P (SynChrom)
Mobile phase: Gradient. A:B 85:15 for 6 min, to 75:25 over 1 min, maintain at 75:25
for 5 min, return to initial conditions over 1 min, re-equilibrate for 13 min. A was
water:ethylene glycol dimethyl ether:trifluoroacetic acid 93:7:0.012. B was isopropanol.
Flow rate: 1
Detector: UV 220
CHROMATOGRAM
Retention time: 11
REFERENCE
Wilks, J.W.; Butler, S.S. Biologic activity of human chorionic gonadotropin following reversed-phase
high-performance liquid chromatography, J.Chromatogr., 1984, 298, 123–130.
ANNOTATED BIBLIOGRAPHY
Pask-Hughes, R.A. Characterisation and purification of some glycoproteins by high-performance liquid
chromatography, J.Chromatogr., 1987, 393, 273–284.
Cilnidipine
Cilnidipine
Molecular formula: C27 H28 N2 O7
Molecular weight: 492.52
135
H
H3C
H3CO
CAS Registry No: 132203-70-4
Merck Index: 13, 2297
N
CH3
O
O
O
O
NO2
SAMPLE
Matrix: blood
Sample preparation: Condition a Bond Elut C18 SPE cartridge with 5 mL MeCN and
10 mL water. Mix 10 µL 10 µg/mL IS in MeCN with 1 mL plasma, add 2 mL MeCN,
vortex for 30 s, centrifuge at 1670 g at 4◦ for 10 min, remove the supernatant, repeat
the extraction with 1 mL MeCN. Add the supernatants to 6 mL water, vortex for 30 s,
add to the SPE cartridge, wash with 2 mL MeCN:water 40:60, elute with 2 mL MeCN.
Evaporate the eluate to dryness under reduced pressure at 40◦ , reconstitute the residue
with 100 µL MeCN, inject a 10 µL aliquot.
HPLC VARIABLES
Column: 60 × 4.6 3 µm Hypersil ODS
Mobile phase: MeCN:100 mM ammonium acetate 60:40
Flow rate: 1
Injection volume: 10
Detector: MS, Hewlett-Packard Model 5988A thermospray quadrupole, vaporizer stem
95◦ , ion source 275◦ , negative ion filament on mode, m/z 375
CHROMATOGRAM
Retention time: 3.7
Internal standard: d3 -cilnidipine (m/z 378) (3.7)
Limit of quantitation: 0.5 ng/mL
OTHER SUBSTANCES
Extracted: metabolites
KEY WORDS
plasma; SPE
REFERENCE
Hatada, K.; Kimura, M.; Ono, I.; Ozaki, M. Determination of a new calcium antagonist and its main
metabolite in plasma by thermospray liquid chromatography-mass spectrometry, J.Chromatogr.,
1992, 583, 116–121.
136
Cimetropium bromide
Cimetropium bromide
Molecular formula: C21 H28 BrNO4
Molecular weight: 438.36
CAS Registry No: 51598-60-8
Merck Index: 13, 2301
N+
CH3
Br−
O
OH
O
O
SAMPLE
Matrix: microsomal incubations
Sample preparation: Condition a Sep-Pak C18 SPE cartridge (unspecified). Mix 3.5 mL
microsomal incubation with 1 mL 1% zinc sulfate solution, centrifuge at 3000 g for
15 min, add to the SPE cartridge, wash with 500 µL water, elute with 5 mL MeOH.
Evaporate the eluate to dryness under a stream of nitrogen at 37◦ , reconstitute the
residue with 100 µL MeOH, inject an aliquot.
HPLC VARIABLES
Column: 250 × 4.6 10 µm Spherisorb ODS-2
Mobile phase: MeCN:MeOH:water 20:20:55 or 10:5:55 containing 20 or 35 mM sodium
heptanesulfonate and 100 mM triethylamine, pH 3.7
Flow rate: 1
Detector: MS, VG 70-SEQ, positive ion FAB, collision gas xenon at 8.5 keV
CHROMATOGRAM
Internal standard: glycopyrrolate or benzyl alcohol
REFERENCE
Kajbaf, M.; Jahanshahi, M.; Pattichis, K.; Gorrod, J.W.; Naylor, S. Rapid and efficient purification of
cimetropium bromide and mifentidine drug metabolite mixtures derived from microsomal incubates
for analysis by mass spectrometry, J.Chromatogr., 1992, 575, 75–85.
Cisatracurium besylate
137
Cisatracurium besylate
CH3O
Molecular formula: C65 H82 N2 O18 S2
Molecular weight: 1243.50
CAS Registry No: 96946-42-8
Merck Index: 13, 872
(See also atracurium besylate
in Volume 2)
O
N+
CH3O
O
O
O
CH3
2
OCH3
N+
OCH3
H3C
SO3−
CH3O
OCH3
OCH3
OCH3
SAMPLE
Matrix: blood, gastric contents
Sample preparation: Immediately vortex 500 µL plasma, serum, whole blood, or
gastric contents with 20 µL 50 mM sulfuric acid, store at -20◦ (if required), add 20 µL
10 µg/mL IS in MeOH, add 1 mL MeCN, vortex, let stand for 10 min, centrifuge at
2000 g for 5 min. Evaporate the supernatant to 100–150 µL under a stream of nitrogen,
inject a 3 µL aliquot.
HPLC VARIABLES
Column: 150 × 1 3.5 µm X-Terra MS C18
Mobile phase: Gradient. A was 2 mM pH 3 ammonium formate buffer. B was MeCN:2
mM pH 3 ammonium formate buffer 90:10. A:B 85:15 for 2 min, to 57:43 over 8 min,
re-equilibrate at initial conditions for 1 min.
Flow rate: 0.05
Injection volume: 3
Detector: MS, PE Sciex API-100, electrospray, ion spray 5800 V, positive ion mode,
nebulizer gas nitrogen, curtain gas nitrogen, collision gas argon, m/z 464.6, 358.4
CHROMATOGRAM
Retention time: 9.1
Internal standard: ambenonium besylate (m/z 125.0, 411.6)
Limit of detection: 5 ng/mL
Limit of quantitation: 10 ng/mL
OTHER SUBSTANCES
Extracted: laudanosine (m/z 206.1, 358.4) (7.0), mivacurium (m/z 446.6, 402.5) (6.3),
pancuronium (m/z 430.5, 472.5) (6.4), rocuronium (m/z 529.4, 358.4) (5.3), vecuronium
(m/z 557.4, 398.4) (6.8)
KEY WORDS
plasma; serum; whole blood
REFERENCE
Sayer, H.; Quintela, O.; Marquet, P.; Dupuy, J.-L.; Gaulier, J.M.; Lachâtre, G. Identification and quantitation of six non-depolarizing neuromuscular blocking agents by LC-MS in biological fluids,
J.Anal.Toxicol., 2004, 28, 105–110.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 × 4.6 5 µm Spherisorb ODS-2
Column temperature: 40
138
Cisatracurium besylate
Mobile phase: Gradient. A was MeCN:buffer 50:50. B was MeOH. A:B 100:0 for 9 min,
to 0:100 over 2 min, maintain at 0:100 for 4 min. (The buffer was 300 mM ammonium
formate adjusted to pH 5.6 with formic acid.)
Flow rate: 1
Injection volume: 20
Detector: UV 280; MS, Micromass Quattro II, triple quadrupole, ESI, source 150◦ ,
source capillary voltage 2500–3500 V, cone voltage 25–50 V or APCI, positive ion mode
to negative ion mode at 11 min, source 150◦ , probe 550◦ , corona voltage 2500 V, cone
voltage 35 V
CHROMATOGRAM
Retention time: 9.3
Limit of detection: 100 pg/mL (positive ion mode)
OTHER SUBSTANCES
Simultaneous: degradation products, propofol (negative ion mode) (13)
REFERENCE
Wang, P.; Zhang, H.; Stewart, J.T.; Bartlett, M.G. Simultaneous detection of cisatracurium, its degradation products and propofol using positive ion detection followed by negative ion detection in a single
LC/MS run, J.Pharm.Biomed.Anal., 1998, 17, 547–554.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 × 4.6 5 µm Spherisorb ODS-2
Mobile phase: MeCN:buffer 50:50 (The buffer was 300 mM ammonium formate adjusted
to pH 5.2 with formic acid.)
Flow rate: 1
Injection volume: 20
Detector: UV 280; MS, Micromass Quattro II, EI positive ion mode or APCI, scan m/z
150–600, m/z 464
CHROMATOGRAM
Retention time: 8.8
OTHER SUBSTANCES
Simultaneous: degradation products, laudanosine (5, m/z 358), propofol (13, m/z 178)
KEY WORDS
stability-indicating
REFERENCE
Zhang, H.; Wang, P.; Bartlett, M.G.; Stewart, J.T. HPLC determination of cisatracurium besylate and
propofol mixtures with LC-MS identification of degradation products, J.Pharm.Biomed.Anal., 1998,
16, 1241–1249.
ANNOTATED BIBLIOGRAPHY
Bryant, B.J.; James, C.D. Jr.; Cook, D.R.; Harrelson, J.C. High performance liquid chromatographic
assay for cisatracurium and its metabolites in human urine, J.Liq.Chromatogr.Rel.Technol., 1997, 20,
2041–2051.
Xu, Q.A.; Zhang, Y.-P.; Trissel, L.A.; Gilbert, D.L.; Martinez, J.F.; Fox, J.L. Stability of cisatracurium
besylate in vials, syringes, and infusion admixtures, Am.J.Health-Syst.Pharm., 1998, 55, 1037–1041.
Citric acid
Citric acid
Molecular formula: C6 H8 O7
Molecular weight: 192.12
139
COOH
HO
COOH
COOH
CAS Registry No: 77-92-9
Merck Index: 13, 2350
SAMPLE
Matrix: blood
Sample preparation: Condition a 30 × 10 column of DEAE-cellulose (Serva) with 10
vol of 100 mM pH 7.0 sodium perchlorate and 15 vol of water. Mix 6 mL plasma with
6 mL MeCN, centrifuge at 4000 g for 10 min, suspend the pellet in 6 mL MeCN:water
50:50, centrifuge at 4000 g for 10 min. Combine the supernatants, adjust the pH of a
10 mL aliquot to 7.0 with 100 mM NaOH, add to the DEAE-cellulose column, wash with
10 mL water, elute with 10 mL 100 mM perchloric acid. Freeze-dry the eluate overnight
at 0◦ , reconstitute the residue with 500 µL mobile phase, centrifuge at 12000 g for
2 min, inject a 100 µL aliquot.
HPLC VARIABLES
Guard column: 50 × 4 micro-Guard-NH2
Column: 300 × 7.8 Aminex HPX-87 cation-exchange (Bio-Rad)
Mobile phase: 6.5 mM sulfuric acid
Flow rate: 0.5
Injection volume: 100
Detector: UV 210
CHROMATOGRAM
Retention time: 5
OTHER SUBSTANCES
Extracted: adipic acid (12), meglutol (8), 2-oxoglutaric acid (6.4),
KEY WORDS
plasma; SPE
REFERENCE
Lippe, G.; Trevisan, R.; Nosadini, R.; Fabris, R.; Deana, R. 3-Hydroxy-3-methylglutaric, adipic, and
2-oxoglutaric acids measured by HPLC in the plasma from diabetic patients, Clin.Biochem., 1987, 20,
275–279.
SAMPLE
Matrix: plants
Sample preparation: Extract 20 g leaves with 250 mL water at 15 psi for 20 min, filter,
repeat extraction and filtration twice more. Combine the extracts, concentrate to 50 mL
under reduced pressure, add 200 mL EtOH, centrifuge, concentrate the supernatant to
25 mL under reduced pressure, inject a 20 µL aliquot.
HPLC VARIABLES
Column: 250 × 4.6 µBondapak C18
Mobile phase: 8 mM sulfuric acid
Flow rate: 1
Injection volume: 20
Detector: UV 210
140
Citric acid
CHROMATOGRAM
Retention time: 5.7
Limit of quantitation: 1.4 µg
KEY WORDS
leaves
REFERENCE
Jayaprakasha, G.K.; Sakariah, K.K. Determination of organic acids in leaves and rinds of Garcinia
indica (Desr.) by LC, J.Pharm.Biomed.Anal., 2002, 28, 379–384.
SAMPLE
Matrix: solutions
Sample preparation: Inject a 5 µL aliquot.
HPLC VARIABLES
Column: 150 × 4.6 5 µm LC-18
Mobile phase: 0.5% Ammonium dihydrogen phosphate adjusted to pH 2.80 with 1 M
phosphoric acid
Flow rate: 1
Injection volume: 5
Detector: UV 214
CHROMATOGRAM
Retention time: 5
Limit of detection: 30 ng
OTHER SUBSTANCES
Simultaneous: ascorbic acid (3.3, LOD 10 ng), malic acid (2.5, LOD 50 ng), oxalic acid
(1, LOD 10 ng), succinic acid (5.7, 100 ng), tartaric acid (1.7, LOD 10 ng)
REFERENCE
Zhanguo, C.; Jiuru, L. Simultaneous and direct determination of oxalic acid, tartaric acid, malic acid,
vitamin C, citric acid, and succinic acid in Fructus mume by reversed-phase high-performance liquid
chromatography, J.Chromatogr.Sci., 2002, 40, 35–39.
ANNOTATED BIBLIOGRAPHY
Jayaprakasha, G.K.; Jena, B.S.; Sakariah, K.K. Improved liquid chromatographic method for determination of organic acids in leaves, pulp, fruits, and rinds of Garcinia, J.AOAC Int., 2003, 86, 1063–1068.
[SPE]
Nozal, M.J.; Bernal, J.L.; Diego, J.C.; Gómez, L.A.; Higes, M. HPLC determination of low molecular
weight organic acids in honey with series-coupled ion-exclusion columns, J.Liq.Chromatogr.Rel.
Technol., 2003, 26, 1231–1253. [oxalic acid; glucuronic acid; citric acid; galacturonic acid; propionic acid; pyruvic acid; malic acid; citramalic acid; quinic acid; gluconic acid; lactic acid; formic acid;
glutaric acid; fumaric acid; succinic acid; butyric acid]
Pérez-Ruiz, T.; Martı́nez-Lozano, C.; Tomás, V.; Martı́n, J. High-performance liquid chromatographic
separation and quantification of citric, lactic, malic, oxalic and tartaric acids using a post-column
photochemical reaction and chemiluminescence detection, J.Chromatogr.A, 2004, 1026, 57–64.
Podgornik, A.; Barut, M.; Jaksa, S.; Jancar, J.; Strancar, A. Application of very short monolithic columns
for separation of low and high molecular mass substances, J.Liq.Chromatogr.Rel.Technol., 2002, 25,
3099–3116. [citric acid; malic acid; ketoglutaric acid]
Qiu, J.; Jin, X. Development and optimization of organic acid analysis in tobacco with ion chromatography
and suppressed conductivity detection, J.Chromatogr.A, 2002, 950, 81–88. [citric acid; malonic acid;
malic acid; succinic acid; lactic acid; formic acid; acetic acid; pyroglutamic acid]
Citric acid
141
Sanarico, D.; Motta, S.; Bertolini, L.; Antonelli, A. HPLC determination of organic acids in traditional
balsamic vinegar of Reggio Emilia, J.Liq.Chromatogr.Rel.Technol., 2003, 26, 2177–2187. [dextrose;
fructose; citric acid; tartaric acid; gluconic acid; malic acid; succinic acid; lactic acid; acetic acid]
Shui, G.; Leong, L.P. Separation and determination of organic acids and phenolic compounds in fruit
juices and drinks by high-performance liquid chromatography, J.Chromatogr.A, 2002, 977, 89–96.
[vitamin C; catechin; epicatechin; myricetin; quercetin; eugenol; kaempferol; tartaric acid; oxalic acid;
malic acid; ascorbic acid; malonic acid; lactic acid; acetic acid; citric acid; fumaric acid; gallic acid;
hydroxybenzoic acid; p-hydroxybenzoic acid; chlorogenic acid; caffeic acid; syringic acid; ferulic acid;
benzoic acid; ellagic acid; salicylic acid; cinnamic acid]
Skelly, N.E. Separation of aliphatic and aromatic acids, aromatic sulfonates, quaternary ammonium
compounds, and chelating agents on a reversed-phase column without ion pairing, J.Chromatogr.Sci.,
2003, 41, 22–25. [nonoxynol-9; citric acid; benzenesulfonic acid; phthalic acid; hydrobromic acid;
nitrilotriacetic acid; oxalic acid; nitric acid; hydriodic acid; glycolic acid; formic acid; nitrous acid;
cyanuric acid; lactic acid; acetic acid; NTA; benzalkonium; EDTA]
Suárez-Luque, S.; Mato, I.; Huidobro, J.F.; Simal-Lozano, J. Solid-phase extraction procedure to remove
organic acids from honey, J.Chromatogr.B, 2002, 770, 77–82. [malic acid; maleic acid; citric acid;
succinic acid; fumaric acid]
Suárez-Luque, S.; Mato, I.; Huidobro, J.F.; Simal-Lozano, J.; Sancho, M.T. Rapid determination of minority organic acids in honey by high-performance liquid chromatography, J.Chromatogr.A, 2002, 955,
207–214. [malic acid; maleic acid; citric acid; succinic acid; fumaric acid; SPE]
Wei, M.-C.; Chang, C.-T.; Jen, J.-F. Determination of organic acids in fermentation products of milk
with high performance liquid chromatography/on-lined micro-dialysis, Chromatographia, 2001, 54,
601–605. [citric acid; acetic acid; lactic acid; formic acid]
Yamamoto, A.; Kodama, S.; Matsunaga, A.; Inoue, Y.; Aoyama, T.; Kumagai, Y. Characteristics of a
column suitable for capacity gradient chromatography with a borate eluent, The Analyst, 2001, 126,
465–468.
142
Clioquinol
Clioquinol
Molecular formula: C9 H5 ClINO
Molecular weight: 305.50
CAS Registry No: 130-26-7
Merck Index: 13, 5053
OH
I
N
Cl
SAMPLE
Matrix: bile, blood, tissue, urine
Sample preparation: Mix 200 µL plasma, bile, urine, or kidney with 20 µL IS in
MeOH, 200 µL 200 mM disodium EDTA, and 800 µL water. Add 4 mL benzene:pyridine
90:10 (Caution! Benzene is a carcinogen!), shake vigorously for 1 min, centrifuge at
3500 g for 5 min. Evaporate the organic layer to dryness under reduced pressure,
reconstitute the residue with 200 µL mobile phase, inject a 2–20 µL aliquot. This
sample measures free clioquinol. To measure the glucuronide conjugate, wash the
aqueous phase left over from the previous extraction twice with 6 mL portions of
benzene. Discard the benzene. Add IS in MeOH, β-glucuronidase (final concentration
200 U/mL), and 150 µL 1 M pH 5 acetate buffer to the aqueous phase. Shake at 37◦ for
2 h. Add 4 mL benzene:pyridine 90:10, shake vigorously for 1 min, centrifuge at 3500 g
for 5 min. Evaporate the organic layer to dryness under reduced pressure, reconstitute
the residue with 200 µL mobile phase, inject a 2–20 µL aliquot. This sample measures
clioquinol glucuronide. To measure the sulfate conjugate, wash the aqueous phase left
over from the previous extraction twice with 6 mL portions of benzene. Discard the
benzene. Add 6 M HCl to achieve an HCl concentration of 1 M, add IS, heat at 40◦ for
2 h, almost (sic) neutralize with 3 M NaOH, add 4 mL benzene:pyridine 90:10, shake
vigorously for 1 min, centrifuge at 3500 g for 5 min. Evaporate the organic layer to
dryness under reduced pressure, reconstitute the residue with 200 µL mobile phase,
inject a 2–20 µL aliquot. This sample measures clioquinol sulfate.
HPLC VARIABLES
Column: 300 × 3 10 µm Iatrobeads 6cp-2010 (polystyrene-type porous polymer) in a
Pyrex glass column
Column temperature: 37 ± 0.5
Mobile phase: MeOH:n-hexane:100 mM citric acid 86:6:8
Flow rate: 0.75
Injection volume: 2–20
Detector: UV 254
CHROMATOGRAM
Retention time: 9.6
Internal standard: 5,7-dichloro-8-hydroxyquinoline (6.8)
Limit of quantitation: 600 ng/mL
KEY WORDS
kidney; plasma; rabbit
REFERENCE
Hayakawa, K.; Kitada, K.; Hamaki, M.; Miyazaki, M. High-performance liquid chromatographic determination of clioquinol and its conjugates in biological materials, J.Chromatogr., 1982, 229, 159–165.
Clobetasol 17-propionate
143
Clobetasol 17-propionate
O O
Cl
Molecular formula: C25 H32 ClFO5
Molecular weight: 466.98
CAS Registry No: 25122-46-7
Merck Index: 13, 2387
CH3
HO
CH3
O
CH3
F
H
CH3
H
O
SAMPLE
Matrix: formulations
Sample preparation: Condition a 3 mL 500 mg Megabond MF C18 SPE cartridge
(Varian) with 3 mL MeOH and 3 mL water. Sonicate 1 g cosmetic with 10 mL MeOH
or MeOH:dichloromethane 10:90 (depending on what appears visually to give the best
solubility) at 40◦ for 10 min, centrifuge, collect the clear supernatant. Add 5 mL of the
supernatant to the SPE cartridge, wash with 4 mL acetone:water 20:80, wash with
1 mL n-hexane, elute with 4 mL diethyl ether. Evaporate the eluate to dryness under
reduced pressure, reconstitute the residue with 5 mL (or more) MeOH, inject a 10 µL
aliquot.
HPLC VARIABLES
Column: 250 × 4.6 5 µm endcapped Purospher RP-18
Column temperature: 25
Mobile phase: Isocratic.MeCN:water 60:40. Gradient. MeCN:water from 25:75 to 90:10
over 30 min, maintain at 90:10 for 10 min.
Flow rate: 1
Injection volume: 10
Detector: UV 239
CHROMATOGRAM
Retention time: k′ 3.41 (isocratic); 23.4 min (gradient)
Limit of detection: 100 ng/mL
OTHER SUBSTANCES
Simultaneous: alclometasone dipropionate (isocratic k′ 2.55; gradient retention time
(min) 21.0; LOD 0.3 µg/mL), amcinonide (isocratic k′ 3.18; gradient retention time (min)
22.6; LOD 0.1 µg/mL), betamethasone (isocratic k′ 0.18; gradient retention time (min)
11.8; LOD 0.1 µg/mL), betamethasone-17-acetate (isocratic k′ 0.73; gradient retention
time (min) 15.4; LOD 0.3 µg/mL), betamethasone-17-benzoate (isocratic k′ 2.04; gradient
retention time (min) 20.6; LOD 0.3 µg/mL), betamethasone-17-propionate-21-stearate
(isocratic k′ >13; gradient retention time (min) >35; LOD 0.5 µg/mL), betamethasone17-propionate-21-butyrate (isocratic k′ 5.91; gradient retention time (min) 26.1; LOD
0.4 µg/mL), betamethasone-17-valerate-21-acetate (isocratic k′ 4.41; gradient retention
time (min) 23.1; LOD 0.4 µg/mL), betamethasone-17-valerate (isocratic k′ 2.32; gradient retention time (min) 21.4; LOD 0.3 µg/mL), betamethasone-17,21-dipropionate
(isocratic k′ 4.00; gradient retention time (min) 24.2; LOD 0.4 µg/mL), betamethasone17,21-diacetate (isocratic k′ 1.81; gradient retention time (min) 20.5; LOD 0.3 µg/mL),
betamethasone-17,21-divalerate (isocratic k′ 10.82; gradient retention time (min) 28.0;
LOD 0.4 µg/mL), betamethasone-21-acetate (isocratic k′ 0.77; gradient retention time
(min) 15.6; LOD 0.3 µg/mL), betamethasone propionate (isocratic k′ 0.82; gradient retention time (min) 17.1; LOD 0.3 µg/mL), clobetasone butyrate (isocratic k′ 5.45; gradient
retention time (min) 26.3; LOD 0.1 µg/mL), cortisone (isocratic k′ 0.18; gradient retention
time (min) 11.1; LOD 0.6 µg/mL), cortisone acetate (isocratic k′ 0.73; gradient retention
time (min) 15.2; LOD 0.6 µg/mL), dehydrocorticosterone (isocratic k′ 4.27; gradient
144
Clobetasol 17-propionate
retention time (min) 22.3; LOD 0.5 µg/mL), deoxymethasone (isocratic k′ 0.64; gradient
retention time (min) 14.2; LOD 0.2 µg/mL), dexamethasone (isocratic k′ 0.27; gradient retention time (min) 11.9; LOD 0.1 µg/mL), dexamethasone-21-acetate (isocratic k′
0.91; gradient retention time (min) 16.1; LOD 0.2 µg/mL), dexamethasone isonicotinate
(isocratic k′ 1.05; gradient retention time (min) 17.7; LOD 0.4 µg/mL), dexamethasone pivalate (isocratic k′ 3.45; gradient retention time (min) 24.1; LOD 0.3 µg/mL),
dexamethasone valerate (isocratic k′ 3.00; gradient retention time (min) 21.6; LOD
0.3 µg/mL), diflucortolone valerate (isocratic k′ 4.73; gradient retention time (min) 23.3;
LOD 0.3 µg/mL), fludrocortisone acetate (isocratic k′ 0.59; gradient retention time (min)
14.1; LOD 0.3 µg/mL), flumethasone pivalate (isocratic k′ 2.68; gradient retention time
(min) 21.2; LOD 0.3 µg/mL), fluocinolone acetonide (isocratic k′ 0.91; gradient retention
time (min) 13.4; LOD 0.3 µg/mL), fluocinonide (isocratic k′ 1.45; gradient retention time
(min) 20.5; LOD 0.1 µg/mL), fluocortin butyl ester (isocratic k′ 5.59; gradient retention
time (min) 24.6; LOD 0.3 µg/mL), fluocortolone caproate (isocratic k′ 6.59; gradient
retention time (min) 25.1; LOD 0.3 µg/mL), fluocortolone pivalate (isocratic k′ 4.50;
gradient retention time (min) 23.6; LOD 0.3 µg/mL), fluorometholone (isocratic k′ 0.59;
gradient retention time (min) 14.4; LOD 0.1 µg/mL), 9-α-fluoroprednisolone (isocratic k′
0.18; gradient retention time (min) 10.0; LOD 0.1 µg/mL), 9-α-fluoroprednisolone acetate
(isocratic k′ 0.50; gradient retention time (min) 13.9; LOD 0.2 µg/mL), flurandrenolide
(isocratic k′ 0.50; gradient retention time (min) 13.5; LOD 0.1 µg/mL), halcinonide
(isocratic k′ 1.64; gradient retention time (min) 20.6; LOD 0.1 µg/mL), hydrocortisone
(isocratic k′ 0.18; gradient retention time (min) 10.0; LOD 0.4 µg/mL), hydrocortisone17-butyrate (isocratic k′ 1.09; gradient retention time (min) 17.7; LOD 0.6 µg/mL),
hydrocortisone-21-acetate (isocratic k′ 0.77; gradient retention time (min) 15.3; LOD
0.6 µg/mL), hydrocortisone pivalate (isocratic k′ 2.27; gradient retention time (min)
20.4; LOD 0.8 µg/mL), methylprednisolone (isocratic k′ 0.55; gradient retention time
(min) 13.5; LOD 0.1 µg/mL), mometasone furoate (isocratic k′ 3.05; gradient retention
time (min) 22.0; LOD 0.2 µg/mL), prednisolone-21-acetate (isocratic k′ 0.60; gradient
retention time (min) 13.6; LOD 0.2 µg/mL), prednisolone acetonide (isocratic k′ 0.50;
gradient retention time (min) 13.0; LOD 0.3 µg/mL), prednisolone pivalate (isocratic
k′ 2.05; gradient retention time (min) 19.7; LOD 0.3 µg/mL), triamcinolone (isocratic
k′ 0.14; gradient retention time (min) 7.2; LOD 0.1 µg/mL), triamcinolone acetonide
(isocratic k′ 0.50; gradient retention time (min) 13.9; LOD 0.2 µg/mL), triamcinolone
diacetate (isocratic k′ 0.45; gradient retention time (min) 13.9; LOD 0.3 µg/mL)
KEY WORDS
cosmetics; SPE
REFERENCE
Gagliardi, L.; De Orsi, D.; Del Giudice, M.R.; Gatta, F.; Porrà, R.; Chimenti, P.; Tonelli, D. Development
of a tandem thin-layer chromatography-high-performance liquid chromatography method for the
identification and determination of corticosteroids in cosmetic products, Anal.Chim.Acta, 2002, 457,
187–198.
SAMPLE
Matrix: formulations
Sample preparation: Mix 1 g of a topical product, 5 mL 100 mM pH 4 citrate buffer
saturated with NaCl, and 10 mL ethyl acetate, shake vigorously by hand to ensure
that no large clumps stick to the tube, mix with the tube on its side on an oscillating
shaker for 15 min. Remove the upper ethyl acetate layer and wash with citrate buffer as
before. Dry the ethyl acetate over anhydrous sodium sulfate and evaporate to dryness.
Dissolve the residue in a mixture of 10 mL heptane and 5 mL MeCN:water 90:10.
Remove the upper heptane layer and extract it with 2 mL MeCN:water 90:10. Combine
the MeCN/water layers and evaporate them to dryness, dissolve the residue in 300 µL
MeOH, filter (0.45 µm nylon), inject a 5 µL aliquot.
HPLC VARIABLES
Guard column: 15 × 3.2 7 µm Brownlee NewGuard C18
Clobetasol 17-propionate
145
Column: 75 × 4.6 3.5 µm Symmetry C18 (Waters)
Mobile phase: Gradient. MeCN:water 18:82 for 2 min, to 82:18 over 12 min, maintain
at 82:18 for 3 min, re-equilibrate at initial conditions for 12 min.
Flow rate: 1
Injection volume: 5
Detector: UV 240
CHROMATOGRAM
Retention time: 11.06
Limit of detection: 0.001%
OTHER SUBSTANCES
Simultaneous: alclometasone 17,21-dipropionate (10.93), amcinonide (10.90), beclomethasone 17,21-dipropionate (11.90), betamethasone (6.52), betamethasone 21-acetate
(8.47), betamethasone 17-benzoate (10.28), betamethasone 17,21-dipropionate (11.42),
betamethasone 17-valerate (10.25), budesonide (8.55, 8.69 (epimers)), cortisone (5.62),
cortisone 21-acetate (8.07), dexamethasone (6.57), dexamethasone 21-acetate (8.68),
desonide (6.99), desoximetasone (7.60), desoxycorticosterone acetate (10.90), desoxycorticosterone pivalate (14.45), diflorasone 17,21-diacetate (9.81), fluocinolone acetonide
(7.38), fluocinonide (9.79), flurandrenolide (7.36), fludrocortisone 21-acetate (7.77),
fluorometholone (7.67), flumethasone 21-pivalate (11.20), flunisolide (7.14), fluprednisolone (5.46), fluticasone 17-propionate (11.19), halcinonide (10.72), halobetasol propionate (10.98), hydrocortisone (5.50), hydrocortisone 21-acetate (7.65), hydrocortisone
17-butyrate (8.66), hydrocortisone 21-cypionate (12.54), hydrocortisone 17-valerate
(9.53), mometasone 17-furoate (11.24), methylprednisolone (6.31), methylprednisolone
21-acetate (8.34), meprednisone (6.55), prednisolone (5.37), prednisolone 21-acetate
(7.47), prednisolone 21-tebuate (11.01), paramethasone 21-acetate (8.64), prednicarbate (10.72), prednisone (5.46), triamcinolone (4.15), triamcinolone acetonide (7.04),
triamcinolone 16,21-diacetate (7.49), triamcinolone hexacetonide (13.12)
KEY WORDS
body wash, cream, gel, lotion, shampoo, spray
REFERENCE
Reepmeyer, J.C. Screening for corticosteroids in topical pharmaceuticals by HPLC with a scanning
ultraviolet detector, J.Liq.Chromatogr.Rel.Technol., 2001, 24, 693–709.
SAMPLE
Matrix: solutions
Sample preparation: The receptor solution for skin diffusion experiments consisted
of 4% bovine serum albumin (BSA) in phosphate-buffered saline. Mix receptor solution
with an equal volume of 2 µg/mL IS in MeCN, let stand for 3 h, centrifuge for 20 min,
inject a 50 µL aliquot of the supernatant.
HPLC VARIABLES
Guard column: 4 × 3 C18 (ODS) (Phenomenex)
Column: 150 × 4.6 5 µm Luna C18 (2) (Phenomenex)
Mobile phase: MeCN:MeOH:100 mM pH 3 potassium dihydrogen phosphate 50:10:40
Flow rate: 1
Injection volume: 50
Detector: UV 239
CHROMATOGRAM
Internal standard: propyl paraben
146
Clobetasol 17-propionate
REFERENCE
Mueller, B.; Anissimov, Y.G.; Roberts, M.S. Unexpected clobetasol propionate profile in human stratum
corneum after topical application in vitro, Pharm.Res., 2003, 20, 1835–1837.
ANNOTATED BIBLIOGRAPHY
Marini, R.D.; Pantella, A.; Bimazubute, M.A.; Chiap, P.; Hubert, P.; Crommen, J. Optimisation and
validation of a generic method for the LC assay of six corticosteroids and salicylic acid in dermopharmaceutical forms, Chromatographia, 2002, 55, 263–269. [salicylic acid; methyl paraben;
propyl paraben; triamcinolone acetonide; hydrocortisone acetate; betamethasone valerate; clobetasol
propionate; clobetasone butyrate; betamethasone dipropionate]
Reepmeyer, J.C.; Revelle, L.K.; Vidavsky, I. Detection of clobetasol propionate as an undeclared steroid
in zinc pyrithione formulations by high-performance liquid chromatography with rapid-scanning
ultraviolet spectroscopy and mass spectrometry, J.Chromatogr.A, 1998, 828, 239–246.
Tsai, J.-C.; Cheng, C.-L.; Tsai, Y.-F.; Sheu, H.-M.; Chou, C.-H. Evaluation of in vivo bioequivalence
methodology for topical clobetasol 17-propionate based on pharmacodynamic modeling using Chinese
skin, J.Pharm.Sci., 2004, 93, 207–217. [betamethasone dipropionate is internal standard]
147
Clopidogrel
Clopidogrel
Molecular formula: C16 H16 ClNO2 S
Molecular weight: 321.83
O
OCH3
N
S
Cl
CAS Registry No: 113665-84-2
Merck Index: 13, 2421
SAMPLE
Matrix: microsomal incubations
Sample preparation: Add 200 µL microsomal incubation to 100 µL ice-cold MeCN,
centrifuge, inject a 200 µL aliquot of the supernatant.
HPLC VARIABLES
Column: 150 × 4.6 5 µm Resolve C18 (Waters)
Mobile phase: Gradient. A:B from 17:83 to 43:57 over 30 min, to 100:0 over 15 min. A
was MeCN. B was MeCN:10 mM ammonium acetate 10:90.
Flow rate: 1
Injection volume: 200
Detector: UV 220; MS, Micromass VG Fisons Platform single quadrupole electrospray,
m/z 322.2
CHROMATOGRAM
Retention time: 26
REFERENCE
Clarke, T.A.; Waskell, L.A. The metabolism of clopidogrel is catalyzed by human cytochrome P450 3A
and is inhibited by atorvastatin, Drug Metab.Dispos., 2003, 31, 53–59.
SAMPLE
Matrix: formulations
Sample preparation: Weigh out an amount of pulverized tablet containing 75 mg of
clopidogrel, sonicate with 40 mL MeCN for 20 min, make up to 50 mL with MeCN,
centrifuge at 2890 g for 5 min, dilute a 1 mL aliquot to 100 mL with water. Remove a
1 mL aliquot of this solution and dilute to 10 mL with 100 ng/mL IS in mobile phase,
inject a 5 µL aliquot.
HPLC VARIABLES
Column: 250 × 2.1 5 µm BDS C8
Mobile phase: MeCN:10 mM pH 3.0 sodium phosphate buffer 65:35
Flow rate: 0.3
Injection volume: 5
Detector: UV 235
CHROMATOGRAM
Retention time: 3.08
Internal standard: naproxen (6.28)
Limit of detection: 120 ng/mL
Limit of quantitation: 390 ng/mL
KEY WORDS
stability-indicating; tablets
148
Clopidogrel
REFERENCE
Mitakos, A.; Panderi, I. A validated LC method for the determination of clopidogrel in pharmaceutical
preparations, J.Pharm.Biomed.Anal., 2002, 28, 431–438.
ANNOTATED BIBLIOGRAPHY
Reist, M.; Roy-De Vos, M.; Montseny, J.P.; Mayer, J.M.; Carrupt, P.-A.; Berger, Y.; Testa, B. Very slow
chiral inversion of clopidogrel in rats: a pharmacokinetic and mechanistic investigation, Drug
Metab.Dispos., 2000, 28, 1405–1410. [The metabolite clopidogrel acid is measured in rat plasma
by chiral HPLC with derivatization.]
149
Clopidol
Clopidol
Molecular formula: C7 H7 Cl2 NO
Molecular weight: 192.05
H3C
N
Cl
CH3
Cl
OH
CAS Registry No: 2971-90-6
Merck Index: 13, 2422
SAMPLE
Matrix: feed
Sample preparation: Moisten 10 g pulverized feed with 5 mL water for 1 min, add
45 mL DMF:water 95:5, shake horizontally for 1 h. Remove a 10 mL aliquot of the
supernatant and centrifuge at 3000 rpm for 5 min. Add 5 mL of the supernatant to a
non-pretreated 6 mL 1 g Isolute A1-B SPE cartridge, discard the first 1 mL, collect the
next 2 mL, inject a 10 µL aliquot.
HPLC VARIABLES
Guard column: 20 × 4.6 5 µm Supelguard LC-18
Column: 250 × 4.6 5 µm Supelcosil LC-18
Mobile phase: Gradient. MeCN:10 mM pH 4.6 acetate buffer from 0:100 to 30:70 over
10 min, to 80:20 over 5 min, maintain at 80:20 for 4 min, return to initial conditions
over 1 min, re-equilibrate for 4 min.
Flow rate: 1.2
Injection volume: 10
Detector: UV 265 for 12 min, UV 345 for rest of the run
CHROMATOGRAM
Retention time: 9
Limit of quantitation: 2 µg/g
OTHER SUBSTANCES
Extracted: nicarbazin (17.5)
KEY WORDS
SPE
REFERENCE
Dusi, G.; Faggionato, E.; Gamba, V.; Baiguera, A. Determination of nicarbazin and clopidol in poultry
feeds by liquid chromatography, J.Chromatogr.A, 2000, 882, 79–84.
SAMPLE
Matrix: tissue
Sample preparation: Fill a 400 × 20 glass tube fitted with a glass wool plug and a
PTFE stopcock about a third full with MeOH, add 15 g neutral alumina (70–230 mesh),
drain the solvent to the top of the column. Fill a 200 × 12 glass tube fitted with a
glass wool plug and a PTFE stopcock with a slurry of Dowex 1-X8 anion-exchange resin
(acetate form, 100–200 mesh) in MeOH to a bed height of 15 mm after settling, drain
the solvent to the top of the bed. Place the alumina column on top of the Dowex column.
Homogenize 10 g minced tissue, 20 g anhydrous sodium sulfate, and 50 mL MeCN at
10000 rpm for 2 min, add the supernatant to the alumina tube. Re-extract the residue
with 50 mL MeCN, centrifuge, add to the columns, rinse the centrifuge tube with 20 mL
MeOH, and add the rinse to the columns. Allow the liquid to flow through the columns
at 3 mL/min. When the various solutions have passed through, discard the alumina
column and elute the Dowex column with 20 mL 0.5% acetic acid in MeOH. Evaporate
150
Clopidol
the eluate to dryness under reduced pressure at 60◦ , reconstitute the residue with 1 mL
MeOH, inject an aliquot.
HPLC VARIABLES
Column: 150 × 4.6 Inertsil ODS
Mobile phase: MeCN:water 20:80
Flow rate: 0.8
Injection volume:
Detector: MS, Finnigan MAT TSQ-7000 quadrupole, atmospheric pressure chemical
ionization, m/z 190, corona voltage 1.8 kV, corona current 5.0 µA, capillary temperature
220◦ , electron multiplier 1400 V, vaporizer temperature 400◦
CHROMATOGRAM
Retention time: 3.47
Limit of detection: 5 ng/g
Limit of quantitation: 10 ng/g
KEY WORDS
chicken; muscle; SPE
REFERENCE
Pang, G.-F.; Cao, Y.-Z.; Fan, C.-L.; Zhang, J.-J.; Li, X.-M.; Wang, C. Determination of clopidol residues
in chicken tissues by high-performance liquid chromatography-mass spectrometry, J.Chromatogr.A,
2000, 882, 85–88.
ANNOTATED BIBLIOGRAPHY
Malisch, R. Multimethode zur Bestimmung der Ruckstande von Chemotherapeutica, Antiparasitica und Wachstumsforderern in Lebensmitt tierischer Herkunft. 3. Mitteilung: Bestimmung von
Chloramphenicol und Meticlorpindol [Method for the simultaneous determination of residues of
chemotherapeutic agents, antiparasitic agents and growth promoters in food of animal origin. 3. Determination of chloramphenicol and meticlorpindol], Z.Lebensm.-Unters.-Forsch., 1987, 184, 467–477.
[clopidol; chloramphenicol]
Mattern, E.M.; Kan, C.A.; van Gend, H.W. An automated HPLC determination of meticlorpindol in
eggs with UV absorbance detection, using on-line dialysis and pre-concentration as sample clean-up;
occurrence in and carry over to eggs, Z.Lebensm.-Unters.-Forsch., 1990, 190, 25–30.
Pang, G.-F.; Cao, Y.-Z.; Fan, C.-L.; Zhang, J.-J.; Li, X.-M.; Jia, X.; Song, W.-B. Determination of clopidol
residues in chicken tissues by liquid chromatography: Part I. Optimization of analytical conditions and
comparison with AOAC gas chromatography method, J.AOAC Int., 2001, 84, 1337–1342. [SPE]
Pang, G.-F.; Cao, Y.-Z.; Fan, C.-L.; Zhang, J.-J.; Li, X.-M.; Zhang, Z.-Y. Determination of clopidol residues
in chicken tissues by liquid chromatography: Part II. Distribution and depletion of clopidol in chicken
tissues, J.AOAC Int., 2001, 84, 1343–1346. [SPE]
Pang, G.-F.; Cao, Y.-Z.; Fan, C.-L.; Zhang, J.-J.; Li, X.-M.; MacNeil, J.D. Determination of clopidol
residues in chicken tissues by liquid chromatography : collaborative study, J.AOAC Int., 2003,
86, 685–693.
151
Cloricromen
Cloricromen
Molecular formula: C20 H26 ClNO5
Molecular weight: 395.88
CAS Registry No: 68206-94-0
Merck Index: 13, 2431
O
H3C
O
Cl
O
O
O
N
CH3
CH3
CH3
SAMPLE
Matrix: aqueous humor
Sample preparation: Vortex 100 µL aqueous humor with 100 µL 4.8 µg/mL IS in
MeCN containing 0.6% perchloric acid for 1 min, centrifuge at 10000 g for 5 min, filter
(0.2 µm) the supernatant, inject a 20 µL aliquot of the supernatant.
HPLC VARIABLES
Guard column: 7.5 × 4.6 5 µm Hypersil ODS C18
Column: 150 × 4.6 5 µm Hypersil ODS C18
Column temperature: 25
Mobile phase: Gradient. MeCN:buffer from 10:90 to 53:47 over 13 min, return to initial
conditions over 5 min, re-equilibrate for 3 min. (The buffer was 1% triethylamine
adjusted to pH 3.5 with phosphoric acid.)
Flow rate: 1
Injection volume: 20
Detector: UV 318
CHROMATOGRAM
Retention time: 11.25
Internal standard: timolol maleate (6.48)
Limit of quantitation: 10 ng/mL
OTHER SUBSTANCES
Extracted: metabolite
KEY WORDS
pharmacokinetics; rabbit
REFERENCE
Maltese, A.; Bucolo, C. Simultaneous determination of cloricromene and its active metabolite in rabbit
aqueous humor by high-performance liquid chromatography, J.Chromatogr.B, 2002, 767, 153–158.
152
Clorsulon
Clorsulon
Molecular formula: C8 H8 Cl3 N3 O4 S2
Molecular weight: 380.66
O
Cl
O
S
NH2
Cl
Cl
CAS Registry No: 60200-06-8
Merck Index: 13, 2435
S
NH2 O
NH2
O
SAMPLE
Matrix: milk
Sample preparation: Matrix Solid-Phase Dispersion (MSPD) – Blend 500 µL milk
with 2 g 40 µm Bondesil octadecylsilyl silica (Analytichem) in a mortar and pestle until
a homogeneous mixture is obtained, place the mixture in the barrel of an 8 mL syringe
fitted with a frit, compress the matrix using the syringe plunger, wash with 3 mL hexane;
when all the hexane has eluted, place under vacuum for 5 s. Place this column on top of
a 6 mL 1 g Supelclean LC-Florisil SPE cartridge (prewashed with diethyl ether), elute
with three 3 mL portions of diethyl ether. Remove the C18 column and elute the Florisil
with 2 mL ether. Combine all the ether eluates, evaporate to dryness under a stream of
nitrogen at 40◦ , reconstitute the residue with 1 mL mobile phase, vortex, filter, inject a
200 µL aliquot. Solid-Phase Extraction (SPE) – Condition a 6 mL 1 g Mega Bond Elut
cyclohexyl SPE cartridge with two 3 mL portions of MeOH and two 3 mL portions of
water. Vortex 6 g milk with 6 mL 200 mg/mL hydroxylamine hydrochloride in water, add
4 mL MeOH, shake on a rotating shaker at 180 rpm for 30 min, centrifuge at 8000 rpm
for 30 min. Add 10 mL of the supernatant to the SPE cartridge, elute at not more than
2 drops/s, discard the effluent, wash with two 4 mL portions of water, elute with two
4 mL portions of MeCN. Evaporate the eluate to dryness under a stream of nitrogen
at 60◦ , reconstitute the residue with 1 mL mobile phase, vortex for 5 s, filter (0.2 µm
PTFE), inject a 50 µL aliquot. (SPE method found in Schenck,F.J.; Wagner,R.; Bargo,W.
Determination of clorsulon residues in milk using a solid-phase extraction cleanup and
liquid chromatographic determination. J.Liq.Chromatogr. 1993, 16, 513–520, other
details are identical.)
HPLC VARIABLES
Column: 150 × 4.6 3 µm Econosphere ODS
Mobile phase: MeCN:10 mM pH 7.0 potassium phosphate buffer 25:75
Flow rate: 1
Injection volume: 50–200
Detector: UV 265
CHROMATOGRAM
Retention time: 11.5
Limit of detection: 5 ng/g (SPE, S/N = 4)
Limit of quantitation: 50 ng/g (MSPD), 15 ng/g (SPE)
KEY WORDS
matrix solid-phase dispersion; SPE
REFERENCE
Schenck, F.J.; Barker, S.A.; Long, A.R. Matrix solid phase dispersion (MSPD) isolation and liquid chromatographic determination of clorsulon in milk, J.Liq.Chromatogr., 1991, 14, 2827–2834.
Colistin
153
Colistin
Molecular formula: C58 H105 N16 Na5 O28 S5 (colistin A sodium
methanesulfonate)
Molecular weight: 1749.82
CAS Registry No: 1066-17-7, 8068-28-8 (sodium
methanesulfonate), 1264-72-8 (sulfate)
Merck Index: 13, 2503
SAMPLE
Matrix: blood
Sample preparation: Condition a 100 mg C18 SPE cartridge (Baker) with 1 mL MeOH
and 1 mL carbonate buffer. Mix 20 µL 5 µg/mL IS in water with 250 µL plasma, add
50 µL MeOH:10% trichloroacetic acid 50:50, vortex for 1 min, centrifuge at 1000 g at 4◦
for 10 min. Remove the supernatant, add 10 µL 1 M NaOH, vortex for 1 min, add 250 µL
MeOH:10 mM HCl 50:50, vortex for 1 min, add to the SPE cartridge, wash with 1 mL
carbonate buffer, add 30 µL 100 mM 9-fluorenylmethyl chloroformate in MeCN, let
stand for 10 min, dry by drawing air through the cartridge, elute with 900 µL acetone.
Add the eluate to 600 µL 200 mM boric acid solution, vortex for 2 min, inject a 50 µL
aliquot. (Prepare the carbonate buffer by adjusting the pH of 1% sodium bicarbonate
solution to 10 with 10% NaOH.)
HPLC VARIABLES
Column: 250 × 4.6 5 µm Ultrasphere C18
Mobile phase: MeCN:THF:water 87:4:13
Flow rate: 1
Injection volume: 50
Detector: F ex 260 em 315
CHROMATOGRAM
Retention time: 21.8 (colistin B), 26.1 (colistin A)
Internal standard: netilmicin sulfate (17.1)
Limit of quantitation: 100 ng/mL
OTHER SUBSTANCES
Noninterfering: aztreonam, ceftazidime, ciprofloxacin, meropenem, piperacillin, ticarcillin, tobramycin
KEY WORDS
derivatization; plasma; SPE
REFERENCE
Li, J.; Milne, R.W.; Nation, R.L.; Turnidge, J.D.; Coulthard, K.; Johnson, D.W. A simple method for the
assay of colistin in human plasma, using pre-column derivatization with 9-fluorenylmethyl chloroformate in solid-phase extraction cartridges and reversed-phase high-performance liquid chromatography,
J.Chromatogr.B, 2001, 761, 167–175.
SAMPLE
Matrix: blood, sputum, urine
Sample preparation: Plasma, sputum. Add 1 mL plasma or sonicated sputum to
1.1 mL water and 200 µL 3 M perchloric acid, vortex for 10 s, centrifuge at 3000 g at 4◦
for 25 min. Remove 1.4 mL of the supernatant and add it to 200 µL 1.5 M KOH, vortex,
add 200 µL 100 mM HCl, vortex, add 100 µL 9% sodium carbonate, vortex, add 400 µL
20 mg/mL dansyl chloride, vortex, heat at 54◦ ; after 5 min, add 100 µL 150 mg/mL
154
Colistin
proline, heat at 54◦ for another 55 min, vortex, heat at 54◦ for 1 h, cool. Remove a 2 mL
aliquot and add it to 1 mL ethyl acetate, rotate for 10 min (Roto-torque Model 7637
setting #5), centrifuge at 3000 rpm for 10 min, inject an aliquot of the top layer. Urine.
Dilute urine 0–20 fold with 100 mM pH 6.8 sodium phosphate buffer. Add 1 mL diluted
urine to 1 mL water and 200 µL 3 M perchloric acid, vortex for 10 s. Remove 1.4 mL and
add it to 200 µL 1.5 M KOH, vortex, add 200 µL 100 mM HCl, vortex, add 100 µL 9%
sodium carbonate, vortex, add 400 µL 20 mg/mL dansyl chloride, vortex, heat at 57◦ ;
after 5 min, add 100 µL 300 mg/mL proline, heat at 57◦ for another 55 min, vortex, heat
at 57◦ for 1 h, cool. Remove a 2 mL aliquot and add it to 1 mL ethyl acetate, rotate for
10 min (Roto-torque Model 7637 setting #5), centrifuge at 3000 rpm for 2 min, inject an
aliquot of the top layer.
HPLC VARIABLES
Guard column: 40 µm Vydac pellicular resin
Column: 150 × 4.6 Eclipse XDB-C8
Mobile phase: Gradient. A:B from 40:60 to 80:20 over 15 min, maintain at 80:20 for
3 min. A was 0.1% trifluoroacetic acid in MeCN. B was 0.1% trifluoroacetic acid in water.
Flow rate: 1
Injection volume: 50
Detector: F ex 350 em 500
CHROMATOGRAM
Limit of quantitation: 5 µg/mL
KEY WORDS
derivatization; pharmacokinetics; plasma
REFERENCE
Reed, M.D.; Stern, R.C.; O’Riordan, M.A.; Blumer, J.L. The pharmacokinetics of colistin in patients with
cystic fibrosis, J.Clin.Pharmacol., 2001, 41, 645–654.
ANNOTATED BIBLIOGRAPHY
Cabanes, A.; Cajal, Y.; Haro, I.; Garcia Anton, J.M.; Arboix, M.; Reig, F. Gentamicin determination in
biological fluids by HPLC, using tobramycin as internal standard, J.Liq.Chromatogr., 1991, 14,
1989–2010. [gentamicin; tobramycin is internal standard; plasma; urine; rabbit; derivatization with
o-phthalaldehyde; fluorescence detection; LOD 300 ng/mL; kanamycin; colistin]
Gmur, D.J.; Bredl, C.R.; Steele, S.J.; Cai, S.; VanDevanter, D.R.; Nardella, P.A. Determination of
polymyxin E1 in rat plasma by high-performance liquid chromatography, J.Chromatogr.B, 2003,
789, 365–372. [SPE; derivatization; fluorescence detection; LOQ 50 ng/mL; rat; dog]
Le Brun, P.P.H.; de Graaf, A.I.; Vinks, A.A.T.M.M. High-performance liquid chromatographic method for
the determination of colistin in serum, Ther.Drug Monit., 2000, 22, 589–593. [colistin; derivatization;
polymyxin]
155
Cypermethrin
Cypermethrin
Molecular formula: C22 H19 Cl2 NO3
Molecular weight: 416.30
CH3
CH3
Cl
Cl
O
O
O
CN
CAS Registry No: 52315-07-8
Merck Index: 13, 2796
SAMPLE
Matrix: blood, milk
Sample preparation: Activate silica gel 60 in air at 130◦ for 5 h, hydrate by adding
water equivalent to 10% of its weight, keep in a sealed container for 30 min before
use. Prepare a chromatographic column with 4 g of this material and wash with 1 mL
n-hexane:diethyl ether 90:10. Acidify 10 mL or 10 g whole blood or whole milk with 1.0 M
HCl to approximately pH 4.0, add 50 mL MeCN, shake mechanically for 30 min, filter
(Whatman paper No. 42 or 44). Add 25 mL MeCN to the residue, shake for 15 min, filter
(Whatman paper No. 42 or 44). Combine the filtrates and add them to 15 mL n-hexane,
shake for 1 min, repeat twice. Retain the acetonitrile phases. Add 45 mL MeCN to
the hexane phases, shake for 1 min. Combine all the acetonitrile layers, evaporate to
dryness under a stream of nitrogen at 50◦ . Dissolve the residue in 10 mL n-hexane.
Add to the silica column, elute with 7 mL n-hexane:diethyl ether 90:10, evaporate the
eluate to dryness at room temperature, dissolve the residue in 1.0 mL MeCN, filter
(0.45 µm cellulose membrane), inject an aliquot of the filtrate. (All diethyl ether should
be peroxide free.)
HPLC VARIABLES
Column: 250 × 4.6 Nucleosil 120-5 C18
Mobile phase: MeCN:water 80:20
Flow rate: 1
Injection volume: 20
Detector: UV 266
CHROMATOGRAM
Retention time: 8.6, 8.9
Limit of detection: 1 ng/g
OTHER SUBSTANCES
Extracted: cyhalothrin (7.7), deltamethrin (9.8), flumethrin (3.8, 4.1)
KEY WORDS
cow; SPE; whole blood
REFERENCE
Bissacot, D.Z.; Vassilieff, I. HPLC determination of flumethrin, deltamethrin, cypermethrin, and
cyhalothrin residues in the milk and blood of lactating dairy cows, J.Anal.Toxicol., 1997, 21, 397–402.
Dalfopristin
H
N
Molecular formula: C34 H50 N4 O9 S
Molecular weight: 690.86
CAS Registry No: 112362-50-2
Merck Index: 13, 2831
CH3
O
CH3
OH
CH3
O
CH3
CH3
CH3
N
O
H
S
O
O
N
O
O
N
O
SAMPLE
Matrix: blood
Sample preparation: Condition a CN SPE cartridge (Lida-Interchim) with 1 mL
MeOH, 1 mL water, and 1 mL buffer. Add 1 mL 3.8% sodium citrate and 2.5 mL
250 mM HCl to 10 mL whole blood. Shake gently by hand and centrifuge at 2000 g at
4◦ for 15 min. Add 1 mL buffer and 50 µL 100 µg/mL IS in MeOH to 1.35 mL acidified
plasma. Vortex for a few seconds, centrifuge at 4000 g at 4◦ for 5 min. Add either
supernatant to the SPE cartridge and dry the SPE cartridge with 3 mL air. Elute with
500 µL MeOH:water 70:30 containing 3.5 mM pentane sulfonic acid, inject an aliquot.
(The buffer was a mixture of 85 mM pH 3.0 citric acid monohydrate containing 81 mM
NaOH and 60 mM HCl.)
HPLC VARIABLES
Guard column: 10 µm µBondapak C18
Column: 125 × 4.6 5 µm Kromasil C18 (Higgins Analytical)
Mobile phase: Gradient. MeCN:buffer 30:70 for 11 min, then 32:68 for 4 min (step
gradient), to 40:60 over 0.6 min, maintain at 40:60 for 0.4 min, at 38:62 for 18 min (step
gradient), at 80:20 for 2 min (step gradient), re-equilibrate at 30:70 for 9 min. (Prepare
buffer by adding 800 µL 70% perchloric acid to 1 L water.)
Flow rate: 0.5 for 11 min, 1 for 25 min, 0.5 for 9 min
Injection volume: 500
Detector: UV 235
CHROMATOGRAM
Retention time: 12.7
Internal standard: dimethylamino-3-propyl thiomethylene-5 virginiamycin S (31.0)
Limit of quantitation: 25 pg/mL
OTHER SUBSTANCES
Extracted: metabolites, pristinamycin II A (24.1), quinupristin (22.1)
KEY WORDS
plasma; SPE; whole blood
REFERENCE
Le Liboux, A.; Pasquier, O.; Montay, G. Simultaneous high-performance liquid chromatographic determination of quinupristin, dalfopristin and their main metabolites in human plasma, J.Chromatogr.B,
1998, 708, 161–168.
SAMPLE
Matrix: formulations
Sample preparation: Inject a 5–10 µL aliquot of the infusion solution.
156
HPLC Methods for Recently Approved Pharmaceuticals. by George Lunn
Copyright 2005 John Wiley & Sons, Ltd. ISBN: 0-471-66941-5
Dalfopristin
157
HPLC VARIABLES
Column: 125 × 4 5 µm LiChrospher-100 RP18
Column Temperature: 40 ± 1
Mobile phase: Gradient. A:B from 0:100 to 66:34 over 42.5 min, return to initial
conditions over 1.5 min, re-equilibrate for 5 min. A was MeCN:buffer 65:35. B was
MeCN:buffer 20:80. The buffer was 30 mM potassium dihydrogen phosphate adjusted
to pH 2.9 with phosphoric acid.
Flow rate: 1.1
Injection volume: 5–10
Detector: UV 254
CHROMATOGRAM
Retention time: 8.5
Limit of quantitation: 0.05%
OTHER SUBSTANCES
Simultaneous: impurities, quinupristin (LOQ 0.12%) (23.9, 23.1, 27.0)
KEY WORDS
infusion; injection; stability-indicating
REFERENCE
Vasselle, B.; Gousset, G.; Bounine, J.-P. Development and validation of a high-performance liquid chromatographic stability-indicating method for the analysis of Synercid in quality control, stability and
compatibility studies, J.Pharm.Biomed.Anal., 1999, 19, 641–657.
ANNOTATED BIBLIOGRAPHY
Abdel-Hamid, M.E.; Phillips, O.A. LC-MS/MS determination of Synercid injections, J.Pharm.Biomed.
Anal., 2003, 32, 1167–1174. [pristinamycin; quinupristin; dalfopristin]
158
Dalteparin
Dalteparin
Molecular weight: 4000–6000
CAS Registry No: 9041-08-1 (Na salt)
Merck Index: 13, 2832
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 10 mg/mL solution in mobile phase, stir for 4 h, filter
(0.2 µm) while centrifuging, inject a 200 µL aliquot.
HPLC VARIABLES
Guard column: Ohpak SB-G
Column: 300 × 8 Shodex Ohpak SB-803 HQ
Mobile phase: 100 mM pH 7 ammonium acetate buffer containing 0.05% sodium azide
(Caution! Sodium azide is highly toxic and can form shock-sensitive and highly explosive
azides when it comes in contact with heavy metals! Sodium azide should not be
discharged to the plumbing system!)
Flow rate: 0.8
Injection volume: 200
Detector: Refractive Index; Light Scattering Detector (miniDAWN, Wyatt Technology,
Santa Barbara CA) (detector measures scattered light intensity (690 nm) at three angles
(45◦ , 90◦ , 135◦ )
CHROMATOGRAM
Retention time: 10
REFERENCE
Knobloch, J.E.; Shaklee, P.N. Absolute molecular weight distribution of low-molecular-weight heparins
by size-exclusion chromatography with multiangle laser light scattering detection, Anal.Biochem.,
1997, 245, 231–241.
Daptomycin
159
Daptomycin
Molecular formula: C72 H101 N17 O26
Molecular weight: 1620.67
CAS Registry No: 103060-53-3
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 mm long DuPont C8
Mobile phase: MeCN:0.5% pH 3.5 ammonium dihydrogen phosphate 38:62
Flow rate: 1.5
Detector: UV 214
CHROMATOGRAM
Retention time: 25
OTHER SUBSTANCES
Simultaneous: anhydrodaptomycin (35), β-asp daptomycin (23)
REFERENCE
Kirsch, L.E.; Molloy, R.M.; Debono, M.; Baker, P.; Farid, K.Z. Kinetics of the aspartyl transpeptidation
of daptomycin, a novel lipopeptide antibiotic, Pharm.Res., 1989, 6, 387–393.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 × 4.6 5 µm Zorbax 300SB-C8
Mobile phase: MeCN:50 mM pH 5 phosphate buffer 29:71
Flow rate: 1
Injection volume: 10
Detector: UV 214
CHROMATOGRAM
Retention time: <30
OTHER SUBSTANCES
Simultaneous: degradation products
REFERENCE
Muangsiri, W.; Kirsch, L.E. The kinetics of the alkaline degradation of daptomycin, J.Pharm.Sci., 2001,
90, 1066–1075.
SAMPLE
Matrix: blood
Sample preparation: Precipitate proteins before analysis (no other details)
HPLC VARIABLES
Guard column: Xterra RP18 (Waters)
Column: Hypersil C8
Mobile phase: MeCN:0.5% ammonium phosphate 32.6:67.4
160
Daptomycin
Flow rate: 1.5
Detector: UV 214
CHROMATOGRAM
Retention time: 14–16
Internal standard: ethyl paraben
Limit of quantitation: 7.5 µg/mL
KEY WORDS
plasma
REFERENCE
Sakoulas, G.; Eliopoulos, G.M.; Alder, J. Thauvin-Eliopoulos,C. Efficacy of daptomycin in experimental
endocarditis due to methicillin-resistant staphylococcus aureus, Antimicrob.Agents Chemother., 2003,
47, 1714–1718.
161
Deferiprone
Deferiprone
CH3
N
CH3
Molecular formula: C7 H9 NO2
Molecular weight: 139.15
CAS Registry No: 30652-11-0
Merck Index: 13, 2878
OH
O
SAMPLE
Matrix: blood, urine
Sample preparation: Add IS to a final concentration of 100 µM and perchloric acid to
a final concentration of 500 mM to plasma or serum, centrifuge at 11 600 g for 10 min,
inject a 50 µL aliquot. Centrifuge urine at 960 g for 20 min, filter (0.45 µm), dilute
10-fold with 50 mM pH 2.0 potassium phosphate buffer, inject a 50 µL aliquot.
HPLC VARIABLES
Guard column: LiChrosorb RP Select B
Column: 5 µm LiChrosorb RP Select B
Mobile phase: Gradient MeOH:50 mM pH 2.0 potassium phosphate buffer 27:73 containing 10 mM octanesulfonic acid for 16 min, to 70:30 over 2 min, maintain at 70:30 for
2 min, return to initial conditions over 2 min, re-equilibrate for 8 min. (Methanol wash
not necessary for urine samples.)
Flow rate: 1.6
Injection volume: 50
Detector: UV 280
CHROMATOGRAM
Retention time: 8.2
Internal standard: 1-ethyl-2-methyl-3-hydroxypyrid-4-one (13.2)
Limit of quantitation: 25 µM
KEY WORDS
plasma; serum
REFERENCE
Goddard, J.G.; Kontoghlorghes, G.J. Development of an HPLC method for measuring orally administered
1-substituted 2-alkyl-3-hydroxypyrid-4-one iron chelators in biological fluids, Clin.Chem., 1990, 36,
5–8.
162
Deflazacort
Deflazacort
O
CH3
O
Molecular formula: C25 H31 NO6
Molecular weight: 441.52
CAS Registry No: 14484-47-0
Merck Index: 13, 2881
O
CH3 N
HO
CH3
H
CH3
O
H
H
O
SAMPLE
Matrix: blood, formulations
Sample preparation: Mix 1 mL serum with 1 µg IS and 1 mL MeOH, vortex for
5 min, centrifuge at 5000 g for 5 min, inject a 20 µL aliquot of the supernatant. Weigh
out amount of crushed tablets containing 10 mg deflazacort, add 7 mL mobile phase,
sonicate for 10 min, make up to 10 mL with mobile phase, centrifuge. Mix an aliquot
of the supernatant with an aliquot of 2 µg/mL IS in mobile phase, dilute with mobile
phase, inject a 20 µL aliquot.
HPLC VARIABLES
Column: 250 × 4.6 5 µm Spherisorb C18
Mobile phase: MeCN:MeOH:67 mM potassium dihydrogen phosphate 27:20:53, adjusted
to pH 6.5 with 3 M NaOH
Flow rate: 0.75
Injection volume: 20
Detector: UV 244
CHROMATOGRAM
Retention time: 12.3
Internal standard: etodolac (5.5)
Limit of detection: 4 ng/mL
Limit of quantitation: 13.6 ng/mL
KEY WORDS
plasma; tablets
REFERENCE
Ozkan, Y.; Savaser, A.; Tas, C.; Uslu, B.; Ozkan, S.A. Drug dissolution studies and determination of
deflazacort in pharmaceutical formulations and human serum samples by RP-HPLC, J.Liq.Chromatogr.
Rel.Technol., 2003, 26, 2141–2156.
SAMPLE
Matrix: blood
Sample preparation: Condition a 3 mL 200 mg Clean-Up C18 SPE cartridge (Worldwide Monitoring) with two 2 mL portions of MeOH and two 2 mL portions of water.
Centrifuge plasma at 2000 rpm for 5 min prior to analysis. Mix 2 mL plasma with 20 µL
5 µg/mL IS in MeCN and 20 µL MeCN, add 1 mL water, vortex until homogeneous,
add to the SPE cartridge, wash with two 2 mL portions of acetone:water 20:80, wash
with 1 mL 50 mM pH 2.7 phosphate buffer, place under vacuum for 3 min, elute with
two 2.5 mL portions of ethyl acetate. Evaporate the eluate to dryness under reduced
pressure at 40◦ , reconstitute the residue with 75 µL mobile phase B, vortex, add 100 µL
50 mM pH 3 phosphate buffer, centrifuge at 5000 rpm for 5 min, inject a 145 µL aliquot.
HPLC VARIABLES
Column: 100 × 2 5 µm YMC Basic
Deflazacort
163
Column temperature: 50
Mobile phase: Gradient. A:B 20:80 for 1 min, to 50:50 over 7 min, to 65:35 over 3 min,
to 100:0 (step gradient), maintain at 100:0 for 1 min, return to initial conditions, reequilibrate for 4 min. A was MeCN:50 mM pH 3.0 potassium dihydrogen phosphate
buffer 50:50. B was MeOH:50 mM pH 3.0 potassium dihydrogen phosphate buffer 20:80.
Flow rate: 0.3
Injection volume: 145
Detector: UV 246
CHROMATOGRAM
Retention time: 13.8
Internal standard: fludrocortisone acetate (12.4)
OTHER SUBSTANCES
Extracted: 21-hydroxydeflazacort (10)
Simultaneous: cortisone, hydrocortisone
KEY WORDS
plasma; SPE; method validated for 21-hydroxydeflazacort rather than deflazacort
REFERENCE
Reynolds, D.L.; Burmaster, S.D.; Eichmeier, L.S. Quantitative determination of 21-hydroxy-deflazacort
in human plasma using gradient semi-microbore liquid chromatography, Biomed.Chromatogr., 1994,
8, 230–235.
ANNOTATED BIBLIOGRAPHY
Ozkan, S.A.; Uslu, B. Rapid HPLC assay for lamivudine in pharmaceuticals and human serum,
J.Liq.Chromatogr.Rel.Technol., 2002, 25, 1447–1456. [deflazacort is internal standard]
164
Desloratadine
Desloratadine
H
N
Molecular formula: C19 H19 ClN2
Molecular weight: 310.83
CAS Registry No: 100643-71-8
Merck Index: 13, 2939
N
Cl
SAMPLE
Matrix: blood
Sample preparation: Mix 1 mL plasma, 100 µL MeOH, 50 µL MeOH containing
20 ng/mL d3 -loratadine and 100 ng/mL d3 -desloratadine, and 50 µL 0.1% ammonium
hydroxide, vortex for 30 s, add 5 mL hexane, vortex for 2 min, centrifuge at 1200 g for
5 min, freeze in dry ice acetone. Evaporate the organic layer to dryness under a stream
of nitrogen at 40◦ , reconstitute the residue with 200 µL 0.1% trifluoroacetic acid in
MeCN, vortex for 2 min, inject a 35 µL aliquot.
HPLC VARIABLES
Column: 50 × 3 5 µm Betasil silica (Keystone)
Mobile phase: MeCN:water:trifluoroacetic acid 90:10:0.1
Flow rate: 0.5
Injection volume: 35
Detector: MS, PE Sciex API 3000 turbo ionspray, positive ion mode, ionspray needle
5 kV, turbo gas temperature 300◦ , auxiliary gas flow 8 L/min, m/z 311–259
CHROMATOGRAM
Retention time: 2
Internal standard: d3 -loratadine (m/z 388–342), d3 -desloratadine (m/z 316–262)
Limit of quantitation: 25 pg/mL
OTHER SUBSTANCES
Extracted: loratadine (LOQ 10 pg/mL, m/z 383–337) (1.2)
Noninterfering: acetaminophen, albuterol, aspirin, caffeine, clonidine, fentanyl, ibuprofen, naltrexone, ritonavir
KEY WORDS
plasma
REFERENCE
Naidong, W.; Addison, T.; Schneider, T.; Jiang, X.; Halls, T.D.J. A sensitive LC/MS/MS method using
silica column and aqueous-organic mobile phase for the analysis of loratadine and descarboethoxyloratadine in human plasma, J.Pharm.Biomed.Anal., 2003, 32, 609–617.
SAMPLE
Matrix: blood
Sample preparation: Mix 1 mL plasma with 20 µL 7.5 µg/mL IS in MeOH:50 mM HCl
20:80 and 200 µL 1 M NaOH, vortex, add 3 mL toluene, shake for 20 min, centrifuge at
2000 g for 10 min, freeze, remove organic layer, repeat extraction. Combine the organic
layers and evaporate to dryness under a stream of nitrogen at 50◦ , reconstitute the
residue with 200 µL MeOH:50 mM HCl 20:80, vortex, mobile phase, inject a 100 µL
aliquot.
HPLC VARIABLES
Column: 150 × 3.9 5 µm Symmetry C18 (Waters)
Desloratadine
165
Column temperature: 35
Mobile phase: Gradient. MeCN:MeOH:50 mM pH 2.0 potassium phosphate buffer from
14:5:81 to 24:3:7.3 over 5.5 min, to 40:3:57 over 4.5 min, return to initial conditions over
4 min, re-equilibrate for 6 min.
Flow rate: 1.2
Injection volume: 100
Detector: F ex 290 em 480
CHROMATOGRAM
Retention time: 3.4
Internal standard: propranolol hydrochloride (8.6)
Limit of detection: 0.25 ng/mL
Limit of quantitation: 0.5 ng/mL
OTHER SUBSTANCES
Extracted: loratadine (11.2)
KEY WORDS
pharmacokinetics; plasma
REFERENCE
Yin, O.Q.P.; Shi, X.; Chow, M.S.S. Reliable and specific high-performance liquid chromatographic method
for simultaneous determination of loratadine and its metabolite in human plasma, J.Chromatogr.B,
2003, 796, 165–172.
ANNOTATED BIBLIOGRAPHY
Rupérez, F.J.; Fernández, H.; Barbas, C. LC determination of loratadine and related impurities, J.Pharm.
Biomed.Anal., 2002, 29, 35–41.
Sutherland, F.C.W.; de Jager, A.D.; Badenhorst, D.; Scanes, T.; Hundt, H.K.L.; Swart, K.J.; Hundt, A.F..
Sensitive liquid chromatography-tandem mass spectrometry method for the determination of loratadine
and its major active metabolite descarboethoxyloratadine in human plasma. J.Chromatogr.A 2001, 914,
37–43.
166
Desogestrel
Desogestrel
Molecular formula: C22 H30 O
Molecular weight: 310.47
CAS Registry No: 54024-22-5
Merck Index: 13, 2943
H3C
OH
H2C
H
H
H
H
H
SAMPLE
Matrix: blood
Sample preparation: Add plasma to a Bakerbond C18 SPE cartridge, wash with
100 mM pH 4.2 ammonium acetate buffer, elute with MeOH. Evaporate the eluate to
dryness under reduced pressure, reconstitute the residue with MeOH, inject an aliquot.
HPLC VARIABLES
Column: 300 × 3.9 µBondapak C18
Column temperature: 50
Mobile phase: Gradient. MeOH:100 mM pH 4.2 ammonium acetate from 10:90 to 90:10
over 30 min
Flow rate: 1.7
Detector: Radioactivity (3 H); UV 254
CHROMATOGRAM
Retention time: 32
OTHER SUBSTANCES
Extracted: metabolites
KEY WORDS
dog; plasma; SPE
REFERENCE
Verhoeven, C.H.J.; Krebbers, S.F.M.; Wagenaars, G.N.; Vos, R.M.E. In vitro and in vivo metabolism of
desogestrel in several species, Drug Metab.Dispos., 1998, 26, 927–936.
Desoximetasone
Desoximetasone
O
CH3
HO
CH3
Molecular formula: C22 H29 FO4
Molecular weight: 376.46
CAS Registry No: 382-67-2
Merck Index: 13, 2947
167
F
H
OH
CH3
H
O
SAMPLE
Matrix: formulations
Sample preparation: Mix 1 g of a topical product, 5 mL 100 mM pH 4 citrate buffer
saturated with NaCl, and 10 mL ethyl acetate, shake vigorously by hand to ensure
that no large clumps stick to the tube, mix with the tube on its side on an oscillating
shaker for 15 min. Remove the upper ethyl acetate layer and wash with citrate buffer as
before. Dry the ethyl acetate over anhydrous sodium sulfate and evaporate to dryness.
Dissolve the residue in a mixture of 10 mL heptane and 5 mL MeCN:water 90:10.
Remove the upper heptane layer and extract it with 2 mL MeCN:water 90:10. Combine
the MeCN/water layers and evaporate them to dryness, dissolve the residue in 300 µL
MeOH, filter (0.45 µm nylon), inject a 5 µL aliquot.
HPLC VARIABLES
Guard column: 15 × 3.2 7 µm Brownlee NewGuard C18
Column: 75 × 4.6 3.5 µm Symmetry C18 (Waters)
Mobile phase: Gradient. MeCN:water 18:82 for 2 min, to 82:18 over 12 min, maintain
at 82:18 for 3 min, re-equilibrate at initial conditions for 12 min.
Flow rate: 1
Injection volume: 5
Detector: UV 240
CHROMATOGRAM
Retention time: 7.60
Limit of detection: 0.001%
OTHER SUBSTANCES
Simultaneous: alclometasone 17,21-dipropionate (10.93), amcinonide (10.90), beclomethasone 17,21-dipropionate (11.90), betamethasone (6.52), betamethasone 21-acetate
(8.47), betamethasone 17-benzoate (10.28), betamethasone 17,21-dipropionate (11.42),
betamethasone 17-valerate (10.25), budesonide (8.55, 8.69 (epimers)), clobetasol 17propionate (11.06), cortisone (5.62), cortisone 21-acetate (8.07), dexamethasone (6.57),
dexamethasone 21-acetate (8.68), desonide (6.99), desoximetasone (7.60), desoxycorticosterone acetate (10.90), desoxycorticosterone pivalate (14.45), diflorasone 17,21-diacetate
(9.81), fluocinolone acetonide (7.38), fluocinonide (9.79), flurandrenolide (7.36), fludrocortisone 21-acetate (7.77), fluorometholone (7.67), flumethasone 21-pivalate (11.20),
flunisolide (7.14), fluprednisolone (5.46), fluticasone 17-propionate (11.19), halcinonide
(10.72), halobetasol propionate (10.98), hydrocortisone (5.50), hydrocortisone 21-acetate
(7.65), hydrocortisone 17-butyrate (8.66), hydrocortisone 21-cypionate (12.54), hydrocortisone 17-valerate (9.53), mometasone 17-furoate (11.24), methylprednisolone (6.31),
methylprednisolone 21-acetate (8.34), meprednisone (6.55), prednisolone (5.37), prednisolone 21-acetate (7.47), prednisolone 21-tebuate (11.01), paramethasone 21-acetate
(8.64), prednicarbate (10.72), prednisone (5.46), triamcinolone (4.15), triamcinolone acetonide (7.04), triamcinolone 16,21-diacetate (7.49), triamcinolone hexacetonide (13.12)
KEY WORDS
body wash, cream, gel, lotion, shampoo, spray
168
Desoximetasone
REFERENCE
Reepmeyer, J.C. Screening for corticosteroids in topical pharmaceuticals by HPLC with a scanning
ultraviolet detector, J.Liq.Chromatogr.Rel.Technol., 2001, 24, 693–709.
SAMPLE
Matrix: urine
Sample preparation: Mix 5 mL urine with 100 ng IS and 400 µL 1 M pH 4.1 sodium
acetate buffer, adjust pH to 5.0, add 600 µL β-glucuronidase solution (10800 U), heat
at 65◦ for 3.5 h or at 37◦ overnight, cool, add 6 mL ethyl acetate, rotate for 10 min.
Remove the organic layer and wash it with 3 mL 1 M NaOH containing 150 mM NaCl
by rotating for 5 min, centrifuge at 1900 g for 30 s. Remove the organic layer and pass it
through an anhydrous sodium sulfate drying column. Evaporate the eluate to dryness
under a stream of nitrogen at 60◦ , reconstitute the residue with 30 µL MeOH, vortex,
inject a 10 µL aliquot.
HPLC VARIABLES
Column: 75 × 4.6 3 µm DB-8 (Supelco)
Column temperature: 25
Mobile phase: Gradient. MeOH:1% acetic acid from 0:100 to 100:0 over 15 min, maintain
at 100:0 for 3 min.
Flow rate: 1
Injection volume: 10
Detector: MS, Finnigan MAT LCQ Classic, APCI, source 450◦ , capillary 150◦ , source
+5 kV for positive ions and – 5 kV for negative ions, collision gas helium, m/z 377
CHROMATOGRAM
Retention time: 27.6
Internal standard: d4 -hydrocortisone (24.4)
Limit of detection: 5 ng/mL
OTHER SUBSTANCES
Extracted: beclomethasone (m/z 409) (26.4), betamethasone (m/z 393) (25.9), deoxycortone (m/z 331) (28.1), 21-deoxydexamethasone (m/z 377) (26.8), dexamethasone (m/z
393) (25.9), dichlorisone (m/z 413) (26.4), fluclorolone acetonide (m/z 487) (27.8), fludrocortisone (m/z 381) (24.2), flumethasone (m/z 411) (25.4), fluocinolone acetonide (m/z
453) (26.4), fluocinonide (m/z 495) (28.5), fluocortolone (m/z 377) (26.8), fluorometholone
(m/z 3.77) (26.6), fluprednisolone (m/z 379) (23.9), flurandrenolide (m/z 437) (26.8),
hydrocortisone (m/z 363) (24.4), isoflupredone (m/z 379) (23.9), methylprednisolone (m/z
375) (26.1), prednisolone (m/z 361) (24.4), prednisone (m/z 359) (23.4), triamcinolone
(m/z 395) (19), triamcinolone acetonide (m/z 435) (29.3)
KEY WORDS
horse
REFERENCE
Tang, P.W.; Law, W.C.; Wan, T.S.M. Analysis of corticosteroids in equine urine by liquid chromatographymass spectrometry, J.Chromatogr.B, 2001, 754, 229–244.
Desoxycorticosterone
Desoxycorticosterone
O
CH3
CH3
Molecular formula: C21 H30 O3
Molecular weight: 330.46
CAS Registry No: 64-85-7
Merck Index: 13, 2917
169
H
OH
H
H
O
SAMPLE
Matrix: blood
Sample preparation: Vortex 500 µL serum with 5 mL diethyl ether for 3 min, cen-
trifuge at 3500 rpm for 5 min. Evaporate the organic layer to dryness at 50◦ , reconstitute
the residue with 50 µL MeOH:water 60:40, vortex for 1 min, sonicate for 1 min, centrifuge at 4000 rpm for 2 min, inject a 20 µL aliquot of the supernatant.
HPLC VARIABLES
Guard column: 50 × 2.1 unspecified
Column: 150 × 6 Shimpack CLC-ODS
Column temperature: 48
Mobile phase: MeOH:THF:water 26:18:56
Flow rate: 1
Injection volume: 20
Detector: UV 254
CHROMATOGRAM
Retention time: 10
Limit of detection: 1 pmol
OTHER SUBSTANCES
Extracted: androstenedione (9.5), corticosterone (7), cortisone (5), 11-deoxycortisol (7.5),
dexamethasone acetate (13), estradiol (F ex 285 em 310) (15), estriol (F ex 285 em
310) (5), estrone (F ex 285 em 310) (17), hydrocortisone (6), hydroxyprogesterone (14),
prednisone acetate (8), progesterone (23), testosterone (11)
KEY WORDS
serum; SPE
REFERENCE
Wei, J.-q.; Wei, J.-l.; Zhou, X.-t.; Cheng, J.-p. Isocratic reversed phase high performance liquid chromatography determination of twelve natural corticosteroids in serum with on-line ultraviolet and
fluorescence detection, Biomed.Chromatogr., 1990, 4, 161–164.
SAMPLE
Matrix: formulations
Sample preparation: Mix 1 g of a topical product, 5 mL 100 mM pH 4 citrate buffer
saturated with NaCl, and 10 mL ethyl acetate, shake vigorously by hand to ensure
that no large clumps stick to the tube, mix with the tube on its side on an oscillating
shaker for 15 min. Remove the upper ethyl acetate layer and wash with citrate buffer as
before. Dry the ethyl acetate over anhydrous sodium sulfate and evaporate to dryness.
Dissolve the residue in a mixture of 10 mL heptane and 5 mL MeCN:water 90:10.
Remove the upper heptane layer and extract it with 2 mL MeCN:water 90:10. Combine
the MeCN/water layers and evaporate them to dryness, dissolve the residue in 300 µL
MeOH, filter (0.45 µm nylon), inject a 5 µL aliquot.
170
Desoxycorticosterone
HPLC VARIABLES
Guard column: 15 × 3.2 7 µm Brownlee NewGuard C18
Column: 75 × 4.6 3.5 µm Symmetry C18 (Waters)
Mobile phase: Gradient. MeCN:water 18:82 for 2 min, to 82:18 over 12 min, maintain
at 82:18 for 3 min, re-equilibrate at initial conditions for 12 min.
Flow rate: 1
Injection volume: 5
Detector: UV 240
CHROMATOGRAM
Retention time: 10.90 (acetate), 14.45 (pivalate)
Limit of detection: 0.001%
OTHER SUBSTANCES
Simultaneous: alclometasone 17,21-dipropionate (10.93), amcinonide (10.90), beclomethasone 17,21-dipropionate (11.90), betamethasone (6.52), betamethasone 21-acetate
(8.47), betamethasone 17-benzoate (10.28), betamethasone 17,21-dipropionate (11.42),
betamethasone 17-valerate (10.25), budesonide (8.55, 8.69 (epimers)), clobetasol 17propionate (11.06), cortisone (5.62), cortisone 21-acetate (8.07), dexamethasone (6.57),
dexamethasone 21-acetate (8.68), desonide (6.99), desoximetasone (7.60), diflorasone
17,21-diacetate (9.81), fluocinolone acetonide (7.38), fluocinonide (9.79), flurandrenolide (7.36), fludrocortisone 21-acetate (7.77), fluorometholone (7.67), flumethasone
21-pivalate (11.20), flunisolide (7.14), fluprednisolone (5.46), fluticasone 17-propionate
(11.19), halcinonide (10.72), halobetasol propionate (10.98), hydrocortisone (5.50),
hydrocortisone 21-acetate (7.65), hydrocortisone 17-butyrate (8.66), hydrocortisone 21cypionate (12.54), hydrocortisone 17-valerate (9.53), mometasone 17-furoate (11.24),
methylprednisolone (6.31), methylprednisolone 21-acetate (8.34), meprednisone (6.55),
prednisolone (5.37), prednisolone 21-acetate (7.47), prednisolone 21-tebuate (11.01),
paramethasone 21-acetate (8.64), prednicarbate (10.72), prednisone (5.46), triamcinolone (4.15), triamcinolone acetonide (7.04), triamcinolone 16,21-diacetate (7.49),
triamcinolone hexacetonide (13.12)
KEY WORDS
body wash, cream, gel, lotion, shampoo, spray
REFERENCE
Reepmeyer, J.C. Screening for corticosteroids in topical pharmaceuticals by HPLC with a scanning
ultraviolet detector, J.Liq.Chromatogr.Rel.Technol., 2001, 24, 693–709.
SAMPLE
Matrix: cell cultures
Sample preparation: Extract cell medium twice with two volumes of ethyl acetate
for 5 min. Evaporate the combined extracts to dryness under a stream of nitrogen,
reconstitute the residue with 100 µL MeCN, inject an aliquot.
HPLC VARIABLES
Guard column: 15 × 3.2 Newguard 7 µm RP-C18
Column: 220 × 4.6 5 µm RP-C18 Spheri (Kontron)
Column temperature: 30
Mobile phase: Gradient. MeCN:water 30:70 for 12 min, to 70:30 over 33 min.
Flow rate: 1.2
Injection volume: 20
Detector: UV 254
CHROMATOGRAM
Retention time: 16.9
Limit of quantitation: 50 ng
Desoxycorticosterone
171
OTHER SUBSTANCES
Extracted: corticosterone (8.3), 18-hydroxydeoxycorticosterone (6.9), progesterone (29.6)
REFERENCE
Matilla, M.J.; Jimenez, M.M.; Montiel, M. Deoxycorticosterone, 18-OH-deoxycorticosterone and corticosterone determination by high performance liquid chromatography in monolayer adrenal cell culture,
Biochem.Int., 1991, 24, 951–957.
ANNOTATED BIBLIOGRAPHY
Sheikh, S.U.; Touchstone, J. HPLC of steroids in non-aqueous mobile phase at subambient temperature,
J.Liq.Chromatogr., 1987, 10, 2489–2496. [column temperature −50◦ ; desoxycorticosterone; estrone;
estradiol; cortisone; hydrocortisone]
Smith, E. Liquid chromatographic determination of desoxycorticosterone acetate in oil injections,
J.Assoc.Off.Anal.Chem., 1979, 62, 812–817.
Wei, J.Q.; Wei, J.L.; Lucarelli, C.; Zhou, X.T.; Wang, D.Q.; Dai, W.J.; Li, S.; Li, S.M.; Liu, R.T. Serum
steroid hormonal profiles by reversed-phase liquid chromatography in patients with 17-hydroxylase
deficiency and in an affected family, Clin.Chem., 1992, 38, 76–82. [cortisone; hydrocortisone; deoxycortisol; androstenedione; testosterone; progesterone; hydroxyprogesterone; desoxycorticosterone]
172
Dexrazoxane
Dexrazoxane
Molecular formula: C11 H16 N4 O4
Molecular weight: 268.27
CAS Registry No: 24584-09-6,
21416-87-5 (racemic)
Merck Index: 13, 8208
O
H
N
O
N
N
N
O
CH3
H
O
SAMPLE
Matrix: blood
Sample preparation: Condition a Sep-Pak Plus C18 SPE cartridge with MeCN and
water. Acidify 1 mL plasma with 50 µL 43% phosphoric acid. Add 70 µL 6 M HCl to
260 µL acidified plasma, centrifuge, remove the supernatant, wash the pellet three
times with 250 µL portions of 10 mM HCl. Combine the supernatant and the washings
and adjust the pH to 6.0 with 80 µL 5 M NaOH, add to the SPE cartridge, wash with
5 mL water, wash with 1 mL hexane, elute with 7.5 mL MeCN. Evaporate the eluate
to dryness under a stream of argon, reconstitute the residue with 130 µL mobile phase
EtOH:MeOH:isopropanol 90:5:5, inject a 50 µL aliquot.
HPLC VARIABLES
Guard column: 3.2 × 1.5 NewGuard silica (Brownlee)
Column: 250 × 4.6 10 µm Chiralcel OD
Mobile phase: EtOH:MeOH:isopropanol:n-hexane 76.5:4.25:4.25:15
Flow rate: 0.5
Injection volume: 50
Detector: UV 207
CHROMATOGRAM
Retention time: 18.5
Limit of quantitation: 2 µM
OTHER SUBSTANCES
Extracted: levrazoxane (16.5)
KEY WORDS
chiral; plasma; pharmacokinetics; rat; SPE
REFERENCE
Hasinoff, B.B.; Aoyama, R.G. Stereoselective metabolism of dexrazoxane (ICRF-187) and levrazoxane
(ICRF-186), Chirality, 1999, 11, 286–290.
SAMPLE
Matrix: blood, urine
Sample preparation: Plasma. Mix 1 mL plasma with 25 µg/mL IS, 100 µL water, and
2 mL MeCN, shake for 10 min, centrifuge at 2500 g for 10 min. Remove the supernatant,
add 20 mL chloroform:2-methyl-2-propanol 90:10 (Caution! Chloroform is a carcinogen!),
shake for 30 min, centrifuge at 2500 g for 10 min. Remove a 15 mL portion of the organic
layer and evaporate it to dryness under a stream of nitrogen at 40◦ , reconstitute the
residue with 500 µL MeOH:100 mM HCl 20:80, inject a 50 µL aliquot. Urine. Mix 1 mL
urine with 250 µg/mL IS, make up to 100 mL with water. Remove a 1 mL portion of this
mixture, add 20 mL chloroform:2-methyl-2-propanol 90:10, shake for 30 min, centrifuge
at 2500 g for 10 min. Remove 15 mL of the organic layer and evaporate it to dryness
under a stream of nitrogen at 40◦ , reconstitute the residue with 500 µL MeOH:100 mM
HCl 20:80, inject a 50 µL aliquot.
Dexrazoxane
173
HPLC VARIABLES
Guard column: 10 × 3 reverse phase (Chrompack)
Column: 300 × 4.6 10 µm µBondapak Phenyl
Mobile phase: MeOH:10 mM pH 4.7 potassium phosphate buffer 20:80
Flow rate: 1
Injection volume: 50
Detector: UV 208
CHROMATOGRAM
Retention time: 6.2
Internal standard: 8-chlorotheophylline (16)
Limit of quantitation: 100 ng/mL (plasma), 10 µg/mL (urine)
OTHER SUBSTANCES
Simultaneous: acetaminophen, 5-bromouracil, caffeine, 5-chlorouracil, 5-fluorocytosine,
5-fluorouracil, phenylethyleneglycol, theophylline
Noninterfering: codeine, cyclophosphamide, doxorubicin, indomethacin, methylparaben, phenacetin, phenazone, prednisone, triamcinolone acetate, triamcinolone
KEY WORDS
plasma; pharmacokinetics
REFERENCE
Rosing, H.; van Gijn, R.; ten Bokkel Huinink, W.W.; Beijnen, J.H. High performance liquid chromatographic analysis of the cardioprotective agent dexrazoxane in human plasma and urine,
J.Liq.Chromatogr.Rel.Technol., 1997, 20, 583–601.
ANNOTATED BIBLIOGRAPHY
Hasinoff, B.B.; Aoyama, R.G. Relative plasma levels of the cardioprotective drug dexrazoxane and its
two active ring-opened metabolites in the rat, Drug Metab.Dispos., 1999, 27, 265–268.
174
Dextran
Dextran
CAS Registry No: 9004-54-0
Merck Index: 13, 2965
SAMPLE
Matrix: blood
Sample preparation: Dilute plasma 4 times with 100 mM pH 12 sodium glycinate
buffer, filter (Amicon Centrifree) while centrifuging. Lyophilize the ultrafiltrate and
reconstitute the residue with 125 µL pH 7 ammonium phosphate buffer, inject a 100 µL
aliquot.
HPLC VARIABLES
Column: Tosohaas 2000SWXL + Tosohaas 2500PWXL in series
Mobile phase: 100 mM pH 6 sodium acetate buffer
Flow rate: 0.8
Injection volume: 100
Detector: Radioactivity (3 H)
CHROMATOGRAM
Retention time: 18
KEY WORDS
plasma; rat
REFERENCE
Hartman, N.R.; Johns, D.G.; Mitsuya, H. Pharmacokinetic analysis of dextran sulfate in rats as pertains
to its clinical usefulness for therapy of HIV infection, AIDS Res.Hum.Retroviruses, 1990, 6, 805–812.
SAMPLE
Matrix: blood
Sample preparation: Filter serum (0.45 µm), inject an aliquot.
HPLC VARIABLES
Guard column: 48 × 4.6 (ES Industries)
Column: 300 × 7.8 5 µm Chromega diol size-exclusion (ES Industries)
Column temperature: 40
Mobile phase: Buffer containing 25 mM potassium dihydrogen phosphate, 25 mM
dipotassium hydrogen phosphate, and 50 mM potassium chloride, pH ca. 6.8
Flow rate: 1
Injection volume: 10
Detector: UV 525 following post-column reaction with an 18.5 µg/mL solution of 1,9dimethylmethylene blue in water pumped at 0.5 mL/min. The column effluent mixed
with the dye solution in a 3.1 µL mixing tee and flowed through a 28.8 cm length of
0.254 mm ID tubing to the detector.
CHROMATOGRAM
Retention time: 7
Limit of detection: 300 ng/mL
Limit of quantitation: 8 µg/mL
KEY WORDS
post-column reaction; rat; serum
Dextran
175
REFERENCE
Maderich, A.B.; Sugita, E.T. Size-exclusion chromatographic determination of dextran sulfate in rat
serum, J.Chromatogr., 1993, 620, 137–142.
ANNOTATED BIBLIOGRAPHY
Hemmelder, M.H.; de Jong, P.E.; De Zeeuw, D. A comparison of analytic procedures for measurement
of fractional dextran clearances, J.Lab.Clin.Med., 1998, 132, 390–403. [RI detection; detection by
reaction of fractions with anthrone reagent]
176
Diacerein
Diacerein
O
COOH
Molecular formula: C19 H12 O8
Molecular weight: 368.29
CAS Registry No: 13739-02-1
Merck Index: 13, 2979
CH3
O
O
O
O
CH3
O
SAMPLE
Matrix: blood, urine
Sample preparation: Mix 1 mL plasma or urine with 1 mL MeCN, centrifuge, inject a
40 µL aliquot of the supernatant.
HPLC VARIABLES
Column: Nucleosil C18
Mobile phase: MeCN:pH 2.2 McIlvaine buffer 47:53
Flow rate: 1
Injection volume: 40
Detector: UV 432
CHROMATOGRAM
Limit of detection: 100 ng/mL
KEY WORDS
determined as rhein, the active metabolite; pharmacokinetics; plasma
REFERENCE
Debord, P.; Louchahi, K.; Tod, M.; Cournot, A.; Perret, G.; Petitjean, O. Influence of renal function on the
pharmacokinetics of diacerein after a single oral dose, Eur.J.Drug Metab.Pharmacokinet., 1994, 19,
13–19.
Dichloroacetic acid
177
Dichloroacetic acid
Molecular formula: C2 H2 Cl2 O2
Cl2CHCOOH
Molecular weight: 128.94
CAS Registry No: 79-43-6
Merck Index: 13, 3075
SAMPLE
Matrix: blood
Sample preparation: Inject 15–25 µL serum directly.
HPLC VARIABLES
Column: Asahipak GS-320 gel permeation
Mobile phase: MeCN:10 mM pH 4.0 ammonium acetate buffer 10:90
Flow rate: 2
Injection volume: 15–25
Detector: UV 220
CHROMATOGRAM
Retention time: 12
Limit of detection: 5 µg/mL
KEY WORDS
pharmacokinetics; serum
REFERENCE
Sakakihara, Y.; Nakamura, G.; Tokoeda, Y.; Abe, T.; Kamoshita, S. A rapid microassay for dichloroacetate in serum by gel-permeation chromatography, Eur.J.Clin.Chem.Clin.Biochem., 1994, 32, 79–83.
178
Dichlorophen
Dichlorophen
OH
OH
Cl
Cl
Molecular formula: C13 H10 Cl2 O2
Molecular weight: 269.13
CAS Registry No: 97-23-4
Merck Index: 13, 3096
SAMPLE
Matrix: solutions
Sample preparation: Inject a 10–40 µL aliquot of a 500 µg/mL solution in MeOH.
HPLC VARIABLES
Column: Zorbax C8
Column temperature: 31
Mobile phase: Gradient. A:B from 0:100 to 100:0 over 30 min. A was MeCN:water:phosphoric acid 89.9:10:0.1. B was 0.1% phosphoric acid.
Flow rate: 2
Injection volume: 10–40
Detector: UV 230
CHROMATOGRAM
Retention time: 21.9
OTHER SUBSTANCES
Simultaneous: acetaminophen (8.2), acetophenetidine (14.6), aminophylline (8.8), amobarbital (15.5), antipyrine (12.9), aprobarbital (12.3), aspirin (13.6), barbital (9.4),
benzoic acid (13.2), butabarbital (13.1), butethal (14.2), caffeine (10.2), chloramphenicol
(14.4), chlorothiazide (9.1), chlorpropamide (17.7), colchicine (15.3), cortisone (15.0),
coumarin (15.1), cyclothiazide (18.6), cyheptamide (17.8), danazol (19.3), danthron
(22.3), dapsone (13.1), diethylstilbestrol (21.5), dronabinol (26.3), estradiol (19.4),
estriol, 15.0), estrone (20.3), ethosuximide (9.8), eugenol (18.8), fenoprofen (19.2), fluorouracil, 3.0), fluoxymesterone (15.0), furosemide (17.0), gitoxigenin (16.0), glutethimide
(16.3), guaiacol (11.4), hexobarbital (15.1), hippuric acid (9.6), hydrocortisone (14.4),
hydroquinone (3.3), ibuprofen (22.4), indomethacin (22.00, isocarbostyril (11.8), mefenamic acid (23.7), methocarbamol (12.6), methyl salicylate (19.3), methyldopa (6.4),
methylparaben (12.8), methyltestosterone (19.6), niacin (3.9), nitrofurantoin (11.2),
normethsuximide (14.5), oxyphenbutazone (19.8), paraxanthine (8.7), pentobarbital
(15.2), phenylbutazone (23.4), piperonyl butoxide (26.1), prednisolone (15.2), prednisone (15.5), primidone (11.3), probenecid (20.5), progesterone (21.8), propylparaben
(18.3), pyrithydione (11.2), pyrocatechol (l4.6), reserpine (22.5), resorcinol (4.3), saccharin (8.0), salicylamide (11.0), salicylic acid (15.0), secobarbital (15.7), stanozolol
(19.2), sulfacetamide (8.4), sulfadimethoxine (7.7), sulfaethidole (13.4), sulfamerazine
(10.1), sulfamethazine (7.0), sulfamethizole (11.5), sulfamethoxazole (13.6), sulfanilamide (4.0), sulfapyridine (9.6), sulfisoxazole (14.2), sulindac (19.1), testosterone acetate
(20.5), testosterone 17 β-cypionate (25.2), testosterone enanthate (25.0), testosterone
propionate, 24.1), theobromine (8.3), thiobarbituric acid (2.4), thiosalicylic acid (15.4),
tolbutamide, 18.8), tolmetin (18.7), triamcinolone (13.8), triamcinolone acetonide (17.8),
warfarin (20.0)
REFERENCE
Hill, D.W.; Langner, K.J. HPLC photodiode array UV detection for toxicological drug analysis, J.Liq.
Chromatogr., 1987, 10, 377–409.
179
Diclazuril
Diclazuril
Molecular formula: C17 H9 Cl3 N4 O2
Molecular weight: 407.64
CAS Registry No: 101831-37-2
Merck Index: 13, 3106
CN
Cl
O
H
Cl
Cl
N
N
N
O
SAMPLE
Matrix: blood
Sample preparation: Condition a Mega Bond Elut C18 SPE cartridge with 2 mL MeOH
and 2 mL 100 mM pH 6.0 phosphate buffer. Mix 20 µL 100 µg/mL IS in DMF:water
50:50 with ? mL plasma, add 2 mL 100 mM pH 6.0 phosphate buffer, mix, adjust pH to
6.0, add to the SPE cartridge, allow to pass through taking at least 2 min, wash with
2 mL 100 mM pH 6.0 phosphate buffer, wash with 2 mL 1 M acetic acid, wash with
2 mL hexane (allow cartridge to dry for 5–10 min after each wash). Elute with 4 mL
MeOH:conc. HCl 95:5. Evaporate the eluate to dryness under a stream of nitrogen at
40◦ , reconstitute the residue with 100 µL DMF, vortex, sonicate, add 100 µL water,
vortex, sonicate, inject a 20 µL aliquot.
HPLC VARIABLES
Guard column: Alltech C18
Column: 150 × 4.6 5 µm Ultrasphere ODS
Mobile phase: MeCN:MeOH:buffer 20:43.2:36.8 (The buffer was 0.5% ammonium
acetate containing 10 mM tetrabutylammonium hydrogen sulfate.)
Flow rate: 1
Injection volume: 20
Detector: UV 280
CHROMATOGRAM
Retention time: 13
Internal standard: R062646 (diclazuril with methyl α to cyano group, Janssen) (14.5)
Limit of detection: 10 ng/mL
KEY WORDS
horse; pharmacokinetics; plasma; SPE; use silanized glassware
REFERENCE
Dirikolu, L.; Lehner, F.; Nattrass, C.; Bentz, B.G.; Woods, W.E.; Carter, W.G.; Karpiesiuk, W.; Jacobs, J.;
Boyles, J.; Harkins, J.D.; Granstrom, D.E.; Tobin, T. Diclazuril in the horse: its identification and
detection and preliminary pharmacokinetics, J.Vet.Pharmacol.Ther., 1999, 22, 374–379.
SAMPLE
Matrix: feed
Sample preparation: Mix 50 g ground feed with 1 mL 50 µg/mL IS in DMF and
200 mL acidified MeOH, stir overnight. Remove 20 mL of the supernatant and dilute
with 20 mL water, add to a Mega Bond C18 SPE cartridge, wash with 25 mL acidified
MeOH:water 65:35, elute with 25 mL acidified MeOH:water 80:20. Evaporate the eluate
to dryness at 60◦ , reconstitute the residue with 1 mL DMF and 1.5 mL water, filter
(0.45 µm), inject a 20 µL aliquot. (Acidified MeOH was 5 mL conc. HCl in 1 L MeOH.)
HPLC VARIABLES
Column: 100 × 4.6 3 µm Hypersil ODS or BDS
Mobile phase: Gradient. MeCN:MeOH:10 mM tetrabutylammonium hydrogen sulfate
20:20:60 for 10 min, to 20:35:45 over 30 min, flush column with MeCN for 10 min.
180
Diclazuril
Flow rate: 2
Injection volume: 20
Detector: UV 280
CHROMATOGRAM
Retention time: 24
Internal standard: R062646 (diclazuril with methyl α to cyano group, Janssen) (26)
Limit of quantitation: 1 µg/g
OTHER SUBSTANCES
Simultaneous: degradant
KEY WORDS
SPE
REFERENCE
De Kock, J.; de Smet, M.; Sneyers, R. Determination of diclazuril in animal feed by liquid chromatography, J.Chromatogr., 1992, 606, 141–146.
ANNOTATED BIBLIOGRAPHY
Fontaine, A.; Haustraete, K. Liquid-chromatographic determination of diclazuril in premix and supplemented feed: Interlaboratory study, J.AOAC Int., 1994, 77, 1359–1361. [SPE]
Mortier, L.; Daeseleire, E.; Delahaut, P. Simultaneous detection of five coccidiostats in eggs by liquid
chromatography-tandem mass spectrometry, Anal.Chim.Acta, 2003, 483, 27–37. [diclazuril; dimetridazole; halofuginone; nicarbazin; robenidine]
181
Dihydrotachysterol
Dihydrotachysterol
CH3
H3C
CH3
Molecular formula: C28 H46 O
Molecular weight: 398.66
CAS Registry No: 67-96-9
Merck Index: 13, 3202
CH3
H
CH3
H
H3C
OH
SAMPLE
Matrix: blood
Sample preparation: Mix 1 mL serum with 2 mL 50 ng/mL IS in EtOH, centrifuge at
1500 g for 15 min, add to an activated (by an unspecified method) Chromabond C18
ec SPE cartridge, wash with 3 mL EtOH:500 mM ammonium acetate 2:1, wash with
1 mL water, elute with 5 mL MeCN. Evaporate the eluate to dryness under a stream of
nitrogen at 45◦ , reconstitute the residue with 90 µL MeCN, inject a 50 µL aliquot.
HPLC VARIABLES
Column: 250 × 3 Nucleosil 100-5 C18 AB
Mobile phase: MeCN:water:acetic acid 95:5:5
Flow rate: 0.75
Injection volume: 50
Detector: UV 252
CHROMATOGRAM
Retention time: 15.4
Internal standard: vitamin D2 (17)
Limit of quantitation: 0.5 ng/mL
KEY WORDS
pharmacokinetics; serum
REFERENCE
Koytchev, R.; Alken, R.G.; Vagaday, M.; Kunter, U.; Kirkov, V. Differences in the bioavailability of
dihydrotachysterol preparations, Eur.J.Clin.Pharmacol., 1994, 47, 81–84.
SAMPLE
Matrix: blood
Sample preparation: Condition a Sep-Pak C18 SPE cartridge with 20 mL MeOH
and 10 mL water. Mix 5 mL plasma with 5 mL freshly charcoal-washed MeCN, let
stand for 1 h with occasional vortexing, centrifuge at 7000 g for 15 min. Add the
supernatant to 2.5 mL 200 mM pH 5.6 acetate buffer, add to the SPE cartridge, wash
with 3 mL MeOH:water 60:40, elute with 6 mL MeOH. Evaporate the eluate to dryness
under a stream of nitrogen or under reduced pressure, reconstitute the residue with
hexane:isopropanol:MeOH 91:7:2, inject an aliquot. (SPE procedure from: Coldwell,R.D.;
Trafford,D.J.H.; Makin,H.L.J.; Varley,M.J.; Kirk,D.N. Specific mass fragmentographic
assay for 25,26-dihydroxyvitamin D in human plasma using a deuterated internal
standard. J.Chromatogr. 1985, 338, 289–302.)
HPLC VARIABLES
Column: 250 × 6.2 Zorbax-SIL
182
Dihydrotachysterol
Mobile phase: Hexane:isopropanol:MeOH 91:7:2
Flow rate: 1.8–2
Detector: UV 254
KEY WORDS
normal phase; plasma; rat; SPE
REFERENCE
Qaw, F.; Calverley, M.J.; Schroeder, N.J.; Trafford, D.J.; Makin, H.L.; Jones, G. In vivo metabolism of
the vitamin D analog, dihydrotachysterol. Evidence for formation of 1α,25- and 1β,25-dihydroxydihydrotachysterol metabolites and studies of their biological activity, J.Biol.Chem., 1993, 268,
282–292.
ANNOTATED BIBLIOGRAPHY
Taylor, A.; Bikle, D.D.; Norman, M.E. Serum dihydrotachysterol levels and biological action in normal
man, J.Clin.Endocrinol.Metab., 1988, 67, 198–202. [normal phase]
Dimethyl sulfoxide
183
Dimethyl sulfoxide
Molecular formula: C2 H6 OS
(CH3)2SO
Molecular weight: 78.13
CAS Registry No: 67-68-5
Merck Index: 13, 3285
SAMPLE
Matrix: bulk
Sample preparation: Dissolve 40 mg bisnafide drug substance in 20 mL water with
sonication and gentle heating, inject a 200 µL aliquot. (Dimethyl sulfoxide is determined
as an impurity in bisnafide drug substance.)
HPLC VARIABLES
Column: Zorbax Rx C8
Column temperature: 45 ± 2
Mobile phase: Gradient. A:B 100:0 for 10 min, to 0:100 (step gradient) maintain at
0:100 for 5 min, 100:0 for 15 min. A was 10 mM NaCl containing 0.10% phosphoric acid.
B was MeCN:10 mM NaCl containing 0.10% phosphoric acid 90:10.
Flow rate: 0.8
Injection volume: 200
Detector: UV 215
CHROMATOGRAM
Retention time: 7
Limit of detection: 51 ng/mL
Limit of quantitation: 219 ng/mL
REFERENCE
Walker, J.T.; Paolini, D.L.; Segretario, J. Quantitation of residual dimethylsulfoxide in a drug substance
(bisnafide) by reversed-phase high-performance liquid chromatography, J.Chromatogr.Sci., 1996, 34,
513–516.
SAMPLE
Matrix: tissue
Sample preparation: Extract 1 g tissue with 10 mL MeOH:water 10:90 for at least
6 h (no other details), dilute an aliquot 10 fold with MeOH:water 10:90, centrifuge for
5 min, inject an aliquot.
HPLC VARIABLES
Guard column: 5 µm Spheri-18 C18 (Brownlee)
Column: 250 × 4.6 5 µm Spherisorb ODS2 C18
Mobile phase: MeOH:water 10:90
Flow rate: 1
Injection volume: 20
Detector: UV 214
CHROMATOGRAM
Retention time: 3.5
Limit of quantitation: 500 ng/mL
184
Dimethyl sulfoxide
KEY WORDS
myocardium; pig
REFERENCE
Carpenter, J.F.; Dawson, P.E. Quantitation of dimethyl sulfoxide in solutions and tissues by highperformance liquid chromatography, Cryobiology, 1991, 28, 210–215.
Dinitolmide
Dinitolmide
O
185
NH2
CH3
Molecular formula: C8 H7 N3 O5
Molecular weight: 225.16
CAS Registry No: 148-01-6
Merck Index: 13, 3297
O2 N
NO2
SAMPLE
Matrix: feed
Sample preparation: Mix 5 g ground feed with 50 mL MeCN:water 85:15, heat at 50◦
with frequent shaking for 10 min, shake on a wrist-action shaker at room temperature
for 10 min, filter, inject a 20 µL aliquot of the filtrate.
HPLC VARIABLES
Column: 250 × 4.9 5 µm Partisil
Mobile phase: MeCN:dichloromethane 50:50
Flow rate: 2
Injection volume: 20
Detector: UV 270
CHROMATOGRAM
Retention time: 3.5
Limit of quantitation: 20 µg/g
OTHER SUBSTANCES
Noninterfering: amprolium, avoparcin, decoquinate, dimetridazole, ethopabate, furazolidone, halquinol, monensin, nifursol, nitrovin, robenidine, vitamin A, vitamin D3 ,
vitamin E, zinc bacitracin
Interfering: sulfaquinoxaline (can be resolved using MeCN:dichloromethane 30:70)
KEY WORDS
normal phase
REFERENCE
Burns, I.W.; Jones, A.D. Determination of 3,5-dinitro-o-toluamide in poultry feedstuffs and pre-mixes by
high-performance liquid chromatography, The Analyst, 1980, 105, 509–512.
186
Dipivefrin
Dipivefrin
Molecular formula: C19 H29 NO5
Molecular weight: 351.44
CAS Registry No: 52365-63-6
Merck Index: 13, 3372
OH
H
N
O
CH3
CH3
CH3
CH3
O
O
CH3
CH3
CH3
O
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: reversed phase
Mobile phase: MeOH:20 mM pH 7.9 Tris buffer 80:20
Flow rate: 1.6
Injection volume: 30
Detector: UV ?
CHROMATOGRAM
Retention time: 12–16
REFERENCE
Tamaru, R.D.; Davis, W.L.; Anderson, J.A. Comparison of ocular disposition of free pivalic acid and
pivalic acid esterified in dipivefrin, Arch.Ophthalmol., 1983, 101, 1127–1129.
Dithiazanine iodide
Dithiazanine iodide
Molecular formula: C23 H23 IN2 S2
Molecular weight: 518.49
CAS Registry No: 514-73-8
Merck Index: 13, 3405
S
S
187
I−
N+
N
CH3
H3C
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 150 × 4.6 5 µm TSKgel ODS 80TM
Mobile phase: MeCN:25 mM pH 6.5 imidazole buffer 60:40 containing 10 mM sodium
1-propanesulfonate
Flow rate: 1
Injection volume:
Detector: Chemiluminescence, the column effluent mixed with MeCN containing 0.25 mM
bis(4-nitro-2-(3,6,9-trioxadecyloxycarbonyl)phenyl) oxalate (TDPO) and 25 mM hydrogen peroxide pumped at 1.3 mL/min and the mixture flowed into the flow cell and the
chemiluminescence was measured using a red-sensitive photomultiplier (Hamamatsu
R2228)
CHROMATOGRAM
Retention time: 14
Limit of detection: 0.19 fmole
OTHER SUBSTANCES
Simultaneous: methylene blue (3.5)
REFERENCE
Kimoto, K.; Gohda, R.; Murayama, K.; Santa, T.; Fukushima, T.; Homma, H.; Imai, K. Sensitive detection of near-infrared fluorescent dyes using high-performance liquid chromatography with peroxalate
chemiluminescence detection system, Biomed.Chromatogr., 1996, 10, 189–190.
188
Docarpamine
Docarpamine
O
H3C
O
Molecular formula: C21 H30 N2 O8 S
Molecular weight: 470.54
CAS Registry No: 74639-40-0
Merck Index: 13, 3430
O
H3C
O
O
O
O
H
H
N
N
CH3
SCH3
O
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 × 4.6 10 µm Cosmosil 5C18-MS
Column temperature: 50
Mobile phase: Gradient. MeCN:10 mM pH 4.2 potassium phosphate buffer 0:100 for
2 min, to 75:25 over 7.5 min, maintain at 75:25 for 5.5 min. Isocratic. MeCN:buffer 40:60
Flow rate: 1.5
Detector: UV (wavelength not specified)
CHROMATOGRAM
Retention time: 11.4 (gradient) or 6.5 (isocratic)
OTHER SUBSTANCES
Simultaneous: acetazolamide (7.9), acyclovir (7.0), allopurinol (7.3), aniracetam (8.6),
benazepril (11.5), betamethasone (10.8), camostat mesylate (8.8), carbamazepine (10.8),
cilazapril (10.7), cimetidine (7.5), clofibrate (15.0), clonazepam (11.3), cyclophosphamide
(9.9), delapril (11.9), dexamethasone (10.9), diazepam (12.5), digoxin (9.0), dilazep
(10.6), diltiazem (10.2), eperisone (8.7), ethosuximide (9.0), fenbufen (11.8), fluconazole (9.2), flutamide (12.7), fominoben (12.6), hydrocortisone acetate (12.1), imidapril
(10.1), indomethacin (12.6), irinotecan (9.2), maprotiline (10.5), methotrexate (8.0),
nefiracetam (9.7), nifedipine (11.9), nitrazepam (11.2), pentobarbital (10.9), phenobarbital (10.0), phenytoin (10.8), pindolol (8.3), pranlukast (13.4), pranoprofen (10.4),
prednisolone (10.3), primidone (8.9), quinapril (10.5), spironolactone (12.4), sulpiride
(7.6), sulthiame (9.3), tolbutamide (11.6), tranilast (10.5), triamcinolone (11.2), warfarin
(12.0), zonisamide (9.5) (gradient retention times; isocratic conditions may differ)
REFERENCE
Sugiyama, T.; Matsuyama, R.; Usui, S.; Katagiri, Y.; Hirano, K. Selection of mobile phases in highperformance liquid chromatographic determination for medicines, Biol.Pharm.Bull., 2000, 23, 274–278.
189
Dofetilide
Dofetilide
H
N
S
Molecular formula: C19 H27 N3 O5 S2
Molecular weight: 441.57
CAS Registry No: 115256-11-6
Merck Index: 13, 3443
O
O
O
S
H3C
N
O
CH3
O
N
CH3
H
SAMPLE
Matrix: blood
Sample preparation: Dilute plasma with an equal volume of MeOH:water 10:90
containing 1 M monochloroacetic acid, centrifuge at 4◦ at 3000 rpm for 1 h, inject a
200 µL aliquot onto column A and elute to waste with mobile phase A, backflush column
A with mobile phase A, backflush the contents of column A onto column B with mobile
phase C, elute column B with mobile phase C. Wash column A with mobile phase B and
wash column B with mobile phase D
HPLC VARIABLES
Column: A 50 × 1 50 µm HTLC Turbo C18 (Cohesive Technologies); B 33 × 2.1 HTLC
HiRes C18 (Cohesive Technologies) (Fit a 5 µm filter before column A and a 2 µm filter
before column B.)
Mobile phase: A 0.01% trifluoroacetic acid in water; B MeCN:0.1% aqueous ammonia
90:10; C MeCN:MeOH:20 mM ammonium acetate 45:45:10; D MeCN:THF 50:50
Flow rate: A 5; B 1
Injection volume: 200
Detector: MS, PE Sciex API 2000 triple quadrupole, positive ion mode, TurboIonspray,
nebulizer gas nitrogen at 25 psi, applied voltage 5200 V, collision energy 50 V, turbo
gas 100◦ and 40 psi, a flow splitter was used to deliver 50 µL/mL to the detector, m/z
442–198
CHROMATOGRAM
Retention time: 1.1
Limit of quantitation: 5 ng/mL
OTHER SUBSTANCES
Extracted: doxazosin (m/z 452–344) (1.1)
KEY WORDS
column-switching; dog; plasma
REFERENCE
Chassaing, C.; Luckwell, J.; Macrae, P.; Saunders, K.; Wright, P.; Venn, R. Direct analysis of crude
plasma samples by turbulent flow chromatography/tandem mass spectrometry, Chromatographia,
2001, 53, 122–130.
SAMPLE
Matrix: urine
Sample preparation: Mix 1 mL urine with 20 µL 5 µg/mL IS in MeOH, add 1 mL
100 mM pH 7.4 phosphate buffer, mix, add 5 mL MTBE, rotate at 30 rpm for 10 min,
centrifuge at 1730 g for 5 min. Remove the organic layer and extract it with 1 mL
20 mM phosphoric acid, centrifuge at 1730 g for 5 min. Add the aqueous layer to 2 mL
100 mM pH 7.4 phosphate buffer, extract with 5 mL MTBE, centrifuge at 1730 g for
5 min. Evaporate the organic layer to dryness under a stream of nitrogen, reconstitute
the residue with 100 µL mobile phase, inject an 80 µL aliquot.
190
Dofetilide
HPLC VARIABLES
Column: 250 × 4.6 5 µm Spherisorb silica
Mobile phase: MeCN:20 mM pH 7.0 ammonium phosphate buffer 35:65
Flow rate: 1
Injection volume: 80
Detector: UV 230
CHROMATOGRAM
Retention time: 8.5
Internal standard: UK-69,308
(N,N-bis(4-methanesulfonamidophenethyl)methyl-
amine) (10)
Limit of quantitation: 2.5 ng/mL
OTHER SUBSTANCES
Extracted: metabolite
REFERENCE
Walker, D.K.; Smith, D.A.; Stopher, D.A. Liquid-liquid extraction and high-performance liquid chromatography for the determination of a novel antidysrhythmic agent (UK-68,798) in human urine,
J.Chromatogr., 1991, 568, 475–480.
191
Dolasetron
Dolasetron
O
N
Molecular formula: C19 H20 N2 O3
Molecular weight: 324.37
CAS Registry No: 115956-12-2
Merck Index: 13, 3445
O
O
N
H
SAMPLE
Matrix: blood, urine
Sample preparation: Plasma. Mix 1 mL plasma with IS and 100 µL 5 M citric acid,
add 1 mL 2 M sodium carbonate, extract with 4 mL ethyl acetate:n-hexane 75:25 for
30 min. Remove the organic layer and extract it with 1 mL 100 mM HCL for 15 min,
centrifuge, discard the organic layer. Add 1 mL 2 M sodium carbonate to the aqueous
layer, extract with 4 mL ethyl acetate:n-hexane 75:25, evaporate the organic layer to
dryness under a stream of nitrogen, reconstitute with 150 µL mobile phase, inject a
100 µL aliquot. Urine. Dilute urine 1:100 with water. Mix 1 mL diluted urine with IS
and 1 mL 2 M sodium carbonate, extract with 4 mL ethyl acetate:n-hexane 75:25 for
30 min. Remove the organic layer and extract it with 1 mL 100 mM HCL for 15 min,
centrifuge, discard the organic layer. Add 1 mL 2 M sodium carbonate to the aqueous
layer, extract with 4 mL ethyl acetate:n-hexane 75:25, evaporate the organic layer to
dryness under a stream of nitrogen, reconstitute with 150 µL mobile phase, inject a
100 µL aliquot.
HPLC VARIABLES
Guard column: 4 × 6 10 µm µBondapak C18
Column: 150 × 4.6 5 µm Ultrasphere IP C18
Column temperature: 30
Mobile phase: MeCN:n-butanol:buffer 5:6:89 (The buffer was 50 mM sodium dihydrogen
phosphate adjusted to pH 2.5 with orthophosphoric acid.)
Flow rate: 0.7
Injection volume: 100
Detector: F ex 285 em 345
CHROMATOGRAM
Retention time: 7.6
Internal standard: trans-octahydro-3-hydroxy-2,6-methano-2H-quinolizin-8-yl-1H-5methyl-indole-3-carboxylate (MDL 101,8588; Marion Merrell Dow Research Institute,
Strasbourg) (17.0)
Limit of quantitation: 10 nM (plasma), 50 nM (urine)
OTHER SUBSTANCES
Extracted: metabolite
KEY WORDS
pharmacokinetics; plasma
192
Dolasetron
REFERENCE
Huebert, N.D.; Schwartz, J.J.; Zeidler, L.; Schwach, V.; Haegele, K.D. Simultaneous measurement of
dolasetron and its major metabolite, MDL 74,156, in human plasma and urine, J.Chromatogr.B, 1996,
685, 291–297.
SAMPLE
Matrix: formulations
Sample preparation: Prepare a liquid suspension by crushing twelve 50 mg tablets,
slowly add 60 mL of Ora-Plus:Ora-Sweet SF 50:50, dilute to 10 µg/mL with MeCN:water
24:76, shake for 15 s, centrifuge at 1000 rpm for 2 min, inject a 5 µL aliquot.
HPLC VARIABLES
Column: 150 × 4.6 3 µm Spherisorb CN
Column temperature: 30
Mobile phase: MeCN:buffer 24:76 (The buffer was 50 mM ammonium acetate adjusted
to pH 7.5 with dilute ammonium hydroxide.)
Flow rate: 0.8
Injection volume: 5
Detector: UV 280
CHROMATOGRAM
Retention time: 6.9
KEY WORDS
stability-indicating; suspension
REFERENCE
Johnson, C.E.; Wagner, D.S.; Bussard, W.E. Stability of dolasetron in two oral liquid vehicles, Am.J.
Health-Syst.Pharm., 2003, 60, 2242–2244.
ANNOTATED BIBLIOGRAPHY
McElvain, J.S.; Vandiver, V.J.; Eichemeier, L.S. Validation of a reversed-phase HPLC method for directly
quantifying the enantiomers of MDL 74,156, the primary metabolite of dolasetron mesylate, in human
plasma, J.Pharm.Biomed.Anal., 1997, 15, 513–521. [HPLC of metabolites only]
Reith, M.K.; Sproles, G.D.; Cheng, L.K. Human metabolism of dolasetron mesylate, a 5-HT3 receptor
antagonist, Drug Metab.Dispos., 1995, 23, 806–812. [column temp 30; metabolites; urine]
Sanwald, P.; Huebert, N.D.; Haegele, K.D. Simultaneous measurement of the major metabolites of
dolasetron mesilate in human urine using solid-phase extraction and high-performance liquid chromatography, J.Chromatogr.B, 1994, 661, 101–107.
Donepezil
Donepezil
Molecular formula: C24 H29 NO3
Molecular weight: 379.49
CAS Registry No: 120014-06-4
Merck Index: 13, 3453
O
193
N
H3CO
H3CO
SAMPLE
Matrix: blood
Sample preparation: Add 100 µL 200 ng/mL IS in 1 mM HCl and 5 mL n-hexane:isopropanol 97:3 to 1 mL plasma, shake for 5 min, centrifuge at 1800 g for 1 min. Remove
the organic layer and add it to 200 µL 1 mM HCl, shake for 1 min, centrifuge at 1800 g
for 1 min, inject a 75 µL aliquot of the aqueous layer.
HPLC VARIABLES
Column: 150 × 2.1 5 µm Bioptick AV-1 (GL Sciences)
Mobile phase: MeOH:10 mM formic acid 25:75
Flow rate: 0.2
Injection volume: 75
Detector: MS, Finnigan MAT TSQ7000, source 4.5 kV, capillary 200◦ , sheath gas nitrogen 70 psi, auxiliary gas nitrogen, collision gas argon 1.5 mtorr 40 eV, m/z 380–91
CHROMATOGRAM
Retention time: 8 (R-enantiomer), 13 (S-enantiomer)
Internal standard: d7 -donepezil (m/z 387–98)
Limit of detection: 7.7 pg
Limit of quantitation: 20 pg/mL
KEY WORDS
chiral; pharmacokinetics; plasma
REFERENCE
Matsui, K.; Oda, Y.; Nakata, H.; Yoshimura, T. Simultaneous determination of donepezil (aricept)
enantiomers in human plasma by liquid chromatography-electrospray tandem mass spectrometry,
J.Chromatogr.B, 1999, 729, 147–155.
SAMPLE
Matrix: blood
Sample preparation: Add 100 µL 3 µg/mL IS in 1 mM HCl and 500 µL 100 mM NaOH
to 1 mL plasma, vortex for 10 s, add 4 mL n-hexane:isopropanol 97:3, shake for 10 min,
centrifuge at 620 g at 4◦ for 10 min. Remove the organic layer and add it to 75 µL
100 mM HCl, mix at 100 cycles/min for 10 min, centrifuge at 1710 g at 4◦ for 5 min.
Carefully remove the upper organic layer, then remove traces of organic solvent with a
stream of air, inject a 50 µL aliquot of the aqueous layer.
HPLC VARIABLES
Column: 150 × 4.6 5 µm STR ODS-II (Shinwa)
Column temperature: 30 (in body of paper) or 40 (in abstract)
Mobile phase: MeCN:20 mM pH 4.6 phosphate buffer:6 M perchloric acid 40:59.5:0.5
Flow rate: 1
Injection volume: 50
Detector: UV 315
194
Donepezil
CHROMATOGRAM
Retention time: 5.1
Internal standard: cisapride (7.1)
Limit of quantitation: 3 ng/mL
OTHER SUBSTANCES
Simultaneous: alprazolam, chlorpromazine, diazepam, levomepromazine, nitrazepam
Noninterfering: haloperidol, risperidone
KEY WORDS
pharmacokinetics; plasma
REFERENCE
Yasui-Furukori, N.; Furuya, R.; Takahata, T.; Tateishi, T. Determination of donepezil, an acetylcholinesterase inhibitor, in human plasma by high-performance liquid chromatography with ultraviolet
absorbance detection, J.Chromatogr.B, 2002, 768, 261–265.
ANNOTATED BIBLIOGRAPHY
Kaddoumi, A.; Mori, M.; Nanashima, K.; Kono, M.; Nakashima, K. High performance liquid chromatographic determination of mazindol in human plasma, The Analyst, 2001, 126, 1963–1968. [donepezil
is internal standard]
Matsui, K.; Mishima, M.; Nagai, Y.; Yuzuriha, T.; Yoshimura, T. Absorption, distribution, metabolism,
and excretion of donepezil (Aricept) after a single oral administration to rat, Drug Metab.Dispos., 1999,
27, 1406–1414.
Pappa, H.; Farrú, R.; Vilanova, P.O.; Palacios, M.; Pizzorno, M.T. A new HPLC method to determine
Donepezil hydrochloride in tablets, J.Pharm.Biomed.Anal., 2002, 27, 177–182.
195
Doxefazepam
Doxefazepam
HO
Molecular formula: C17 H14 ClFN2 O3
Molecular weight: 348.76
CAS Registry No: 40762-15-0
Merck Index: 13, 3467
O
N
OH
N
Cl
F
SAMPLE
Matrix: blood
Sample preparation: Condition a Supelclean LC-18 SPE cartridge with 1 mL MeOH
and 2 mL water. Mix 1 mL plasma with 100 µL 2 µg/mL IS in MeOH, add to the SPE
cartridge, wash with 2 column volumes of water, wash with 100 µL MeOH, elute with
400 µL MeOH. Evaporate the eluate to dryness under a stream of nitrogen, reconstitute
the residue with 200 µL mobile phase, inject a 25 µL aliquot.
HPLC VARIABLES
Guard column: 20 × 4 Supelguard C18
Column: 300 × 4 µBondapak C18
Mobile phase: MeOH:water 20:80
Flow rate: 2
Injection volume: 25
Detector: UV 280
CHROMATOGRAM
Retention time: 5.2
Internal standard: diazepam (3.1)
Limit of quantitation: 100 ng/mL
KEY WORDS
plasma; SPE
REFERENCE
Carlucci, G. A high-performance liquid chromatographic method for the determination of doxefazepam
in human plasma using a solid-phase extraction column, J.Liq.Chromatogr., 1988, 11, 1559–1568.
196
Doxercalciferol
Doxercalciferol
CH3
H3C
CH3
CH3
Molecular formula: C28 H44 O2
CH3
Molecular weight: 412.65
CAS Registry No: 54573-75-0
Merck Index: 13, 3470
H
[1α-Hydroxyergocalciferol]
CH2
HO
OH
SAMPLE
Matrix: blood
Sample preparation: Condition a 60 mg Oasis HLB SPE cartridge with 2 mL ethyl
acetate, 2 mL MeOH, and 2 mL water. Condition a 500 mg Bond Elut silica SPE
cartridge with 4 mL chloroform:MeOH 30:1 (Caution! Chloroform is a carcinogen!) and
4 mL chloroform. Mix 10 µL 40 ng/mL IS in EtOH with 1 mL plasma, let stand for
15 min, add this mixture to 1 mL MeCN in another tube, rinse the first tube with
250 µL MeCN and add it to the plasma/MeCN mixture. Vortex for 30 s, centrifuge at
1500 g for 10 min. Add 2 mL water to the supernatant and pass the mixture through the
Oasis SPE cartridge. Wash with 2 mL water, wash with 2 mL MeOH:water 70:30, wash
with 1 mL hexane, elute with 1 mL ethyl acetate. Evaporate the eluate to dryness under
a stream of nitrogen, reconstitute the residue with two 200 µL portions of chloroform.
Add the chloroform layers to the silica SPE cartridge, wash with 3 mL chloroform, wash
with 2.2 mL chloroform:MeOH 40:1, elute with 2 mL chloroform:MeOH 30:1. Evaporate
the eluate to dryness, place the residue under reduced pressure for 10 min, reconstitute
the residue with 25 µL 100 µg/mL DMEQTAD in ethyl acetate, let stand at room
temperature for 30 min, add another 25 µL 100 µg/mL DMEQTAD in ethyl acetate, let
stand at room temperature for 1 h, add 40 µL EtOH, evaporate to dryness, reconstitute
with 10 µL acetic anhydride and 20 µL pyridine, heat at 50◦ for 1 h, add 40 µL EtOH,
evaporate the solvent, dissolve the residue in 40 µL mobile phase, inject a 15 µL aliquot.
(DMEQTAD is 4-[2-(6,7-dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalyl)ethyl]-1,2,4triazoline-3,5-dione and can be purchased from Wako.)
HPLC VARIABLES
Column: 150 × 4.6 4 µm YMC J’sphere ODS H-80
Column temperature: 40
Mobile phase: MeCN:water 92:8
Flow rate: 1
Injection volume: 15
Detector: MS, ThermoQuest LCQ, APCI, positive ion mode, collision gas helium, source
current 5 µA, heated capillary 225◦ , vaporizer 475◦ , capillary 3 V, tube lens offset 15 V
CHROMATOGRAM
Retention time: 6.8
Internal standard: d4 -doxercalciferol (6.7)
Limit of detection: 6.3 fmol
Limit of quantitation: 25 pg/mL
KEY WORDS
derivatization; plasma; SPE; Doxercalciferol can be determined without derivatization
using MeOH:water 95:5, retention time 5.4 min, LOD 200 pg/injection.
Doxercalciferol
197
REFERENCE
Higashi, T.; Awada, D.; Shimada, K. Liquid chromatography-mass spectrometric method combined with
derivatization for determination of 1α-hydroxyvitamin D3 in human plasma, J.Chromatogr.B, 2002,
772, 229–238.
SAMPLE
Matrix: cell cultures
Sample preparation: Precipitate cells with MeOH. Homogenize 1 vol of cells with 2
vol of MeOH, add 1 vol of chloroform (Caution! Chloroform is a carcinogen!), homogenize
(blender or Polytron) for 2 min, add 1 vol of chloroform, homogenize for 30 s, add 1 vol
of water, homogenize for 30 s, centrifuge, extract solids with 0.4 vol chloroform:MeOH
50:50. Combine the liquids and separate the chloroform layer. Pass the chloroform
layer through anhydrous sodium sulfate and evaporate under reduced pressure at
40◦ , reconstitute, inject an aliquot. (Current Protocols in Food Analytical Chemistry,
Wrolstad,R.E. (ed), Wiley, New York, 2003, page D.1.1.5; after Bligh,E.G.; Dyer,W.J.
A rapid method of total lipid extraction and purification. Can.J.Biochem.Physiol. 1959,
37, 911–917.))
HPLC VARIABLES
Column: 80 × 6.2 3 µm Zorbax SIL
Mobile phase: Hexane:isopropanol:MeOH 91:7:2
Flow rate: 1.5
Detector: UV 265
CHROMATOGRAM
Retention time: 5.6
OTHER SUBSTANCES
Simultaneous: metabolites
KEY WORDS
normal phase
REFERENCE
Strugnell, S.; Byford, V.; Makin, H.L.; Moriarty, R.M.; Gilardi, R.; LeVan, L.W.; Knutson, J.C.; Bishop,
C.W.; Jones, G. 1α,24(S)-dihydroxyvitamin D2 : a biologically active product of 1α-hydroxyvitamin D2
made in the human hepatoma, Hep3B, Biochem.J., 1995, 310, 233–241.
198
Dropropizine
Dropropizine
N
N
OH
OH
Molecular formula: C13 H20 N2 O2
Molecular weight: 236.31
CAS Registry No: 17692-31-8, 99291-24-4
(levodropropizine)
Merck Index: 13, 3486
SAMPLE
Matrix: blood
Sample preparation: Mix 200 µL plasma or serum with 10 ng IS and 200 µL 100 mM
pH 8.9 disodium hydrogen phosphate, add 5 mL chloroform:isopropanol 90:10 (Caution!
Chloroform is a carcinogen!), vortex for 2 min, centrifuge at 700 g for 10 min. Evaporate
4 mL of the organic layer to dryness under a stream of nitrogen, reconstitute the residue
with 500 µL water, inject a 100 µL aliquot.
HPLC VARIABLES
Column: 150 × 4.6 5 µm Bio-Gel PRP 70-5 poly(styrene-divinylbenzene) (Bio-Rad)
Mobile phase: MeOH:THF:100 mM pH 3 potassium phosphate buffer 30:0.5:70
Flow rate: 0.5
Injection volume: 100
Detector: F ex 240 em 350 (slits 18 and 30 nm, respectively)
CHROMATOGRAM
Retention time: 10
Internal standard: p-methoxylevodropropizine (15)
Limit of detection: 1–2 ng/mL
Limit of quantitation: 3.1 ng/mL
OTHER SUBSTANCES
Noninterfering: acetaminophen, amitriptyline, amobarbital, amphetamine, aprobarbital, atropine, barbital, benzoylecgonine, benztropine, butabarbital, caffeine, carbamazepine, carisoprodol, chlorpheniramine, chlorpromazine, chlorprothixene, cimetidine, cocaine, codeine, dextromethorphan, diazepam, dihydrocodeine, diphenhydramine,
diphenoxylate, diphenylhydantoin, disopyramide, doxepin, doxylamine, emetine, erythromycin, ethinamate, ethylmorphine, flurazepam, glutethimide, hydrocodone, hydrocortisone, hydromorphone, hydroxyzine, imipramine, lidocaine, loxapine, meperidine,
meprobamate, methadone, methamphetamine, methapyrilene, methaqualone, methocarbamol, methylphenidate, morphine, naloxone, nicotine, nordiazepam, nortriptyline, orphenadrine, oxycodone, papaverine, pentazocine, pentobarbital, phenacetin,
phencyclidine, phenmetrazine, phenobarbital, phenolphthalein, phentermine, phenylpropanolamine, phenytoin, phetidine, prazepam, procainamide, procaine, propoxyphene,
propranolol, protriptyline, pseudoephedrine, pyrilamine, quinine, salicylamide, secobarbital, spironolactone, strychnine, terpin hydrate, thioridazine, thiothixene, triamterene,
trifluperazine, triflupromazine, trihexyphenidyl, trimeprazine, trimethoprim, trimetobenzamide
KEY WORDS
plasma; serum
REFERENCE
Tagliaro, F.; Moffa, M.; De Battisti, Z.; Smith, F.P.; Gentile, M. High-performance liquid chromatographic
determination of levodropropizine in human plasma with fluorometric detection, J.Chromatogr.B, 1996,
685, 165–170.
199
Drospirenone
Drospirenone
O
H3C
Molecular formula: C24 H30 O3
Molecular weight: 366.49
CAS Registry No: 67392-87-4
Merck Index: 13, 3488
CH3
H
O
H
H
O
SAMPLE
Matrix: blood
Sample preparation: Vortex 3 mL plasma with 3 mL n-hexane:toluene 50:50 for 1 min,
centrifuge at 1200 g for 5 min, repeat the extraction. Combine the organic layers,
evaporate them to dryness under a stream of nitrogen, reconstitute the residue with
200 µL mobile phase, inject a 150 µL aliquot. To increase sensitivity, extract 5 mL
plasma twice with 2 mL aliquots of n-hexane:toluene 50:50.
HPLC VARIABLES
Column: 250 × 4.6 10 µm LiChrosorb RP-18
Mobile phase: MeOH:water 60:40
Flow rate: 2
Injection volume: 150
Detector: UV 254
CHROMATOGRAM
Retention time: 13.4
Limit of detection: <5 ng/mL
OTHER SUBSTANCES
Extracted: spirenone (9.2)
KEY WORDS
pharmacokinetics; plasma
REFERENCE
Krause, W.; Jakobs, U. Determination of plasma levels of spirorenone, a new aldosterone antagonist, and
one of its metabolites by high-performance liquid chromatography, J.Chromatogr., 1982, 230, 37–45.
200
Droxicam
Droxicam
O
O
N
N
Molecular formula: C16 H11 N3 O5 S
Molecular weight: 357.35
CAS Registry No: 90101-16-9
Merck Index: 13, 3491
O
S
O
N
CH3
O
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 × 4 5 µm LiChrospher 60 RP-Select B
Mobile phase: MeOH:water:acetic acid 48:45:7
Flow rate: 1.1
Injection volume: 20
Detector: UV 340
OTHER SUBSTANCES
Simultaneous: isoxicam, piroxicam
KEY WORDS
Droxicam cannot be detected in plasma, but it can be chromatographed under the
above conditions.
REFERENCE
Maya, M.T.; Pais, J.P.; Morais, J.A. A rapid method for the determination of piroxicam in plasma by
high-performance liquid chromatography, J.Pharm.Biomed.Anal., 1995, 13, 319–322.
201
Droxidopa
Droxidopa
Molecular formula: C9 H11 NO5
Molecular weight: 213.19
CAS Registry No: 23561-95-8
Merck Index: 13, 3492
OH
HO
HO
COOH
NH2
SAMPLE
Matrix: solutions
HPLC VARIABLES
Guard column: 50 × 4 Develosil ODS-10
Column: 250 × 4 Develosil ODS-5
Mobile phase: MeCN:buffer 10:90 (The buffer was 50 mM pH 3.4 sodium dihydrogen
phosphate containing 50 mM trichloroacetic acid, 200 µg/mL sodium dodecylsulfate,
and 10 µg/mL disodium EDTA.)
Flow rate: 0.6
Detector: E, Coulochem 5100A, conditioning cell Model 5021 + 400 mV, analytical cell
Model 5011, first electrode −100 mV, second electrode (recording electrode) −400 mV
CHROMATOGRAM
Retention time: 4
REFERENCE
Naoi, M.; Nagatsu, T. Uptake of L-threo-dihydroxyphenylserine into human brain synaptosomes,
J.Neural Transm., 1987, 70, 51–61.
Ebrotidine
Br
H
H2N
Molecular formula: C14 H17 BrN6 O2 S3
Molecular weight: 477.43
CAS Registry No: 100981-43-9
Merck Index: 13, 3518
N
N
NH2
S
N
S
N
S
O
O
SAMPLE
Matrix: bulk
Sample preparation: Inject a 20 µL aliquot of an 0.2% solution in MeOH.
HPLC VARIABLES
Column: 250 × 4 10 µm Spherisorb CN
Column temperature: 30
Mobile phase: MeCN:10 mM sodium dihydrogen phosphate 35:65
Flow rate: 1.5
Injection volume: 20
Detector: UV 245
CHROMATOGRAM
Retention time: 11
OTHER SUBSTANCES
Simultaneous: impurities
REFERENCE
Albet, C.; Fernandez, J.M.; Castello, J.M.; Sacristan, A.; Ortiz, J.A. Physicochemical properties, analytical determinations and stability of ebrotidine, Arzneimittelforschung, 1997, 47, 435–438.
SAMPLE
Matrix: urine
Sample preparation: Vortex 1 mL urine with 100 µL 1 M NaOH, add 5 mL dichloromethane:isopropanol 90:10, shake mechanically for 15 min, centrifuge at 2700 g for
5 min. Evaporate 4 mL of the organic layer to dryness under a stream of nitrogen at
40◦ , reconstitute the residue with 200 µL MeOH, vortex, filter (0.45 µm), inject a 25 µL
aliquot.
HPLC VARIABLES
Column: 300 × 3.9 10 µm µBondapak C18
Mobile phase: Gradient. MeCN:buffer from 20:80 to 35:65 over 30 min (The buffer was
5 mM 1-hexanesulfonic acid adjusted to pH 3 with glacial acetic acid.)
Flow rate: 1.5
Injection volume: 25
Detector: UV 235
CHROMATOGRAM
Retention time: 24
Limit of detection: 97 ng/mL
OTHER SUBSTANCES
Extracted: metabolites
Simultaneous: acetaminophen, amoxicillin, aspirin, caffeine, cimetidine, codeine, diazepam, omeprazole, ranitidine, theophylline
Interfering: imipramine
202
HPLC Methods for Recently Approved Pharmaceuticals. by George Lunn
Copyright 2005 John Wiley & Sons, Ltd. ISBN: 0-471-66941-5
Ebrotidine
203
KEY WORDS
SPE
REFERENCE
Rozman, E.; Galcerán, M.T.; Albet, C. Determination of ebrotidine and its metabolites in human urine
by reversed-phase ion-pair high-performance liquid chromatography, J.Chromatogr.B, 1997, 688,
107–115.
ANNOTATED BIBLIOGRAPHY
Rozman, E.; Galceran, M.T.; Anglada, L.; Albet, C. Investigation of the metabolism of ebrotidine in
human urine by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry,
Drug Metab.Dispos., 1995, 23, 976–981. [APCI].
Rozman, E.; Galcerán, M.T.; Albet, C. Ebrotidine and its metabolites studied by mass spectrometry with
electrospray ionization. Comparison of tandem and in-source fragmentation, Rapid Commun.Mass
Spectrom., 1995, 9, 1492–1498.
204
Edaravone
Edaravone
Molecular formula: C10 H10 N2 O
Molecular weight: 174.20
CAS Registry No: 89-25-8
Merck Index: 13, 6746
O
N
N
CH3
SAMPLE
Matrix: blood, urine
Sample preparation: Plasma. Add 25 µL 15 µg/mL IS in MeOH, 500 µL 750 mM
pH 5.0 sodium acetate buffer containing 40 mg/mL sodium metabisulfite, and 10 mg
β-glucuronidase/arylsulfatase (Limpet acetone powder type 1:Platela vulgata (Sigma))
to 500 µL plasma, heat at 37◦ for 2 h, add 100 mg NaCl, add 1.2 mL chloroform:EtOH
90:10 (Caution! Chloroform is a carcinogen!), vortex for 2 min, centrifuge at 1800 g
for 20 min. Remove the organic layer and add it to 50 µL 100 mM HCl. Evaporate to
dryness under a stream of nitrogen at room temperature, reconstitute the residue with
75 µL mobile phase, wash with 25 µL n-hexane, inject a 20 µL aliquot of the lower layer.
Urine. Add 25 µL 500 µg/mL IS in MeOH, 500 µL 750 mM pH 5.0 sodium acetate buffer
containing 40 mg/mL sodium metabisulfite, and 10 mg β-glucuronidase/arylsulfatase
(Limpet acetone powder type 1: Platela vulgata (Sigma)) to 500 µL urine, heat at 37◦ for
2 h, add 200 mg NaCl, add 2 mL dichloromethane:isopropanol 90:10, vortex for 1.5 min,
centrifuge at 1800 g for 30 min. Remove the organic layer, evaporate to dryness under
a stream of nitrogen at room temperature, reconstitute the residue with 200 µL mobile
phase (keep in a dry ice bath), vortex for 15 s, inject a 20 µL aliquot. (Wash NaCl with
extraction mixture before use.)
HPLC VARIABLES
Column: 150 × 3.9 Nova-Pak C18
Mobile phase: MeOH:250 mM pH 5.0 sodium acetate buffer 30:70
Flow rate: 1
Injection volume: 20
Detector: UV 254
CHROMATOGRAM
Retention time: 7
Internal standard: phenacetin (10)
Limit of quantitation: 50 ng/mL
OTHER SUBSTANCES
Extracted: antipyrine (5)
Noninterfering: albuterol, aspirin, chlorpropamide, chlorthalidone, codeine, diazepam,
diclofenac, dipyrone, enalapril, furosemide, heparin, nifedipine, phenytoin, ranitidine,
warfarin
Interfering: aminophylline, dapsone
KEY WORDS
plasma
REFERENCE
Lanchote, V.L.; Ping, W.C.; Santos, S.R.C.J. Determination of antipyrine and metabolites in plasma of a
patient with mild renal failure, Ther.Drug Monit., 1997, 19, 705–710.
SAMPLE
Matrix: urine
Edaravone
205
Sample preparation: Centrifuge urine, mix a 100 µL aliquot with 1 mL acetate buffer
and 50 µL water containing 5000–6000 IU β-glucuronidase and 150–200 IU sulfatase
from Helix pomatia, Sigma) (hydrolysis conditions not specified but 3 h at 37◦ is
recommended by St.Peter,J.V.; Awni,W.M. J.Chromatogr. 1989, 494, 424–427), add
8 mL dichloromethane, add 1 g NaCl, extract on a linear agitator for 10 min, centrifuge
at 1500 g for 5 min, remove the organic layer and add it to 1 mL washing buffer, extract
on a linear agitator for 5 min, centrifuge at 1500 g for 5 min, remove the organic layer
and add it to 500 µL water. Evaporate the dichloromethane to dryness under a stream
of nitrogen at 37◦ , add 500 µL MeOH, homogenize, inject a 20 µL aliquot. (Stock acetate
buffer was 27 g/L sodium acetate trihydrate containing 3 mL/L glacial acetic acid. Store
at 4◦ . Prepare the working acetate buffer (pH 4.9) fresh each day by adding 2% sodium
metabisulfite to the stock solution. Washing buffer was 500 mM disodium hydrogen
phosphate containing 35 g/L NaCl adjusted to pH 7.8 with 38% KOH.)
HPLC VARIABLES
Guard column: 20 × 4.6 10 µm C8
Column: 150 × 4.6 5 µm Ultrasphere C8
Mobile phase: MeCN:buffer 15:85 (The buffer was 5 mL/L glacial acetic acid containing
3 g/L sodium acetate trihydrate and 0.1 mM (20 µL/L) n-decylamine, pH 3.8–3.9.)
Flow rate: 1
Injection volume: 20
Detector: UV 242
CHROMATOGRAM
Retention time: 11
Internal standard: pyramidon (5)
Limit of detection: 1 µg/mL
Limit of quantitation: 15 µg/mL
REFERENCE
Palette, C.; Cordonnier, P.; Naline, E.; Advenier, C.; Pays, M. High-performance liquid chromatographic
method for the determination of the three main oxidative and 3-carboxylic antipyrine metabolites in
human urine, J.Chromatogr., 1991, 563, 103–113.
ANNOTATED BIBLIOGRAPHY
Ali, H.A.; el-Yazigi, A.; Sieck, J.O.; Dossing, M.; Saour, J.; Raines, D.A.; Ernst, P. Elimination studies of
antipyrine and its metabolites in healthy Saudi Arabians, Hum.Exp.Toxicol., 1994, 13, 658–662.
Mikati, M.A.; Szabo, G.K.; Pylilo, R.J.; LeDuc, B.W.; Browne, T.R.; Greenblatt, D.J. Improved highperformance liquid chromatographic assay of antipyrine, hydroxymethylantipyrine, 4-hydroxyantipyrine and norantipyrine in urine, J.Chromatogr., 1988, 433, 305–311.
Nakagawa, A.; Nakamura, K.; Ishizaki, T.; Chiba, K. Automated high-performance liquid chromatographic method for the determination of antipyrine and its metabolites in urine. Some preliminary
results obtained from smokers and non-smokers, J.Chromatogr., 1982, 231, 349–360.
St.Peter, J.V.; Awni, W.M. Modified high-performance liquid chromatographic assay for antipyrine and
its three major metabolites in urine, J.Chromatogr., 1989, 494, 424–427.
206
EDTA
EDTA
Molecular formula: C10 H16 N2 O8
COOH
HOOC
N
N
COOH
HOOC
Molecular weight: 292.24
CAS Registry No: 60-00-4
Merck Index: 13, 3546
SAMPLE
Matrix: blood
Sample preparation: Place 0.5 cm2 of a dried blood stain in 50–100 µL 25 mM copper(II) sulfate, let stand for 3 h, vortex, centrifuge at 3000–9000 rpm for 10 min, filter
(0.2 µm), inject a 25 µL aliquot. Alternatively, dilute 200 µL whole blood with 2 mL
water, mix with an equal volume of 50 mM copper(II) sulfate, centrifuge for 7 min, inject
an aliquot of the supernatant.
HPLC VARIABLES
Column: Hamilton PRP X-100
Mobile phase: MeOH:3 mM sulfuric acid 5:95
Flow rate: 2
Injection volume: 25
Detector: UV 243
CHROMATOGRAM
Retention time: 6
Limit of detection: 5 ppm
KEY WORDS
complexation; derivatization; dried blood; whole blood
REFERENCE
Miller, M.L.; McCord, B.R.; Martz, R.; Budowle, B. The analysis of EDTA in dried bloodstains by electrospray LC-MS-MS and ion chromatography, J.Anal.Toxicol., 1997, 21, 521–528.
SAMPLE
Matrix: blood
Sample preparation: Place 0.5 cm2 of a dried blood stain with 25 µL water in an
Ultrafree-MC centrifugal filter with a PTTK polysulfone membrane (cutoff 30 000 Da),
let stand for 45 min, centrifuge for 10 min, inject an aliquot of the filtrate.
HPLC VARIABLES
Column: 150 × 2.1 Hamilton PRP-1
Mobile phase: MeCN:water:ammonium hydroxide 80:20:0.03
Flow rate: 0.3
Detector: MS, Finnigan MAT TSQ700, triple-stage quadrupole, electrospray, collision
gas argon, positive ion mode, spray voltage 4 kV, sheath gas 90 psi, interface capillary
200◦ , collision offset – 20 V, m/z 293
CHROMATOGRAM
Retention time: 0.9
EDTA
207
KEY WORDS
dried blood
REFERENCE
Miller, M.L.; McCord, B.R.; Martz, R.; Budowle, B. The analysis of EDTA in dried bloodstains by electrospray LC-MS-MS and ion chromatography, J.Anal.Toxicol., 1997, 21, 521–528.
208
Efavirenz
Efavirenz
H
N
Molecular formula: C14 H9 ClF3 NO2
Molecular weight: 315.68
CAS Registry No: 154598-52-4
Merck Index: 13, 3552
Cl
O
O
F3C
SAMPLE
Matrix: blood
Sample preparation: Condition an Extrasep C18 SPE cartridge (Lida) with 2 mL
MeOH and 2 mL water. Dilute 500 µL serum with 500 µL water, add to the SPE
cartridge, wash with 500 µL water, elute with 1 mL MeOH. Evaporate the eluate to
dryness with vortexing under reduced pressure at 40◦ and reconstitute the residue with
300 µL MeOH, inject a 10 µL aliquot.
HPLC VARIABLES
Column: two 150 × 4.6 3 µm Luna C18 columns in series
Column temperature: 60
Mobile phase: Gradient. MeCN:4 mM sulfuric acid from 8:92 to 63:37 over 45 min,
maintain at 63:37 for 5 min.
Flow rate: 0.85
Injection volume: 10
Detector: UV 265 for 31 min then UV 240
CHROMATOGRAM
Retention time: 51
Limit of detection: 62 ng/mL
OTHER SUBSTANCES
Extracted: delavirdine (25.5, LOD 110 ng/mL), indinavir (24.5, LOD 210 ng/mL), nelfinavir (33.5, LOD 400 ng/mL), nevirapine (23.5, LOD 84 ng/mL), ritonavir (50.5, LOD
510 ng/mL), saquinavir (35, LOD 100 ng/mL)
KEY WORDS
serum; SPE
REFERENCE
Simon, V.A.; Thiam, M.D.; Lipford, L.C. Determination of serum levels of thirteen human immunodeficiency virus-suppressing drugs by high-performance liquid chromatography, J.Chromatogr.A, 2001,
913, 447–453.
SAMPLE
Matrix: blood
Sample preparation: Mix 1 mL plasma with 200 µL 10 µg/mL IS in water, add 200 µL
100 mM NaOH, mix, add 4 mL diethyl ether, shake for 5 min, centrifuge at 2500 rpm for
5 min. Evaporate the organic layer to dryness under a stream of nitrogen, reconstitute
the residue with 200 µL initial mobile phase, inject a 100 µL aliquot.
HPLC VARIABLES
Column: 250 × 4.6 Stability RP18 (CIL, France)
Mobile phase: Gradient. MeCN:50 mM pH 5.65 phosphate buffer from 36:64 to 64:36
over 25 min, to 80:20 (step gradient), maintain at 80:20 for 10 min, re-equilibrate at
initial conditions for 5 min.
Flow rate: 1.5
Efavirenz
209
Injection volume: 100
Detector: UV 240 for 5 min, UV 215 for 22 min, UV 260 for rest of run
CHROMATOGRAM
Retention time: 19.9
Internal standard: JR051012 (Janssen Cilag) (28.2)
Limit of quantitation: 50 ng/mL
OTHER SUBSTANCES
Extracted: amprenavir (11.2), indinavir (8.5), lopinavir (18.9), nelfinavir (24.1), nevirapine (3.3), ritonavir (17.6), saquinavir (16.7)
Noninterfering: acetaminophen, amineptine, amphotericin B, aspirin, bromazepam,
buspirone, citalopram, clobazam, diazepam, didanosine, fluconazole, flunitrazepam, fluvoxamine, hydroxyitraconazole, isoniazid, itraconazole, lamivudine, loprazolam, lorazepam, metronidazole, minalcipram, nordiazepam, omeprazole, paroxetine, pyrimethamine, rifampin, sertraline, stavudine, sulfadiazine, trimethoprim, venlafaxine, zalcitabine, zidovudine, zolpidem, zopiclone
KEY WORDS
plasma
REFERENCE
Titier, K.; Lagrange, F.; Péhourcq, F.; Edno-Mcheik, L.; Moore, N.; Molimard, M. High-performance liquid chromatographic method for the simultaneous determination of the six HIV-protease inhibitors
and two non-nucleoside reverse transcriptase inhibitors in human plasma, Ther.Drug Monit., 2002,
24, 417–424.
SAMPLE
Matrix: bulk, formulations
Sample preparation: Inject a 35 µL aliquot of a solution in MeCN:water 52.5:47.5.
HPLC VARIABLES
Column: 150 × 4.6 Zorbax SB-CN
Column temperature: 40
Mobile phase: Gradient. A:B from 40:60 to 50:50 over 16 min, to 65:35 over 7 min, to
70:30 over 5 min, to 80:20 over 1 min, maintain at 80:20 for 2 min, return to initial
conditions over 1 min, re-equilibrate for 8 min. A was MeOH:water:trifluoroacetic acid
90:10:0.05. B was MeOH:water:trifluoroacetic acid 10:90:0.05.
Flow rate: 1.5
Injection volume: 35
Detector: UV 250
CHROMATOGRAM
Retention time: 15
Limit of detection: 0.01%
Limit of quantitation: 0.05%
OTHER SUBSTANCES
Simultaneous: impurities
KEY WORDS
capsules; stability-indicating
210
Efavirenz
REFERENCE
Montgomery, E.R.; Edmanson, A.L.; Cook, S.C.; Hovsepian, P.K. Development and validation of a
reverse-phase HPLC method for analysis of efavirenz and its related substances in the drug substance
and in a capsule formulation, J.Pharm.Biomed.Anal., 2001, 25, 267–284.
ANNOTATED BIBLIOGRAPHY
Aarnoutse, R.E.; Grintjes, K.J.T.; Telgt, D.S.C.; Stek, M. Jr.; Hugen, P.W.H.; Reiss, P.; Koopmans, P.P.;
Hekster, Y.A.; Burger, D.M. The influence of efavirenz on the pharmacokinetics of a twice-daily
combination of indinavir and low-dose ritonavir in healthy volunteers, Clin.Pharmacol.Ther., 2002,
71, 57–67.
Aymard, G.; Legrand, M.; Trichereau, N.; Diquet, B. Determination of twelve antiretroviral agents in
human plasma sample using reversed-phase high-performance liquid chromatography, J.Chromatogr.B,
2000, 744, 227–240. [amprenavir; efavirenz; indinavir; nelfinavir; ritonavir; saquinavir; abacavir;
didanosine; lamivudine; stavudine; nevirapine; zidovudine]
Balani, S.K.; Kauffman, L.R.; deLuna, F.A.; Lin, J.H. Nonlinear pharmacokinetics of efavirenz (DMP266), a potent HIV-1 reverse transcriptase inhibitor, in rats and monkeys, Drug Metab.Dispos., 1999,
27, 41–45.
Cociglio, M.; Hillaire-Buys, D.; Peyriègre, H.; Alric, R. Performance analysis of a rapid HPLC determination with the solvent demixing extraction of HIV antiproteases and efavirenz in plasma,
J.Chromatogr.Sci., 2003, 41, 80–86. [efavirenz; indinavir; amprenavir; ritonavir; saquinavir; nelfinavir]
Fan, B.; Bartlett, M.G.; Stewart, J.T. Determination of lamivudine/stavudine/efavirenz in human serum
using liquid chromatography/electrospray tandem mass spectrometry with ionization polarity switch,
Biomed.Chromatogr., 2002, 16, 383–389. [aprobarbital is internal standard; SPE]
Faux, J.; Venisse, N.; Le Moal, G.; Dupuis, A.; Bouquet, S. Simultaneous determination of six HIV
protease inhibitors, one metabolite, and two non-nucleoside reverse transcriptase inhibitors in
human plasma by isocratic reversed-phase liquid chromatography after solid-phase extraction, Chromatographia, 2003, 58, 421–426. [amprenavir; indinavir; lopinavir; nelfinavir; ritonavir; saquinavir;
efavirenz; nevirapine; prazepam is internal standard; SPE]
Keil, K.; Frerichs, V.A.; DiFrancesco, R.; Morse, G. Reverse phase high-performance liquid chromatography method for the analysis of amprenavir, efavirenz, indinavir, lopinavir, nelfinavir and its active
metabolite (M8), ritonavir, and saquinavir in heparinized human plasma, Ther.Drug Monit., 2003, 25,
340–346.
Langmann, P.; Schirmer, D.; Väth, T.; Zilly, M.; Klinker, H. High-performance liquid chromatographic
method for the determination of HIV-1 non-nucleoside reverse transcriptase inhibitor efavirenz in
plasma of patients during highly active antiretroviral therapy, J.Chromatogr.B, 2001, 755, 151–156.
Lavison, G.; Thiébaut, D. Evaluation of a ristocetin bonded stationary phase for subcritical fluid chromatography of enantiomers, Chirality, 2003, 15, 630–636. [warfarin; efavirenz; chiral; thalidomide]
Marzolini, C.; Telenti, A.; Buclin, T.; Biollaz, J.; Decosterd, L.A. Simultaneous determination of the HIV
protease inhibitors indinavir, amprenavir, saquinavir, ritonavir, nelfinavir and the non-nucleoside
reverse transcriptase inhibitor efavirenz by high-performance liquid chromatography after solid-phase
extraction, J.Chromatogr.B, 2000, 740, 43–58.. [SPE]
Marzolini, C.; Béguin, A.; Telenti, A.; Schreyer, A.; Buclin, T.; Biollaz, J.; Decosterd, L.A. Determination
of lopinavir and nevirapine by high-performance liquid chromatography after solid-phase extraction:
application for the assessment of their transplacental passage at delivery, J.Chromatogr.B, 2002, 774,
127–140. [SPE; clozapine is internal standard]
Matthews, C.Z.; Woolf, E.J.; Mazenko, R.S.; Haddix-Wiener, H.; Chavez-Eng, C.M.; Constanzer, M.L.;
Doss, G.A.; Matuszewski, B.K. Determination of efavirenz, a selective non-nucleoside reverse transcriptase inhibitor, in human plasma using HPLC with post-column photochemical derivatization and
fluorescence detection, J.Pharm.Biomed.Anal., 2002, 28, 925–934.
Maurin, M.B.; Rowe, S.M.; Blom, K.; Pierce, M.E. Kinetics and mechanism of hydrolysis of efavirenz,
Pharm.Res., 2002, 19, 517–521.
Mutlib, A.E.; Chen, H.; Nemeth, G.; Gan, L.; Christ, D.D. Liquid chromatography/mass spectrometry
and high-field nuclear magnetic resonance characterization of novel mixed diconjugates of the
non-nucleoside human immunodeficiency virus-1 reverse transcriptase inhibitor, efavirenz, Drug
Metab.Dispos., 1999, 27, 1045–1056.
Poirier, J.-M.; Robidou, P.; Jaillon, P. Simultaneous determination of the six HIV protease inhibitors
(amprenavir, indinavir, lopinavir, nelfinavir, ritonavir, and saquinavir) plus M8 nelfinavir metabolite
Efavirenz
211
and the nonnucleoside reverse transcription inhibitor efavirenz in human plasma by solid-phase
extraction and column liquid chromatography, Ther.Drug Monit., 2002, 24, 302–309.
Proust, V.; Toth, K.; Hulin, A.; Taburet, A.-M.; Gimenez, F.; Singlas, E. Simultaneous high-performance
liquid chromatographic determination of the antiretroviral agents amprenavir, nelfinavir, ritonavir,
saquinavir, delavirdine and efavirenz in human plasma, J.Chromatogr.B, 2000, 742, 453–458.
Rentsch, K.M. Sensitive and specific determination of eight antiretroviral agents in plasma by highperformance liquid chromatography-mass spectrometry, J.Chromatogr.B, 2003, 788, 339–350. [SPE;
amprenavir; efavirenz; indinavir; lopinavir; nelfinavir; nevirapine; ritonavir; saquinavir]
Rezk, N.L.; Tidwell, R.R.; Kashuba, A.D.M. Simple and rapid quantification of the non-nucleoside reverse
transcriptase inhibitors nevirapine, delavirdine, and efavirenz in human blood plasma using highperformance liquid chromatography with ultraviolet absorbance detection, J.Chromatogr.B, 2002, 774,
79–88. [SPE]
Sarasa-Nacenta, M.; López-Púa, Y.; López-Cortés, L.F.; Mallolas, J.; Gatell, J.M.; Carné, X. Determination of efavirenz in human plasma by high-performance liquid chromatography with ultraviolet
detection, J.Chromatogr.B, 2001, 763, 53–59.
Simon, V.A.; Thiam, M.D.; Lipford, L.C. Determination of serum levels of thirteen human immunodeficiency virus-suppressing drugs by high-performance liquid chromatography, J.Chromatogr.A,
2001, 913, 447–453. [zalcitabine; lamivudine; stavudine; didanosine; zidovudine; nevirapine; abacavir;
indinavir; delavirdine; nelfinavir; saquinavir; ritonavir; efavirenz]
Störmer, E.; von Moltke, L.L.; Perloff, M.D.; Greenblatt, D.J. Differential modulation of P-glycoprotein
expression and activity by non-nucleoside HIV-1 reverse transcriptase inhibitors in cell culture,
Pharm.Res., 2002, 19, 1038–1045. [verapamil; efavirenz; nevirapine; delavirdine]
Tribut, O.; Arvieux, C.; Michelet, C.; Chapplain, J.-M.; Allain, H.; Bentué-Ferrer, D. Simultaneous quantitative assay of six HIV protease inhibitors, one metabolite, and two non-nucleoside reverse transcriptase inhibitors in human plasma by isocratic reversed-phase liquid chromatography, Ther.Drug Monit.,
2002, 24, 554–562. [nevirapine; efavirenz; indinavir; amprenavir; nelfinavir; ritonavir; lopinavir;
saquinavir]
Turner, M.L.; Reed-Walker, K.; King, J.R.; Acosta, E.P. Simultaneous determination of nine antiretroviral compounds in human plasma using liquid chromatography, J.Chromatogr.B, 2003, 784, 331–341.
[indinavir; nelfinavir; saquinavir; ritonavir; amprenavir; delavirdine; efavirenz; lopinavir]
Usami, Y.; Oki, T.; Nakai, M.; Sagisaka, M.; Kaneda, T. A simple HPLC method for simultaneous determination of lopinavir, ritonavir and efavirenz, Chem.Pharm.Bull., 2003, 26, 715–718.
Veldkamp, A.I.; van Heeswijk, R.P.G.; Meenhorst, P.L.; Mulder, J.W.; Lange, J.M.A.; Beijnen, J.H.;
Hoetelmans, R.M.W. Quantitative determination of efavirenz (DMP 266), a novel non-nucleoside
reverse transcriptase inhibitor, in human plasma using isocratic reversed-phase high-performance
liquid chromatography with ultraviolet detection, J.Chromatogr.B, 1999, 734, 55–61.
Villani, P.; Pregnolato, M.; Banfo, S.; Rettani, M.; Burroni, D.; Seminari, E.; Maserati, R.; Regazzi, M.B.
High-performance liquid chromatography method for analyzing the antiretroviral agent efavirenz in
human plasma, Ther.Drug Monit., 1999, 21, 346–350. [saquinavir is internal standard]
Villani, P.; Feroggio, M.; Gianelli, L.; Bartoli, A.; Montagna, M.; Maserati, R.; Regazzi, M.B. Antiretrovirals: simultaneous determination of five protease inhibitors and three nonnucleoside transcriptase
inhibitors in human plasma by a rapid high-performance liquid chromatography-mass spectrometry
assay, Ther.Drug Monit., 2001, 23, 380–388. [saquinavir; indinavir; ritonavir; nelfinavir; amprenavir;
nevirapine; delavirdine; efavirenz]
Ward, B.A.; Gorski, J.C.; Jones, D.R.; Hall, S.D.; Flockhart, D.A.; Desta, Z. The cytochrome P450 2B6
(CYP2B6) is the main catalyst of efavirenz primary and secondary metabolism: implication for
HIV/AIDS therapy and utility of efavirenz as a substrate marker of CYP2B6 catalytic activity,
J.Pharmacol.Exp.Ther., 2003, 306, 287–300.
Weissburg, R.P.; Montgomery, E.R.; Junnier, L.A.; Segretario, J.; Cook, S.; Hovsepian, P.K. Investigation
of critical factors for the resolution of SR695, a key impurity, from efavirenz in the reversed-phase
assay of efavirenz dosage forms, J.Pharm.Biomed.Anal., 2002, 28, 45–56.
Xu, J.Q.; Aubry, A.-F. Impurity profiling of non-nucleoside reverse transcriptase inhibitors by HPLC
using a porous graphitic carbon stationary phase, Chromatographia, 2003, 57, 67–71.
212
Efrotomycin
Efrotomycin
Molecular formula:
C59 H88 N2 O20
Molecular weight:
1145.33
CAS Registry No:
56592-32-6
Merck Index:
13, 3556
HO
CH3O
H3C
OCH3
O
O
H3C
O
OCH3
OH O
CH3
CH3
CH3
OH
CH3
H
OCH3
O
N
O
OH
O
CH3
CH3
OH OH
O
CH3
HO
O
N
CH3
SAMPLE
Matrix: feed
Sample preparation: Condition a 2.8 mL Bond-Elut NH2 SPE cartridge with 1 mL
MeCN:dichloromethane 50:50 saturated with 50 mM pH 7.5 potassium phosphate
buffer. Shake 100 g blended homogenized feed with 320 mL 50 mM pH 7.5 potassium
phosphate buffer in a 950 mL amber bottle for 30 min, add 280 mL MeCN, shake for
15 min, centrifuge a 30 mL aliquot at 2500 rpm for 10 min. Remove a 5 mL aliquot
and add it to 10 mL dichloromethane and 20 mL MeCN:dichloromethane 50:50 saturated with 50 mM pH 7.5 potassium phosphate buffer, shake for 30 min, centrifuge at
2500 rpm for 10 min. Add 10 mL of the lower phase to the SPE cartridge, wash with
2 mL MeOH, wash with 2 mL ethyl acetate, wash with 2 mL hexane, draw air through
the cartridge for 5 min, elute with 1 mL elution solvent, inject a 100 µL aliquot of the
eluate. (The elution solvent was 250 mM pH 5 phosphate buffer containing 1.5 M LiCl
saturated with MeCN. Prepare by mixing 500 mL of this aqueous phase with 250 mL
MeCN.)
HPLC VARIABLES
Column: 150 × 4.6 Zorbax C8
Column temperature: 55
Mobile phase: MeCN:MeOH:water:85% phosphoric acid 32.5:22.5:57.5:1 (Adjust with
MeCN or water to give a k’ of 19–22.)
Flow rate: 2
Injection volume: 100
Detector: UV 335
CHROMATOGRAM
Retention time: 10
Limit of quantitation: 2 ppm
KEY WORDS
SPE
REFERENCE
Stong, J.D. Determination of efrotomycin in feeds by high-performance liquid chromatography, Analyst,
1986, 111, 853–855.
Egualen
Egualen
213
CH3
CH3
SO3H
Molecular formula: C15 H18 O3 S
Molecular weight: 278.37
CAS Registry No: 99287-30-6
CH3
SAMPLE
Matrix: blood
Sample preparation: Prepare plasma using a Sep-Pak C18 SPE cartridge (no further
details).
HPLC VARIABLES
Column: 250 × 4.6 TSK-gel ODS-80TM
Mobile phase: Gradient. MeCN:20 mM pH 6.0 phosphate buffer 20:80 for 10 min, to
30:70 over 10 min, maintain at 30:70 for 15 min.
Flow rate: 1
Detector: Radioactivity (14 C)
OTHER SUBSTANCES
Extracted: metabolites
KEY WORDS
pharmacokinetics; plasma; SPE
REFERENCE
Sato, M.; Suzaka, H.; Miyazaki, H. Sex-related differences in urinary excretion of egualen sodium in rats,
Drug Metab.Dispos., 2000, 28, 21–27.
214
Eletriptan
Eletriptan
H
N
Molecular formula: C22 H26 N2 O2 S
Molecular weight: 382.53
CAS Registry No: 143322-58-1
Merck Index: 13, 3577
N
S
O
O
CH3
SAMPLE
Matrix: blood, saliva
Sample preparation: Mix 570 µL plasma or saliva with 130 µL 1 (plasma) or 0.1
(saliva) M monochloroacetic acid containing 500 ng/mL IS. Place 690 µL of this mixture
in the donor channel of a dialyzer fitted with a15 kDa cuprophan membrane (regenerated
cellulose, Enka, Germany), pass 3500 µL recipient solvent in 500 µL pulsed bursts
through the acceptor channel over 4 min, pass the recipient solvent through column
A, wash column A with 200 µL MeCN:water 60:40, wash with 800 µL donor solvent,
backflush the contents of column A onto column B with the mobile phase, monitor the
effluent from column B. (The donor solvent was 10 mM pH 7.0 potassium phosphate
buffer. Recipient solvent was MeOH:10 mM pH 7.0 potassium phosphate buffer 10:90.
After each run, purge the donor channel with 1.5 mL donor solvent. Purge the HPLC
system with 14 mL of donor and recipient solvent. Condition column A with 200 µL
recipient solvent.)
HPLC VARIABLES
Column: A 5 × 4.6 10 µm Hypersil C1; B 100 × 4.6 5 µm Kromasil C1
Mobile phase: MeCN:500 mM pH 3.5 potassium phosphate buffer:water 30:6:64 (Add
20 mM diethylamine hydrochloride to the buffer/water mixture before adding the
MeCN.)
Flow rate: 1
Detector: UV 225
CHROMATOGRAM
Retention time: 5
Internal standard: UK-136,509 (7)
Limit of quantitation: 0.5 ng/mL
OTHER SUBSTANCES
Extracted: metabolite
KEY WORDS
column-switching; dialysis; pharmacokinetics; plasma
REFERENCE
Cooper, J.D.H.; Muirhead, D.C.; Taylor, J.E. Determination of eletriptan in plasma and saliva using
automated sequential trace enrichment of dialysate and high-performance liquid chromatography,
J.Pharm.Biomed.Anal., 1999, 21, 787–796.
215
Emtricitabine
Emtricitabine
Molecular formula: C8 H10 FN3 O3 S
Molecular weight: 247.25
CAS Registry No: 143491-57-0
Merck Index: 13, 3597
NH2
F
HO
S
N
N
O
O
SAMPLE
Matrix: blood, CSF, urine
Sample preparation: Plasma. Mix 100 µL serum, 50 µL 20 µg/mL IS, and 50 µL 2 M
perchloric acid, centrifuge at 5000 g for 5 min, add 50 µL 2 M KOH, mix well, centrifuge
at 5000 g for 5 min, inject a 15–200 µL aliquot. CSF. Mix 100 µL CSF with 20 µL IS
solution and 80 µL water, inject a 100 µL aliquot. Urine. Dilute 50 µL urine and 100 µL
IS solution to 1 mL with water, inject a 15–100 µL aliquot.
HPLC VARIABLES
Column: 150 × 4.6 5 µm Hypersil octadecanoylsulfate (sic) (Alltech)
Mobile phase: MeOH:40 mM pH 2.5 potassium phosphate buffer 5.5:94.5
Flow rate: 2
Injection volume: 15–200
Detector: UV 279
CHROMATOGRAM
Retention time: 5.5
Internal standard: 3′ deoxy-2′ ,3′ -didehydrothymidine (9.5)
Limit of quantitation: 100 ng/mL
OTHER SUBSTANCES
Extracted: 2,3′ -dideoxy-5-fluoro-3′ -thiauridine (FTU) (8.3)
KEY WORDS
monkey; pharmacokinetics; plasma
REFERENCE
Schinazi, R.F.; Boudinot, F.D.; Ibrahim, S.S.; Manning, C.; McClure, H.M.; Liotta, D.C. Pharmacokinetics and metabolism of racemic 2′ ,3′ -dideoxy-5-fluoro-3′ -thiacytidine in rhesus monkeys, Antimicrob.Agents Chemother., 1992, 36, 2432–2438.
SAMPLE
Matrix: cell cultures
Sample preparation: Extract cells twice with 4 mL portions of MeOH:water 60:40
at – 70◦ overnight, centrifuge at 2000 g for 10 min. Evaporate the methanol from a
500 µL aliquot under a stream of nitrogen, adjust the volume to 200 µL with water,
inject a 180 µL aliquot.
HPLC VARIABLES
Column: 250 × 4.6 10 µm Partisil SAX
Mobile phase: Gradient. A:B 100:0 for 10 min, to 0:100 over 65 min. A was 8 mM pH
3.5 potassium phosphate buffer. B was 1 M pH 3.5 potassium phosphate buffer.
Flow rate: 1
Injection volume: 180
Detector: Radioactivity (3 H)
216
Emtricitabine
CHROMATOGRAM
Retention time: 5
OTHER SUBSTANCES
Extracted: emtricitabine monophosphate (25), emtricitabine diphosphate (40), emtricitabine triphosphate (57)
KEY WORDS
peripheral blood mononuclear cells
REFERENCE
Darque, A.; Valette, G.; Rousseau, F.; Wang, L.H.; Sommadossi, J.-P.; Zhou, X.-J. Quantitation of intracellular triphosphate for emtricitabine in peripheral blood mononuclear cells from human immunodeficiency virus-infected patients, Antimicrob.Agents Chemother., 1999, 43, 2245–2250.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 × 4.6 10 µm Chiralpak AS
Mobile phase: Isopropanol
Flow rate: 0.8
Detector: UV 270
CHROMATOGRAM
Retention time: 5.9 (emtricitabine), 9.5 ((+)-enantiomer)
KEY WORDS
chiral
REFERENCE
Schinazi, R.F.; McMillan, A.; Cannon, D.; Mathis, R.; Lloyd, R.M.; Peck, A.; Sommadossi, J.-P.; St. Clair,
M.; Wilson, J.; Furman, P.A.; Painter, G.; Choi, W.-B.; Liotta, D.C. Selective inhibition of human
immunodeficiency viruses by racemates and enantiomers of cis-5-fluoro-1-[2-(hydroxymethyl)-1,3oxathiolan-5-yl]cytosine, Antimicrob.Agents Chemother., 1992, 36, 2423–2431.
ANNOTATED BIBLIOGRAPHY
Cass, Q.B.; Watanabe, C.S.F.; Rabi, J.A.; Bottari, P.Q.; Costa, M.R.; Nascimento, R.M.; Cruz, J.E.D.;
Ronald, R.C. Polysaccharide-based chiral phase under polar organic mode of elution in the determination of the enantiomeric purity of emtricitabine an anti-HIV analogue nucleoside, J.Pharm.Biomed.
Anal., 2003, 33, 581–587.
Paff, M.T.; Averett, D.R.; Prus, K.L.; Miller, W.H.; Nelson, D.J. Intracellular metabolism of (-) and (+)cis-5-fluoro-1-[2-(hydroxymethyl)- 1,3-oxathiolan-5-yl]cytosine in HepG2 derivative 2.2.15 (Subclone
P5A) cells, Antimicrob.Agents Chemother., 1994, 38, 1230–1238.
Enoxaparin sodium
217
Enoxaparin sodium
Molecular weight: ca. 4500
CAS Registry No: 9041-08-1
Merck Index: 13, 3621
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 300 × 3.2 Superdex (Pharmacia PC 3.2/30)
Mobile phase: 200 mM NaCl
Flow rate: 0.1
Detector: Refractive Index
CHROMATOGRAM
Retention time: 12–16
REFERENCE
Intes, O.; Renault, J.-H.; Sinquin, C.; Zèches-Hanrot, M.; Nuzillard, J.-M. Fractionation of low-molecularmass heparin by centrifugal partition chromatography in the ion-exchange displacement mode,
J.Chromatogr.A, 2001, 918, 47–57.
218
Entacapone
Entacapone
Molecular formula: C14 H15 N3 O5
Molecular weight: 305.29
CAS Registry No: 130929-57-6
Merck Index: 13, 3626
O
HO
N
CN
HO
CH3
CH3
NO2
SAMPLE
Matrix: blood, urine
Sample preparation: Plasma. Add 50 µL 50 mM pH 7.2 phosphate buffer to 1 mL
plasma, vortex, add 100 µL 2 M HCl, add 6 mL n-hexane:ethyl acetate 50:50, vortex for
2 min, centrifuge at 3500 g for 5 min. Remove 5 mL of the organic layer, evaporate it to
dryness under a stream of nitrogen at 40◦ , reconstitute the residue with 500 µL DMSO,
inject a 20 µL aliquot. Urine. Add 50 µL 50 mM pH 7.2 phosphate buffer to 1 mL urine,
vortex, add 100 µL 2 M HCl, vortex, add 5 mL n-hexane:ethyl acetate 75:25, vortex for
2 min, centrifuge at 3500 g for 5 min. Remove 4 mL of the organic layer and add it to
1 mL 50 mM pH 7.2 phosphate buffer, vortex for 2 min, centrifuge at 3500 g for 5 min,
inject a 30 µL aliquot of the aqueous layer. (Protect from light during preparation.)
HPLC VARIABLES
Guard column: µBondapak Guard-PAC C18
Column: 250 × 4 10 µm Lichrosorb C18
Column temperature: 35 (plasma), 28 (urine)
Mobile phase: MeOH:THF:buffer A 50:5:63 (plasma) or MeCN:THF:buffer B 50:5:125
(urine) (Buffer A was 50 mM sodium dihydrogen phosphate containing 20 mM citric
acid and 0.25 mM EDTA, adjusted to pH 2.0 with phosphoric acid. Buffer B was
50 mM sodium dihydrogen phosphate containing 20 mM citric acid and 0.25 mM EDTA,
adjusted to pH 3.0 with 10 M NaOH.)
Flow rate: 1.5
Injection volume: 20 (plasma), 30 (urine)
Detector: E, Bioanalytical Systems LC-4B, glassy carbon electrode 700 mV, Ag/AgCl
reference electrode
CHROMATOGRAM
Retention time: 7.5 (plasma), 10.7 (urine)
Limit of quantitation: 10 ng/mL
OTHER SUBSTANCES
Extracted: Z-isomer (5.3 (plasma), 9.1 (urine))
Noninterfering: carbidopa, 3,4-dihydroxyphenylacetic acid, homovanillic acid, levodopa, 3-O-methyldopa
KEY WORDS
pharmacokinetics; plasma
REFERENCE
Karlsson, M.; Wikberg, T. Liquid chromatographic determination of a new catechol-O-methyltransferase
inhibitor, entacapone, and its Z-isomer in human plasma and urine, J.Pharm.Biomed.Anal., 1992, 10,
593–600.
ANNOTATED BIBLIOGRAPHY
Keski-Hynnilä, H.; Kurkela, M.; Elovaara, E.; Antonio, L.; Magdalou, J.; Luukkanen, L.; Taskinen, J.;
Kostiainen, R. Comparison of electrospray, atmospheric pressure chemical ionization, and atmospheric
pressure photoionization in the identification of apomorphine, dobutamine, and entacapone phase II
Entacapone
219
metabolites in biological samples, Anal.Chem., 2002, 74, 3449–3457. [urine; microsomal incubations;
entacapone metabolites only]
Luukkanen, L.; Kilpelainen, I.; Kangas, H.; Ottoila, P.; Elovaara, E.; Taskinen, J. Enzyme-assisted synthesis and structural characterization of nitrocatechol glucuronides, Bioconjug.Chem., 1999, 10,
150–154. [HPLC of metabolites only]
Wikberg, T.; Ottoila, P.; Taskinen, J. Identification of major urinary metabolites of the catechol-Omethyltransferase inhibitor entacapone in the dog, Eur.J.Drug Metab.Pharmacokinet., 1993, 18,
359–367.
Wikberg, T.; Vuorela, A.; Ottoila, P.; Taskinen, J. Identification of major metabolites of the catechol-Omethyltransferase inhibitor entacapone in rats and humans, Drug Metab.Dispos., 1993, 21, 81–92.
Wikberg, T.; Vuorela, A. Metabolite profiles of two [14 C]-labelled catechol O-methyltransferase inhibitors,
nitecapone and entacapone, in rat and mouse urine and rat bile, Eur.J.Drug Metab.Pharmacokinet.,
1994, 19, 125–135. [HPLC of metabolites only]
220
Eperisone
Eperisone
Molecular formula: C17 H25 NO
O
N
H3C
CH3
Molecular weight: 259.39
CAS Registry No: 64840-90-0
Merck Index: 13, 3637
SAMPLE
Matrix: blood
Sample preparation: Mix 1 mL water, 100 µL 1 µg/mL IS in MeOH, and 1 mL 1 M
pH 7.2 phosphate buffer with 100 µL plasma, add 4 mL diethyl ether, extract, repeat
extraction. Combine the organic layers and add them to 200 µL 100 mM HCl, extract,
inject a 70 µL aliquot of the aqueous layer.
HPLC VARIABLES
Column: 250 × 4.6 YMC pack A-303
Mobile phase: MeCN:5 mM pH 2.5 sodium dodecyl sulfate 50:50
Flow rate: 1
Injection volume: 70
Detector: UV 256
CHROMATOGRAM
Internal standard: tolperisone HCl
Limit of quantitation: 2 ng/mL
KEY WORDS
plasma
REFERENCE
Matsunaga, M.; Uemura, Y.; Yonemoto, Y.; Kanai, K.; Etoh, H.; Tanaka, S.; Atsuta, Y.; Nishizawa, Y.;
Yamanishi, Y. Long-lasting muscle relaxant activity of eperisone hydrochloride after percutaneous
administration in rats, Jpn.J.Pharmacol., 1997, 73, 215–220.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 × 4.6 10 µm Cosmosil 5C18-MS
Column temperature: 50
Mobile phase: Gradient. MeCN:10 mM pH 4.2 potassium phosphate buffer 0:100 for
2 min, to 75:25 over 7.5 min, maintain at 75:25 for 5.5 min. Isocratic. MeCN:buffer 20:80
Flow rate: 1.5
Detector: UV (wavelength not specified)
CHROMATOGRAM
Retention time: 8.7 (gradient) or 5.8 (isocratic)
OTHER SUBSTANCES
Simultaneous: acetazolamide (7.9), acyclovir (7.0), allopurinol (7.3), aniracetam (8.6),
benazepril (11.5), betamethasone (10.8), camostat mesylate (8.8), carbamazepine (10.8),
cilazapril (10.7), cimetidine (7.5), clofibrate (15.0), clonazepam (11.3), cyclophosphamide
(9.9), delapril (11.9), dexamethasone (10.9), diazepam (12.5), digoxin (9.0), dilazep
(10.6), diltiazem (10.2), docarpamine (11.4), ethosuximide (9.0), fenbufen (11.8), fluconazole (9.2), flutamide (12.7), fominoben (12.6), hydrocortisone acetate (12.1), imidapril
Eperisone
221
(10.1), indomethacin (12.6), irinotecan (9.2), maprotiline (10.5), methotrexate (8.0),
nefiracetam (9.7), nifedipine (11.9), nitrazepam (11.2), pentobarbital (10.9), phenobarbital (10.0), phenytoin (10.8), pindolol (8.3), pranlukast (13.4), pranoprofen (10.4),
prednisolone (10.3), primidone (8.9), quinapril (10.5), spironolactone (12.4), sulpiride
(7.6), sulthiame (9.3), tolbutamide (11.6), tranilast (10.5), triamcinolone (11.2), warfarin
(12.0), zonisamide (9.5) (gradient retention times; isocratic conditions may differ)
REFERENCE
Sugiyama, T.; Matsuyama, R.; Usui, S.; Katagiri, Y.; Hirano, K. Selection of mobile phases in highperformance liquid chromatographic determination for medicines, Biol.Pharm.Bull., 2000, 23, 274–278.
ANNOTATED BIBLIOGRAPHY
Nakamura, K.; Fujima, H.; Kitagawa, H.; Wada, H.; Makino, K. Preparation and chromatographic characteristics of a chiral-recognizing perphenylated cyclodextrin column, J.Chromatogr.A, 1995, 694,
111–118. [ibuprofen; chlorpheniramine; acetylpheneturide; alprenolol; arotinolol; atenolol; benzoin;
biperiden; bunitrolol; chlormezanone; chlorphenesin; eperisone; flavanone; oxprenolol; phenylethyl
alcohol; phenylethylamine; pindolol; proglumide; propranolol; trihexyphenidyl]
222
Eplerenone
Eplerenone
CH3
CH3 OH
Molecular formula: C24 H30 O6
Molecular weight: 414.49
CAS Registry No: 107724-20-9
O
O
H
O
CO2CH3
SAMPLE
Matrix: urine
Sample preparation: Urine. Condition a 1 mL 100 mg Bond Elut C18 SPE cartridge
with 2 mL MeCN and 2 mL water. Vortex 500 µL urine and 500 µL 1 µg/mL IS in
20 mM pH 7.4 ammonium acetate, add to the SPE cartridge, wash with 3 mL water,
elute with 250 µL MeCN. Vortex the eluate with 250 µL 20 mM pH 7.4 ammonium
acetate buffer, inject a 20 µL aliquot. Plasma. Condition a 1 mL 100 mg Bond Elut C18
SPE cartridge with 2 mL MeOH and 2 mL water. Centrifuge plasma at 2000 g at 4◦ for
10 min. Vortex 400 µL of the supernatant and 400 µL 500 ng/mL IS in water, add to the
SPE cartridge, wash with 2 mL water, elute with 500 µL MeCN. Evaporate the eluate
to dryness under a stream of nitrogen at room temperature, reconstitute the residue
with 100 µL mobile phase, inject a 10 µL aliquot. (Plasma preparation from Zhang,J.Y.;
Fast,D.M.; Breau,A.P. Development and validation of a liquid chromatography-tandem
mass spectrometric assay for Eplerenone and its hydrolyzed metabolite in human
plasma. J.Chromatogr.B 2003, 787, 333–344.)
HPLC VARIABLES
Column: 50 × 2.1 5 µm Zorbax XDB-C8
Mobile phase: MeCN:water 40:60 containing 10 mM ammonium acetate, pH 7.4
Flow rate: 0.1
Injection volume: 20 (urine), 10 (plasma)
Detector: MS, PE Sciex API-III Plus quadrupole, ionspray, positive ionization, ionspray
interface 4400 V, orifice 67 V, nebulizer gas nitrogen at 60 psi, curtain gas nitrogen at
1.8 L/min, collision gas argon, m/z 415–163
CHROMATOGRAM
Retention time: 3.2
Internal standard: 13 C2 H3 -eplerenone (m/z 419–163)
Limit of quantitation: 50 ng/mL (urine), 10 ng/mL (plasma)
OTHER SUBSTANCES
Extracted: metabolite
KEY WORDS
pharmacokinetics; SPE
REFERENCE
Zhang, J.Y.; Fast, D.M.; Breau, A.P. A validated SPE-LC-MS/MS assay for Eplerenone and its hydrolyzed
metabolite in human urine, J.Pharm.Biomed.Anal., 2003, 31, 103–115.
ANNOTATED BIBLIOGRAPHY
Cook, C.S.; Berry, L.M.; Kim, D.H.; Burton, E.G.; Hribar, J.D.; Zhang, L. Involvement of CYP3A in the
metabolism of eplerenone in humans and dogs: Differential metabolism by CYP3A4 and CYP3A5,
Drug Metab.Dispos., 2002, 30, 1344–1351. [radioactivity detection (14 C); LC-MS]
Cook, C.S.; Zhang, L. Atypical dose-route-dependent food effects of eplerenone in the dog: Presence of
food effects following intravenous dosing and lack of food effects of following oral dosing, J.Pharm.Sci.,
2002, 91, 607–614.
Eplerenone
223
Cook, C.S.; Berry, L.M.; Bible, R.H.; Hribar, J.D.; Hajdu, E.; Liu, N.W. Pharmacokinetics and metabolism
of [14 C]eplerenone after oral administration to humans, Drug Metab.Dispos., 2003, 31, 1448–1455.
[radioactivity detection; LC-MS]
Cook, C.S.; Zhang, L.; Ames, G.B.; Fischer, J.; Zhang, J.; Levin, S. Single-and repeated-dose pharmacokinetics of eplerenone, a selective aldosterone receptor blocker, in rats, Xenobiotica, 2003, 33, 305–321.
[LC-MS; SPE]
224
Epoprostenol
Epoprostenol
HOOC
O
Molecular formula: C20 H32 O5
Molecular weight: 352.46
CAS Registry No: 35121-78-9, 61849-14-7 (Na salt)
Merck Index: 13, 7967
H
H
CH3
OH
OH
SAMPLE
Matrix: blood
Sample preparation: Pack a polypropylene column with 1 g Hi-Flosil (80/100) C18
material (Applied Science) in an MeCN slurry, add 5 mL 10% dimethyldichlorosilane
in toluene, wash with 15 mL toluene, wash with 15 mL MeCN. Just prior to use,
equilibrate with 15 mL 2 mM sodium borate adjusted to pH 10 with NaOH. Mix 1 mL
whole blood with 10 mg/mL sodium carbonate solution, keep on ice, centrifuge at 2000 g
at 4◦ for 10 min, add a 500 µL aliquot to the SPE column, wash with two 3 mL portions
of 2 mM pH 10 sodium borate, elute with three 3 mL portions of MeCN:2 mM pH 10
sodium borate 40:60. Collect the eluate in a container containing 200 µL 200 mM NaOH.
Evaporate the eluate to dryness under reduced pressure at 40◦ , reconstitute the residue
with 1 mL 2 mM pH 9.1 sodium borate, inject a 100 µL aliquot.
HPLC VARIABLES
Column: 250 × 4 MCH-10
Mobile phase: Gradient. MeCN:2 mM pH 9.1 sodium borate from 12:88 to 27:73 over
7 min.
Flow rate: 2
Injection volume: 100
Detector: UV 200; Radioactivity (3 H)
CHROMATOGRAM
Retention time: 6
OTHER SUBSTANCES
Extracted: 6-ketoprostaglandin F1α (2)
KEY WORDS
SPE; whole blood
REFERENCE
Skrinska, V.; Lucas, F.V. Isolation of prostacyclin from whole blood, Prostaglandins, 1981, 22, 365–375.
Eprosartan
Eprosartan
225
N
H3C
Molecular formula: C23 H24 N2 O4 S
Molecular weight: 424.52
CAS Registry No: 133040-01-4
Merck Index:: 13, 3664
S
N
COOH
COOH
SAMPLE
Matrix: blood
Sample preparation: Condition a 1 mL 100 mg phenyl SPE cartridge (Analytichem)
with 2 mL MeOH and 2 mL water. Add 500 µL 100 mM pH 3.5 citrate buffer to 500 µL
plasma containing 200 ng IS, vortex briefly, centrifuge at 8800 g for 5 min. Add the
supernatant to the SPE cartridge, wash with 2 mL 50 mM acetic acid, dry the cartridge
by passing air through it for 45 s, wash with 1 mL ethyl acetate containing 0.1%
triethylamine, dry with air for 45 s, elute with 2 mL MeOH:50 mM acetic acid 90:10.
Evaporate the eluate to dryness under nitrogen at 45◦ , reconstitute the residue with
125 µL mobile phase, vortex, centrifuge at 1875 g for 10 min, inject a 50 µL aliquot of
the supernatant.
HPLC VARIABLES
Guard column: 20 × 2 5 µm BDS-Hypersil C18
Column: 150 × 2 5 µm BDS-Hypersil C18
Mobile phase: THF:50 mM pH 3.5 citrate buffer 32:68
Flow rate: 0.25
Injection volume: 50
Detector: UV 300
CHROMATOGRAM
Retention time: 9
Internal standard: SB-200062 [(E)-3-[2-butyl-1-[(4-carboxy-phenyl)methyl]-1H-imidazol-5-yl]-2-[(2-thienyl)-ethyl]propenoic acid] (12.3)
Limit of quantitation: 10 ng/mL
KEY WORDS
pharmacokinetics; plasma; SPE
REFERENCE
Lundberg, D.E. Jr.; Person, C.R.; Knox, S.; Cyronak, M.J. Determination of SK&F 108566 (Teveten) in
human plasma by reversed-phase high-performance liquid chromatography, J.Chromatogr.B, 1998,
707, 328–333.
SAMPLE
Matrix: urine
Sample preparation: Mix 100 µL urine with 200 µL 500 ng/mL IS in MeCN, vortex,
centrifuge at 11 000 g, inject a 1 µL aliquot of the supernatant.
HPLC VARIABLES
Column: 20 × 2 3 µm Hypersil APS-2 amino
Mobile phase: MeCN:10 mM pH 3.0 ammonium formate 80:20
Flow rate: 0.25
Injection volume: 1
Detector: MS, PE Sciex API-III Plus, ionspray, positive ion mode, split column effluent
so 50 µL/min enters MS
226
Eprosartan
CHROMATOGRAM
Retention time: 0.42
Internal standard: unspecified (0.44)
Limit of quantitation: 50 ng/mL
KEY WORDS
SPE
REFERENCE
Martin, D.E.; Chapelsky, M.C.; Ilson, B.; Tenero, D.; Boike, S.C.; Zariffa, N.; Jorkasky, D.K. Pharmacokinetics and protein binding of eprosartan in healthy volunteers and in patients with varying degrees of
renal impairment, J.Clin.Pharmacol., 1998, 38, 129–137.
227
Eptazocine
Eptazocine
Molecular formula: C15 H21 NO
Molecular weight:: 231.33
CAS Registry No: 72522-13-5
Merck Index:: 13, 3668
H
N
CH3
HO
CH3
SAMPLE
Matrix: blood
Sample preparation: Add 100 µL 100 ng/mL IS in MeOH to 50 µL plasma, add 300 µL
water, add 20 µL 25% ammonium hydroxide, vortex gently for 1 min, add 400 µL ethyl
acetate, vortex for 5 min, centrifuge at 10 000 g for 10 min. Evaporate the organic layer
to dryness under a stream of nitrogen at 38◦ , reconstitute the residue with 200 µL
MeOH, inject a 20 µL aliquot.
HPLC VARIABLES
Column: 150 × 4.6 5 µm Cosmosil 5C18-MS
Column temperature: 25
Mobile phase: MeOH:70 mM pH 3.0 sodium phosphate buffer containing 5 mM sodium
heptylsulfonate 45:55
Flow rate: 0.8
Injection volume: 20
Detector: F ex 278 em 324
CHROMATOGRAM
Retention time: 4.9
Internal standard: 7-methyleptazocine (7.2)
Limit of detection: 5 ng/mL
Limit of quantitation: 10 ng/mL
KEY WORDS
pharmacokinetics; plasma; rat
REFERENCE
Suzuki, T.; Shimizu, R.; Suganuma, T.; Nishino, J.; Tomono, K.; Hanano, M.; Watanabe, J. Pharmacokinetic/pharmacodynamic relationship of eptazocine, a narcotic-antagonist analgesic, in rats, Biol.Pharm.
Bull., 2000, 23, 1504–1510.
228
Eptifibatide
Eptifibatide
Molecular formula: C35 H49 N11 O9 S2
Molecular weight: 831.98
CAS Registry No: 188627-80-7
Merck Index: 13, 3670
SAMPLE
Matrix: formulations
Sample preparation: Dilute oral solution with MeCN:water 70:30, inject a 100 µL
aliquot.
HPLC VARIABLES
Column: 150 × 4.6 5 µm Pinnacle octyl amine (C8) (Restek)
Mobile phase: Gradient. MeCN:buffer 17:83 for 15 min, to 100:0 over 5 min, return
to initial conditions over 2 min. (The buffer was 0.1% trifluoroacetic acid in water
containing 0.1% triethylamine.)
Flow rate: 1
Injection volume: 100
Detector: UV 220
CHROMATOGRAM
Retention time: 10.1
Limit of quantitation: 500 ng/mL
OTHER SUBSTANCES
Simultaneous: degradants
KEY WORDS
oral solution
REFERENCE
Zhao, L.; Yalkowsky, S.H. Stabilization of eptifibatide by cosolvents, Int.J.Pharm., 2001, 218, 43–56.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 30 mm long C18 (Perkin-Elmer)
Mobile phase: Gradient. MeCN:water:trifluoroacetic acid from 80:20:0.1 to 75:25:0.1
(sic) over 5 min.
Detector: UV 280
REFERENCE
Schachter, D.M.; Kohn, J. A synthetic polymer matrix for the delayed or pulsatile release of water-soluble
peptides, J.Control.Rel., 2002, 78, 143–153.
Erdosteine
Erdosteine
Molecular formula: C8 H11 NO4 S2
O
S
229
H
N
S
COOH
O
Molecular weight: 249.31
CAS Registry No: 84611-23-4
Merck Index: 13, 3677
SAMPLE
Matrix: blood
Sample preparation: Mix 100 µL plasma, 500 µL water, 560 µL 100 mM pH 9.3 borate
buffer, 10 µL 100 µM IS in water, and 420 µL 762 µg/mL DBD-F in MeCN, let stand
at room temperature for 30 min (to derivatize thiol in metabolites and IS), evaporate
the MeCN under reduced pressure, add 2 mL ethyl acetate to the remaining solution,
mix vigorously, centrifuge at 3000 rpm for 2 min. Wash the aqueous layer three more
times with ethyl acetate. Adjust the pH of the aqueous layer to 1–2 with ca. 1.5 mL
100 mM HCl, extract three times with 2 mL portions of ethyl acetate. Combine the
organic layers and evaporate them to dryness under reduced pressure, reconstitute
the residue with 100 µL MeCN. Remove a 50 µL aliquot, add 5 µL DPPA, add 45 µL
17.8 mg/mL R-(−)-DBD-Apy in MeCN, let stand at room temperature for 2 h (to derivatize carboxylic acid), inject an aliquot. (DBD-F is 4-(N, N-dimethylaminosulfonyl)-7fluoro-2,1,3-benzoxadiazole. R-(−)-DBD-Apy is R-(−)-4-(N, N-dimethylaminosulfonyl)7-(3-aminopyrrolidin-1-yl)-2,1,3-benzoxadiazole. DPPA is diphenyl phosphoryl azide.
They are all available from Tokyo Kasei or TCI America (www.tciamerica.com).)
HPLC VARIABLES
Column: 150 × 4.6 5 µm Ultron VX-ODS
Column temperature: 40
Mobile phase: Gradient. MeCN:water:trifluoroacetic acid 30:70:0.1 for 10 min, to
37:63:0.1 over 25 min, to 43:57:0.1 over 30 min.
Flow rate: 1
Detector: F ex 468 em 563
CHROMATOGRAM
Retention time: 11
Internal standard: captopril (41)
Limit of detection: 0.22 pmol
OTHER SUBSTANCES
Extracted: metabolites
KEY WORDS
derivatization; plasma; rat
REFERENCE
Muramatsu, N.; Toyo’oka, T.; Yamaguchi, K.; Kobayashi, S. High-performance liquid chromatographic
determination of erdosteine and its optical active metabolite utilizing a fluorescent chiral tagging
reagent, R-(−)-4-(N,N-dimethylaminosulfonyl)-7-(3-aminopyrrolidin-1-yl)-2,1,3-benzoxadiazole, J.
Chromatogr.B, 1998, 719, 177–189.
230
Ergotamine
Ergotamine
H
O
Molecular formula: C33 H35 N5 O5
Molecular weight: 581.66
CAS Registry No: 113-15-5
Merck Index: 13, 3696
HO
N
H3C O
H
N
N
O
O
N
H
CH3
N
H
SAMPLE
Matrix: blood
Sample preparation: Mix 1 mL whole blood with 20 µL 1 µg/mL dibenzepin in
MeOH:water 50:50, add 300 µL pH 11 Tris buffer, mix, add 500 µL butyl acetate,
vortex for 2 min, centrifuge. Preserve the aqueous layer (A). Remove the organic layer
and add it to 75 µL 10 mM ammonium acetate buffer containing 0.1% formic acid (pH
3.2), evaporate (?). Add 75 µL MeCN, sonicate for 5 min, centrifuge at 5000 rpm for
5 min, keep the extract as B. Add 20 µL 1 µg/mL enalapril in MeOH:water 50:50 and
120 mg NaCl to the aqueous layer (A), mix, add 500 µL pH 3 phosphate buffer, add
600 µL 8.5% phosphoric acid, add 5 mL dichloromethane:isopropanol 95:5, shake at
250 cycles/min in a bench-top shaker for 30 min, centrifuge at 5000 rpm for 5 min.
Remove the lower organic layer and evaporate it to dryness under a stream of air at
45◦ . Reconstitute the residue with 150 µL initial mobile phase, sonicate, centrifuge,
combine with extract B, inject a 30 µL aliquot. (Sample preparation from Gergov,M.;
Robson,J.N.; Ojanperä,I.; Heinonen,O.P.; Vuori,E. Simultaneous screening and quantitation of 18 antihistamine drugs in blood by liquid chromatography ionspray tandem
mass spectrometry. Forensic Sci.Inter. 2001, 121, 108–115.)
HPLC VARIABLES
Guard column: 40 mm long 4 µm Purospher RP-18 LiChro Cart 4-4
Column: 100 × 2.1 4 µm Genesis C18 (Jones Chromatography)
Column temperature: 35
Mobile phase: Gradient. MeCN:buffer from 20:80 to 100:0 over 10 min, maintain at
0:100 for 3 min, re-equilibrate at initial conditions for 5 min. (The buffer was 10 mM
ammonium acetate containing 0.1% formic acid (pH 3.2).)
Flow rate: 0.2
Injection volume: 30
Detector: MS, PE Sciex API 365 triple-stage quadrupole LC-MS-MS, PE Sciex TurboIonSpray interface, positive ion mode, needle voltage 5.2 kV, nebulizer gas air at 60 psi,
curtain gas nitrogen at 40 psi, collision cell gas nitrogen at 40 psi, turbo ionspray heater
375◦ , heater gas flow 7 L/min
CHROMATOGRAM
Retention time: 5.5
Internal standard: dibenzepin, enalapril
Limit of detection: 5 ng/mL
OTHER SUBSTANCES
Extracted: acebutolol (3.8, LOD 0.1 µg/mL), acrivastine (5.7, LOD <0.02 µg/mL),
alprazolam (6.1, LOD <0.02 µg/mL), alprenolol (5.4, LOD 0.01 µg/mL), amantadine
(3.4, LOD 0.1 µg/mL), amiloride (2.0, LOD 0.1 µg/mL), aminophenazone (2.8, LOD
<5 µg/mL), amiodarone (10.2, LOD 0.05 µg/mL), amitriptyline (6.6, LOD <0.02 µg/mL),
Ergotamine
231
astemizole (5.8, LOD <0.02 µg/mL), atenolol (1.7, LOD 0.30 µg/mL), azacyclonol (5.1,
LOD 0.02 µg/mL), benzhexol (6.6, LOD <0.02 µg/mL), benzoylecgonine (3.3, LOD
0.01 µg/mL), betaxolol (5.5, LOD 0.01 µg/mL), biperidine (6.2, LOD <0.02 µg/mL),
bisoprolol (5.0, LOD <0.02 µg/mL), brompheniramine (5.3, LOD 0.002 µg/mL),
bupivacaine (5.1, LOD <0.02 µg/mL), buprenorphine (5.9, LOD 0.01 µg/mL),
buspirone (5.1, LOD 0.002 µg/mL), caffeine (2.8, LOD 1 µg/mL), carbamazepine
(6.1, LOD <0.02 µg/mL), carbinoxamine (5.1, LOD 0.002 µg/mL), carisoprodol
(6.7, LOD <5 µg/mL), carvedilol (6.2, LOD <0.02 µg/mL), celiprolol (4.3, LOD
0.05 µg/mL), cetirizine (6.3, LOD 0.05 µg/mL), chlorcyclizine (6.6, LOD <0.02 µg/mL),
chlordiazepoxide (5.7, LOD <0.02 µg/mL), chlormezanone (5.8, LOD <5 µg/mL),
chloroquine (2.7, LOD 0.02 µg/mL), chlorpheniramine (5.1, LOD 0.002 µg/mL),
chlorpromazine (7.0, LOD 0.02 µg/mL), chlorpropamide (6.7, LOD <5 µg/mL),
chlorprothixene (7.0, LOD <0.02 µg/mL), cinnarizine (7.9, LOD <0.02 µg/mL),
citalopram (5.7, LOD <0.02 µg/mL), clemastine (7.7, LOD 0.02 µg/mL), clobazam
(7.3, LOD <0.02 µg/mL), clobutinol (5.3, LOD 0.02 µg/mL), clomethiazole (6.2,
LOD 0.5 µg/mL), clomipramine (7.1, LOD <0.02 µg/mL), clonazepam (6.6, LOD
<0.02 µg/mL), clonidine (2.8, LOD 0.1 µg/mL), clozapine (5.6, LOD <0.02 µg/mL),
cocaine (4.6, LOD <0.02 µg/mL), codeine (2.5, LOD 0.1 µg/mL), coumatetralyl
(8.4, LOD 0.05 µg/mL), cyclizine (5.8, LOD <0.02 µg/mL), dextropropoxyphene
(6.6, LOD 0.05 µg/mL), demoxepam (5.8, LOD 0.02 µg/mL), dextromethorphan
(5.5, LOD <0.02 µg/mL), diazepam (8.1, LOD 0.02 µg/mL), diltiazem (5.8,
LOD <0.02 µg/mL), diphenhydramine (5.7, LOD <0.02 µg/mL), dipyridamole (5.4,
LOD 0.005 µg/mL), disopyramine (4.4, LOD <0.02 µg/mL), dixyrazine (6.8, LOD
0.005 µg/mL), doxapram (4.8, LOD <0.02 µg/mL), doxepin (5.9, LOD <0.02 µg/mL),
ebastine (9.6, LOD 0.005 µg/mL), embutramide (6.7, LOD 0.005 µg/mL), ethenzamide
(5.0, LOD 0.05 µg/mL), ethylmorphine (3.2, LOD 0.05 µg/mL), ethylparathion
(9.7, LOD <5 µg/mL), etodroxizine (6.4, LOD <0.02 µg/mL), felodipine (9.6, LOD
0.02 µg/mL), fenazepam (7.5, LOD <0.02 µg/mL), fenfluramine (5.3, LOD <0.02 µg/mL),
fenkamfamine (5.1, LOD <0.02 µg/mL), fentanyl (5.5, LOD <0.02 µg/mL), fexofenadine
(6.3, LOD <0.02 µg/mL), flecainide (5.9, LOD <0.02 µg/mL), fluconazole (4.0, LOD
0.1 µg/mL), flumazenil (5.2, LOD <0.02 µg/mL), flunitrazepam (7.1, LOD 0.002 µg/mL),
fluoxetine (6.8, LOD 0.1 µg/mL), flupentixol (7.5, LOD 0.18 µg/mL), fluvoxamine
(6.3, LOD 0.02 µg/mL), glibenclamide (8.5, LOD <0.02 µg/mL), glipizide (6.8,
LOD <0.05 µg/mL), haloperidol (6.1, LOD <0.02 µg/mL), histapyrrodine (6.3, LOD
0.02 µg/mL), hydrocodone (3.0, LOD 0.05 µg/mL), hydroxychloroquine (2.4, LOD
<0.3 µg/mL), hydroxyzine (6.3, LOD <0.02 µg/mL), imipramine (6.4, LOD 0.05 µg/mL),
indomethacin (8.6, LOD 0.05 µg/mL), isoniazid (2.2, LOD 3 µg/mL), isradipine (8.6, LOD
0.05 µg/mL), ketamine (3.6, LOD <0.05 µg/mL), ketobemidone (3.3, LOD <0.05 µg/mL),
ketoprofen (7.3, LOD 0.1 µg/mL), ketorolac (6.2, LOD 0.05 µg/mL), labetalol (4.9, LOD
0.05 µg/mL), lamotrigine (4.0, LOD 0.1 µg/mL), levocabastine (5.8, LOD 0.01 µg/mL),
levomepromazine (6.5, LOD 0.02 µg/mL), lidocaine (3.7, LOD <0.05 µg/mL), loratadine
(9.3, LOD 0.002 µg/mL), lorazepam (6.6, LOD 0.02 µg/mL), lormetazepam (7.4,
LOD <0.02 µg/mL), LSD (4.7, LOD <0.02 µg/mL), malathion (8.9, LOD 10 µg/mL),
maprotiline (6.4, LOD <0.02 µg/mL), MDMA (3.3, LOD 0.02 µg/mL), meclozine (8.5,
LOD <0.02 µg/mL), medazepam (6.3, LOD <0.02 µg/mL), meloxicam (7.1, LOD
0.01 µg/mL), melperone (5.0, LOD <0.02 µg/mL), meperidine (4.7, LOD <0.02 µg/mL),
mepivacaine (3.7, LOD <0.02 µg/mL), meprobamate (4.9, LOD 0.1 µg/mL), mesoridazine
(5.4, LOD <0.02 µg/mL), methamphetamine (3.3, LOD 0.05 µg/mL), methadone (6.7,
LOD <0.02 µg/mL), methylparathion (8.6, LOD 10 µg/mL), methylphenidate (4.2,
LOD <0.02 µg/mL), metoclopramide (3.8, LOD <0.02 µg/mL), metoprolol (4.1, LOD
0.02 µg/mL), metronidazole (2.6, LOD 1 µg/mL), mexiletine (4.4, LOD 0.05 µg/mL),
mianserin (5.7, LOD <0.02 µg/mL), midazolam (5.9, LOD <0.02 µg/mL), mirtazapine
(4.4, LOD <0.02 µg/mL), mizolastine (5.5, LOD 0.01 µg/mL), moclobemide (3.7,
LOD 0.05 µg/mL), molindone (4.0, LOD <0.02 µg/mL), monoacetylmorphine (2.7,
LOD 0.1 µg/mL), morphine (2.0, LOD 0.1 µg/mL), nicotine (2.2, LOD 0.05 µg/mL),
nifedipine (7.5, LOD 0.02 µg/mL), nikethamide (3.6, LOD <0.02 µg/mL), nitrazepam
(6.5, LOD <0.02 µg/mL), nizatidine (1.7, LOD 1 µg/mL), nomifensine (4.6, LOD
<0.02 µg/mL), nortriptyline (6.4, LOD <0.02 µg/mL), norverapamil (6.2, LOD 1 µg/mL),
noscapine (5.0, LOD <0.02 µg/mL), olanzapine (3.0, LOD 0.05 µg/mL), ondansetron
(4.6, LOD <0.02 µg/mL), orphenadrine (6.1, LOD <0.02 µg/mL), oxazepam (6.3, LOD
232
Ergotamine
<0.02 µg/mL), oxcarbazepine (5.3, LOD 0.02 µg/mL), oxprenolol (4.7, LOD 0.02 µg/mL),
oxycodone (2.8, LOD 0.05 µg/mL), papaverine (4.8, LOD <0.02 µg/mL), acetaminophen
(2.5, LOD <5 µg/mL), paroxetine (6.2, LOD 0.02 µg/mL), pemoline (3.3, LOD
0.05 µg/mL), pentazocine (5.0, LOD <0.02 µg/mL), pentifylline (7.3, LOD <5 µg/mL),
pentoxyverine (6.6, LOD <0.02 µg/mL), perphenazine (6.9, LOD 0.002 µg/mL),
phenazone (3.9, LOD 0.05 µg/mL), phencyclidine (5.3, LOD 0.05 µg/mL), pheniramine
(4.1, LOD 0.02 µg/mL), phenylbutazone (9.0, LOD <5 µg/mL), phenylpropanolamine
(2.5, LOD 0.3 µg/mL), phenytoin (6.1, LOD 0.05 µg/mL), pindolol (3.3, LOD 0.05 µg/mL),
piroxicam (6.6, LOD 0.02 µg/mL), pitofenone (5.4, LOD <0.02 µg/mL), pizotifen (6.5,
LOD <0.02 µg/mL), practolol (1.8, LOD 0.1 µg/mL), prazosin (4.1, LOD 0.05 µg/mL),
prilocaine (3.8, LOD <0.02 µg/mL), primidone (4.0, LOD <5 µg/mL), procainamide
(2.2, LOD 0.05 µg/mL), prochlorperazine (7.5, LOD 0.02 µg/mL), promazine (6.2,
LOD <0.02 µg/mL), promethazine (6.0, LOD 0.05 µg/mL), propafenone (6.3, LOD
<0.02 µg/mL), propranolol (5.4, LOD 0.02 µg/mL), propyphenazone (6.6, LOD
0.50 µg/mL), pseudoephedrine (2.6, LOD 1 µg/mL), quinine (4.2, LOD 0.02 µg/mL),
ranitidine (1.8, LOD 0.1 µg/mL), risperidone (4.9, LOD <0.02 µg/mL), rocurone
(3.8, LOD 0.1 µg/mL), ropivacaine (4.6, LOD <0.02 µg/mL), salicylamide (4.2, LOD
<5 µg/mL), selegiline (4.1, LOD 0.05 µg/mL), sertindole (7.2, LOD <0.02 µg/mL),
sertraline (6.8, LOD 0.02 µg/mL), sulindac (6.5, LOD 0.02 µg/mL), simazine (6.0, LOD
0.1 µg/mL), sincocaine (6.5, LOD <0.02 µg/mL), sisapride (5.9, LOD <0.02 µg/mL),
sotalol (2.1, LOD 0.1 µg/mL), strychnine (5.3, LOD 0.05 µg/mL), sulpiride (1.9, LOD
0.1 µg/mL), sulthiame (4.1, LOD 0.05 µg/mL), temazepam (7.2, LOD <0.02 µg/mL),
terbutaline (2.3, LOD 0.1 µg/mL), terfenadine (8.1, LOD 0.002 µg/mL), terodiline
(6.7, LOD <0.02 µg/mL), tetracaine (5.7, LOD <0.02 µg/mL), dronabinol (12.3,
LOD 0.05 µg/mL), tetrahydrozoline (3.6, LOD 0.1 µg/mL), theobromine (2.3, LOD
<5 µg/mL), theophylline (2.4, LOD <5 µg/mL), thioridazine (7.5, LOD 0.02 µg/mL),
timolol (3.8, LOD 0.05 µg/mL), thiothixene (6.7, LOD 0.02 µg/mL), tolbutamide (7.1,
LOD <5 µg/mL), toremifene (8.7, LOD 0.02 µg/mL), tramadol (4.2, LOD 0.02 µg/mL),
trazodone (5.2, LOD <0.02 µg/mL), triamterene (3.2, LOD 0.1 µg/mL), triazolam (6.7,
LOD 0.002 µg/mL), trimeprazine (6.4, LOD <0.02 µg/mL), trimethoprim (3.1, LOD
0.05 µg/mL), trimipramine (6.7, LOD <0.02 µg/mL), venlafaxine (4.9, LOD 0.02 µg/mL),
verapamil (6.5, LOD <0.02 µg/mL), warfarin (7.9, LOD <0.02 µg/mL), yohimbine (4.5,
LOD <0.02 µg/mL), zolpidem (4.7, LOD <0.02 µg/mL), zopiclone (4.0, LOD 0.1 µg/mL)
KEY WORDS
whole blood
REFERENCE
Gergov, M.; Ojanperä, I.; Vuori, E. Simultaneous screening for 238 drugs in blood by liquid chromatography-ionspray tandem mass spectrometry with multiple-reaction monitoring, J.Chromatogr.B, 2003,
795, 41–53.
SAMPLE
Matrix: blood
Sample preparation: Rotate 2 mL plasma and 5 mL diethyl ether for 30 min, centrifuge, repeat the extraction. Combine the organic layers and evaporate them to
dryness under a stream of nitrogen at room temperature, reconstitute the residue with
100 µL MeOH, inject a 50 µL aliquot.
HPLC VARIABLES
Guard column: 15 × 3.2 7 µm silica Newguard
Column: 100 × 4.6 5 µm Spheri-5 silica
Mobile phase: MeOH:water:acetic acid:MeCN 40:2:10 (sic)
Flow rate: 1
Injection volume: 50
Detector: F ex 322 em 405
Ergotamine
233
CHROMATOGRAM
Retention time: 7.5
Internal standard: ergotamine
OTHER SUBSTANCES
Extracted: lisuride (12)
KEY WORDS
ergotamine is IS in original paper; plasma; protect from light
REFERENCE
Wolthers, B.G.; Verhagen Kamerbeek, W.D.J.; van Beusekom, C.M.; Elshof, F.; de Ruyter Buitenhuis, A.W.; Brunt, E.P.R.; Lakke, J.P.W.F. Quantitative determination of the dopamine agonist lisuride
in plasma using high-performance liquid chromatography with fluorescence detection, J.Chromatogr.,
1993, 622, 33–38.
SAMPLE
Matrix: blood
Sample preparation: Vortex 500 µL plasma, 200 µL 2.5 M potassium carbonate, and
3 mL diethyl ether. Remove a 2 mL aliquot of the organic layer and add it to 100 µL
50 mM sulfuric acid, vortex for 1 min, inject a 50 µL aliquot of the aqueous layer.
HPLC VARIABLES
Column: 250 × 4 5 µm Hypersil ODS RP-C18
Mobile phase: MeCN:MeOH:water 8:56:36 containing 200 mg (per 500 mL (?)) sodium
heptanesulfonate
Flow rate: 1
Injection volume: 50
Detector: UV 254
CHROMATOGRAM
Retention time: 7
Internal standard: ergotamine
OTHER SUBSTANCES
Extracted: bromocriptine (13)
KEY WORDS
ergotamine is IS in original paper; plasma; rabbit
REFERENCE
Degim, I.T.; Acartürk, F.; Erdogan, D.; Lortlar, N.D. Transdermal administration of bromocriptine,
Biol.Pharm.Bull., 2003, 26, 501–505.
234
Ertapenem
Ertapenem
Molecular formula: C22 H25 N3 O7 S
Molecular weight: 475.52
CAS Registry No: 153832-46-3
Merck Index: 13, 3706
OH
H
H H
N
CH3
H3C
S
O
N
COOH
O
N
COOH
H
SAMPLE
Matrix: blood
Sample preparation: Filter (Amicon Centrifree) 1 mL plasma while centrifuging at
1500 g for 15 min, mix an aliquot of the filtrate with an equal volume of buffer, inject a
50 µL aliquot. (Prepare the buffer by dissolving 15.5 g sodium 2-[N-morpholino]ethane
sulfonate (MES) and 5.58 g 2-[N-morpholino]ethane sulfonic acid in 100 mL water.
Dilute this stock solution 10-fold before use, pH 6.5.)
HPLC VARIABLES
Column: 100 × 4.6 5 µm Hypersil C18
Mobile phase: MeOH:25 mM phosphate buffer 10:90, apparent pH 6.5
Flow rate: 2
Injection volume: 50
Detector: UV 300
CHROMATOGRAM
Retention time: 5.6
Limit of quantitation: 250 ng/mL
KEY WORDS
pharmacokinetics; plasma; ultrafiltrate
REFERENCE
Musson, D.G.; Birk, K.L.; Kitchen, C.J.; Zhang, J.; Hsieh, J.Y.K.; Fang, W.; Majumdar, A.K.; Rogers, J.D.
Assay methodology for the quantitation of unbound ertapenem, a new carbapenem antibiotic, in human
plasma, J.Chromatogr.B, 2003, 783, 1–9.
SAMPLE
Matrix: blood
Sample preparation: Mix 50 µL 50 µg/mL IS with 200 µL serum, add 800 µL MeCN,
vortex briefly, centrifuge at 2600 g for 10 min. remove the supernatant and add it to
2.5 mL dichloromethane, vortex briefly, centrifuge, inject an aliquot of the aqueous
layer.
HPLC VARIABLES
Column: 100 × 4.6 5 µm C18 (Keystone)
Mobile phase: MeOH:25 mM pH 6.5 phosphate buffer 9.5:100
Flow rate: 1
Detector: UV 300
CHROMATOGRAM
Retention time: 10.5
Internal standard: meropenem (14.9)
Limit of quantitation: 150 ng/mL
Ertapenem
235
KEY WORDS
mouse; pharmacokinetics; serum
REFERENCE
Xuan, D.; Banevicius, M.; Capitano, B.; Kim, M.-K.; Nightingale, C.; Nicolau, D. Pharmacodynamic
assessment of ertapenem (MK-0826) against streptococcus pneumoniae in a murine neutropenic
thigh infection model, Antimicrob.Agents Chemother., 2002, 46, 2990–2995.
ANNOTATED BIBLIOGRAPHY
Kitchen, C.J.; Musson, D.G.; Fisher, A.L. Column-switching technique for the sensitive determination
of ertapenem in human cerebrospinal fluid using liquid chromatography and ultraviolet absorbance
detection, J.Chromatogr.B, 2004, 799, 9–14.
McQuade, M.S.; Van Nostrand, V.; Schariter, J.; Kanike, J.D.; Forsyth, R.J. Stability and compatibility
of reconstituted ertapenem with commonly used i.v. infusion and coinfusion solutions, Am.J.HealthSyst.Pharm., 2004, 61, 38–45.
Musson, D.G.; Kitchen, C.J.; Hsieh, J.Y.-K.; Birk, K.L. Modified high-performance liquid chromatographic method for the determination of ertapenem in human urine: enhanced selectivity and
automation, J.Chromatogr.B, 2002, 779, 341–346.
Musson, D.G.; Majumdar, A.; Birk, K.; Holland, S.; Wickersham, P.; Li, S.X.; Mistry, G.; Fisher, A.; Waldman, S.; Greenberg, H.; Deutsch, P.; Rogers, J.D. Pharmacokinetics of intramuscularly administered
ertapenem, Antimicrob.Agents Chemother., 2003, 47, 1732–1735.
Sajonz, P.; Natishan, T.K.; Wu, Y.; Williams, J.M.; Pipik, B.; DiMichele, L.; Novak, T.; Pitzenberger, S.;
Dubost, D.; Almarsson, O. Preparation, isolation, and characterization of dimeric degradation products of the 1β-methylcarbapenem antibiotic, ertapenem, J.Liq.Chromatogr.Rel.Technol., 2001, 24,
2999–3015.
236
Ethopabate
Ethopabate
Molecular formula: C12 H15 NO4
Molecular weight: 237.25
CAS Registry No: 59-06-3
Merck Index: 13, 3781
O
O
H3C
OCH3
N
O
CH3
H
SAMPLE
Matrix: blood
Sample preparation: Vortex 100 µL MeOH:water 80:20 and 400 µL trichloroacetic
acid solution with 500 µL plasma, centrifuge at 4000 rpm for 4 min, filter (Spin-X) the
supernatant. Mix the filtrate with an equal quantity of water and inject a 30 µL aliquot.
(Prepare trichloroacetic acid solution as follows. Dissolve 85 g trichloroacetic acid in
15 mL water. Store this solution in the refrigerator. Dilute 150 µL of this solution with
100 mL acetone.)
HPLC VARIABLES
Column: 250 × 4.6 5 µm Supelcosil LC-ABZ + Plus
Mobile phase: MeCN:10 mM ammonium acetate 35:65
Flow rate: 1
Injection volume: 30
Detector: MS, PE Sciex API 100 single quadrupole, turbo ionspray, 50 µL/min flowed
into detector, m/z 238.2
CHROMATOGRAM
Retention time: 7.5
Limit of quantitation: 15 ng/mL
KEY WORDS
chicken; plasma
REFERENCE
Hormazábal, V.; Yndestad, M. Determination of amprolium, ethopabate, lasalocid, monensin, narasin,
and salinomycin in chicken tissues, plasma, and egg using liquid chromatography-mass spectrometry,
J.Liq.Chromatogr.Rel.Technol., 2000, 23, 1585–1598.
Ethyl icosapentate
237
Ethyl icosapentate
Molecular formula: C22 H34 O2
Molecular weight: 330.51
CAS Registry No: 73310-10-8
CO2Et
SAMPLE
Matrix: solutions
Sample preparation: Inject a 2 µL aliquot.
HPLC VARIABLES
Column: 125 × 4 4 µm Supersphere Si 60
Column temperature: 27
Mobile phase: n-Hexane:diethyl ether 98:2
Flow rate: 1.8
Injection volume: 2
Detector: UV 213
CHROMATOGRAM
Retention time: 2
Internal standard: 2,4-dinitrochlorobenzene (6)
KEY WORDS
normal phase
REFERENCE
Teraoka, R.; Otsuka, M.; Matsuda, Y. Chemical stability of ethyl icosapentate against autoxidation. I.
Effect of temperature on oxidation kinetics, Pharm.Res., 1992, 9, 1673–1676.
238
Etonogestrel
Etonogestrel
H3C
H
Molecular formula: C22 H28 O2
Molecular weight: 324.46
CAS Registry No: 54048-10-1
Merck Index: 13, 3916
OH
H
H2C
H
H
H
O
SAMPLE
Matrix: cell culture
Sample preparation: Extract medium twice with 2 mL diethyl ether. Evaporate the
extracts to dryness, reconstitute the residue with MeOH, inject an aliquot.
HPLC VARIABLES
Column: 250 × 4.6 Ultratech 5-ODS (HPLC Technology)
Mobile phase: MeCN:MeOH:0.5% pH 3.0 ammonium dihydrogen phosphate 15:47:38
Flow rate: 1.2
Detector: UV 214
CHROMATOGRAM
Retention time: 13
OTHER SUBSTANCES
Extracted: metabolites
REFERENCE
Wild, M.J.; Rudland, P.S.; Back, D.J. Metabolism of the oral contraceptive steroids ethynylestradiol,
norgestimate and 3-ketodesogestrel by a human endometrial cancer cell line (HEC-1A) and endometrial
tissue in vitro, J.Steroid Biochem.Mol.Biol., 1993, 45, 407–420.
SAMPLE
Matrix: microsomal incubations
HPLC VARIABLES
Guard column: Novapak C18
Column: 150 × 3.9 Novapak C18
Column temperature: 50
Mobile phase: Gradient. MeCN:MeOH:water from 5:20:75 to 8:32:60 over 5 min, to
14:56:30 over 10 min, to 18:72:10 over 2 min.
Flow rate: 1
Detector: Radioactivity (3 H); UV 254
CHROMATOGRAM
Retention time: 16
OTHER SUBSTANCES
Extracted: metabolites
KEY WORDS
dog; human; liver; rabbit, rat
REFERENCE
Verhoeven, C.H.J.; Krebbers, S.F.M.; Wagenaars, G.N.; Vos, R.M.E. In vitro and in vivo metabolism of
desogestrel in several species, Drug Metab.Dispos., 1998, 26, 927–936.
Etonogestrel
239
SAMPLE
Matrix: solutions
Sample preparation: Extract 15 mL aqueous solution with dichloromethane, evaporate the extract to dryness, take up the residue in 3 mL mobile phase, inject a 50 µL
aliquot.
HPLC VARIABLES
Column: C18
Mobile phase: MeOH:water 82:18
Injection volume: 50
Detector: UV 242
CHROMATOGRAM
Internal standard: progesterone
Limit of quantitation: 50 ng/mL
OTHER SUBSTANCES
Simultaneous: ethinyl estradiol (F ex 200 em 300), mestranol (F ex 200 em 300)
REFERENCE
de Leede, L.G.; Govers, C.P.M.; de Nijs, H. A multi-compartment vaginal ring system for independently
adjustable release of contraceptive steroids, Contraception, 1986, 34, 589–602.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 150 × 3.9 Novapak C18
Column temperature: 30
Mobile phase: MeCN:water 30:70
Flow rate: 1.5
Injection volume: 10
Detector: UV 205
OTHER SUBSTANCES
Simultaneous: ethinyl estradiol
REFERENCE
van Laarhoven, J.A.H.; Kruft, M.A.B.; Vromans, H. In vitro release properties of etonogestrel and ethinyl
estradiol from a contraceptive vaginal ring, Int.J.Pharm., 2002, 232, 163–173.
240
Etoricoxib
Etoricoxib
CH3
N
Molecular formula: C18 H15 ClN2 O2 S
Molecular weight: 358.84
CAS Registry No: 202409-33-4
O
O
S
CH3
N
Cl
SAMPLE
Matrix: blood
Sample preparation: Condition a 1 mL 30 mg Oasis HLB SPE cartridge with 1 mL
MeOH and 1 mL water. Mix 200 µL plasma with 20 µL 500 ng/mL IS in water, centrifuge at 12 000 g, add to the SPE cartridge, wash with 1 mL MeOH:water 5:95, dry
under reduced pressure for 5 min, elute with 2 mL MeCN:ethyl acetate 50:50. Evaporate the eluate to dryness under reduced pressure at 45◦ , reconstitute the residue with
200 µL mobile phase, inject a 15 µL aliquot.
HPLC VARIABLES
Column: 30 × 2 5 µm Nucleodur C18
Mobile phase: MeCN:water 90:10
Flow rate: 0.3
Injection volume: 15
Detector: MS, PE Sciex API 3000 triple quadrupole, turbo ionspray, positive ion
mode 5400 V and 400◦ , nebulizer gas nitrogen at 1.49 L/min, curtain gas nitrogen
at 1.25 L/min, collision gas nitrogen, collision energy 43 eV, m/z 359.0–280.1
CHROMATOGRAM
Retention time: 0.5
Internal standard: phenazone (m/z 189.0–104.0, 33 eV) (0.5)
Limit of quantitation: 0.2 ng/mL
KEY WORDS
pharmacokinetics; plasma
REFERENCE
Bräutigam, L.; Nefflen, J.U.; Geisslinger, G. Determination of etoricoxib in human plasma by liquid
chromatography-tandem mass spectrometry with electrospray ionisation, J.Chromatogr.B, 2003, 788,
309–315.
SAMPLE
Matrix: solutions
Sample preparation: Inject a 10 µL aliquot of a 100 µg/mL solution in MeCN:water
50:50.
HPLC VARIABLES
Column: 150 × 4.6 3 µm YMC AQ-ODS
Column temperature: 35
Mobile phase: Gradient. MeCN:buffer 28:72 for 11 min, to 70:30 over 19 min, to 90:10
over 5 min. (The buffer was 10 mM potassium dihydrogen phosphate adjusted to pH 3.1
with 2.2 mM phosphoric acid.)
Flow rate: 1
Injection volume: 10
Detector: UV 220
Etoricoxib
241
CHROMATOGRAM
Retention time: 12
Limit of detection: 0.02%
Limit of quantitation: 0.04%
OTHER SUBSTANCES
Simultaneous: degradants, impurities
KEY WORDS
stability-indicating; robust
REFERENCE
Hartman, R.; Abrahim, A.; Clausen, A.; Mao, B.; Crocker, L.S.; Ge, Z. Development and validation of an
HPLC method for the impurity and quantitative analysis of etoricoxib, J.Liq.Chromatogr.Rel.Technol.,
2003, 26, 2551–2566.
ANNOTATED BIBLIOGRAPHY
Kassahun, K.; McIntosh, I.S.; Shou, M.; Walsh, D.J.; Rodeheffer, C.; Slaughter, D.E.; Geer, L.A.; Halpin,
R.A.; Agrawal, N.; Rodrigues, A.D. Role of human liver cytochrome P4503A in the metabolism of
etoricoxib, a novel cyclooxygenase-2 selective inhibitor, Drug Metab.Dispos., 2001, 29, 813–820.
Niederberger, E.; Tegeder, I.; Schäfer, C.; Seegel, M.; Grösch, S.; Geisslinger, G. Opposite effects of rofecoxib on nuclear factor-kappaB and activating protein-1 activation, J.Pharmacol.Exp.Ther., 2003, 304,
1153–1160. [etoricoxib is internal standard; post-column photochemical derivatization]
Rodrigues, A.D.; Halpin, R.A.; Geer, L.A.; Cui, D.; Woolf, E.J.; Matthews, C.Z.; Gottesdiener, K.M.; Larson, P.J.; Lasseter, K.C.; Agrawal, N.G.B. Absorption, metabolism, and excretion of etoricoxib, a potent
and selective cyclooxygenase-2 inhibitor, in healthy male volunteers, Drug Metab.Dispos., 2003, 31,
224–232.
Rose, M.J.; Agrawal, N.; Woolf, E.J.; Matuszewski, B.K. Simultaneous determination of unlabeled and
carbon-13-labeled etoricoxib, a new cyclooxygenase-2 inhibitor, in human plasma using HPLC-MS/MS,
J.Pharm.Sci., 2002, 91, 405–416. [SPE]
242
Etorphine
Etorphine
Molecular formula: C25 H33 NO4
Molecular weight: 411.53
CAS Registry No: 14521-96-1
Merck Index: 13, 3919
HO
O
N
H3CO
H3C
HO
CH3
CH3
SAMPLE
Matrix: blood, urine
Sample preparation: Add 3 mL 200 mM sodium carbonate solution containing
100 ng/mL IS and 3 mL 1-chlorobutane to 3 mL blood or urine, shake for 3 min,
centrifuge at 4500 rpm for 5 min, repeat the extraction. Combine the organic layers and
add them to 100 µL 50 mM sulfuric acid, shake for 3 min, centrifuge at 3500 rpm for
3 min, inject a 20 µL aliquot of the aqueous layer.
HPLC VARIABLES
Guard column: 10 × 4 Spherisorb S5ODS2
Column: 150 × 4.6 Spherisorb S50D/CN
Mobile phase: MeCN:water 20:80
Flow rate: 2
Injection volume: 20
Detector: UV 220
CHROMATOGRAM
Retention time: 3.77
Internal standard: pentazocine (6.31)
Limit of quantitation: 2 ng/mL
KEY WORDS
whole blood
REFERENCE
Elliott, S.P.; Hale, K.A. Analysis of etorphine in postmortem samples by HPLC with UV diode-array
detection, Forensic Sci.Int., 1999, 101, 9–16.
243
Exemestane
Exemestane
CH3 O
CH3
H
Molecular formula: C20 H24 O2
Molecular weight: 296.40
CAS Registry No: 107868-30-4
Merck Index: 13, 3944
H
H
O
CH2
SAMPLE
Matrix: blood
Sample preparation: Mix 1 mL plasma with 100 µL 1 µg/mL IS in water, add 600 µL
500 mM pH 7.4 potassium phosphate buffer, mix, add 3 mL dichloromethane:isooctane
40:60, shake for 10 min, centrifuge at 1200 g for 15 min, repeat the extraction. Combine
the organic layers and evaporate them to dryness under a stream of nitrogen at 37◦ ,
reconstitute the residue with 200 µL MeCN:water 50:50, inject a 150 µL aliquot.
HPLC VARIABLES
Guard column: 37–53 µm pellicular ODS
Column: 125 × 4.6 5 µm Lichrocart RP18
Mobile phase: MeCN:50 mM pH 4.5 potassium phosphate buffer 35:65
Flow rate: 1.5
Injection volume: 150
Detector: UV 247
CHROMATOGRAM
Retention time: 14
Internal standard: norgestrel (17)
Limit of detection: 2 ng
Limit of quantitation: 10 ng/mL
OTHER SUBSTANCES
Extracted: metabolite
KEY WORDS
pharmacokinetics; plasma
REFERENCE
Breda, M.; Pianezzola, E.; Strolin Benedetti, M.S. Determination of exemestane, a new aromatase
inhibitor, in plasma by high-performance liquid chromatography with ultraviolet detection, J.Chromatogr., 1993, 620, 225–231.
SAMPLE
Matrix: blood
Sample preparation: Condition a 2 mL 50 mg C2 end-capped SPE cartridge (in a 96
well plate format) with two 1 mL portions of MeCN and two 1 mL portions of water.
Vortex 500 µL plasma with 50 µL 1.11 µg/mL IS in MeOH:water 50:50 and 500 µL
water, add to the SPE cartridge, wash with 1 mL MeCN:water 10:90, dry under vacuum
for 30 min, elute with two 150 µL portions of MeCN containing 0.1% trifluoroacetic
acid. Centrifuge the eluate at 1500 rpm for 2 min and inject an 80 µL aliquot of the
supernatant.
HPLC VARIABLES
Guard column: 15 × 3.2 7 µm New Guard RP8 Aquapore Octyl
Column: 150 × 4.6 5 µm Zorbax SB C8
Column temperature: 45
244
Exemestane
Mobile phase: MeCN
Flow rate: 1
Injection volume: 80
Detector: MS, PE Sciex API 300 triple quadrupole, APCI, nebulizer probe 375◦ , collision
energy 30 eV, m/z 297–121
CHROMATOGRAM
Retention time: 2.3
Internal standard: 13 C3 -exemestane (m/z 300–123) (2.3)
Limit of quantitation: 50 pg/mL
KEY WORDS
plasma; SPE
REFERENCE
Cenacchi, V.; Barattè, S.; Cicioni, P.; Frigerio, E.; Long, J.; James, C. LC-MS-MS determination of
exemestane in human plasma with heated nebulizer interface following solid-phase extraction in
the 96 well plate format, J.Pharm.Biomed.Anal., 2000, 22, 451–460.
ANNOTATED BIBLIOGRAPHY
Del Nero, S.; Di Somma, M.; Vigevani, A. High-performance liquid chromatographic analysis of FCE
24304 (6-methylenandrosta-1,4-diene-3,17-dione) and FCE 24928 (4-aminoandrosta-1,4,6-triene-3,17dione), two new aromatase inhibitors, J.Chromatogr., 1992, 593, 25–28.
Ezetimibe
Ezetimibe
245
OH
OH
Molecular formula: C24 H21 F2 NO3
Molecular weight: 409.42
CAS Registry No: 163222-33-1
Merck Index: 13, 3949
F
N
O
F
SAMPLE
Matrix: bile
Sample preparation: Extract bile with 1.5 vol of MeCN.
HPLC VARIABLES
Column: 150 × 4.6 5 µm Inertsil C8
Mobile phase: Gradient. MeCN:100 mM pH 6 ammonium acetate from 30:70 to 100:0
over 40 min (concave gradient (Waters Expert-Ease Curve #10)).
Flow rate: 1
Detector: UV 245; Radioactivity (3 H)
CHROMATOGRAM
Retention time: 40
OTHER SUBSTANCES
Extracted: metabolites
KEY WORDS
rat
REFERENCE
Van Heek, M.; Farley, C.; Compton, D.S.; Hoos, L.; Alton, K.B.; Sybertz, E.J.; Davis, H.R. Jr. Comparison
of the activity and disposition of the novel cholesterol absorption inhibitor, SCH58235, and its
glucuronide, SCH60663. Br.J.Pharmacol., 2000, 129, 1748–1754.
SAMPLE
Matrix: blood
Sample preparation: For unconjugated ezetimibe, add 100 µL 513 ng/mL IS in water
and 1 mL water to 200 µL plasma, add 8 mL 1-chlorobutane. For total ezetimibe, add
100 µL 513 ng/mL IS in water, 500 µL 500 mM pH 5.0 sodium acetate buffer, and
50 µL β-glucuronidase (100 000 U/mL) to 200 µL plasma, heat at 50◦ for 1 h, add
500 µL 1 M sodium borate solution, add 8 mL 1-chlorobutane. Shake each mixture for
15 min and centrifuge at 491 g for 10 min. Evaporate the organic layer to dryness under
reduced pressure, reconstitute the residue with 500 µL MeOH, evaporate to dryness,
reconstitute with 50 µL MeOH, inject an aliquot.
HPLC VARIABLES
Column: 100 × 4.6 10 µm Spherisorb ODS-2
Mobile phase: MeOH:25 mM ammonium acetate 90:10
Flow rate: 1.5
Detector: MS, PE Sciex API-III, positive ion mode, m/z 392.3–133.1
246
Ezetimibe
CHROMATOGRAM
Internal standard: SCH 58053 ((+)-7-(4-chlorophenyl)-2-(4-fluorophenyl)-7-hydroxy3(R)-4-hydroxyphenyl)-2-azaspiro[3.5]nonan-1-one (enantiomer A)) (m/z 434.2–216.1)
Limit of quantitation: 1 ng/mL (unconjugated), 5.02 ng/mL (total)
KEY WORDS
pharmacokinetics; plasma
REFERENCE
Patrick, J.E.; Kosoglou, T.; Stauber, K.L.; Alton, K.B.; Maxwell, S.E.; Zhu, Y.; Statkevich, P.; Iannucci, R.;
Chowdhury, S.; Affrime, M.; Cayen, M.N. Disposition of the selective cholesterol absorption inhibitor
ezetimibe in healthy male subjects, Drug Metab.Dispos., 2002, 30, 430–437.
Fadrozole
N
N
CN
Molecular formula: C14 H13 N3
Molecular weight: 223.27
CAS Registry No: 102676-47-1
Merck Index: 13, 3958
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: Chiralcel OD
Mobile phase: Hexane:isopropanol 70:30
Detector: UV
CHROMATOGRAM
Retention time: (+)-enantiomer elutes first, α = 1.40
KEY WORDS
chiral
REFERENCE
Furet, P.; Batzl, C.; Bhatnagar, A.; Francotte, E.; Rihs, G.; Lang, M. Aromatase inhibitors: synthesis,
biological activity, and binding mode of azole-type compounds, J.Med.Chem., 1993, 36, 1393–1400.
HPLC Methods for Recently Approved Pharmaceuticals. by George Lunn
Copyright 2005 John Wiley & Sons, Ltd. ISBN: 0-471-66941-5
247
248
Falecalcitriol
Falecalcitriol
H3C H
CH3
CF3
H
Molecular formula: C27 H38 F6 O3
Molecular weight: 524.58
CAS Registry No: 83805-11-2
OH
CF3
H
CH2
HO
H
H
OH
SAMPLE
Matrix: cell suspensions
Sample preparation: Condition a Sep-Pak silica SPE cartridge with n-hexane:isopro-
panol 80:20. Sonicate 2 (?) mL cell suspension at 0◦ for 2 min, add 1 mL THF, add 4 mL
ethyl acetate, vortex, centrifuge at 2200 rpm at 4◦ for 5 min, extract the aqueous layer
four times with 4 mL portions of ethyl acetate. Dry the combined organic layers over
anhydrous sodium sulfate, evaporate to dryness, reconstitute with n-hexane:isopropanol
96:4, pass through the SPE cartridge. Evaporate the eluate to dryness under a stream
of nitrogen, reconstitute the residue with 200 µL toluene:EtOH 50:50, inject an aliquot.
HPLC VARIABLES
Column: 150 × 6 Sumipax ODS A212
Mobile phase: MeCN:THF:water 45:5:50
Flow rate: 1.5
Detector: Radioactivity (3 H)
CHROMATOGRAM
Retention time: 60
OTHER SUBSTANCES
Extracted: metabolites
KEY WORDS
SPE
REFERENCE
Miyahara, T.; Gomyo, S.; Ueda, Y.; Ohyama, Y.; Sigeno, C.; Kozakai, A.; Takamura, T.; Yamazaki, R.;
Higuchi, S.; Yamamoto, M.; Sakuma, T.; Nemoto, N. Metabolism of 26,27-hexafluoro-1α,25-dihydroxyvitamin D3 and 26,27-hexafluoro-1α,23(S)25−trihydroxyvitamin D3 in ROS17/2.8 cells transfected
with a plasmid expressing CYP24, Xenobiotica, 2000, 30, 1055–1062.
SAMPLE
Matrix: enzyme incubations
Sample preparation: Extract enzyme incubation with 3 vol dichloromethane, evaporate the extract to dryness, dissolve the residue in MeCN, inject an aliquot.
HPLC VARIABLES
Column: 300 × 4 µBondapak C18
Column temperature: 50
Mobile phase: Gradient. MeCN:water from 50:50 to 100:0 over 15 (?) min.
Flow rate: 1
Detector: UV 265
Falecalcitriol
249
CHROMATOGRAM
Retention time: 12
OTHER SUBSTANCES
Extracted: metabolites
REFERENCE
Hayashi, K.; Akiyoshi-Shibata, M.; Sakaki, T.; Yabusaki, Y. Rat CYP24 catalyses 23S-hydroxylation of
26,26,26,27,27,27-hexafluorocalcitriol in vitro, Xenobiotica, 1998, 28, 457–463.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 × 4.6 Zorbax Sil
Mobile phase: n-Hexane:dichloromethane:methanol 49:48:3
Flow rate: 1.5
Detector: UV 254
CHROMATOGRAM
Retention time: 12
OTHER SUBSTANCES
Simultaneous: metabolites
KEY WORDS
normal phase
REFERENCE
Imanishi, Y.; Inaba, M.; Seki, H.; Koyama, H.; Nishizawa, Y.; Morii, H.; Otani, S. Increased biological
potency of hexafluorinated analogs of 1,25-dihydroxyvitamin D3 on bovine parathyroid cells, J.Steroid
Biochem.Mol.Biol., 1999, 70, 243–248.
ANNOTATED BIBLIOGRAPHY
Harada, M.; Miyahara, T.; Kajita-Kondo, S.; Kozakai, A.; Higuchi, S.; Otomo, S.; Kozuka, H. Differences in metabolism between 26,26,26,27,27,27-hexafluoro-1α,25-dihydroxyvitamin D3 and 1α,25dihydroxyvitamin D3 in cultured neonatal mouse calvaria, Res.Commun.Mol.Pathol.Pharmacol., 1994,
86, 183–193.
Miyahara, T.; Harada, M.; Kozakai, A.; Matsumoto, M.; Hashimoto, K.; Inoue, H.; Yoda, K.; Nakatsu, T.;
Kajita, S.; Yamazaki, R.; Higuchi, S.; Kozuka, H.; Nemoto, N. Comparison of 26,27-hexafluoro-1α,25dihydroxyvitamin D3 and 1α,25-dihydroxyvitamin D3 on the resorption of bone explants ex vivo.
J.Nutr.Sci.Vitaminol., 1999, 45, 239–249.
250
Fenoxycarb
Fenoxycarb
O
Molecular formula: C17 H19 NO4
H
O
Molecular weight: 301.34
CAS Registry No: 72490-01-8
Merck Index: 13, 4013
N
O
CH3
O
SAMPLE
Matrix: fruit, vegetables
Sample preparation: Blend 500 mg chopped fruit or vegetable with 500 mg 45–55 µm
C8 reversed-phase material in a mortar and pestle for 5 min, place in a 100 × 9 glass
column, elute with 10 mL MeCN:dichloromethane 40:60. Concentrate the eluate to
500 µL under a stream of nitrogen, inject a 5 µL aliquot.
HPLC VARIABLES
Guard column: 10 × 4.6 5 µm LiChrosorb RP-8
Column: 150 × 4.6 3 µm Spherisorb C8
Mobile phase: Gradient. MeOH:water 50:50 for 15 min, to 70:30 over 5 min, maintain
at 70:30 for 5 min, to 90:10 over 5 min, maintain at 90:10 for 5 min.
Flow rate: 0.5
Injection volume: 5
Detector: MS, HP, electrospray, positive mode, gas 350◦ , drying gas flow 13 L/min,
nebulizer gas pressure 30 psi, capillary 4000 V, m/z 302.1
CHROMATOGRAM
Retention time: 30
Limit of detection: 5 ng/g
OTHER SUBSTANCES
Extracted: carbaryl (19), carbofuran (17), diethofencarb (24), ethiofencarb (20), fenobucarb (24), isoprocarb (21), methiocarb (26), metholcarb (13.5), oxamyl (4.5), pirimicarb
(22), propoxur (16), thiobencarb (32.5)
KEY WORDS
matrix solid phase dispersion
REFERENCE
Fernández, M.; Picó, Y.; Mañes, J. Determination of carbamate residues in fruits and vegetables by
matrix solid-phase dispersion and liquid chromatography-mass spectrometry, J.Chromatogr.A, 2000,
871, 43–56.
ANNOTATED BIBLIOGRAPHY
Fernández, M.; Picó, Y.; Mañes, J. Simultaneous determination of carbamate and organophosphorus
pesticides in honeybees by liquid chromatography-mass spectrometry, Chromatographia, 2003, 58,
151–158. [very similar to above method]
251
Fenticonazole
Fenticonazole
Molecular formula: C24 H20 Cl2 N2 OS
N
Cl
N
S
Molecular weight: 455.41
CAS Registry No: 72479-26-6
Merck Index: 13, 4033
O
Cl
SAMPLE
Matrix: blood
Sample preparation: Vortex 50 µL plasma with 100 µL 2 ng/mL IS in MeCN, centrifuge at 12 000 rpm for 2 min, inject a 20 µL aliquot of the supernatant.
HPLC VARIABLES
Guard column: 15 × 3.2 Brownlee C18
Column: 50 × 4.6 3 µm Shandon BDS C8
Column temperature: 45
Mobile phase: MeCN:buffer 70:30 (The buffer was 10 mM ammonium formate adjusted
to pH 3.5 with formic acid.)
Flow rate: 1
Injection volume: 20
Detector: MS, PE Sciex API-III+ triple quadrupole, APCI, positive ion, heated nebulizer
interface, nebulizer probe 500◦ , nebulizer gas nitrogen 0.6 L/min, curtain gas nitrogen at
0.8 L/min, auxiliary gas nitrogen 2.0 L/min, nebulizer gas pressure 70 psi, m/z 455–199
CHROMATOGRAM
Retention time: 2.25
Internal standard: econazole (m/z 381–125) (1.5)
Limit of quantitation: 0.49 ng/mL
OTHER SUBSTANCES
Simultaneous: clindamycin (1)
KEY WORDS
plasma
REFERENCE
Speed, W.; Long, J.M.; Simmonds, R.J.; Enos, T.A. The development and validation of a high performance
liquid chromatography (HPLC)/tandem mass spectrometry assay for fenticonazole in human plasma
and comparison with an HPLC-UV method, Rapid Commun.Mass Spectrom., 1995, 9, 1452–1456.
SAMPLE
Matrix: blood
Sample preparation: Liquid–liquid extraction of 1 mL basified plasma (not otherwise
specified), inject an 80 µL aliquot of the supernatant.
HPLC VARIABLES
Guard column: Guard Pak µBondapak C18
Column: 150 × 4.6 4 µm phenyl
Column temperature: 40
Mobile phase: MeCN:buffer 76:24 (The buffer was 120 mM acetic acid containing
0.02 mM sodium bisulfate.)
Flow rate: 1.5
Injection volume: 80
Detector: UV 254
252
Fenticonazole
CHROMATOGRAM
Retention time: 12.9
Internal standard: miconazole (9.9)
Limit of quantitation: 5 ng/mL
OTHER SUBSTANCES
Simultaneous: clindamycin (1)
KEY WORDS
plasma
REFERENCE
Speed, W.; Long, J.M.; Simmonds, R.J.; Enos, T.A. The development and validation of a high performance
liquid chromatography (HPLC)/tandem mass spectrometry assay for fenticonazole in human plasma
and comparison with an HPLC-UV method, Rapid Commun.Mass Spectrom., 1995, 9, 1452–1456.
Fexofenadine
Fexofenadine
CH3
CH3
COOH
Molecular formula: C32 H39 NO4
Molecular weight: 501.65
CAS Registry No: 83799-24-0
Merck Index: 13, 4101
253
N
OH
OH
SAMPLE
Matrix: blood
Sample preparation: Mix 1 mL whole blood with 20 µL 1 µg/mL dibenzepin in
MeOH:water 50:50, add 300 µL pH 11 Tris buffer, mix, add 500 µL butyl acetate,
vortex for 2 min, centrifuge. Preserve the aqueous layer (A). Remove the organic layer
and add it to 75 µL 10 mM ammonium acetate buffer containing 0.1% formic acid (pH
3.2), evaporate (?). Add 75 µL MeCN, sonicate for 5 min, centrifuge at 5000 rpm for
5 min, keep the extract as B. Add 20 µL 1 µg/mL enalapril in MeOH:water 50:50 and
120 mg NaCl to the aqueous layer (A), mix, add 500 µL pH 3 phosphate buffer, add
600 µL 8.5% phosphoric acid, add 5 mL dichloromethane:isopropanol 95:5, shake at
250 cycles/min in a bench-top shaker for 30 min, centrifuge at 5000 rpm for 5 min.
Remove the lower organic layer and evaporate it to dryness under a stream of air at
45◦ . Reconstitute the residue with 150 µL initial mobile phase, sonicate, centrifuge,
combine with extract B, inject a 30 µL aliquot. (Sample preparation from Gergov,M.;
Robson,J.N.; Ojanperä,I.; Heinonen,O.P.; Vuori,E. Simultaneous screening and quantitation of 18 antihistamine drugs in blood by liquid chromatography ionspray tandem
mass spectrometry. Forensic Sci.Inter. 2001, 121, 108–115.)
HPLC VARIABLES
Guard column: 40 mm long 4 µm Purospher RP-18 LiChro Cart 4-4
Column: 100 × 2.1 4 µm Genesis C18 (Jones Chromatography)
Column temperature: 35
Mobile phase: Gradient. MeCN:buffer from 20:80 to 100:0 over 10 min, maintain at
0:100 for 3 min, re-equilibrate at initial conditions for 5 min. (The buffer was 10 mM
ammonium acetate containing 0.1% formic acid (pH 3.2).)
Flow rate: 0.2
Injection volume: 30
Detector: MS, PE Sciex API 365 triple-stage quadrupole LC-MS-MS, PE Sciex Turbo Ion
Spray interface, positive ion mode, needle voltage 5.2 kV, nebulizer gas air at 60 psi,
curtain gas nitrogen at 40 psi, collision cell gas nitrogen at 40 psi, turbo ionspray heater
375◦ , heater gas flow 7 L/min
CHROMATOGRAM
Retention time: 6.3
Internal standard: dibenzepin, enalapril
Limit of detection: <20 ng/mL
OTHER SUBSTANCES
Extracted: acebutolol (3.8, LOD 0.1 µg/mL), acrivastine (5.7, LOD <0.02 µg/mL),
alprazolam (6.1, LOD <0.02 µg/mL), alprenolol (5.4, LOD 0.01 µg/mL), amantadine
(3.4, LOD 0.1 µg/mL), amiloride (2.0, LOD 0.1 µg/mL), aminophenazone (2.8, LOD
<5 µg/mL), amiodarone (10.2, LOD 0.05 µg/mL), amitriptyline (6.6, LOD <0.02 µg/mL),
astemizole (5.8, LOD <0.02 µg/mL), atenolol (1.7, LOD 0.30 µg/mL), azacyclonol (5.1,
LOD 0.02 µg/mL), benzhexol (6.6, LOD <0.02 µg/mL), benzoylecgonine (3.3, LOD
0.01 µg/mL), betaxolol (5.5, LOD 0.01 µg/mL), biperidine (6.2, LOD <0.02 µg/mL),
bisoprolol (5.0, LOD <0.02 µg/mL), brompheniramine (5.3, LOD 0.002 µg/mL),
254
Fexofenadine
bupivacaine (5.1, LOD <0.02 µg/mL), buprenorphine (5.9, LOD 0.01 µg/mL),
buspirone (5.1, LOD 0.002 µg/mL), caffeine (2.8, LOD 1 µg/mL), carbamazepine
(6.1, LOD <0.02 µg/mL), carbinoxamine (5.1, LOD 0.002 µg/mL), carisoprodol
(6.7, LOD <5 µg/mL), carvedilol (6.2, LOD <0.02 µg/mL), celiprolol (4.3, LOD
0.05 µg/mL), cetirizine (6.3, LOD 0.05 µg/mL), chlorcyclizine (6.6, LOD <0.02 µg/mL),
chlordiazepoxide (5.7, LOD <0.02 µg/mL), chlormezanone (5.8, LOD <5 µg/mL),
chloroquine (2.7, LOD 0.02 µg/mL), chlorpheniramine (5.1, LOD 0.002 µg/mL),
chlorpromazine (7.0, LOD 0.02 µg/mL), chlorpropamide (6.7, LOD <5 µg/mL),
chlorprothixene (7.0, LOD <0.02 µg/mL), cinnarizine (7.9, LOD <0.02 µg/mL),
citalopram (5.7, LOD <0.02 µg/mL), clemastine (7.7, LOD 0.02 µg/mL), clobazam
(7.3, LOD <0.02 µg/mL), clobutinol (5.3, LOD 0.02 µg/mL), clomethiazole (6.2,
LOD 0.5 µg/mL), clomipramine (7.1, LOD <0.02 µg/mL), clonazepam (6.6, LOD
<0.02 µg/mL), clonidine (2.8, LOD 0.1 µg/mL), clozapine (5.6, LOD <0.02 µg/mL),
cocaine (4.6, LOD <0.02 µg/mL), codeine (2.5, LOD 0.1 µg/mL), coumatetralyl
(8.4, LOD 0.05 µg/mL), cyclizine (5.8, LOD <0.02 µg/mL), dextropropoxyphene
(6.6, LOD 0.05 µg/mL), demoxepam (5.8, LOD 0.02 µg/mL), dextromethorphan
(5.5, LOD <0.02 µg/mL), diazepam (8.1, LOD 0.02 µg/mL), diltiazem (5.8,
LOD <0.02 µg/mL), diphenhydramine (5.7, LOD <0.02 µg/mL), dipyridamole (5.4,
LOD 0.005 µg/mL), disopyramine (4.4, LOD <0.02 µg/mL), dixyrazine (6.8, LOD
0.005 µg/mL), doxapram (4.8, LOD <0.02 µg/mL), doxepin (5.9, LOD <0.02 µg/mL),
ebastine (9.6, LOD 0.005 µg/mL), embutramide (6.7, LOD 0.005 µg/mL), ergotamine
(5.5, LOD 0.005 µg/mL), ethenzamide (5.0, LOD 0.05 µg/mL), ethylmorphine (3.2,
LOD 0.05 µg/mL), ethylparathion (9.7, LOD <5 µg/mL), etodroxizine (6.4, LOD
<0.02 µg/mL), felodipine (9.6, LOD 0.02 µg/mL), fenazepam (7.5, LOD <0.02 µg/mL),
fenfluramine (5.3, LOD <0.02 µg/mL), fenkamfamine (5.1, LOD <0.02 µg/mL),
fentanyl (5.5, LOD <0.02 µg/mL), flecainide (5.9, LOD <0.02 µg/mL), fluconazole
(4.0, LOD 0.1 µg/mL), flumazenil (5.2, LOD <0.02 µg/mL), flunitrazepam (7.1, LOD
0.002 µg/mL), fluoxetine (6.8, LOD 0.1 µg/mL), flupentixol (7.5, LOD 0.18 µg/mL),
fluvoxamine (6.3, LOD 0.02 µg/mL), glibenclamide (8.5, LOD <0.02 µg/mL), glipizide
(6.8, LOD <0.05 µg/mL), haloperidol (6.1, LOD <0.02 µg/mL), histapyrrodine (6.3,
LOD 0.02 µg/mL), hydrocodone (3.0, LOD 0.05 µg/mL), hydroxychloroquine (2.4, LOD
<0.3 µg/mL), hydroxyzine (6.3, LOD <0.02 µg/mL), imipramine (6.4, LOD 0.05 µg/mL),
indomethacin (8.6, LOD 0.05 µg/mL), isoniazid (2.2, LOD 3 µg/mL), isradipine (8.6, LOD
0.05 µg/mL), ketamine (3.6, LOD <0.05 µg/mL), ketobemidone (3.3, LOD <0.05 µg/mL),
ketoprofen (7.3, LOD 0.1 µg/mL), ketorolac (6.2, LOD 0.05 µg/mL), labetalol (4.9, LOD
0.05 µg/mL), lamotrigine (4.0, LOD 0.1 µg/mL), levocabastine (5.8, LOD 0.01 µg/mL),
levomepromazine (6.5, LOD 0.02 µg/mL), lidocaine (3.7, LOD <0.05 µg/mL), loratadine
(9.3, LOD 0.002 µg/mL), lorazepam (6.6, LOD 0.02 µg/mL), lormetazepam (7.4,
LOD <0.02 µg/mL), LSD (4.7, LOD <0.02 µg/mL), malathion (8.9, LOD 10 µg/mL),
maprotiline (6.4, LOD <0.02 µg/mL), MDMA (3.3, LOD 0.02 µg/mL), meclozine (8.5,
LOD <0.02 µg/mL), medazepam (6.3, LOD <0.02 µg/mL), meloxicam (7.1, LOD
0.01 µg/mL), melperone (5.0, LOD <0.02 µg/mL), meperidine (4.7, LOD <0.02 µg/mL),
mepivacaine (3.7, LOD <0.02 µg/mL), meprobamate (4.9, LOD 0.1 µg/mL), mesoridazine
(5.4, LOD <0.02 µg/mL), methamphetamine (3.3, LOD 0.05 µg/mL), methadone (6.7,
LOD <0.02 µg/mL), methylparathion (8.6, LOD 10 µg/mL), methylphenidate (4.2,
LOD <0.02 µg/mL), metoclopramide (3.8, LOD <0.02 µg/mL), metoprolol (4.1, LOD
0.02 µg/mL), metronidazole (2.6, LOD 1 µg/mL), mexiletine (4.4, LOD 0.05 µg/mL),
mianserin (5.7, LOD <0.02 µg/mL), midazolam (5.9, LOD <0.02 µg/mL), mirtazapine
(4.4, LOD <0.02 µg/mL), mizolastine (5.5, LOD 0.01 µg/mL), moclobemide (3.7,
LOD 0.05 µg/mL), molindone (4.0, LOD <0.02 µg/mL), monoacetylmorphine (2.7,
LOD 0.1 µg/mL), morphine (2.0, LOD 0.1 µg/mL), nicotine (2.2, LOD 0.05 µg/mL),
nifedipine (7.5, LOD 0.02 µg/mL), nikethamide (3.6, LOD <0.02 µg/mL), nitrazepam
(6.5, LOD <0.02 µg/mL), nizatidine (1.7, LOD 1 µg/mL), nomifensine (4.6, LOD
<0.02 µg/mL), nortriptyline (6.4, LOD <0.02 µg/mL), norverapamil (6.2, LOD 1 µg/mL),
noscapine (5.0, LOD <0.02 µg/mL), olanzapine (3.0, LOD 0.05 µg/mL), ondansetron
(4.6, LOD <0.02 µg/mL), orphenadrine (6.1, LOD <0.02 µg/mL), oxazepam (6.3, LOD
<0.02 µg/mL), oxcarbazepine (5.3, LOD 0.02 µg/mL), oxprenolol (4.7, LOD 0.02 µg/mL),
oxycodone (2.8, LOD 0.05 µg/mL), papaverine (4.8, LOD <0.02 µg/mL), acetaminophen
(2.5, LOD <5 µg/mL), paroxetine (6.2, LOD 0.02 µg/mL), pemoline (3.3, LOD
0.05 µg/mL), pentazocine (5.0, LOD <0.02 µg/mL), pentifylline (7.3, LOD <5 µg/mL),
Fexofenadine
255
pentoxyverine (6.6, LOD <0.02 µg/mL), perphenazine (6.9, LOD 0.002 µg/mL),
phenazone (3.9, LOD 0.05 µg/mL), phencyclidine (5.3, LOD 0.05 µg/mL), pheniramine
(4.1, LOD 0.02 µg/mL), phenylbutazone (9.0, LOD <5 µg/mL), phenylpropanolamine
(2.5, LOD 0.3 µg/mL), phenytoin (6.1, LOD 0.05 µg/mL), pindolol (3.3, LOD 0.05 µg/mL),
piroxicam (6.6, LOD 0.02 µg/mL), pitofenone (5.4, LOD <0.02 µg/mL), pizotifen (6.5,
LOD <0.02 µg/mL), practolol (1.8, LOD 0.1 µg/mL), prazosin (4.1, LOD 0.05 µg/mL),
prilocaine (3.8, LOD <0.02 µg/mL), primidone (4.0, LOD <5 µg/mL), procainamide
(2.2, LOD 0.05 µg/mL), prochlorperazine (7.5, LOD 0.02 µg/mL), promazine (6.2,
LOD <0.02 µg/mL), promethazine (6.0, LOD 0.05 µg/mL), propafenone (6.3, LOD
<0.02 µg/mL), propranolol (5.4, LOD 0.02 µg/mL), propyphenazone (6.6, LOD
0.50 µg/mL), pseudoephedrine (2.6, LOD 1 µg/mL), quinine (4.2, LOD 0.02 µg/mL),
ranitidine (1.8, LOD 0.1 µg/mL), risperidone (4.9, LOD <0.02 µg/mL), rocurone
(3.8, LOD 0.1 µg/mL), ropivacaine (4.6, LOD <0.02 µg/mL), salicylamide (4.2, LOD
<5 µg/mL), selegiline (4.1, LOD 0.05 µg/mL), sertindole (7.2, LOD <0.02 µg/mL),
sertraline (6.8, LOD 0.02 µg/mL), sulindac (6.5, LOD 0.02 µg/mL), simazine (6.0, LOD
0.1 µg/mL), sincocaine (6.5, LOD <0.02 µg/mL), sisapride (5.9, LOD <0.02 µg/mL),
sotalol (2.1, LOD 0.1 µg/mL), strychnine (5.3, LOD 0.05 µg/mL), sulpiride (1.9, LOD
0.1 µg/mL), sulthiame (4.1, LOD 0.05 µg/mL), temazepam (7.2, LOD <0.02 µg/mL),
terbutaline (2.3, LOD 0.1 µg/mL), terfenadine (8.1, LOD 0.002 µg/mL), terodiline
(6.7, LOD <0.02 µg/mL), tetracaine (5.7, LOD <0.02 µg/mL), dronabinol (12.3,
LOD 0.05 µg/mL), tetrahydrozoline (3.6, LOD 0.1 µg/mL), theobromine (2.3, LOD
<5 µg/mL), theophylline (2.4, LOD <5 µg/mL), thioridazine (7.5, LOD 0.02 µg/mL),
timolol (3.8, LOD 0.05 µg/mL), thiothixene (6.7, LOD 0.02 µg/mL), tolbutamide (7.1,
LOD <5 µg/mL), toremifene (8.7, LOD 0.02 µg/mL), tramadol (4.2, LOD 0.02 µg/mL),
trazodone (5.2, LOD <0.02 µg/mL), triamterene (3.2, LOD 0.1 µg/mL), triazolam (6.7,
LOD 0.002 µg/mL), trimeprazine (6.4, LOD <0.02 µg/mL), trimethoprim (3.1, LOD
0.05 µg/mL), trimipramine (6.7, LOD <0.02 µg/mL), venlafaxine (4.9, LOD 0.02 µg/mL),
verapamil (6.5, LOD <0.02 µg/mL), warfarin (7.9, LOD <0.02 µg/mL), yohimbine (4.5,
LOD <0.02 µg/mL), zolpidem (4.7, LOD <0.02 µg/mL), zopiclone (4.0, LOD 0.1 µg/mL)
KEY WORDS
whole blood
REFERENCE
Gergov, M.; Ojanperä, I.; Vuori, E. Simultaneous screening for 238 drugs in blood by liquid chromatography-ion spray tandem mass spectrometry with multiple-reaction monitoring, J.Chromatogr.B,
2003, 795, 41–53.
SAMPLE
Matrix: formulations
Sample preparation: Extract tablets with MeOH, centrifuge, dilute the supernatant
with mobile phase, inject an aliquot.
HPLC VARIABLES
Column: 150 × 4.6 5 µm Eclipse XDB C8
Mobile phase: MeCN:MeOH:buffer 20:20:60 (The buffer was 1% triethylamine adjusted
to pH 3.7 with concentrated orthophosphoric acid.)
Flow rate: 1.2
Injection volume: 10
Detector: UV 210
CHROMATOGRAM
Retention time: 14
Internal standard: 5-methyl-2-nitrophenol (12)
Limit of detection: 0.12–0.18 µg/mL
Limit of quantitation: 0.5–0.6 µg/mL
256
Fexofenadine
OTHER SUBSTANCES
Simultaneous: impurities
KEY WORDS
robust; tablets; validated
REFERENCE
Radhakrishna, T.; Reddy, G.O. Simultaneous determination of fexofenadine and its related compounds
by HPLC, J.Pharm.Biomed.Anal., 2002, 29, 681–690.
ANNOTATED BIBLIOGRAPHY
Dresser, G.K.; Bailey, D.G.; Leake, B.F.; Schwarz, U.I.; Dawson, P.A.; Freeman, D.J.; Kim, R.B. Fruit
juices inhibit organic anion transporting polypeptide-mediated drug uptake to decrease the oral
availability of fexofenadine, Clin.Pharmacol.Ther., 2002, 71, 11–20.
Hamman, M.A.; Bruce, M.A.; Haehner-Daniels, D.; Hall, S.D. The effect of rifampin administration on
the disposition of fexofenadine, Clin.Pharmacol.Ther., 2001, 69, 114–121. [SPE]
Hofmann, U.; Seiler, M.; Drescher, S.; Fromm, M.F. Determination of fexofenadine in human plasma
and urine by liquid chromatography-mass spectrometry, J.Chromatogr.B, 2002, 766, 227–233. [SPE]
Kim, R.B.; Leake, B.F.; Choo, E.F.; Dresser, G.K.; Kubba, S.V.; Schwarz, U.I.; Taylor, A.; Xie, H.-G.; McKinsey, J.; Zhou, S.; Lan, L.-B.; Schuetz, J.D.; Schuetz, E.G.; Wilkinson, G.R. Identification of functionally variant MDR1 alleles among European Americans and African Americans, Clin.Pharmacol.Ther.,
2001, 70, 189–199.
Milne, R.W.; Larsen, L.A.; Jorgensen, K.L.; Bastlund, J.; Stretch, G.R.; Evans, A.M. Hepatic disposition of fexofenadine: Influence of the transport inhibitors erythromycin and dibromosulphothalein,
Pharm.Res., 2000, 17, 1511–1515.
Putnam, W.S.; Ramanathan, S.; Pan, L.; Takahashi, L.H.; Benet, L.Z. Functional characterization of
monocarboxylic acid, large neutral amino acid, bile acid and peptide transporters, and P-glycoprotein
in MDCK and Caco-2 cells, J.Pharm.Sci., 2002, 91, 2622–2635. [column-switching]
Robbins, D.K.; Castles, M.A.; Pack, D.J.; Bhargava, V.O.; Weir, S.J. Dose proportionality and comparison
of single and multiple dose pharmacokinetics of fexofenadine (MDL 16455) and its enantiomers in
healthy male volunteers, Biopharm.Drug Dispos., 1998, 19, 455–463.
Soldner, A.; Christians, U.; Susanto, M.; Wacher, V.J.; Silverman, J.A.; Benet, L.Z. Grapefruit juice activates P-glycoprotein-mediated drug transport, Pharm.Res., 1999, 16, 478–485. [losartan; felodipine;
nifedipine; fexofenadine; column-switching]
Tannergren, C.; Petri, N.; Knutson, L.; Hedeland, M.; Bondesson, U.; Lennernäs, H. Multiple transport mechanisms involved in the intestinal absorption and first-pass extraction of fexofenadine,
Clin.Pharmacol.Ther., 2003, 74, 423–436. [SPE; LC-MS; UV detection]
257
Flomoxef
Flomoxef
Molecular formula: C15 H18 F2 N6 O7 S2
Molecular weight: 496.47
CAS Registry No: 99665-00-6
Merck Index: 13, 4132
OCH3
H
O
S
F
F
N
O
N
H
S
O
COOH
N
N
N N
HO
SAMPLE
Matrix: blood
Sample preparation: Condition a 3 mL 200 mg Bond-Elut C18 SPE cartridge with
two 3 mL portions of MeOH and two 3 mL portions of 5% acetic acid. Mix 500 µL
serum with 500 µL 10% acetic acid, add to the SPE cartridge, wash with 3 mL water,
elute with four 500 µL portions of MeOH:water 60:40. Evaporate the combined eluates
to dryness under a stream of nitrogen, reconstitute the residue with 250 µL water,
filter (0.22 µm). Inject a 100 µL aliquot onto column A and elute to waste with mobile
phase A. After an unspecified time, backflush the contents of column A onto column
B using mobile phase B. Monitor the effluent from column B. (Sato,K.; Kobayashi,K.;
Moore,C.M.; Mizuno,Y.; Katsumata,Y. Semi-quantitative analysis of cefaclor in human
serum by capillary high performance liquid chromatography/fast atom bombardment
mass spectrometry. Forensic Sci.Int. 1993, 59, 71–77.)
HPLC VARIABLES
Column: A 30 × 0.5 10 µm Develosil PhA (phenethyl); B 150 × 0.5 5 µm Develosil PhA
(phenethyl)
Mobile phase: A 10 mM ammonium acetate:glycerol 99.5:0.5, adjusted to pH 5 with
acetic acid; B MeOH:water:acetic acid:glycerol 40:59:0.5:0.5 (pH ca. 3)
Flow rate:: A 0.05; B 0.004
Injection volume: 100
Detector: MS, JMS DX303 double focusing, xenon FAB ion source, gun current 10 mA,
voltage 3 kV, positive ion mode
CHROMATOGRAM
Retention time: 18.3
LIMIT OF DETECTION:
200–1000 ng
OTHER SUBSTANCES
Extracted: cefaclor (14.4), cefamandole (31.0), cefazolin (19.7), cefbuperazone (20.8),
cefixime (19.2), cefmenoxime (18.8), cefmetazole (21.4), cefoperazone (24.2), cefotaxime
(16.1), cefotetan (16.8), cefotiam (11.7), cefpiramide (20.9), cefsulodin (12.2), ceftazidime (12.0), ceftizoxime (15.6), ceftriaxone (16.7), cefuroxime (16.9), cefuzonam
(32.0), cephalexin (14.8), cephaloglycine (15.7), cephaloridine (15.6), cephalothin (34.7),
latamoxef (15.1)
KEY WORDS
column-switching; serum; SPE
REFERENCE
Kobayashi, K.; Sato, K.; Mizuno, Y.; Katsumata, Y. Capillary high-performance liquid chromatographyfast atom bombardment mass spectrometry of 24 cephem antibiotics, J.Chromatogr.B, 1996, 677,
275–290.
258
Florfenicol
Florfenicol
OH
H
Cl
N
Cl
Molecular formula: C12 H14 Cl2 FNO4 S
Molecular weight: 358.22
CAS Registry No: 73231-34-2
Merck Index: 13, 4135
H3C
S
O
F
O
O
SAMPLE
Matrix: blood, CSF
Sample preparation: Condition a 3 mL 500 mg Bond-Elut C18 SPE cartridge with
2 mL MeOH and 2 mL water. Mix 250 µL plasma or CSF with 750 µL water and 40 µL
8 µg/mL IS in MeOH:water 2:98 or 400 µg/mL IS in MeOH, vortex. Add to the SPE
cartridge, wash with 2 mL MeCN:water 15:85, wash with 3 mL hexane, elute with 3 mL
MeCN. Evaporate the eluate to dryness under reduced pressure. Dissolve the residue
in 500 µL MeCN:water 22:78, filter (0.45 µm polyvinylidene difluoride), inject a 100 µL
aliquot.
HPLC VARIABLES
Column: 250 × 4.6 5 µm Hypersil ODS
Column temperature: 40
Mobile phase: MeCN:50 mM ammonium acetate 22:78
Flow rate: 1.5
Injection volume: 100
Detector: UV 224
CHROMATOGRAM
Retention time: 6
Internal standard: chloramphenicol (7.50)
Limit of detection: 20 ng/mL
Limit of quantitation: 30 ng/mL
KEY WORDS
calf; cow; pharmacokinetics; plasma; SPE
REFERENCE
de Craene, B.A.; Deprez, P.; D’Haese, E.; Nelis, H.J.; Van den Bossche, W.; De Leenheer, P. Pharmacokinetics of florfenicol in cerebrospinal fluid and plasma of calves, Antimicrob.Agents Chemother., 1997,
41, 1991–1995.
SAMPLE
Matrix: water
Sample preparation: Inject a 25 µL aliquot.
HPLC VARIABLES
Column: 150 × 3.9 4 µm Nova-Pak C18
Column temperature: 30
Mobile phase: MeCN:buffer 15:85 (The buffer was 3.28 g sodium acetate and 1.01 g
sodium 1-heptanesulfonate in 1 L water, adjusted to pH 4.6 with phosphoric acid.)
Flow rate: 1
Injection volume: 25
Detector: UV 224
CHROMATOGRAM
Retention time: 8
Florfenicol
259
Limit of detection: 0.1 µg/mL
Limit of quantitation: 0.2 µg/mL
OTHER SUBSTANCES
Simultaneous: degradants, thiamphenicol (3.3)
REFERENCE
Hayes, J.M.; Eichman, J.; Katz, T.; Gilewicz, R. Stability of florfenicol in drinking water, J.AOAC Int.,
2003, 86, 22–29.
ANNOTATED BIBLIOGRAPHY
De Wasch, K.; Van Hoof, N.; Poelmans, S.; Okerman, L.; Courtheyn, D.; Ermens, A.; Cornelis, M.; De Brabander, H.F. Identification of ‘‘unknown analytes’’ in injection sites: a semi-quantitative interpretation,
Anal.Chim.Acta, 2003, 483, 387–399.
Hormazabal, V.; Steffenak, I.; Yndestad, M. Simultaneous determination of residues of florfenicol and the
metabolite florfenicol amine in fish tissues by high-performance liquid chromatography, J.Chromatogr.,
1993, 616, 161–165.
Hormazabal, V.; Steffenak, I.; Yndestad, M. Simultaneous extraction and determination of florfenicol and the metabolite florfenicol amine in sediment by high-performance liquid chromatography,
J.Chromatogr.A, 1996, 724, 364–366.
Liu, J.; Fung, K.-F.; Chen, Z.; Zeng, Z.; Zhang, J. Pharmacokinetics of florfenicol in healthy pigs and in
pigs experimentally infected with Actinobacillus pleuropneumoniae, Antimicrob.Agents Chemother.,
2003, 47, 820–823.
Lobell, R.D.; Varma, K.J.; Johnson, J.C.; Sams, R.A.; Gerken, D.F.; Ashcraft, S.M. Pharmacokinetics of
florfenicol following intravenous and intramuscular doses to cattle, J.Vet.Pharmacol.Ther., 1994, 17,
253–258. [thiamphenicol is internal standard]
Nagata, T.; Saeki, M. Simultaneous determination of thiamphenicol, florfenicol and chloramphenicol
residues in muscles of animals and cultured fish by liquid chromatography, J.Liq.Chromatogr., 1992,
15, 2045–2056. [LOD 0.01 ppm; column temp 55◦ ; SPE]
Vue, C.; Schmidt, L.J.; Stehly, G.R.; Gingerich, W.H. Liquid chromatographic determination of florfenicol
in the plasma of multiple species of fish, J.Chromatogr.B, 2002, 780, 111–117.
260
Fludrocortisone
Fludrocortisone
O
HO
Molecular formula: C21 H29 FO5
Molecular weight: 380.45
CAS Registry No: 127-31-1, 514-36-3 (21-acetate)
Merck Index: 13, 4156
CH3
OH
OH
CH3
F
H
H
O
SAMPLE
Matrix: blood
Sample preparation: Condition a 3 mL 500 mg Sep-Pak Vac C18 SPE cartridge with
3 mL MeOH and 3 mL water. Mix 500 µL 200 mM pH 3.85 acetate buffer with 1 mL
serum, add 400 µL 2.5 µM IS in mobile phase, centrifuge, add to the SPE cartridge, wash
with 3 mL acetone:water 20:80, wash with 3 mL water, wash with 3 mL hexane, elute
with 3 mL diethyl ether. Vortex the eluate with 1 mL 200 mM NaOH, centrifuge. Evaporate the organic layer to dryness under a stream of nitrogen, reconstitute the residue
with 250 µL mobile phase, place on a rotary mixer for 5 min, inject a 60 µL aliquot.
(Fludrocortisone is IS. Extraction from serum has not been strictly demonstrated.)
HPLC VARIABLES
Column: 250 × 4.6 5 µm Spherex C18 (Phenomenex)
Mobile phase: MeOH:THF:water 3:25:72
Flow rate: 1
Injection volume: 60
Detector: UV 254
CHROMATOGRAM
Retention time: 15.90
Internal standard: fludrocortisone (15.90)
Limit of detection: 5 nM
Limit of quantitation: 10 nM
OTHER SUBSTANCES
Extracted: 11-deoxycortisol (22.10), dexamethasone (29.85), hydrocortisone (12.85),
methylprednisolone (21.00), prednisolone (12.00)
KEY WORDS
fludrocortisone is IS in original method; serum; SPE
REFERENCE
McWhinney, B.C.; Ward, G.; Hickman, P.E. Improved HPLC method for simultaneous analysis of cortisol, 11-deoxycortisol, prednisolone, methylprednisolone, and dexamethasone in serum and urine,
Clin.Chem., 1996, 42, 979–981.
SAMPLE
Matrix: blood
Sample preparation: Condition a 3 mL 200 mg Clean-Up C18 SPE cartridge (Worldwide Monitoring) with two 2 mL portions of MeOH and two 2 mL portions of water.
Centrifuge plasma at 2000 rpm for 5 min prior to analysis. Mix 2 mL plasma with
40 µL MeCN, add 1 mL water, vortex until homogeneous, add to the SPE cartridge,
wash with two 2 mL portions of acetone:water 20:80, wash with 1 mL 50 mM pH 2.7
phosphate buffer, place under vacuum for 3 min, elute with two 2.5 mL portions of ethyl
acetate. Evaporate the eluate to dryness under reduced pressure at 40◦ , reconstitute the
residue with 75 µL mobile phase B, vortex, add 100 µL 50 mM pH 3 phosphate buffer,
centrifuge at 5000 rpm for 5 min, inject a 145 µL aliquot.
Fludrocortisone
261
HPLC VARIABLES
Column: 100 × 2 5 µm YMC Basic
Column temperature: 50
Mobile phase: Gradient. A:B 20:80 for 1 min, to 50:50 over 7 min, to 65:35 over 3 min,
to 100:0 (step gradient), maintain at 100:0 for 1 min, return to initial conditions, reequilibrate for 4 min. A was MeCN:50 mM pH 3.0 potassium dihydrogen phosphate
buffer 50:50. B was MeOH:50 mM pH 3.0 potassium dihydrogen phosphate buffer 20:80.
Flow rate: 0.3
Injection volume: 145
Detector: UV 246
CHROMATOGRAM
Retention time: 12.4
Internal standard: fludrocortisone acetate
OTHER SUBSTANCES
Extracted: deflazacort (13.8), 21-hydroxydeflazacort (10)
Simultaneous: cortisone, hydrocortisone
KEY WORDS
fludrocortisone acetate is internal standard; plasma; SPE
REFERENCE
Reynolds, D.L.; Burmaster, S.D.; Eichmeier, L.S. Quantitative determination of 21-hydroxy-deflazacort
in human plasma using gradient semi-microbore liquid chromatography, Biomed.Chromatogr., 1994,
8, 230–235.
262
Fluprostenol
Fluprostenol
HO
COOH
Molecular formula: C23 H29 F3 O6
Molecular weight: 458.47
CAS Registry No: 40666-16-8
Merck Index: 13, 4220
O
HO
OH
CF3
SAMPLE
Matrix: blood
Sample preparation: Condition a 1 mL 100 mg IST Isolute ODS SPE cartridge (Jones
Chromatography) with 1 mL MeOH and 1 mL MeOH:water:formic acid 3:97:0.1. Vortex
200 µL plasma with 200 pg IS for 30 s, add to the SPE cartridge, wash with 1 mL
MeOH:water:formic acid 3:97:0.1, wash with 1 mL MeOH:water 25:75, elute with 1 mL
MeOH:water 90:10. Evaporate the eluate to dryness under a stream of nitrogen at 35◦ ,
reconstitute the residue with 120 µL MeOH:water 30:70, inject a 25 µL aliquot.
HPLC VARIABLES
Column: 10 × 2.1 3 µm Symmetry Sentry Guard C18 (Waters)
Mobile phase: MeOH:water:formic acid 38:62:0.1 containing 70 µg/mL ammonium
acetate
Flow rate: 0.4
Injection volume: 25
Detector: MS, PE Sciex API-III Plus triple quadrupole, TurboIonSpray, positive ionization, ESI 3800 V, orifice 40 V, nebulizer gas nitrogen at 62 psi, TurboProbe 475◦ ,
TurboProbe nitrogen at 8 L/min, collision gas argon, m/z 476–279
CHROMATOGRAM
Retention time: 0.63
Internal standard: 3,3,4,4-2 H4 -fluprostenol (m/z 480–283) (0.63)
Limit of quantitation: 25 pg/mL
KEY WORDS
pharmacokinetics; plasma; rat; SPE
REFERENCE
Eichhold, T.H.; Kuhlenbeck, D.L.; Baker, T.R.; Stella, M.E.; Amburgey, J.S.; deLong, M.A.; Hartke, J.R.;
Cruze, C.A.; Pierce, S.A.; Wehmeyer, K.R. Use of short high-performance liquid chromatography
columns and tandem-mass spectrometry for the rapid analysis of a prostaglandin analog, fluprostenol,
in rat plasma, J.Chromatogr.B, 2000, 741, 213–220.
SAMPLE
Matrix: blood
Sample preparation: Condition a SPEC 3 mL 15 mg MP1 nonpolar reversed-phase/
strong cation exchange SPE Cartridge (Ansys) with 500 µL MeOH and 500 µL 40 mM
formic acid. Mix 1 mL plasma with 15 µL 20 ng/mL IS and 1 mL 100 mM formic
acid, add to the SPE cartridge, rinse tube with 0.5–1 mL water, add rinse to the
SPE cartridge, wash with 500 µL water, dry under vacuum, wash with two 500 µL
portions of toluene:dichloromethane 60:40, dry under vacuum, elute with 600 µL
toluene:methylformate 20:80. Evaporate the eluate to dryness under a stream of nitrogen at 30◦ , reconstitute the residue with 125 µL MeOH:water 50:50, inject a 35 µL
aliquot.
HPLC VARIABLES
Column: 150 × 2 5 µm Phenomenex Columbus C18
Fluprostenol
263
Mobile phase: MeOH:5 mM pH 6.3 ammonium formate 70:30
Flow rate: 0.2
Injection volume: 35
Detector: MS, Micromass Quattro LC, electrospray, capillary 3.0 kV, sample cone 40 V,
extraction cone 2 V, RF lens 0.3 V, source temperature 125◦ , drying gas 250◦ , MS1
parameters LM resolution 14, HM resolution 14, ion energy, 1.2 V, entrance and exit set
to 0 and 1, collision energy 30 eV, MS2 parameters LM resolution 15.0, HM resolution
15.0, ion energy 1.2 V, multiplier 650 V, nebulizing gas 75 L/h, drying gas 570 L/h,
collision gas argon, m/z 457–161
CHROMATOGRAM
Retention time: 5.3
Internal standard: AL-5848X (tetradeutero travoprost free acid) (m/z 461–161)
Limit of quantitation: 10 pg/mL
KEY WORDS
plasma; SPE
REFERENCE
McCue, B.A.; Cason, M.M.; Curtis, M.A.; Faulkner, R.D.; Dahlin, D.C. Determination of travoprost and
travoprost free acid in human plasma by electrospray HPLC/MS/MS, J.Pharm.Biomed.Anal., 2002,
28, 199–208.
264
Flurandrenolide
Flurandrenolide
HO
Molecular formula: C24 H33 FO6
Molecular weight: 436.51
CAS Registry No: 1524-88-5
Merck Index: 13, 4223
CH3
HO
CH3
H
H
O
CH3
O
CH3
O
H
O
F
SAMPLE
Matrix: formulations
Sample preparation: Mix 1 g of a topical product, 5 mL 100 mM pH 4 citrate buffer
saturated with NaCl, and 10 mL ethyl acetate, shake vigorously by hand to ensure
that no large clumps stick to the tube, mix with the tube on its side on an oscillating
shaker for 15 min. Remove the upper ethyl acetate layer and wash with citrate buffer as
before. Dry the ethyl acetate over anhydrous sodium sulfate and evaporate to dryness.
Dissolve the residue in a mixture of 10 mL heptane and 5 mL MeCN:water 90:10.
Remove the upper heptane layer and extract it with 2 mL MeCN:water 90:10. Combine
the MeCN/water layers and evaporate them to dryness, dissolve the residue in 300 µL
MeOH, filter (0.45 µm nylon), inject a 5 µL aliquot.
HPLC VARIABLES
Guard column: 15 × 3.2 7 µm Brownlee NewGuard C18
Column: 75 × 4.6 3.5 µm Symmetry C18 (Waters)
Mobile phase: Gradient. MeCN:water 18:82 for 2 min, to 82:18 over 12 min, maintain
at 82:18 for 3 min, re-equilibrate at initial conditions for 12 min.
Flow rate: 1
Injection volume: 5
Detector: UV 240
CHROMATOGRAM
Retention time: 7.36
Limit of detection: 0.001%
OTHER SUBSTANCES
Simultaneous: alclometasone
17,21-dipropionate (10.93), amcinonide (10.90),
beclomethasone 17,21-dipropionate (11.90), betamethasone (6.52), betamethasone 21acetate (8.47), betamethasone 17-benzoate (10.28), betamethasone 17,21-dipropionate
(11.42), betamethasone 17-valerate (10.25), budesonide (8.55, 8.69 (epimers)), clobetasol
17-propionate (11.06), cortisone (5.62), cortisone 21-acetate (8.07), dexamethasone
(6.57), dexamethasone 21-acetate (8.68), desonide (6.99), desoximetasone (7.60),
desoxycorticosterone acetate (10.90), desoxycorticosterone pivalate (14.45), diflorasone
17,21-diacetate (9.81), fluocinolone acetonide (7.38), fluocinonide (9.79), fludrocortisone
21-acetate (7.77), fluorometholone (7.67), flumethasone 21-pivalate (11.20), flunisolide
(7.14), fluprednisolone (5.46), fluticasone 17-propionate (11.19), halcinonide (10.72),
halobetasol propionate (10.98), hydrocortisone (5.50), hydrocortisone 21-acetate (7.65),
hydrocortisone 17-butyrate (8.66), hydrocortisone 21-cypionate (12.54), hydrocortisone
17-valerate (9.53), mometasone 17-furoate (11.24), methylprednisolone (6.31),
methylprednisolone 21-acetate (8.34), meprednisone (6.55), prednisolone (5.37),
prednisolone 21-acetate (7.47), prednisolone 21-tebuate (11.01), paramethasone
21-acetate (8.64), prednicarbate (10.72), prednisone (5.46), triamcinolone (4.15),
triamcinolone acetonide (7.04), triamcinolone 16,21-diacetate (7.49), triamcinolone
hexacetonide (13.12)
KEY WORDS
body wash, cream, gel, lotion, shampoo, spray
Flurandrenolide
265
REFERENCE
Reepmeyer, J.C. Screening for corticosteroids in topical pharmaceuticals by HPLC with a scanning
ultraviolet detector, J.Liq.Chromatogr.Rel.Technol., 2001, 24, 693–709.
SAMPLE
Matrix: urine
Sample preparation: Mix 5 mL urine with 100 ng IS and 400 µL 1 M pH 4.1 sodium
acetate buffer, adjust pH to 5.0, add 600 µL β-glucuronidase solution (10800 U), heat
at 65◦ for 3.5 h or at 37◦ overnight, cool, add 6 mL ethyl acetate, rotate for 10 min.
Remove the organic layer and wash it with 3 mL 1 M NaOH containing 150 mM NaCl
by rotating for 5 min, centrifuge at 1900 g for 30 s. Remove the organic layer and pass it
through an anhydrous sodium sulfate drying column. Evaporate the eluate to dryness
under a stream of nitrogen at 60◦ , reconstitute the residue with 30 µL MeOH, vortex,
inject a 10 µL aliquot.
HPLC VARIABLES
Column: 75 × 4.6 3 µm DB-8 (Supelco)
Column temperature: 25
Mobile phase: Gradient. MeOH:1% acetic acid from 0:100 to 100:0 over 15 min, maintain
at 100:0 for 3 min.
Flow rate: 1
Injection volume: 10
Detector: MS, Finnigan MAT LCQ Classic, APCI, source 450◦ , capillary 150◦ , source
+5 kV for positive ions and – 5 kV for negative ions, collision gas helium, m/z 437
CHROMATOGRAM
Retention time: 26.8
Internal standard: d4 -hydrocortisone (24.4)
Limit of detection: 5 ng/mL
OTHER SUBSTANCES
Extracted: beclomethasone (m/z 409) (26.4), betamethasone (m/z 393) (25.9), deoxycortone (m/z 331) (28.1), 21-deoxydexamethasone (m/z 377) (26.8), desoximetasone (m/z
377) (27.6), dexamethasone (m/z 393) (25.9), dichlorisone (m/z 413) (26.4), fluclorolone
acetonide (m/z 487) (27.8), fludrocortisone (m/z 381) (24.2), flumethasone (m/z 411) (25.4),
fluocinolone acetonide (m/z 453) (26.4), fluocinonide (m/z 495) (28.5), fluocortolone (m/z
377) (26.8), fluorometholone (m/z 3.77) (26.6), fluprednisolone (m/z 379) (23.9), hydrocortisone (m/z 363) (24.4), isoflupredone (m/z 379) (23.9), methylprednisolone (m/z 375)
(26.1), prednisolone (m/z 361) (24.4), prednisone (m/z 359) (23.4), triamcinolone (m/z
395) (19), triamcinolone acetonide (m/z 435) (29.3)
KEY WORDS
horse
REFERENCE
Tang, P.W.; Law, W.C.; Wan, T.S.M. Analysis of corticosteroids in equine urine by liquid chromatographymass spectrometry, J.Chromatogr.B, 2001, 754, 229–244.
ANNOTATED BIBLIOGRAPHY
Gagliardi, L.; De Orsi, D.; Del Giudice, M.R.; Gatta, F.; Porrà, R.; Chimenti, P.; Tonelli, D. Development
of a tandem thin-layer chromatography-high-performance liquid chromatography method for the
identification and determination of corticosteroids in cosmetic products, Anal.Chim.Acta, 2002, 457,
187–198. [hydrocortisone; hydrocortisone acetate; hydrocortisone butyrate; hydrocortisone pivalate;
prednisolone pivalate; prednisolone acetonide; prednisolone acetate; fluorocortolone pivalate; clobetasone butyrate; flumethasone pivalate; fluorocortolone caproate; prednisolone; fluorocortolone;
clobetasone; flumethasone; triamcinolone; triamcinolone diacetate; triamcinolone acetonide; clobetasol
266
Flurandrenolide
propionate; clobetasol; fluocortin; fluocortin butyl ester; fludrocortisone; fludrocortisone acetate; dexamethasone; dexamethasone acetate; dexamethasone pivalate; dexamethasone isonicotinate; dexamethasone valerate; betamethasone; betamethasone acetate; betamethasone benzoate; betamethasone
propionate butyrate; betamethasone propionate stearate; betamethasone propionate; betamethasone
dipropionate; betamethasone valerate; betamethasone divalerate; betamethasone valerate acetate;
fluocinolone; fluocinolone acetonide; fluocinonide; cortisone; cortisone acetate; amcinonide; flurandrenolide; fluorometholone; methylprednisolone; halcinonide; deoxymethasone; diflucortolone; valerate;
dehydrocorticosterone; fluoroprednisolone; beclomethasone; alclometasone; mometasone; fluoroprednisolone acetate; beclomethasone dipropionate; alclometasone dipropionate; mometasone furoate]
Tang, P.W.; Law, W.C.; Wan, T.S.M. Analysis of corticosteroids in equine urine by liquid chromatographymass spectrometry, J.Chromatogr.B, 2001, 754, 229–244. [triamcinolone; prednisone; fluprednisolone;
isoflupredone; fludrocortisone; hydrocortisone; prednisolone; flumethasone; betamethasone; dexamethasone; methylprednisolone; dichlorisone; beclomethasone; fluocinolone acetonide; fluocinolone;
fluorometholone; fluocortolone; desoximetasone; triamcinolone acetonide; flurandrenolide; fluclorolone
acetonide; fluclorolone; deoxycortone; fluocinonide; flucloronide]
267
Flurithromycin
Flurithromycin
Molecular formula: C37 H66 FNO13
O
H3C
H3C
F
OH
CH3
OH
O
O
N
O HO
H3C
O
CH3
H3C
CH3
OH
Molecular weight: 751.92
CAS Registry No: 82664-20-8
O
CH3
CH3
OCH3
CH3
O
CH3
OH
CH3
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 × 4.6 10 µm Lichrosorb RP18
Column temperature: 35
Mobile phase: MeCN:buffer 68:32 (The buffer was 10 mM potassium dihydrogen phosphate adjusted to pH 7 with 20% NaOH.)
Flow rate: 1.5
Detector: UV 214
CHROMATOGRAM
Retention time: 3.9 (hemiketal), 5.6 (ketone)
REFERENCE
Colombo, N.; Depaoli, A.; Gobetti, M.; Saorin, M.G. Analytical-physical profile of the novel macrolide
antibiotic flurithromycin ethylsuccinate, Arzneimittelforschung, 1994, 44, 850–855.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 125 × 4 3 µm LKB Spherisorb ODS-2
Mobile phase: MeCN:MeOH:100 mM pH 7.0 ammonium acetate 65:20:15
Flow rate: 0.5
Injection volume: 10–50
Detector: UV 235
CHROMATOGRAM
Retention time: 5.1
OTHER SUBSTANCES
Simultaneous: azithromycin (4.5), clarithromycin (7.6), erythromycin (5.4), oleandomycin (4.4), roxithromycin (7.9)
REFERENCE
Lingerfelt, B.; Champney, W.S. Macrolide and ketolide antibiotic separation by reversed phase high
performance liquid chromatography, J.Pharm.Biomed.Anal., 1999, 20, 459–469.
268
Flurogestone acetate
Flurogestone acetate
O
HO
Molecular formula: C23 H31 FO5
Molecular weight: 406.49
CAS Registry No: 2529-45-5
Merck Index: 13, 4226
CH3
CH3
O
CH3
CH3
H
O
F
H
O
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 300 × 3.9 10 µm µBondapak C18
Mobile phase: MeOH:water 50:50
Flow rate: 2
Detector: UV 254
CHROMATOGRAM
Retention time: 21
Internal standard: testosterone (32)
OTHER SUBSTANCES
Simultaneous: degradants
REFERENCE
Kabadi, M.B.; Valia, K.H.; Chien, Y.W. Intravaginal controlled administration of flurogestone acetate
I: development of a stability-indicating liquid chromatographic method and stability kinetics of
flurogestone acetate, J.Pharm.Sci., 1984, 73, 1461–1464.
SAMPLE
Matrix: tissue
Sample preparation: Condition a 100 mg C18 SPE cartridge (Varian) with MeOH and
10 mM pH 8.5 Tris buffer. Fill a 22 mL vessel (from bottom to top) with 5 g alumina
(dried at 120◦ for 48 h before use), 6 g anhydrous sodium sulfate, and 2 g melted
(microwave) kidney fat. There is filter paper between the alumina and sodium sulfate.
Pass hexane down through the layers at 60◦ at 1500 psi followed by MeCN at 50◦ at
1500 psi. Store the MeCN at – 20◦ for 30 min to precipitate fat, filter through a plug
of glass wool, evaporate to dryness under a stream of nitrogen at 40◦ , reconstitute the
residue with 2 mL MeOH, mix with 5 mL water, add to the SPE cartridge, wash with
2 mL 20 mM pH 8.5 Tris buffer, wash with 2 mL MeOH:water 40:60, elute with 2 mL
MeOH. Evaporate the eluate to dryness under a stream of nitrogen at 40◦ , reconstitute
the residue with 75 µL MeCN, add 75 µL 0.5% formic acid, mix, inject a 75 µL aliquot.
(The extraction is done in a Dionex ASE system (advanced solvent extraction).)
HPLC VARIABLES
Column: 150 × 3 5 µm Symmetry (Waters)
Column temperature: 40
Mobile phase: Gradient. A:B from 45:55 to 100:0 over 12 min. A was MeCN:water:formic
acid 10:90:0.5. B was MeCN:water:formic acid 90:10:0.5.
Flow rate: 0.4
Injection volume: 75
Detector: MS, Micromass Quattro Ultima, positive electrospray, capillary 2.5 kV, cone
40 V, source 120◦ , cone gas 189 L/h, desolvation gas 652 L/h, m/z 407.2–267.4–225.4
Flurogestone acetate
269
CHROMATOGRAM
Retention time: 4.5
Limit of detection: <2 ng/g
OTHER SUBSTANCES
Extracted: chloromadinone acetate (m/z 405.2–309.2–345.3) (8.6), chlorotestosterone
acetate (m/z 365.2–305.2–323.3) (12.2), delmadinone acetate (m/z 403.2–205.1–181.1)
(7.8), medroxyprogesterone acetate (m/z 387.2–327.3–285.3) (8.8), megestrol acetate
(m/z 385.2–267.3–325.3) (8.4), melengestrol acetate (m/z 397.2–279.3–279.3) (8.8)
KEY WORDS
fat; kidney; SPE
REFERENCE
Hooijerink, H.; van Bennekom, E.O.; Nielen, M.W.F. Screening for gestagens in kidney fat using
accelerated solvent extraction and liquid chromatography electrospray tandem mass spectrometry,
Anal.Chim.Acta, 2003, 483, 51–59.
ANNOTATED BIBLIOGRAPHY
Kabadi, M.B.; Chien, Y.W. Intravaginal controlled administration of flurogestone acetate II: development
of an in vitro system for studying the intravaginal release and permeation of flurogestone acetate,
J.Pharm.Sci., 1984, 73, 1464–1468.
270
Fluticasone propionate
Fluticasone propionate
S
F
Molecular formula: C25 H31 F3 O5 S
Molecular weight: 500.58
CAS Registry No: 80474-14-2
Merck Index: 13, 4237
CH3
HO
CH3
F
H
O O
CH3
O
CH3
H
O
F
SAMPLE
Matrix: blood
Sample preparation: The SPE cartridges were contained in 96 well MicroLute II SPE
blocks (Porvair Sciences) with each cell packed with 50 mg Varian C18. Condition each
cell with 400 µL MeOH and 400 µL water. Centrifuge plasma at 1000 g for 10 min.
Mix 500 µL plasma with 500 µL 1 ng/mL IS in 100 mM pH 7.4 ammonium formate
buffer, wash with 400 µL water, wash with 400 µL MeOH:water 40:60 (40% aqueous
methanol), elute with 200 µL MeOH. Evaporate the eluate to dryness under a stream of
heated nitrogen, reconstitute the residue with 100 µL MeOH:25 mM pH 5 ammonium
formate 50:50, inject an 80 µL aliquot.
HPLC VARIABLES
Column: 150 × 4.6 5 µm ResElut C8 BD (Varian)
Column temperature: 40
Mobile phase: MeOH:25 mM pH 5 ammonium formate 80:20
Flow rate: 1
Injection volume: 80
Detector: MS, PE Sciex API-III Plus triple quadrupole, column effluent split 1:30 so
that 336 µL/min enters MS (?), TurboIonspray 500◦ , positive ion electrospray 5000 V,
nebulizer gas nitrogen at 400 kPa, auxiliary gas nitrogen at 2.0 L/min, collision gas
argon, collision energy 25 eV, m/z 501–313
CHROMATOGRAM
Retention time: 3.0
Internal standard: 13 C3 -fluticasone propionate (m/z 504–313)
Limit of quantitation: 20 pg/mL
KEY WORDS
pharmacokinetics; plasma; SPE
REFERENCE
Callejas, S.L.; Biddlecombe, R.A.; Jones, A.E.; Joyce, K.B.; Pereira, A.I.; Pleasance, S. Determination of
the glucocorticoid fluticasone propionate in plasma by automated solid-phase extraction and liquid
chromatography-tandem mass spectrometry, J.Chromatogr.B, 1998, 718, 243–250.
SAMPLE
Matrix: bulk
Sample preparation: Inject a 200 µL aliquot of a 6.7 mg/mL solution in mobile phase.
HPLC VARIABLES
Column: 250 × 4.6 5 µm Inertsil ODS-2
Column temperature: 40
Fluticasone propionate
271
Mobile phase: Gradient. A:B from 45:55 to 60:40 over 25 min, to 75:25 over 25 min. A
was MeCN. B was deuterated water containing 0.05% trifluoroacetic acid.
Flow rate: 1
Injection volume: 200
Detector: UV 239 connected with NMR, Bruker AMX-600, stop-flow mode, 600.14 MHz;
MS, Trio 1000 single quadrupole, thermospray, source 200◦ , orifice 200◦ , source ion
repeller 100 V
CHROMATOGRAM
Retention time: 14
OTHER SUBSTANCES
Simultaneous: impurities
REFERENCE
Mistry, N.; Ismail, I.M.; Smith, M.S.; Nicholson, J.K.; Lindon, J.C. Characterisation of impurities in bulk
drug batches of fluticasone propionate using directly coupled HPLC-NMR spectroscopy and HPLC-MS,
J.Pharm.Biomed.Anal., 1997, 16, 697–705.
SAMPLE
Matrix: formulations
Sample preparation: Mix 1 g of a topical product, 5 mL 100 mM pH 4 citrate buffer
saturated with NaCl, and 10 mL ethyl acetate, shake vigorously by hand to ensure
that no large clumps stick to the tube, mix with the tube on its side on an oscillating
shaker for 15 min. Remove the upper ethyl acetate layer and wash with citrate buffer as
before. Dry the ethyl acetate over anhydrous sodium sulfate and evaporate to dryness.
Dissolve the residue in a mixture of 10 mL heptane and 5 mL MeCN:water 90:10.
Remove the upper heptane layer and extract it with 2 mL MeCN:water 90:10. Combine
the MeCN/water layers and evaporate them to dryness, dissolve the residue in 300 µL
MeOH, filter (0.45 µm nylon), inject a 5 µL aliquot.
HPLC VARIABLES
Guard column: 15 × 3.2 7 µm Brownlee NewGuard C18
Column: 75 × 4.6 3.5 µm Symmetry C18 (Waters)
Mobile phase: Gradient. MeCN:water 18:82 for 2 min, to 82:18 over 12 min, maintain
at 82:18 for 3 min, re-equilibrate at initial conditions for 12 min.
Flow rate: 1
Injection volume: 5
Detector: UV 240
CHROMATOGRAM
Retention time: 11.19 (17-propionate)
Limit of detection: 0.001%
OTHER SUBSTANCES
Simultaneous: alclometasone
17,21-dipropionate (10.93), amcinonide (10.90),
beclomethasone 17,21-dipropionate (11.90), betamethasone (6.52), betamethasone 21acetate (8.47), betamethasone 17-benzoate (10.28), betamethasone 17,21-dipropionate
(11.42), betamethasone 17-valerate (10.25), budesonide (8.55, 8.69 (epimers)), clobetasol
17-propionate (11.06), cortisone (5.62), cortisone 21-acetate (8.07), dexamethasone
(6.57), dexamethasone 21-acetate (8.68), desonide (6.99), desoximetasone (7.60),
desoxycorticosterone acetate (10.90), desoxycorticosterone pivalate (14.45), diflorasone
17,21-diacetate (9.81), fluocinolone acetonide (7.38), fluocinonide (9.79), flurandrenolide
(7.36), fludrocortisone 21-acetate (7.77), fluorometholone (7.67), flumethasone 21pivalate (11.20), flunisolide (7.14), fluprednisolone (5.46), halcinonide (10.72),
halobetasol propionate (10.98), hydrocortisone (5.50), hydrocortisone 21-acetate
(7.65), hydrocortisone 17-butyrate (8.66), hydrocortisone 21-cypionate (12.54),
272
Fluticasone propionate
hydrocortisone 17-valerate (9.53), mometasone 17-furoate (11.24), methylprednisolone
(6.31), methylprednisolone 21-acetate (8.34), meprednisone (6.55), prednisolone (5.37),
prednisolone 21-acetate (7.47), prednisolone 21-tebuate (11.01), paramethasone
21-acetate (8.64), prednicarbate (10.72), prednisone (5.46), triamcinolone (4.15),
triamcinolone acetonide (7.04), triamcinolone 16,21-diacetate (7.49), triamcinolone
hexacetonide (13.12)
KEY WORDS
body wash; cream; gel; lotion; shampoo; spray
REFERENCE
Reepmeyer, J.C. Screening for corticosteroids in topical pharmaceuticals by HPLC with a scanning
ultraviolet detector, J.Liq.Chromatogr.Rel.Technol., 2001, 24, 693–709.
ANNOTATED BIBLIOGRAPHY
Bernal, J.L.; Del Nozal, M.J.; Martı́n, M.T.; Diez-Masa, J.C.; Cifuentes, A. Quantitation of active ingredients and excipients in nasal sprays by high-performance liquid chromatography, capillary electrophoresis and UV spectroscopy, J.Chromatogr.A, 1998, 823, 423–431. [beclomethasone dipropionate; fluticasone dipropionate; beclomethasone; fluticasone; benzalkonium; benzalkonium chloride; phenylethyl
alcohol]
Borchard, G.; Cassará, M.L.; Roemelé, P.E.H.; Florea, B.I.; Junginger, H.E. Transport and local metabolism of budesonide and fluticasone propionate in a human bronchial epithelial cell line (Calu-3),
J.Pharm. Sci., 2002, 91, 1561–1567.
Krishnaswami, S.; Möllmann, H.; Derendorf, H.; Hochhaus, G. A sensitive LC-MS/MS method for the
quantification of fluticasone propionate in human plasma, J.Pharm.Biomed.Anal., 2000, 22, 123–129.
[SPE]
Laugher, L.; Noctor, T.G.; Barrow, A.; Oxford, J.M.; Phillips, T. An improved method for the determination of fluticasone propionate in human plasma, J.Pharm.Biomed.Anal., 1999, 21, 749–758.
[SPE]
Li, Y.N.; Tattam, B.N.; Brown, K.F.; Seale, J.P. A sensitive method for the quantification of fluticasone propionate in human plasma by high-performance liquid chromatography/atmospheric pressure
chemical ionisation mass spectrometry, J.Pharm.Biomed.Anal., 1997, 16, 447–452.
Li, Y.N.; Tattam, B.; Brown, K.F.; Seale, J.P. Quantification of epimeric budesonide and fluticasone
propionate in human plasma by liquid chromatography-atmospheric pressure chemical ionization
tandem mass spectrometry, J.Chromatogr.B, 2001, 761, 177–185. [SPE]
273
Flutrimazole
Flutrimazole
N
F
N
Molecular formula: C22 H16 F2 N2
Molecular weight: 346.37
CAS Registry No: 119006-77-8
Merck Index: 13, 4241
F
SAMPLE
Matrix: blood, feces, urine
Sample preparation: Mix 500 µL plasma or urine with 500 µL MeOH, shake, centrifuge, inject a 50 µL aliquot. Homogenize feces with an equal volume of MeOH,
centrifuge at 5000 rpm for 20 min, inject a 100 µL aliquot.
HPLC VARIABLES
Guard column: µBondapak C18
Column: 300 × 3.9 10 µm µBondapak C18
Mobile phase: MeOH:5 mM pH 7 phosphate buffer 75:25
Flow rate: 1
Injection volume: 50–100
Detector: UV 225
CHROMATOGRAM
Retention time: 10
OTHER SUBSTANCES
Extracted: clotrimazole (11.2), imidazole (3.3)
KEY WORDS
dog; plasma
REFERENCE
Conte, L.; Ramis, J.; Mis, R.; Vilageliu, J.; Basi, N.; Forn, J. Pharmacokinetic study of [14 C]flutrimazole
after oral and intravenous administration in dogs. Comparison with clotrimazole, Arzneimittelforschung,
1992, 42, 854–858.
274
Fomepizole
Fomepizole
H
N
N
Molecular formula: C4 H6 N2
Molecular weight: 82.10
CAS Registry No: 7554-65-6
Merck Index: 13, 4252
H3C
SAMPLE
Matrix: blood
Sample preparation: Condition a 100 mg Bond Elut SCX SPE cartridge with two 1 mL
portions of MeOH and two 1 mL portions of 5 mM HCl. Mix 200 µL plasma with 800 µL
6.25 µM IS in 5 mM HCl, add to the SPE cartridge, wash with 1 mL MeOH:5 mM
HCl 5:95, elute with 1 mL MeOH:250 mM pH 7.4 potassium phosphate buffer, inject a
100 µL aliquot.
HPLC VARIABLES
Guard column: 30 × 4 Nucleosil 120-5C18
Column: 100 × 4 Nucleosil 120-5C18
Mobile phase: MeOH:5 mM pH 6.0 potassium phosphate buffer 20:80
Flow rate: 0.8
Injection volume: 100
Detector: UV 220
CHROMATOGRAM
Retention time: 7
Internal standard: 3-methylpyrazole (6)
Limit of quantitation: 2.5 µM
KEY WORDS
plasma; SPE
REFERENCE
Diczfalusy, U.; Eklöf, R. Determination of 4-methylpyrazole in plasma using solid phase extraction and
HPLC, Biomed.Chromatogr., 1987, 2, 226–227.
SAMPLE
Matrix: blood, dialysate
Sample preparation: Mix 200 µL plasma with 10 µL 300 µg/mL IS in water, add
100 µL 15% trichloroacetic acid, mix, centrifuge at 10 000 g for 4 min. Mix a 150 µL
aliquot of the supernatant with 100 µL 500 mM disodium hydrogen phosphate solution
(pH ca. 7), inject a 50 µL aliquot. Inject a 200 µL aliquot of dialysate directly.
HPLC VARIABLES
Guard column: 4 × 4 5 µm LiChrospher 60 RP-select B
Column: 125 × 4 5 µm LiChrospher 100 RP-18
Column temperature: 40
Mobile phase: MeCN:5 mM pH 6 potassium phosphate buffer 7.5:92.5
Flow rate: 1.5
Injection volume: 50–200
Detector: UV 220
CHROMATOGRAM
Retention time: 4.9
Internal standard: 3-methylpyrazole (4.1)
Fomepizole
275
Limit of quantitation: 300 ng/mL (plasma), 50 ng/mL (dialysate)
KEY WORDS
plasma
REFERENCE
Jobard, E.; Turcant, A.; Harry, P.; Le Bouil, A.; Allain, P. High-performance liquid chromatographic
determination of 4-methylpyrazole in plasma and in dialysate, J.Chromatogr.B, 1997, 695, 444–447.
ANNOTATED BIBLIOGRAPHY
McMartin, K.E.; Collins, T.D.; Hewlett, T.P. High pressure liquid chromatographic assay of 4-methylpyrazole. Measurements of plasma and urine levels, J.Toxicol.Clin.Toxicol., 1984, 22, 133–148.
276
Fomivirsen
Fomivirsen
Molecular formula: C204 H263 N63 O114 P20 S20
Molecular weight: 6682.46
CAS Registry No: 144245-52-3, 160369-77-7 (Na salt)
Merck Index: 13, 4254
SAMPLE
Matrix: tissue, vitreous humor
Sample preparation: Heat retina sample with 2 mg/mL proteinase K in buffer at
37◦ for 16–24 h, wash twice with phenol:chloroform 50:50 (Caution! Chloroform is
a carcinogen!), wash with chloroform, evaporate to dryness under reduced pressure,
suspend in water, inject an aliquot. Vitreous humor is similar, but buffer is not used.
(Caution! Chloroform is a carcinogen! Buffer was 20 mM pH 8.0 Tris-HCl containing
0.5% Non-Idet P-40 (NP-40), 20 mM EDTA, and 100 mM NaCl.)
HPLC VARIABLES
Column: 100 × 4.6 Gen Pak Fax strong anion exchange
Mobile phase: Gradient. A was MeOH:86 mM pH 8.0 Tris-HCl 20:80. B was 86 mM pH
8.0 Tris-HCl containing 1.5 M NaBr. A:B 100:0 for 5 min, to 40:60 over 45 min.
Flow rate: 0.5
Detector: Radioactivity (14 C)
CHROMATOGRAM
Retention time: 50
OTHER SUBSTANCES
Extracted: metabolites
KEY WORDS
retina
REFERENCE
Leeds, J.M.; Henry, S.P.; Truong, L.; Zutshi, A.; Levin, A.A.; Kornbrust, D. Pharmacokinetics of a potential human cytomegalovirus therapeutic, a phosphorothioate oligonucleotide, after intravitreal injection
in the rabbit, Drug Metab.Dispos., 1997, 25, 921–926.
277
Fondaparinux
Fondaparinux
SO3Na
Molecular formula: C31 H43 N3 Na10 O49 S8
Molecular weight: 1728.08
CAS Registry No: 114870-03-0
Merck Index: 13, 4257
O
O
SO3Na
OH
O
O
SO3Na
O
OSO3Na
COONa
O
O
O
HO
H
SO3Na
O
N
H
O
OCH3
N
O
OH
OH
O
COONa
OH
O
SO3Na
SO3Na
OH
N
H
SO3Na
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: HR 5/5 mono-Q (Pharmacia)
Mobile phase: Gradient. A was 500 mM NaCl. B was 2 M NaCl. A:B from 100:0 to 0:100
over 25 min.
Flow rate: 1
Detector: Radioactivity (35 S)
CHROMATOGRAM
Retention time: 18
REFERENCE
Lieu, C.; Shi, J.; Donat, F.; Van Horn, R.; Brian, W.; Newton, J.; Delbressine, L.; Vos, R. Fondaparinux
sodium is not metabolised in mammalian liver fractions and does not inhibit cytochrome P450-mediated
metabolism of concomitant drugs, Clin.Pharmacokinet., 2002, 41(Suppl. 2), 19–26.
278
Formestane
Formestane
CH3 O
CH3
H
Molecular formula: C19 H26 O3
Molecular weight: 302.41
CAS Registry No: 566-48-3
Merck Index: 13, 4264
H
H
O
OH
SAMPLE
Matrix: urine
Sample preparation: Add 500 µL β-glucuronidase (Boehringer Mannheim) to 5 mL
urine, heat at 37◦ for 22 h, extract three times with 10 mL portions of ethyl acetate.
Combine the extracts and evaporate to dryness, reconstitute the residue with 100 µL
MeOH, inject a 20 µL aliquot.
HPLC VARIABLES
Column: 150 × 4.6 Apex II 5ODS (Jones)
Mobile phase: MeOH:water:formic acid 60:40:0.01
Flow rate: 1.2
Injection volume: 20
Detector: MS, Finnigan MAT TSQ 700 triple quadrupole, thermospray, discharge-on
mode (1000 V), positive ion mode, source block 200◦ , vaporizer 100◦ , repeller 50 V, m/z
303
CHROMATOGRAM
Retention time: 8.1
OTHER SUBSTANCES
Extracted: metabolites
REFERENCE
Poon, G.K.; Jarman, M.; McCague, R.; Davies, J.H.; Heeremans, C.E.M.; van der Hoeven, R.A.M.; Niessen,
W.M.A.; van der Greef, J. Identification of 4-hydroxyandrost-4-ene-3,17-dione metabolites in prostatic
cancer patients by liquid chromatography-mass spectrometry, J.Chromatogr., 1992, 576, 235–244.
Formoterol
Formoterol
Molecular formula: C19 H24 N2 O4
279
O
H
N
HO
Molecular weight: 344.40
CAS Registry No: 73573-87-2
Merck Index: 13, 4269
CH3
OCH3
N
OH
H
SAMPLE
Matrix: blood
Sample preparation: Condition a 1 mL 100 mg polysulfonic sorbent strong cationexchange SPE cartridge with 2 mL MeOH, 500 µL water, 3 mL 25 mM pH 6.6 phosphate
buffer, and 1 mL water. Mix 1 mL plasma with 50 µL 5 ng/mL IS in water, 750 µL
50 mM pH 6.6 buffer, and 50 µL water, vortex for 5 s. Add to the SPE cartridge,
wash with 2.5 mL 25 mM pH 6.6 phosphate buffer, wash with 500 µL water, wash
with 250 µL MeOH:water 20:80, dry with 2 mL air, elute with two 150 µL portions of
MeOH:pH 6.0 buffer 30:70, dilute the eluate with 200 µL water, mix, filter (0.45 µm
nylon), inject a 300 µL aliquot. (50 mM pH 6.6 Buffer was 50 mM potassium dihydrogen
phosphate:50 mM disodium hydrogen phosphate 62.7:37.3. Prepare 25 mM pH 6.6 buffer
by dilution. The pH 6.0 buffer was 30 mM potassium dihydrogen phosphate:30 mM
disodium hydrogen phosphate 87.7:12.3 containing 15 g/L KCl.)
HPLC VARIABLES
Guard column: 10 × 2 R2 Chromsep
Column: 200 × 3 5 µm Hypersil ODS
Column temperature: 33
Mobile phase: MeCN:MeOH:buffer 5:25:70 (The buffer was 35 mM potassium dihydrogen phosphate:35 mM disodium hydrogen phosphate 87.7:12.3 containing 20 mg/L
EDTA, pH 6.)
Flow rate: 0.4
Injection volume: 300
Detector: E, Model Decade (Antec, Leiden, Netherlands), VT-03 flowcell, working electrode with a 50 µm thickness spacer, potential +0.63 V in DC mode
CHROMATOGRAM
Retention time: 15.6
Internal standard: 3-acetylamino-4-hydroxy-α-[N-[1-methyl-2-(p-methoxyphenyl)
ethyl]aminomethyl]benzyl alcohol (CGP 47086A) (19)
Limit of detection: 4 pM
Limit of quantitation: 3 pg/mL
KEY WORDS
pharmacokinetics; plasma; SPE
REFERENCE
Campestrini, J.; Lecaillon, J.B.; Godbillon, J. Automated and sensitive method for the determination
of formoterol in human plasma by high-performance liquid chromatography and electrochemical
detection, J.Chromatogr.B, 1997, 704, 221–229.
SAMPLE
Matrix: blood
Sample preparation: Condition an Isolute weak cation-exchange SPE cartridge with
MeOH, water, and 100 mM pH 5.0 ammonium acetate buffer. Add plasma to the SPE
cartridge, wash with 100 mM pH 5.0 ammonium acetate buffer, wash with MeOH,
elute with MeOH:water:formic acid 75:23.75:1.25. Evaporate the eluate to dryness,
280
Formoterol
reconstitute the residue with MeOH:water:acetic acid 10:89.5:0.5, inject an 80 µL
aliquot onto column A, elute to waste with mobile phase A, divert the mobile phase
containing formoterol onto column B, elute the contents of column B onto column C with
mobile phase B, monitor the effluent from column C.
HPLC VARIABLES
Column: A 10 × 2.1 Hypersil NH2 + 33 × 2.1 4 µm Genesis CN; B 10 × 2.1 4 µm Genesis
C18; C Hichrom ACE C18
Mobile phase: A MeOH:water:acetic acid 15:84.5:0.5; B MeOH:water:acetic acid 35:64.5:
0.5
Flow rate: 0.22
Injection volume: 80
Detector: MS, Finnigan TSQ 7000, atmospheric pressure ionization, positive ion electrospray
CHROMATOGRAM
Limit of quantitation: 5 pM
KEY WORDS
column-switching; pharmacokinetics; plasma; SPE
REFERENCE
Tronde, A.; Nordén, B.; Marchner, H.; Wendel, A.-K.; Lennernäs, H.; Bengtsson, U.H. Pulmonary absorption rate and bioavailability of drugs in vivo in rats: structure-absorption relationships and physicochemical profiling of inhaled drugs, J.Pharm.Sci., 2003, 92, 1216–1233.
ANNOTATED BIBLIOGRAPHY
Rosenborg, J.; Larsson, P.; Tegnér, K.; Hallström, G. Mass balance and metabolism of [3 H]formoterol in
healthy men after combined i.v. and oral administration-mimicking inhalation, Drug Metab.Dispos.,
1999, 27, 1104–1116.
Fosamprenavir calcium
281
Fosamprenavir calcium
Molecular formula: C25 H36 CaN3 O9 PS
Molecular weight: 625.68
CAS Registry No: 226700-81-8
NH2
O
O
O
S
O
N
H
O
N
O
P
CH3
O−
O−
O
Ca++
CH3
SAMPLE
Matrix: blood
Sample preparation: Vortex 100 µL plasma with 200 µL 1 µg/mL IS in MeCN, centrifuge. Mix a 100 µL aliquot of the supernatant with 100 µL 0.1% formic acid and
400 µL MeCN:water 50:50, inject a 10–20 µL aliquot.
HPLC VARIABLES
Column: 50 × 2 3 µm Phenomenex Aqua C18 or 30 × 2 3 µm Phenomenex Aqua C18
Mobile phase: Gradient. A was MeOH:10 mM pH 3.5 ammonium formate 1.5:98.5. B
was MeCN. A:B 99:1 for 3 min, to 5:95 over 1.5 min, maintain at 5:95 for 1.4 min, return
to initial conditions over 0.1 min, re-equilibrate for 3 min.
Flow rate: 0.325
Injection volume: 10–20
Detector: MS, PE Sciex API2000 turbo ion spray, electrospray ionization, positive ion
mode
CHROMATOGRAM
Internal standard:
13
C6 -amprenavir
OTHER SUBSTANCES
Extracted: amprenavir, nelfinavir
KEY WORDS
plasma
REFERENCE
Huang, L.; Wring, S.A.; Woolley, J.L.; Brouwer, K.R.; Serabjit-Singh, C.; Polli, J.W. Induction of Pglycoprotein and cytochrome P450 3A by HIV protease inhibitors, Drug Metab.Dispos., 2001, 29,
754–760.
SAMPLE
Matrix: blood
Sample preparation: Mix 100 µL plasma with 500 µL EtOH:MeCN 50:50, centrifuge
at 15 800 g at 4◦ for 5 min. Evaporate the supernatant to dryness, reconstitute the
residue with 100 µL initial mobile phase, inject a 10 µL aliquot.
HPLC VARIABLES
Column: 100 × 2 5 µm Luna C18 (Phenomenex)
Mobile phase: Gradient. MeCN:buffer from 20:80 to 95:5 over 5 min, maintain at 95:5
for 4 min. (The buffer was 0.1% aqueous acetic acid adjusted to pH 5.4 with ammonium
hydroxide.)
Flow rate: 0.2
Injection volume: 10
282
Fosamprenavir calcium
Detector: MS, PE Sciex API-365, triple quadrupole, collision energy 30 eV, m/z 586.3–
418.2
CHROMATOGRAM
Retention time: 5.5
Limit of quantitation: 300 pg/mL
OTHER SUBSTANCES
Extracted: amprenavir (506.3–245.2) (7.0)
KEY WORDS
dog; plasma; rat
REFERENCE
Furfine, E.S.; Baker, C.T.; Hale, M.R.; Reynolds, D.J.; Salisbury, J.A.; Searle, A.D.; Studenberg, S.D.;
Todd, D.; Tung, R.D.; Spaltenstein, A. Preclinical pharmacology and pharmacokinetics of GW433908,
a water-soluble prodrug of the human immunodeficiency virus protease inhibitor amprenavir, Antimicrob.Agents Chemother., 2004, 48, 791–798.
Fosinopril
Fosinopril
O
Molecular formula: C30 H46 NO7 P
P
Molecular weight: 563.66
CAS Registry No: 98048-97-6
Merck Index: 13, 4279
O
H3C
O
O
O
283
N
COOH
CH3
CH3
SAMPLE
Matrix: blood
Sample preparation: Condition a 3 mL 200 mg Bond Elut cyclohexyl SPE cartridge
with 3 mL MeOH and 3 mL 0.1 N phosphoric acid. Vortex 1 mL serum with 2 mL 0.2 N
phosphoric acid and an aliquot of EtOH containing 1.5 µg/mL SQ-33055 and 1.5 µg/mL
SQ-27133, add to the SPE cartridge, wash with 3 mL 0.1 N phosphoric acid, wash with
3 mL 10 mM pH 4.6 ammonium acetate buffer, dry under vacuum, elute with 1.5 mL
MeOH containing 10 mM ammonium acetate. Evaporate the eluate to dryness under
a stream of nitrogen at 40◦ , reconstitute the residue with 75 µL initial mobile phase,
inject a 5 µL aliquot. (Use silanized glass or polypropylene containers. Silanize glass
vials by immersing in Prosil-28 working solution (10 mL concentrate diluted to 1 L with
water) for 10 min, rinsing with water several times, drying in air.)
HPLC VARIABLES
Column: 50 × 2 5 µm Asahipak ODP PVA-C18
Mobile phase: Gradient. A was 770 mg ammonium acetate in 750 mL water and 250 mL
MeOH, adjusted to pH 5.5 with glacial acetic acid. B was 770 mg ammonium acetate in
MeOH. A:B from 70:30 to 5:95 over 1 min, maintain at 5:95 for 2 min, return to initial
conditions over 2 min, re-equilibrate for 5 min.
Flow rate: 0.2
Injection volume: 5
Detector: MS, PE Sciex API-III Plus triple quadrupole, turbo ion spray, positive ion,
sprayer 4200 V, orifice 35 V, declustering 4 V, nebulizer gas nitrogen at 65 psi, curtain
gas nitrogen at 1.2 L/min, turbo ion spray nitrogen 4 L/min, collision gas argon 23 eV,
m/z 581.3–436.2
CHROMATOGRAM
Retention time: 4.2
Internal standard: SQ-33055 ((4S)-4-phenyl-1-[[(R)-[(1S)-2-methyl-1-(1-oxopropoxy)
propoxy](4-phenylbutyl)phosphinyl]acetyl]-L-proline) (m/z 575.3–430.2) (4), SQ-27133
((4S)-4-phenylthio-1-[[(R)-(4-phenylbutyl)phosphinyl]acetyl]-L-proline) (m/z 479.2–
416.2) (3.3)
Limit of quantitation: 1.17 ng/mL
OTHER SUBSTANCES
Extracted: fosinoprilat (m/z 453.2–390.2) (3.5)
KEY WORDS
serum; SPE
REFERENCE
Jemal, M.; Mulvana, D.E. Liquid chromatographic-electrospray tandem mass spectrometric method for
the simultaneous quantitation of the prodrug fosinopril and the active drug fosinoprilat in human
serum, J.Chromatogr.B, 2000, 739, 255–271.
284
Fosphenytoin
Fosphenytoin
H
N
Molecular formula: C16 H15 N2 O6 P
Molecular weight: 362.27
CAS Registry No: 93390-81-9
Merck Index: 13, 4280
N
O
O
O
OH
P
OH
O
SAMPLE
Matrix: blood
Sample preparation: Vortex 1 mL plasma, 3 mL MeCN, and 5 µg IS, centrifuge for
10 min, inject an aliquot of the supernatant.
HPLC VARIABLES
Column: 150 × 3.9 µBondapak C18
Mobile phase: MeOH:10 mM pH 4.0 tetrabutylammonium dihydrogen phosphate 47:53
Flow rate: 1.7
Detector: UV 214
CHROMATOGRAM
Internal standard: diphenylphosphate
Limit of quantitation: 100 ng/mL
KEY WORDS
pharmacokinetics; plasma
REFERENCE
Boucher, B.A.; Bombassaro, A.M.; Rasmussen, S.N.; Watridge, C.B.; Achari, R.; Turlapaty, P. Phenytoin prodrug 3-phosphoryloxymethyl phenytoin (ACC-9653): pharmacokinetics in patients following
intravenous and intramuscular administration, J.Pharm.Sci., 1989, 78, 929–932.
SAMPLE
Matrix: blood
Sample preparation: Vortex 100 µL plasma, 100 µL 30 µg/mL IS in MeCN:water 3:97,
and 100 µL 85% phosphoric acid for 10 s, add 2 mL diethyl ether, shake horizontally at
120 cycles/min for 20 min, centrifuge at 1300 g for 10 min, evaporate the supernatant
to dryness under a stream of nitrogen at 40◦ , reconstitute the residue with 200 µL
mobile phase, inject a 50 µL aliquot. Alternatively, filter (Amicon Centrifree with YMT
membrane) 1 mL plasma at 1000 g for 20 min, collect ca. 150 µL, inject a 50 µL aliquot
of the ultrafiltrate.
HPLC VARIABLES
Guard column: Guard-Pak Resolve C18 (Waters)
Column: 150 × 3.9 5 µm Resolve C18 (Waters)
Column temperature: 30
Mobile phase: MeCN:water 20:80 containing 5 mM tetrabutylammonium hydrogen
sulfate, adjusted to pH 2.2–2.5 with 800 µL/L 85% phosphoric acid
Flow rate: 2
Injection volume: 50
Detector: UV 210
CHROMATOGRAM
Retention time: 8.8
Internal standard: 5-(p-methylphenyl)-5-phenylhydantion (11.3)
Limit of detection: 100 ng/mL (extraction), 15 ng/mL (ultrafiltrate)
Fosphenytoin
285
Limit of quantitation: 400 ng/mL (extraction), 30 ng/mL (ultrafiltrate)
OTHER SUBSTANCES
Extracted: phenytoin (5.8)
Noninterfering: N-acetylprocainamide, carbamazepine, diazepam, digoxin, ethosuximide, gentamicin, lithium, lorazepam, phenobarbital, primidone, procainamide, quinidine, theophylline, valproic acid
KEY WORDS
pharmacokinetics; plasma; ultrafiltrate
REFERENCE
Cwik, M.J.; Liang, M.; Deyo, K.; Andrews, C.; Fischer, J. Simultaneous rapid high-performance liquid
chromatographic determination of phenytoin and its prodrug, fosphenytoin in human plasma and
ultrafiltrate, J.Chromatogr.B, 1997, 693, 407–414.
ANNOTATED BIBLIOGRAPHY
Kugler, A.R.; Annesley, T.M.; Nordblom, G.D.; Koup, J.R.; Olson, S.C. Cross-reactivity of fosphenytoin
in two human plasma phenytoin assays, Clin.Chem., 1998, 44, 1474–1480.
286
Frovatriptan
Frovatriptan
Molecular formula: C14 H17 N3 O
Molecular weight: 243.30
CAS Registry No: 158747-02-5,
158930-17-7 (succinate)
O
H
CH3
N
H
NH2
N
H
SAMPLE
Matrix: blood
Sample preparation: Mix 200 µL blood with 40 µL 10 µg/mL IS and 100 µL 100 mM
sodium carbonate, extract with 3 mL chloroform:isopropanol 50:50 (Caution! Chloroform
is a carcinogen!). Evaporate the organic layer to dryness under a stream of nitrogen at
60◦ , reconstitute the residue with 150 µL mobile phase, inject an aliquot.
HPLC VARIABLES
Guard column: 10 × 4.6 4 µm µBondapak C18
Column: 150 × 4.6 5 µm Hypersil BDS C18
Mobile phase: MeCN:10 mM ammonium acetate 6:94, adjusted to pH 4 with glacial
acetic acid
Flow rate: 1
Detector: UV 244
CHROMATOGRAM
Retention time: 5.5
Internal standard: 5,6,7,8-Tetrahydro-6-(ethylamino)carbazole-3-carboxamide (8)
Limit of quantitation: 10 ng/mL
KEY WORDS
dog; mouse; rat; whole blood
REFERENCE
Laugher, L.; Briggs, R.; Doughty, J. Development of an analytical methodology from toxicokinetic to
clinical studies for the anti-migraine drug frovatriptan, Chromatographia, 2000, 52, S113–S119.
SAMPLE
Matrix: blood
Sample preparation: Human. Mix 1 mL blood with 100 µL 200 ng/mL IS and 2 mL
MeCN, centrifuge. Evaporate the supernatant to dryness under a stream of nitrogen at
40◦ , reconstitute the residue with 200 µL mobile phase, inject an aliquot. Rabbit. Mix
250 µL blood with 100 µL 500 ng/mL IS and 2 mL MeCN, centrifuge. Evaporate the
supernatant to dryness under a stream of nitrogen at 40◦ , reconstitute the residue with
250 µL mobile phase, inject an aliquot.
HPLC VARIABLES
Guard column: 10 × 3.2 5 µm Kromasil C8
Column: 50 × 4.6 3 µm Hypersil BDS C8
Mobile phase: MeCN:10 mM pH 4 ammonium acetate buffer 13:87
Flow rate: 1
Injection volume:
Detector: MS, PE Sciex API-III, ionspray, 40 µL/min entered the detector, m/z
244.3–213.0
CHROMATOGRAM
Retention time: 1
Frovatriptan
287
Internal standard: 5,6,7,8-Tetrahydro-6-(ethylamino)carbazole-3-carboxamide (m/z
258.3–213.0) (1)
Limit of quantitation: 200 pg/mL (human), 10 ng/mL (rabbit)
KEY WORDS
human; rabbit; whole blood
REFERENCE
Laugher, L.; Briggs, R.; Doughty, J. Development of an analytical methodology from toxicokinetic to
clinical studies for the anti-migraine drug frovatriptan, Chromatographia, 2000, 52, S113–S119.
288
Fumagillin
Fumagillin
CH3
HO
Molecular formula: C26 H34 O7
Molecular weight: 458.54
CAS Registry No: 23110-15-8
Merck Index: 13, 4307
CH3
H3C
O
OCH3
O
COOH
O
SAMPLE
Matrix: formulations
Sample preparation: Mix 100 µL of a 70 µg/mL solution in 0.9% sodium chloride with
900 µL MeOH, vortex for 15 s, centrifuge for 10 min. Mix 100 µL of the supernatant
with 900 µL MeOH, vortex for 15 s, centrifuge for 10 min, inject a 20 µL aliquot.
HPLC VARIABLES
Column: Zorbax C18
Mobile phase: MeCN:MeOH:water:phosphoric acid 50:20:30:0.1
Flow rate: 0.5
Injection volume: 20
Detector: UV 350
CHROMATOGRAM
Retention time: 3.7
Limit of quantitation: 200 ng/mL
KEY WORDS
ophthalmic solutions; stability-indicating
REFERENCE
Abdel-Rahman, S.M.; Nahata, M.C. Stability of fumagillin in an extemporaneously prepared ophthalmic
solution, Am.J.Health-Syst.Pharm., 1999, 56, 547–550.
SAMPLE
Matrix: tissue
Sample preparation: Mix 2 g cut up tissue with dichloromethane:dioxane:isopropanol
75:16:9 (Caution! Dioxane is a carcinogen!), sonicate, shake, try to divide solid into
smaller pieces with a glass rod, sonicate for 15 min, centrifuge at 5000 rpm for 10 min,
filter. Evaporate the filtrate to 1–1.5 mL under a stream of nitrogen at 55◦ , make up to
2 mL with mobile phase, inject an aliquot.
HPLC VARIABLES
Column: 250 × 4.6 5 µm silica (Perkin Elmer) (Before use, wash column with dichloromethane:acetic acid 95:5.)
Mobile phase: n-Hexane:dichloromethane:dioxane:isopropanol:acetic acid 43:43:9:5:0.1
Flow rate: 2
Detector: UV 340
CHROMATOGRAM
Retention time: 10
Limit of detection: 5 ng/g
Limit of quantitation: 15 ng/g
Fumagillin
289
KEY WORDS
fat; fish; kidney; liver; muscle; normal phase; protect from light
REFERENCE
Fekete, J.; Romvári, Z.; Gebefügi, I.; Kettrup, A. Comparative study on determination of fumagillin in
fish by normal and reversed phase chromatography, Chromatographia, 1998, 48, 48–52.
SAMPLE
Matrix: tissue
Sample preparation: Condition
a Sep-Pak C18 SPE cartridge with 5 mL
dichloromethane and 10 mL MeOH. Mix 2 g cut up tissue with dichloromethane:dioxane:isopropanol 75:16:9 (Caution! Dioxane is a carcinogen!), sonicate,
shake, try to divide solid into smaller pieces with a glass rod, sonicate for 15 min,
centrifuge at 5000 rpm for 10 min, filter. Evaporate the filtrate to 0.1–0.2 mL under a
stream of nitrogen at 55◦ , make up to 2 mL with mobile phase, add to the SPE cartridge,
discard the first 0.5 mL (?), collect the second 0.5 mL, inject a 100 µL aliquot.
HPLC VARIABLES
Column: 250 × 4.6 5 µm Spherisorb ODS
Mobile phase: MeCN:water:bicyclohexylamine 70:30:0.05, adjusted to pH 5.0 with 1 M
phosphoric acid
Flow rate: 2
Injection volume: 100
Detector: UV 340
CHROMATOGRAM
Retention time: 5
Limit of detection: 5 ng/g
Limit of quantitation: 15 ng/g
KEY WORDS
fat; fish; kidney; liver; muscle; protect from light; SPE
REFERENCE
Fekete, J.; Romvári, Z.; Gebefügi, I.; Kettrup, A. Comparative study on determination of fumagillin in
fish by normal and reversed phase chromatography, Chromatographia, 1998, 48, 48–52.
ANNOTATED BIBLIOGRAPHY
Fekete, J.; Romvári, Z.; Szepesi, I.; Morovján, G. Liquid chromatographic determination of the antibiotic
fumagillin in fish meat samples, J.Chromatogr.A, 1995, 712, 378–381. [trout; LOQ 5 ng/g]
Guyonnet, J.; Richard, M.; Hellings, P. Determination of fumagillin in muscle tissue of rainbow trout
using automated ion-pairing liquid chromatography, J.Chromatogr.B, 1995, 666, 354–359. [SPE; LOQ
20 ng/g]
Galantamine
OH
Molecular formula: C17 H21 NO3
Molecular weight: 287.35
O
CAS Registry No: 357-70-0
Merck Index: 13, 4361
H3CO
N
CH3
SAMPLE
Matrix: blood
Sample preparation: Mix 1 mL plasma with 100 µL 10 µg/mL IS in MeOH, 1 mL
saturated KCl solution, and 100 µL 1 M NaOH, vortex, extract twice with 2.5 mL
portions of toluene. Combine the organic layers and evaporate to dryness under a stream
of nitrogen at 65◦ , reconstitute the residue with 100 µL MeOH:10 mM ammonium
acetate:diethylamine 90:10:1, inject an aliquot.
HPLC VARIABLES
Column: 100 × 4.6 3 µm Hypersil C18 BDS
Mobile phase: MeCN:10 mM pH 7 ammonium acetate 15:85
Flow rate: 0.8
Detector: F ex 280 em 310
CHROMATOGRAM
Internal standard: codeine phosphate
Limit of quantitation: 2–10 ng/mL
OTHER SUBSTANCES
Extracted: norgalantamine
KEY WORDS
dog; mouse; pharmacokinetics; plasma; rabbit; rat
REFERENCE
Monbaliu, J.; Verhaeghe, T.; Willems, B.; Bode, W.; Lavrijsen, K.; Meuldermans, W. Pharmacokinetics
of galantamine, a cholinestrase inhibitor, in several animal species, Arzneimittelforschung, 2003, 53,
486–495.
SAMPLE
Matrix: blood
Sample preparation: Mix 200 µL plasma with 100 µL 50 ng/mL IS in 2% bovine
serum albumin in 50 mM pH 7.5 phosphate buffer, add 1 mL 100 mM NaOH, add
500 µL saturated KCl, add 5 mL toluene, extract by over-the-top mixing at 15 rpm for
10 min, centrifuge at 2000 g for 10 min. Evaporate the organic layer to dryness under a
stream of nitrogen at 65◦ , reconstitute the residue with 200 µL mobile phase, inject a
20 µL aliquot.
HPLC VARIABLES
Column: 50 × 4.6 3.5 µm Symmetry Shield C18 (Waters)
Mobile phase: MeCN:10 mM ammonium acetate 15:85
Flow rate: 1.5
Injection volume: 20
Detector: MS, PE Sciex API 3000 triple quadrupole, TurboIonspray, positive ion mode,
ionspray 4500 V, turbo gas nitrogen 350◦ 6.5 L/min, nebulizing gas air, collision gas
nitrogen, orifice 31 V, ring potential 230 V, m/z 288.1–213.0
290
HPLC Methods for Recently Approved Pharmaceuticals. by George Lunn
Copyright 2005 John Wiley & Sons, Ltd. ISBN: 0-471-66941-5
Galantamine
291
CHROMATOGRAM
Retention time: 1.12
Internal standard: 13 C2 H3 -galantamine (292.1–217.0) (1.10)
Limit of quantitation: 1 ng/mL
OTHER SUBSTANCES
Extracted: epigalantamine
KEY WORDS
pharmacokinetics; plasma
REFERENCE
Verhaeghe, T.; Diels, L.; De Vries, R.; De Meulder, M.; de Jong, J. Development and validation of a liquid
chromatographic-tandem mass spectrometric method for the determination of galantamine in human
heparinized plasma, J.Chromatogr.B, 2003, 789, 337–346.
ANNOTATED BIBLIOGRAPHY
Huang, F.; Lasseter, K.C.; Janssens, L.; Verhaeghe, T.; Lau, H.; Zhao, Q. Pharmacokinetic and safety
assessments of galantamine and risperidone after the two drugs are administered alone and together,
J.Clin.Pharmacol., 2002, 42, 1341–1351.
Mannens, G.S.J.; Snel, C.A.W.; Hendrickx, J.; Verhaeghe, T.; Le Jeune, L.; Bode, W.; van Beijsterveldt, L.; Lavrijsen, K.; Leempoels, J.; Van Osselaer, N.; Van Peer, A.; Meuldermans, W. The metabolism and excretion of galantamine in rats, dogs, and humans, Drug Metab.Dispos., 2002, 30,
553–563.
Zhao, Q.; Brett, M.; Van Osselaer, N.; Huang, F.; Raoult, A.; Van Peer, A.; Verhaeghe, T.; Hust, R.
Galantamine pharmacokinetics, safety, and tolerability profiles are similar in healthy Caucasian and
Japanese subjects, J.Clin.Pharmacol., 2002, 42, 1002–1010.
Zhao, Q.; Iyer, G.R.; Verhaeghe, T.; Truyen, L. Pharmacokinetics and safety of galantamine in subjects
with hepatic impairment and healthy volunteers, J.Clin.Pharmacol., 2002, 42, 428–436.
292
Ganirelix
Ganirelix
Molecular formula: C80 H113 ClN18 O13
Molecular weight: 1570.35
CAS Registry No: 124904-93-4
Merck Index: 13, 4380
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 × 4.6 5 µm Zorbax Rx octyl
Mobile phase: MeCN:50 mM pH 2 ammonium phosphate buffer 27.5:72.5
Flow rate: 1
Detector: UV 225
REFERENCE
Strickley, R.G.; Brandl, M.; Chan, K.W.; Straub, K.; Gu, L. High-performance liquid chromatographic
(HPLC) and HPLC-mass spectrometric (MS) analysis of the degradation of the luteinizing hormonereleasing hormone (LH-RH) antagonist RS-26306 in aqueous solution, Pharm.Res., 1990, 7, 530–536.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 × 4.6 5 µm Zorbax Rx octyl
Mobile phase: MeCN:100 mM pH 6 ammonium acetate buffer 42:58
Flow rate: 1.2
Injection volume: 100
Detector: UV 230; MS, Finnigan-MAT TSQ-70, thermospray, vaporizer 65–94◦ , jet 240◦ ,
collector 75–80 V, m/z 1570
CHROMATOGRAM
Retention time: 23
OTHER SUBSTANCES
Simultaneous: degradants
REFERENCE
Strickley, R.G.; Brandl, M.; Chan, K.W.; Straub, K.; Gu, L. High-performance liquid chromatographic
(HPLC) and HPLC-mass spectrometric (MS) analysis of the degradation of the luteinizing hormonereleasing hormone (LH-RH) antagonist RS-26306 in aqueous solution, Pharm.Res., 1990, 7, 530–536.
Gatifloxacin
Gatifloxacin
293
CH3
H
N
OCH3
Molecular formula: C19 H22 FN3 O4
Molecular weight: 375.39
N
CAS Registry No: 112811-59-3
Merck Index: 13, 4388
F
N
COOH
O
SAMPLE
Matrix: blood, urine
Sample preparation: Serum. Mix 200 µL serum with 200 µL 500 µg/mL IS in MeCN:
100 mM phosphoric acid 90:10 and 400 µL MeCN, centrifuge. Mix 200 µL of the
supernatant with 800 µL PIC A solution, inject a 20 µL aliquot. Urine. Dilute 10 µL
urine with 990 µL PIC A solution, inject a 5 µL aliquot. (PIC A solution was 10 mM
tetrabutylammonium phosphate (Waters PIC A) adjusted to pH 3.48.)
HPLC VARIABLES
Guard column: 30 × 4 30–40 µm Perisorb RP-18
Column: 125 × 4 5 µm Nucleosil-100 5C18
Mobile phase: MeCN:10 mM tetrabutylammonium phosphate (Waters PIC A) 17:83,
adjusted to pH 3.48 with ca. 1.3 mL concentrated phosphoric acid.
Flow rate: 1
Injection volume: 5–20
Detector: F ex 295 em 480
CHROMATOGRAM
Retention time: 4.5
Internal standard: trovafloxacin (12.6)
Limit of detection: 30 ng/mL (serum), 1.7 µg/mL (urine)
Limit of quantitation: 60 ng/mL (serum), 3.5 µg/mL (urine)
OTHER SUBSTANCES
Simultaneous: ciprofloxacin (3.6), grepafloxacin (11.1), moxifloxacin (7.7), ofloxacin
(2.7), salicylic acid (22.2), tryptophan (1.9)
Noninterfering: furosemide, sparfloxacin
KEY WORDS
pharmacokinetics; serum
REFERENCE
Borner, K.; Hartwig, H.; Lode, H. Determination of gatifloxacin in human serum and urine by HPLC,
Chromatographia, 2000, 52, S105–S107.
SAMPLE
Matrix: blood, urine
Sample preparation: Serum. Add 50 µL water to 450 µL serum, vortex for 30 s, add
450 µL MeCN:75 mM pH 7.5 phosphate buffer containing 500 µg/mL sodium dodecyl
sulfate 20:80, add 50 µL 30 µg/mL IS solution, vortex for 30 s, filter (Amicon Centrifree)
while centrifuging at 1500 g for 30 min, inject a 20 µL aliquot of the ultrafiltrate. Urine.
Dilute 50 µL urine with 1 mL mobile phase, add 50 µL 30 µg/mL IS solution, vortex for
30 s, inject a 20 µL aliquot.
HPLC VARIABLES
Guard column: 7.5 × 4.6 5 µm Adsorbosphere HS C18
294
Gatifloxacin
Column: 250 × 4.6 5 µm Adsorbosphere HS C18
Mobile phase: MeCN:buffer 50:50 (The buffer was 25 mM citric acid containing 10 mM
sodium dodecyl sulfate and 10 mM tetrabutylammonium acetate.)
Flow rate: 1
Injection volume: 20
Detector: UV 293
CHROMATOGRAM
Retention time: 6.3
Internal standard: ciprofloxacin (5.5)
Limit of quantitation: 100 ng/mL
KEY WORDS
pharmacokinetics; serum; ultrafiltrate
REFERENCE
Overholser, B.R.; Kays, M.B.; Sowinski, K.M. Determination of gatifloxacin in human serum and urine
by high-performance liquid chromatography with ultraviolet detection, J.Chromatogr.B, 2003, 798,
167–173.
ANNOTATED BIBLIOGRAPHY
Grant, E.M.; Nicolau, D.P.; Nightingale, C.; Quintiliani, R. Minimal interaction between gatifloxacin and
oxycodone, J.Clin.Pharmacol., 2002, 42, 928–932. [no HPLC of oxycodone]
Liang, H.; Kays, M.B.; Sowinski, K.M. Separation of levofloxacin, ciprofloxacin, gatifloxacin, moxifloxacin,
trovafloxacin and cinoxacin by high-performance liquid chromatography: application to levofloxacin
determination in human plasma, J.Chromatogr.B, 2002, 772, 53–63.
Wallace, A.W.; Victory, J.M.; Amsden, G.W. Lack of bioequivalence of gatifloxacin when coadministered with calcium-fortified orange juice in healthy volunteers, J.Clin.Pharmacol., 2003, 43, 92–96.
[moxifloxacin is internal standard; fluorescence detection]
295
Gefitinib
Gefitinib
Molecular formula: C22 H24 ClFN4 O3
Molecular weight: 446.90
H3CO
N
N
N
O
N
O
Cl
H
CAS Registry No: 184475-35-2
F
SAMPLE
Matrix: blood
Sample preparation: Vortex 500 µL plasma, 25 µL of 1 µg/mL IS in MeOH, 500 µL
1 M NaOH, and 6 mL MTBE for 2 min, centrifuge for 30 s. Remove 5.5 mL of the organic
layer and evaporate it to dryness under a stream of nitrogen at 20◦ , reconstitute the
residue with 250 µL mobile phase, inject a 15–100 µL aliquot.
HPLC VARIABLES
Guard column: Inertsil ODS3
Column: 150 × 4.6 Inertsil ODS3
Mobile phase: MeCN:1% ammonium acetate 80:20
Flow rate: 1
Injection volume: 15–100
Detector: MS, PE Sciex API-III tandem, APCI, m/z 447.2–128
CHROMATOGRAM
Retention time: 2.5
Internal standard: d8 -gefitinib (m/z 455.4–136) (2.5)
Limit of quantitation: 0.5 ng/mL
KEY WORDS
pharmacokinetics; plasma
REFERENCE
Jones, H.K.; Stafford, L.E.; Swaisland, H.C.; Payne, R. A sensitive assay for ZD1839 (Iressa) in human
plasma by liquid-liquid extraction and high performance liquid chromatography with mass spectrometric detection: validation and use in Phase I clinical trials, J.Pharm.Biomed.Anal., 2002, 29,
221–228.
296
Gemcitabine
Gemcitabine
Molecular formula: C9 H11 F2 N3 O4
Molecular weight: 263.20
CAS Registry No: 95058-81-4
Merck Index: 13, 4397
NH2
N
O
HO
N
O
F
OH F
SAMPLE
Matrix: blood
Sample preparation: Mix 6 mL blood with 100 µL 10 mg/mL tetrahydrouridine, a
cytidine deaminase inhibitor, centrifuge at 1200 g for 15 min to obtain plasma. Vortex
100 µL plasma and 10 µL 40% trichloroacetic acid for 1 min, centrifuge at 10 000 g at
4◦ for 5 min, inject a 25 µL aliquot of the supernatant.
HPLC VARIABLES
Column: 250 × 4.6 5 µm Thermo Hypersil BDS C18
Mobile phase: MeOH:buffer 17:83 (The buffer was 20 mM pH 3.1 ammonium dihydrogen
phosphate buffer containing 10 mM sodium 1-heptanesulfonate.)
Flow rate: 0.8
Injection volume: 25
Detector: UV 272
CHROMATOGRAM
Retention time: 24
Limit of detection: 50 ng/mL
Limit of quantitation: 80 ng/mL
OTHER SUBSTANCES
Extracted: 2′ ,2′ -difluorodeoxyuridine (dFdU) (6)
KEY WORDS
pharmacokinetics; plasma
REFERENCE
Wang, L.-Z.; Goh, B.-C.; Lee, H.-S.; Noordhuis, P.; Peters, G.J. An expedient assay for determination
of gemcitabine and its metabolite in human plasma using isocratic ion-pair reversed-phase highperformance liquid chromatography, Ther.Drug Monit., 2003, 25, 552–557.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 × 4.6 5 µm Zorbax Rx-C8
Mobile phase: 13.8 g/L sodium dihydrogen phosphate containing 2.5 mL/L phosphoric
acid
Flow rate: 1.2
Injection volume: 5
Detector: UV 275
Gemcitabine
297
REFERENCE
Jansen, P.J.; Akers, M.J.; Amos, R.M.; Baertschi, S.W.; Cooke, G.G.; Dorman, D.E.; Kemp, C.A.J.; Maple,
S.R.; McCune, K.A. The degradation of the antitumor agent gemcitabine hydrochloride in an acidic
aqueous solution at pH 3.2 and identification of degradation products, J.Pharm.Sci., 2000, 89, 885–891.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 × 4.6 5 µm Zorbax Rx-C8
Mobile phase: Gradient. A was MeOH:buffer 3:97. B was MeOH:buffer 50:50. A:B 100:0
for 8 min, to 0:100 over 5 min, maintain at 0:100 for 7 min. (The buffer was 13.8 g/L
sodium dihydrogen phosphate containing 2.5 mL/L phosphoric acid.)
Flow rate: 1.2
Injection volume: 5
Detector: UV 205
CHROMATOGRAM
Retention time: 7.5
OTHER SUBSTANCES
Simultaneous: degradants
Interfering: 6-hydroxy-5,6-dihydro-2′ -deoxy-2′ ,2′ -difluorouridine
REFERENCE
Jansen, P.J.; Akers, M.J.; Amos, R.M.; Baertschi, S.W.; Cooke, G.G.; Dorman, D.E.; Kemp, C.A.J.; Maple,
S.R.; McCune, K.A. The degradation of the antitumor agent gemcitabine hydrochloride in an acidic
aqueous solution at pH 3.2 and identification of degradation products, J.Pharm.Sci., 2000, 89, 885–891.
ANNOTATED BIBLIOGRAPHY
Floridia, L.; Pietropaolo, A.M.; Tavazzani, M.; Rubino, F.M.; Colombi, A. Measurement of surface contamination from nucleoside analogue antineoplastic drugs by high-performance liquid chromatography
in occupational hygiene studies of oncologic hospital departments, J.Chromatogr.B, 1999, 724,
325–334. [fluorouracil; bromouracil; iodouracil; cytarabine; gemcitabine; methotrexate; aminopterin;
doxorubicin; daunorubicin; epirubicin; idarubicin]
Freeman, K.B.; Anliker, S.; Hamilton, M.; Osborne, D.; Dhahir, P.H.; Nelson, R.; Allerheiligen, S.R.B.
Validated assays for the determination of gemcitabine in human plasma and urine using highperformance liquid chromatography with ultraviolet detection, J.Chromatogr.B, 1995, 665, 171–181.
[normal phase; LOQ 50 ng/mL; pharmacokinetics]
Keith, B.; Xu, Y.; Grem, J.L. Measurement of the anti-cancer agent gemcitabine in human plasma by
high-performance liquid chromatography, J.Chromatogr.B, 2003, 785, 65–72.
Sparidans, R.W.; Crul, M.; Schellens, J.H.M.; Beijnen, J.H. Isocratic ion-exchange chromatographic
assay for the nucleotide gemcitabine triphosphate in human white blood cells, J.Chromatogr.B,
2002, 780, 423–430.
Yilmaz, B.; Kadioglu, Y.; Aksoy, Y. Simultaneous determination of gemcitabine and its metabolite in
human plasma by high-performance liquid chromatography, J.Chromatogr.B, 2003, 791, 103–109.
298
Gemifloxacin
Gemifloxacin
Molecular formula: C18 H20 FN5 O4
Molecular weight: 389.38
H2N
H3CO
N
CAS Registry No: 175463-14-6
Merck Index: 13, 4400
N
N
N
F
COOH
O
SAMPLE
Matrix: blood
Sample preparation: Vortex 50 µL plasma and 250 µL 250 ng/mL IS in MeCN for 10 s,
shake for 30 min, let stand at room temperature for 10 min, centrifuge at 14 000 g for
15 min, add the supernatant to 200 µL buffer in a silanized tube, inject a 10 µL aliquot.
(The buffer was 10 mM ammonium acetate adjusted to pH 2.5 with trifluoroacetic acid.)
HPLC VARIABLES
Column: 500 × 4.6 5 µm 100 Å PLRP-S
Column temperature: 40
Mobile phase: MeCN:buffer 30:70 (The buffer was 10 mM ammonium acetate adjusted
to pH 2.5 with trifluoroacetic acid.)
Flow rate: 1
Injection volume: 10
Detector: MS, PE Sciex API 300 tandem, heat-assisted nebulization, electrospray, positive ion mode, nebulizer gas at 60 psi, curtain gas at 40 psi, collision gas thickness 4,
auxiliary gas 7 L/min, dwell 400 ms, pause 5 ms, m/z 390–313
CHROMATOGRAM
Retention time: 1
Internal standard: 13 C2 H3 -gemifloxacin (m/z 394–313)
Limit of quantitation: 10 ng/mL
KEY WORDS
pharmacokinetics; plasma
REFERENCE
Doyle, E.; Fowles, S.E.; McDonnell, D.F.; McCarthy, R.; White, S.A. Rapid determination of gemifloxacin in human plasma by high-performance liquid chromatography-tandem mass spectrometry,
J.Chromatogr.B, 2000, 746, 191–198.
SAMPLE
Matrix: solutions
Sample preparation: Inject a 1–3 µL aliquot of a 2 mg/mL solution.
HPLC VARIABLES
Column: 150 × 4 5 µm Crownpak CR(+) (Daicel)
Mobile phase: MeOH:water 15:85 containing 10 mM sulfuric acid
Flow rate: 1.2
Injection volume: 1–3
Detector: UV 272
CHROMATOGRAM
Retention time: 38.88, 58.86 (enantiomers)
Gemifloxacin
299
KEY WORDS
chiral
REFERENCE
Lee, W.; Hong, C.Y. Direct liquid chromatographic enantiomer separation of new fluoroquinolones including gemifloxacin, J.Chromatogr.A, 2000, 879, 113–120.
ANNOTATED BIBLIOGRAPHY
Edelstein, P.H.; Shinzato, T.; Doyle, E.; Edelstein, M.A.C. In vitro activity of gemifloxacin (SB-265805,
LB20304a) against Legionella pneumophila and its pharmacokinetics in guinea pigs with L-pneumophila pneumonia, Antimicrob.Agents Chemother., 2001, 45, 2204–2209. [LC-MS]
Hyun, M.H.; Han, S.C. Liquid chromatographic separation of the enantiomers of fluoroquinolone antibacterials on a chiral stationary phase based on a chiral crown ether, J.Biochem.Biophys.Methods, 2002,
54, 235–243.
Hyun, M.H.; Han, S.C.; Cho, Y.J.; Jin, J.S.; Lee, W. Liquid chromatographic resolution of gemifloxacin
mesylate on a chiral stationary phase derived from crown ether, Biomed.Chromatogr., 2002, 16,
356–360.
Naber, C.K.; Hammer, M.; Kinzig-Schippers, M.; Sauber, C. Söurgel, F.; Bygate, E.A.; Fairless, A.J.;
Machka, K.; Naber, K.G. Urinary excretion and bactericidal activities of gemifloxacin and ofloxacin
after a single oral dose in healthy volunteers, Antimicrob.Agents Chemother., 2001, 45, 3524–3530.
[LC-MS; no HPLC of ofloxacin]
Ramji, J.V.; Austin, N.E.; Boyle, G.W.; Chalker, M.H.; Duncan, G.; Fairless, A.J.; Hollis, F.J.; McDonnell, D.F.; Musick, T.J.; Shardlow, P.C. The disposition of gemifloxacin, a new fluoroquinolone antibiotic, in rats and dogs, Drug Metab.Dispos., 2001, 29, 435–442.
300
Gestodene
Gestodene
H3C
H
H
Molecular formula: C21 H26 O2
Molecular weight: 310.43
CAS Registry No: 60282-87-3
Merck Index: 13, 4425
OH
H
H
H
O
SAMPLE
Matrix: formulations
Sample preparation: Powder 2 tablets, sonicate with 10 mL EtOH for 15 min, shake
mechanically for 20 min, centrifuge. Dilute a 2.5 mL aliquot of the supernatant to 10 mL
with water, inject a 20 µL aliquot.
HPLC VARIABLES
Column: 150 × 3.9 4 µm Nova-Pak C18
Mobile phase: MeCN:MeOH:water 35:15:45
Flow rate: 1
Injection volume: 20
Detector: UV 215
OTHER SUBSTANCES
Extracted: ethinyl estradiol
KEY WORDS
tablets
REFERENCE
Berzas, J.J.; Rodrı́guez, J.; Gastañeda, G. Determination of ethinylestradiol and gestodene in pharmaceuticals by a partial least-squares and principal component regression multivariate calibration,
Anal.Sci., 1997, 13, 1029–1032.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 150 × 4.6 3 µm Hypersil ODS
Mobile phase: Gradient. MeCN:water from 35:65 to 65:35 (time not given).
Injection volume: 50
Detector: UV 242, UV 227
OTHER SUBSTANCES
Simultaneous: gestodene esters
REFERENCE
Lipp, R.; Laurent, H.; Günther, C.; Riedl, J.; Esperling, P.; Täuber, U. Prodrugs of gestodene for matrixtype transdermal drug delivery systems, Pharm.Res., 1998, 15, 1419–1424.
301
Gestrinone
Gestrinone
H3C
H
H
Molecular formula: C21 H24 O2
Molecular weight: 308.41
CAS Registry No: 16320-04-0
Merck Index: 13, 4427
OH
H
O
SAMPLE
Matrix: blood
Sample preparation: Mix 200 µL serum with 50 µL 100 ng/mL IS in MeOH, add 2 mL
diethyl ether, vortex for 2 min, freeze in dry ice/acetone, repeat the extraction. Combine
the organic layers and evaporate to dryness, reconstitute the residue with 200 µL mobile
phase, inject a 20 µL aliquot.
HPLC VARIABLES
Column: 250 × 4.6 5 µm Kromasil C18
Mobile phase: 0.2% Formic acid in MeOH
Flow rate: 1
Injection volume: 20
Detector: MS, PE Sciex API 3000, 100 µL/min entered the MS, electrospray, positive
ion mode, source 200◦ , ionspray 5000 V, nebulizer gas nitrogen at 1 L/min, curtain gas
at 1.25 L/min, orifice 45 V, collision gas nitrogen 30 µtorr, collision energy 45 eV, m/z
309–241
CHROMATOGRAM
Retention time: 3
Internal standard: mifepristone (m/z 430–372) (2.7)
Limit of detection: 0.8 ng/mL (S/N = 3)
Limit of quantitation: 3.5 ng/mL
KEY WORDS
pharmacokinetics; serum; UV 340 can be used with MeOH:water 70:30
REFERENCE
Wang, Q.; Wu, Z.; Wang, Y.; Luo, G.; Wu, E.; Gao, X. Determination of gestrinone in human serum by
liquid chromatography-electrospray tandem mass spectrometry, J.Chromatogr.B, 2000, 746, 151–159.
302
Glycerin
Glycerin
Molecular formula: C3 H8 O3
Molecular weight: 92.09
HO
OH
OH
CAS Registry No: 56-81-5
Merck Index: 13, 4497
SAMPLE
Matrix: blood
Sample preparation: Mix 1 mL plasma with 2 mL 10% trichloroacetic acid, centrifuge
at 2500 rpm for 20 min. Wash the clear supernatant several times with ether until the
aqueous layer becomes neutral. Dry the aqueous layer under a stream of air, reconstitute
with 50 µL water containing 5 µg ethylene glycol, inject a 25 µL aliquot.
HPLC VARIABLES
Column: 20 × 7 5 µm IEX 215 cation-exchange (Toyo Soda)
Mobile phase: Gradient. MeCN:water 76:24 for 30 min, to 0:100 (time not specified)
Flow rate: 0.6
Injection volume: 25
Detector: F ex 412 em 475 following post-column reaction. The column effluent mixed
with 25 mM periodic acid pumped at 0.1 mL/min and the resulting mixture flowed
through an 0.4 mm ID reaction coil at 145◦ for 3 min. The effluent from this coil mixed
with 8% acetylacetone in 2 M ammonium acetate (allowed to stand overnight before
use) pumped at 0.2 mL/min and the mixture flowed through a similar coil at 64◦ to the
detector.
CHROMATOGRAM
Retention time: 26
OTHER SUBSTANCES
Extracted: anhydroglucitol (29), dexteros (43), erythritol (32), ethylene glycol (20)
KEY WORDS
plasma; post-column reaction
REFERENCE
Akanuma, H.; Ogawa, K.; Lee, Y.; Akanuma, Y. Reduced levels of plasma 1,5-anhydroglucitol in diabetic
patients, J.Biochem.(Tokyo), 1981, 90, 157–162.
SAMPLE
Matrix: blood
Sample preparation: Add 2 mL of a slurry of an anion-exchange resin (AG1-X8,
200–400 mesh, hydroxide form, Bio-Rad) in an equal volume of water to an 80 × 10
column. Add 2 mL of a slurry of a cation-exchange resin (AG50-WX8, 200–400 mesh,
hydrogen form, Bio-Rad) in an equal volume of water to an 80 × 10 column. Place the
anion column on top of the cation column. Vortex 1 mL plasma briefly, add 1 mL 0.3
N barium hydroxide, add 1.1 mL 0.3 N zinc sulfate, vortex, centrifuge at 2000 rpm
for 10 min. Add the supernatant to the columns, vortex the pellet with 1 mL water,
centrifuge at 2000 rpm, add the supernatant to the columns, repeat the extraction
of the pellet, add two 1 mL portions of water to the columns. Collect all the eluate
from the columns, add 100 µL 100 mM sucrose, vortex, lyophilize at – 20◦ for 15 h.
Add 5 drops 2,2-dimethoxypropane, mix, dry under air (it is critical that the sample
be completely dry), add 1 mL 30 mg/mL 4-dimethylaminopyridine in pyridine, vortex,
sonicate at 45◦ for 5 min (no longer than 5 min), add 100 µL 25% benzoyl peroxide in
Glycerin
303
butyl acetate, vortex until a white precipitate appears, heat at 45◦ for 15 min, cool to
room temperature, add 50 µL MeOH, vortex at 45◦ for 5 min. Evaporate to dryness
under a stream of air at 45◦ for 1 h, reconstitute the residue with 4 mL chloroform with
vortexing and sonicating (Caution! Chloroform is a carcinogen!), add 2 mL 1 M HCl,
vortex for 15 s, centrifuge at 2000 rpm for 5 min, discard the aqueous layer. Repeat the
HCl wash. Evaporate the chloroform layer to dryness under a stream of air, reconstitute
the residue with 172.8 µL MeCN and 67.2 µL water, vortex, centrifuge for 2 min, inject
a 200 µL aliquot.
HPLC VARIABLES
Column: 250 mm long 5 µm Apex C18 (Jones)
Mobile phase: Gradient. MeCN:water 56:44 for 30 min, to 95:5 (step gradient), maintain
at 95:5 for 10 min, re-equilibria at initial conditions for 20 min.
Flow rate: 1.3
Injection volume: 200
Detector: UV 254
CHROMATOGRAM
Retention time: 26
OTHER SUBSTANCES
Extracted: dextrose (28), sucrose (30)
KEY WORDS
derivatization; plasma; SPE
REFERENCE
Akanuma, H.; Ogawa, K.; Lee, Y.; Akanuma, Y. Reduced levels of plasma 1,5-anhydroglucitol in diabetic
patients, J.Biochem., 1981, 90, 157–162.
304
Guanabenz
Guanabenz
Molecular formula: C8 H8 Cl2 N4
Molecular weight: 231.09
Cl
NH
N
N
Cl
NH2
H
CAS Registry No: 5051-62-7
Merck Index: 13, 4574
SAMPLE
Matrix: blood
Sample preparation: Mix 2 mL serum with 10 µL 10 µg/mL IS in MeOH, add 200 µL
concentrated ammonium hydroxide, add 5 mL dichloromethane, shake on a reciprocating shaker for 30 min, centrifuge at 730 g for 20 min. (If excessive emulsion is present,
mix by inversion and centrifuge again.). Evaporate the organic layer to dryness in a
silanized glass tube under a stream of nitrogen below 40◦ , reconstitute the residue with
80 µL mobile phase, vortex, inject a 25 µL aliquot.
HPLC VARIABLES
Column: 250 × 2 5 µm Luna phenyl hexyl (Phenomenex)
Column temperature: 30
Mobile phase: Gradient. MeCN:water:formic acid 5:95:0.05 for 1 min, to 90:10:0.05
over 4 min, maintain at 90:10:0.05 for 5 min, return to initial conditions over 0.5 min,
re-equilibrate for 4.5 min.
Flow rate: 0.45
Injection volume: 25
Detector: MS, Micromass Quattro II, positive ion mode, collision gas argon 3 µbar,
photomultiplier 750–800 V, source cone 26 V, collision energy – 23 ± 1 V, capillary +
3.1 kV, HV lens 520 V, source 120◦ , m/z 231–172
CHROMATOGRAM
Retention time: 6.19
Internal standard: clenbuterol (277–203) (6.12)
Limit of detection: 0.3 ng/mL
Limit of quantitation: 3 ng/mL
KEY WORDS
horse; pharmacokinetics; serum
REFERENCE
Harkins, J.D.; Dirikolu, L.; Lehner, A.F.; Hughes, C.; Schroedter, D.; Mayer, B.; Bratton, C.; Fisher,
M.V.; Tobin, T. The detection and biotransformation of guanabenz in horses: a preliminary report,
Vet.Ther., 2003, 4, 197–209.
305
Guanadrel
Guanadrel
NH
O
Molecular formula: C10 H19 N3 O2
Molecular weight: 213.28
O
N
NH2
H
CAS Registry No: 40580-59-4
Merck Index: 13, 4575
SAMPLE
Matrix: feed
Sample preparation: Vortex 1 g pulverized feed with 5 mL 50 mM ammonium dihydrogen phosphate, let stand for 15 min, add 1 mL IS solution, add 3 mL MeCN, shake
on a reciprocating shaker at 2000 rpm for 20 min. Add 3 mL supernatant to 1 mL 1 M
sodium bicarbonate, add 2.5 mL MeOH, add 2 mL acetylacetone, mix, heat at 120 ± 5◦
for 100 min, cool, mix, centrifuge at 2000 rpm for 10 min, inject an aliquot of the
supernatant.
HPLC VARIABLES
Column: 250 × 4.6 10 µm LiChrosorb RP-8
Mobile phase: MeCN:THF:50 mM ammonium dihydrogen phosphate 40:10:50
Flow rate: 0.8
Injection volume: 40
Detector: F ex 238 em 360
CHROMATOGRAM
Retention time: 10.5
Internal standard: cyclohexymethylguanidine (18)
Limit of detection: 1 ppm
KEY WORDS
derivatization
REFERENCE
Bombardt, P.A.; Adams, W.J. Liquid chromatographic determination of guanadrel in laboratory animal
diet as the fluorescent acetylacetone derivative, Anal.Chem., 1982, 54, 1087–1090.
Halobetasol propionate
Molecular formula: C25 H31 ClF2 O5
Molecular weight: 484.97
CAS Registry No: 66852-54-8
Merck Index: 13, 4608
Cl
HO
O
O
CH3
CH3
O
CH3
F
H
CH3
H
O
F
SAMPLE
Matrix: formulations
Sample preparation: Mix 1 g of a topical product, 5 mL 100 mM pH 4 citrate buffer
saturated with NaCl, and 10 mL ethyl acetate; shake vigorously by hand to ensure
that no large clumps stick to the tube, mix with the tube on its side on an oscillating
shaker for 15 min. Remove the upper ethyl acetate layer and wash with citrate buffer as
before. Dry the ethyl acetate over anhydrous sodium sulfate and evaporate to dryness.
Dissolve the residue in a mixture of 10 mL heptane and 5 mL MeCN:water 90:10.
Remove the upper heptane layer and extract it with 2 mL MeCN:water 90:10. Combine
the MeCN/water layers and evaporate them to dryness, dissolve the residue in 300 µL
MeOH, filter (0.45 µm nylon), inject a 5 µL aliquot.
HPLC VARIABLES
Guard column: 15 × 3.2 7 µm Brownlee NewGuard C18
Column: 75 × 4.6 3.5 µm Symmetry C18 (Waters)
Mobile phase: Gradient. MeCN:water 18:82 for 2 min, to 82:18 over 12 min, maintain
at 82:18 for 3 min, re-equilibrate at initial conditions for 12 min.
Flow rate: 1
Injection volume: 5
Detector: UV 240
CHROMATOGRAM
Retention time: 10.98
Limit of detection: 0.001%
OTHER SUBSTANCES
Simultaneous: alclometasone 17,21-dipropionate (10.93), amcinonide (10.90), beclomethasone 17,21-dipropionate (11.90), betamethasone (6.52), betamethasone 21-acetate
(8.47), betamethasone 17-benzoate (10.28), betamethasone 17,21-dipropionate (11.42),
betamethasone 17-valerate (10.25), budesonide (8.55, 8.69 (epimers)), clobetasol 17propionate (11.06), cortisone (5.62), cortisone 21-acetate (8.07), dexamethasone (6.57),
dexamethasone 21-acetate (8.68), desonide (6.99), desoximetasone (7.60), desoxycorticosterone acetate (10.90), desoxycorticosterone pivalate (14.45), diflorasone 17,21-diacetate
(9.81), fluocinolone acetonide (7.38), fluocinonide (9.79), flurandrenolide (7.36), fludrocortisone 21-acetate (7.77), fluorometholone (7.67), flumethasone 21-pivalate (11.20),
flunisolide (7.14), fluprednisolone (5.46), fluticasone 17-propionate (11.19), halcinonide (10.72), hydrocortisone (5.50), hydrocortisone 21-acetate (7.65), hydrocortisone
17-butyrate (8.66), hydrocortisone 21-cypionate (12.54), hydrocortisone 17-valerate
(9.53), mometasone 17-furoate (11.24), methylprednisolone (6.31), methylprednisolone
21-acetate (8.34), meprednisone (6.55), prednisolone (5.37), prednisolone 21-acetate
(7.47), prednisolone 21-tebuate (11.01), paramethasone 21-acetate (8.64), prednicarbate (10.72), prednisone (5.46), triamcinolone (4.15), triamcinolone acetonide (7.04),
triamcinolone 16,21-diacetate (7.49), triamcinolone hexacetonide (13.12)
306
HPLC Methods for Recently Approved Pharmaceuticals. by George Lunn
Copyright 2005 John Wiley & Sons, Ltd. ISBN: 0-471-66941-5
Halobetasol propionate
307
KEY WORDS
body wash, cream, gel, lotion, shampoo, spray
REFERENCE
Reepmeyer, J.C. Screening for corticosteroids in topical pharmaceuticals by HPLC with a scanning
ultraviolet detector, J.Liq.Chromatogr.Rel.Technol., 2001, 24, 693–709.
308
Halofuginone
Halofuginone
Molecular formula: C16 H17 BrClN3 O3
Molecular weight: 414.69
CAS Registry No: 55837-20-2
Merck Index: 13, 4611
Br
N
HO
O
N
Cl
O
N
H
SAMPLE
Matrix: blood
Sample preparation: Condition a 1 mL Bond Elut C8 SPE cartridge with 2 mL MeOH
and 8 mL water adjusted to pH 4.3 with acetic acid. Mix 4 mL serum with 8 mL 10%
acetic acid, add to the SPE cartridge, wash with 5 mL water adjusted to pH 4.3 with
acetic acid, wash with 1 mL MeOH:water 35:65 adjusted to pH 4.3 with acetic acid, wash
with 1 mL water adjusted to pH 4.3 with acetic acid, elute with 1 mL MeCN:water:acetic
acid 20:79.9:0.1 containing 2.104 µL/mL decylamine (pH ca. 4.3), inject a 50 µL aliquot
of the eluate. (Use silanized glassware.)
HPLC VARIABLES
Guard column: 30–38 µm CO:PELL
Column: 250 × 4.6 5 µm Supelcosil LC-DB18
Mobile phase: MeCN:buffer:water 22:15:63 containing 210.4 µL/L decylamine, pH ca.
4.75 (The buffer was 250 mM pH 4.3 ammonium acetate.)
Injection volume: 50
Detector: UV 243
CHROMATOGRAM
Retention time: 8
Limit of detection: 1.5 ng/mL
KEY WORDS
chicken; serum; SPE
REFERENCE
Beier, R.C.; Rowe, L.D.; Abd El-Aziz Nasr, M.I.; Elissalde, M.H.; Stanker, L.H. Extraction and HPLC
analysis of halofuginone in chicken serum, J.Liq.Chromatogr., 1994, 17, 2961–2970.
SAMPLE
Matrix: eggs, tissue
Sample preparation: Condition a 3 mL 60 mg Oasis HLB SPE cartridge with 3 mL
MeOH, 3 mL water, and 3 mL 125 mM pH 4.9 ammonium acetate buffer. Mix 2 g minced
liver or homogenized egg with 2 mL 25 mg/mL trypsin in water, vortex, adjust pH to
7–8 with 10% sodium carbonate, shake on an orbital shaker at 40◦ overnight, cool, add
1 mL 10% sodium carbonate, add 10 (liver) or 15 (eggs) mL ethyl acetate, shake on a
mechanical shaker for 3 min, centrifuge at 600 g at 4◦ for 2 min, extract the aqueous
layer again with 10 mL ethyl acetate. Combine the ethyl acetate layers and add to 5 mL
125 mM pH 4.9 ammonium acetate buffer, shake mechanically for 1 min. Shake the
aqueous layer with 5 mL hexane for 20 s, discard the hexane layer. Add the aqueous
layer to the SPE cartridge, wash with 2 mL toluene, push air through the SPE cartridge
at 19 mL/min, elute with 2 mL MeOH. Evaporate the eluate to dryness under a stream
of nitrogen at 40◦ , reconstitute the residue with 500 µL 125 mM pH 4.9 ammonium
acetate buffer, vortex for 30 s, inject a 25 µL aliquot.
HPLC VARIABLES
Column: 250 × 4.6 5 µm Prodigy C18 (Phenomenex)
Halofuginone
309
Mobile phase: MeOH:water:acetic acid 40:59.5:0.5
Flow rate: 1
Injection volume: 25
Detector: MS, Micromass Quattro LC, 0.2 mL/min entered the detector, source 150◦ ,
drying gas nitrogen 500 L/h, nebulizing gas nitrogen 80 L/h, positive mode, collision cell
entrance 0 eV, collision cell exit 2 eV, cone 30 V, collision gas argon 2.3310 mbar, m/z
416–398–138–120–100
CHROMATOGRAM
Retention time: 6.3
Limit of quantitation: 15 ng/g (liver), 5 ng/g (eggs)
KEY WORDS
chicken; liver; SPE
REFERENCE
Yakkundi, S.; Cannavan, A.; Elliott, C.T.; Lövgren, T.; Kennedy, D.G. Development and validation of a
method for the confirmation of halofuginone in chicken liver and eggs using electrospray tandem mass
spectrometry, J.Chromatogr.B, 2003, 788, 29–36.
ANNOTATED BIBLIOGRAPHY
Anderson, A.; Christopher, D.H.; Woodhouse, R.N. Analysis of the anti-coccidial drug, halofuginone,
in chicken feed using gas-liquid chromatography and high-performance liquid chromatography,
J.Chromatogr., 1979, 168, 471–480.
Anderson, A.; Goodall, E.; Bliss, G.W.; Woodhouse, R.N. Analysis of the anti-coccidial drug, halofuginone,
in chicken tissue and chicken feed using high-performance liquid chromatography, J.Chromatogr.,
1981, 212, 347–355.
Holland, D.C.; Manns, R.K.; Roybal, J.E.; Hurlbut, J.A.; Long, A.R. Liquid chromatographic determination of the anticoccidial drug halofuginone hydrobromide in eggs, J.AOAC Int., 1995, 78, 37–40.
Kinabo, L.D.B.; McKellar, Q.A.; Murray, M. Determination of halofuginone in bovine plasma by competing-ion high performance liquid chromatography after solid phase extraction, Biomed.Chromatogr.,
1989, 3, 136–138.
Mortier, L.; Daeseleire, E.; Delahaut, P. Simultaneous detection of five coccidiostats in eggs by liquid
chromatography-tandem mass spectrometry, Anal.Chim.Acta, 2003, 483, 27–37. [diclazuril; dimetridazole; halofuginone; nicarbazin; robenidine]
Tillier, C.; Cagniant, E.; Devaux, P. Determination of halofuginone in poultry feeds by high-performance
liquid chromatography, J.Chromatogr., 1988, 441, 406–416.
Yamamoto, Y.; Kondo, F. Determination of halofuginone and amprolium in chicken muscle and egg by
liquid chromatography, J.AOAC Int., 2001, 84, 43–46. [SPE]
310
Hetastarch
Hetastarch
CAS Registry No: 9004-62-0
Merck Index: 13, 4692
OR′
O
OR
H
O
OH
OR
n
R or R′ = H or CH2CH2OH
SAMPLE
Matrix: blood, lymph
Sample preparation: Mix 2 mL plasma or lymph with 150 µL 85% trichloroacetic acid,
centrifuge. Add 100 µL Tris buffer and 25µL phenol red to 1.5 mL of the supernatant,
neutralize excess acid with 5–25 µL 5 M KOH, inject an aliquot of the supernatant
HPLC VARIABLES
Column: SEC-60 (TosoHaas) + SEC-50 (TosoHaas) + SEC-10 (Bio-Rad)
Mobile phase: pH 4.0 acetate buffer
Detector: Refractive Index
KEY WORDS
plasma; sheep
REFERENCE
Korent, V.A.; Conhaim, R.L.; McGrath, A.M.; DeAngeles, D.A.; Harms, B.A. Molecular distribution of
hetastarch in plasma and lung lymph of unanesthetized sheep, Am.J.Respir.Crit.Care Med., 1997, 155,
1302–1308.
Hydroquinone
311
Hydroquinone
Molecular formula: C6 H6 O2
HO
OH
Molecular weight: 110.11
CAS Registry No: 123-31-9
Merck Index: 13, 4833
SAMPLE
Matrix: blood
Sample preparation: Filter (Amicon Centrifree) whole blood while centrifuging at 3◦
at 3000 g for 1 h, inject a 100 µL aliquot of the ultrafiltrate.
HPLC VARIABLES
Column: 170 × 4.6 5 µm Supelcosil LC-18
Mobile phase: 50 mM pH 4.5 formate buffer
Flow rate: 1
Injection volume: 100
Detector: Radioactivity (14 C); UV (wavelength not specified, other papers have recommended 220–280)
CHROMATOGRAM
Retention time: 7
OTHER SUBSTANCES
Extracted: hydroquinone glucuronide (3), hydroquinone sulfate (5)
KEY WORDS
ultrafiltrate; whole blood
REFERENCE
Deisinger, P.J.; English, J.C. Bioavailability and metabolism of hydroquinone after intratracheal instillation in male rats, Drug Metab.Dispos., 1999, 27, 442–448.
312
Hygromycin B
Hygromycin B
OH
OH
OH
O
Molecular formula: C20 H37 N3 O13
Molecular weight: 527.52
CAS Registry No: 31282-04-9
Merck Index: 13, 4878
NH2
HO
OH O
H3C
OH
O
O
N
H
NH2
HO
O
HO
SAMPLE
Matrix: tissue
Sample preparation: Mix 500 mg homogenized kidney with 2 g 40 µm Bondesil
preparative-grade end-capped cyanopropyl bulk packing material (Analytichem), blend
with mortar and pestle for 2 min. (For a higher degree of purification, use 3 g Bondesil
CN and blend for 3 min.) Place in a chromatography column, wash with 3 mL hexane,
wash with 5 mL ethyl acetate, wash with 5 mL MeOH, wash with 5 mL MeOH:water
50:50, elute with 1 mL water and then with 8 mL 100 mM formic acid, filter (0.45 µm)
a 4.5 mL aliquot of the eluate. Concentrate 4 mL of the filtrate to 75 µL under reduced
pressure, add 7.5 µL 10% pentafluoropropionic acid in water, vortex, filter (0.20 µm),
inject a 22 µL aliquot of the filtrate.
HPLC VARIABLES
Guard column: 10 × 2 3 µm YMCbasic
Column: 100 × 2 3 µm YMCbasic
Mobile phase: Gradient. A was MeCN:water 60:40 containing 20 mM pentafluoropropionic acid. B was MeCN:water 5:95 containing 20 mM pentafluoropropionic acid. A:B
0:100 for 5.1 min, to 32:68 over 0.1 min, maintain at 32:68 for 13.3 min, to 100:0
over 0.1 min, maintain at 100:0 for 5.5 min, return to initial conditions over 0.1 min,
re-equilibrate for 21.8 min.
Flow rate: 0.2 for 18.5 min, 0.22 for 0.1 min, 0.25 for 22.3 min, then 0.2
Injection volume: 22
Detector: MS, PE Sciex API III triple quadrupole, atmospheric pressure ion source,
positive ion, laboratory-constructed ionspray interface (details provided), nebulizer gas
nitrogen at 67 psi, curtain gas nitrogen, collision gas argon, 40 µL/min entered the
detector, m/z 265–177 or 528–352–177
CHROMATOGRAM
Retention time: 5.5
Limit of detection: 0.02 ppm
OTHER SUBSTANCES
Extracted: dihydrostreptomycin (7), gentamicin (13.5–15), neomycin (16), spectinomycin
(5), streptomycin (6.5), tobramycin (12)
KEY WORDS
cow; kidney; MSPD
REFERENCE
McLaughlin, L.G.; Henion, J.D.; Kijak, P.J. Multi-residue confirmation of aminoglycoside antibiotics
and bovine kidney by ion spray high-performance liquid chromatography/tandem mass spectrometry,
Biol.Mass Spectrom., 1994, 23, 417–429.
Iloprost
HOOC
Molecular formula: C22 H32 O4
Molecular weight: 360.49
CAS Registry No: 78919-13-8
Merck Index: 13, 4925
CH3
OH
CH3
OH
SAMPLE
Matrix: blood
Sample preparation: Mix 200 µL plasma with 200 µL 100 ppm 2-naphthoic acid
and 300 µL MeCN, shake well, centrifuge for 10 min, inject a 20 µL aliquot of the
supernatant.
HPLC VARIABLES
Column: 250 × 4.6 5 µm Zorbax C8
Mobile phase: MeCN:MeOH:20 mM pH 3.0 potassium phosphate buffer 30:24:46
Flow rate: 1.7
Injection volume: 20
Detector: UV 210; radioactivity (3 H)
CHROMATOGRAM
Retention time: 14.7, 15.9 (diastereomers)
Internal standard: 2-naphthoic acid (6)
Limit of quantitation: 500 ng (UV), 42 pg (radioactivity detector)
OTHER SUBSTANCES
Extracted: misoprostol (LOQ 1 µg (UV), 12 pg (radioactivity detector)) (18.5, diastereomers not resolved)
KEY WORDS
plasma; rat
REFERENCE
Womack, I.M.; Lee, A.S.; Kamath, B.; Agrawal, K.C.; Kishore, V. A high performance liquid radiochromatographic assay for the simultaneous analysis of iloprost and misoprostol, Prostaglandins, 1996,
52, 249–259.
HPLC Methods for Recently Approved Pharmaceuticals. by George Lunn
Copyright 2005 John Wiley & Sons, Ltd. ISBN: 0-471-66941-5
313
314
Imatinib
Imatinib
Molecular formula: C29 H31 N7 O
N
H3C
N
Molecular weight: 493.60
CAS Registry No: 152459-95-5,
220127-57-1 (mesylate)
Merck Index: 13, 4926
O
H
H
N
N
N
N
N
CH3
SAMPLE
Matrix: blood
Sample preparation: Mix 200 µL plasma with 10 µL 10 µg/mL IS in MeOH, add 1 mL
MeCN, vortex briefly, centrifuge at 12000 g for 5 min. Evaporate a 1 mL aliquot of the
supernatant to dryness under a stream of nitrogen at 27◦ , reconstitute the residue with
100 µL MeOH:water 20:80, vortex briefly, inject a 3 µL aliquot.
HPLC VARIABLES
Column: 50 × 4.6 5 µm Phenomenex Luna C18(2)
Mobile phase: Gradient. MeOH:water:formic acid from 20:80:0.1 to 60:40:0.1 over 6 min,
to 100:0:0.1 over 1 min, maintain at 100:0:0.1 for 2 min, return to initial conditions over
1 min, re-equilibrate for 4 min.
Flow rate: 1 for 6 min, to 2 over 1 min, maintain at 2 for 6 min, to 1 over 0.1 min,
maintain at 1 for 0.9 min
Injection volume: 3
Detector: MS, ThermoFinnigan aQa, electrospray, positive single-ion, 10% of column
effluent entered detector, insert probe 250◦ , ionspray 5000 V, orifice 10 V, nitrogen
75 psi, m/z 493.7
CHROMATOGRAM
Retention time: 4.0
Internal standard: d8 -imatinib (m/z 501.7) (3.9)
Limit of quantitation: 30 ng/mL
OTHER SUBSTANCES
Extracted: CGP 74588 (metabolite) (m/z 479.7) (3.7)
KEY WORDS
pharmacokinetics; plasma
REFERENCE
Parise, R.A.; Ramanathan, R.K.; Hayes, M.J.; Egorin, M.J. Liquid chromatographic-mass spectrometric
assay for quantitation of imatinib and its main metabolite (CGP 74588) in plasma, J.Chromatogr.B,
2003, 791, 39–44.
SAMPLE
Matrix: formulations
Sample preparation: Dissolve capsules in MeOH:water 25:75 containing 40 µg/mL
acetaminophen so as to achieve an imatinib concentration of 2 mg/mL, inject an aliquot.
HPLC VARIABLES
Column: 150 × 4.6 5 µm XTerra C18
Column temperature: 25
Mobile phase: MeOH:water:triethylamine 25:74:1 (Mix 740 mL water with 10 mL triethylamine, adjust pH to 2.4 with 85% phosphoric acid, add 250 mL MeOH, adjust pH
to 2.6 with 85% phosphoric acid, if necessary.)
Imatinib
315
Flow rate: 1
Injection volume: 20
Detector: UV 267
CHROMATOGRAM
Retention time: 7.7
Internal standard: acetaminophen (2.9)
Limit of detection: 10 ng/mL
Limit of quantitation: 100 ng/mL
OTHER SUBSTANCES
Simultaneous: impurity STI 509-00
KEY WORDS
capsules; robust
REFERENCE
Ivanovic, D.; Medenica, M.; Jancic, B.; Malenovic, A. Reversed-phase liquid chromatography analysis of
imatinib mesylate and impurity product in Glivec capsules, J.Chromatogr.B, 2004, 800, 253–258.
ANNOTATED BIBLIOGRAPHY
Bakhtiar, R.; Khemani, L.; Hayes, M.; Bedman, T.; Tse, F. Quantification of the anti-leukemia drug
STI571 (Gleevec) and its metabolite (CGP 74588) in monkey plasma using a semi-automated solid phase
extraction procedure and liquid chromatography-tandem mass spectrometry, J.Pharm.Biomed.Anal.,
2002, 28, 1183–1194. [SPE; LOQ 1 ng/mL]
Bakhtiar, R.; Lohne, J.; Ramos, L.; Khemani, L.; Hayes, M.; Tse, F. High-throughput quantification of
the anti-leukemia drug STI571 (Gleevec) and its main metabolite (CGP 74588) in human plasma
using liquid chromatography-tandem mass spectrometry, J.Chromatogr.B, 2002, 768, 325–340. [LOQ
4 ng/mL]
Hamada, A.; Miyano, H.; Watanabe, H.; Saito, H. Interaction of imatinib mesilate with human
P-glycoprotein, J.Pharmacol.Exp.Ther., 2003, 307, 824–828. [cell cultures; UV detection]
Vivekanand, V.V.; Rao, D.S.; Vaidyanathan, G.; Sekhar, N.M.; Kelkar, S.A.; Puranik, P.R. A validated
LC method for imatinib mesylate, J.Pharm.Biomed.Anal., 2003, 33, 879–889. [bulk; UV detection]
316
Imidocarb
Imidocarb
Molecular formula: C19 H20 N6 O
Molecular weight: 348.40
CAS Registry No: 27885-92-3
Merck Index: 13, 4938
O
H
N
N
H
N
N
H
H
N
N
SAMPLE
Matrix: blood
Sample preparation: Condition a 100 mg Supelclean LC-18 SPE cartridge with 3 mL
MeOH and 2 mL water. Vortex 1 mL plasma with 3 mL 50 mM disodium EDTA solution
for 30 s, add to the SPE cartridge, wash with 2 mL MeOH:water 10:90, dry under vacuum
for 5 min, elute with 3 mL MeCN:2% acetic acid in water containing 25 mM sodium
octanesulfonate 90:10. Evaporate the eluate to dryness under reduced pressure at 40◦ ,
reconstitute the residue with 350 µL mobile phase, vortex, filter (0.45 µm), inject a
50 µL aliquot.
HPLC VARIABLES
Column: 100 × 8 4 µm Nova Pak C18 radial compression
Mobile phase: MeCN:buffer 30:70 (The buffer was 5 mM sodium octanesulfonate containing 0.1% triethylamine adjusted to pH 3.2 with glacial acetic acid.)
Flow rate: 1.5
Detector: UV 254
CHROMATOGRAM
Retention time: 5.4
OTHER SUBSTANCES
Extracted: diminazene (UV 370) (3.7)
KEY WORDS
cow; plasma; SPE; imidocarb is IS in original paper
REFERENCE
Gummow, B.; Du Preez, J.L.; Swan, G.E. Paired-ion extraction and high-performance liquid chromatographic determination of diminazene in cattle plasma: a modified method, Onderstepoort J.Vet.Res.,
1995, 62, 1–4.
SAMPLE
Matrix: tissue
Sample preparation: Homogenize (Ultra-Turrax) 10 g thinly sliced kidney with 2 mL
1 M sodium carbonate and 25 mL acetone for 2 min, sonicate for 3 min, centrifuge
at 4 200 g for 5 min, extract the residue again with 2 mL 1 M sodium carbonate,
25 mL acetone, and 8 mL water. Combine the supernatants, add 50 mL chloroform
(Caution! Chloroform is a carcinogen!), add 20 mL saturated aqueous NaCl, add 2 mL
40% NaOH, shake for 1 min. Filter the lower organic layer through anhydrous sodium
sulfate and phase-separating filter paper (Whatman PS-1), evaporate to dryness under
reduced pressure at 50◦ , reconstitute with three 3 mL portions of MeOH:10 mM pH 7
sodium acetate containing 10 mM sodium trifluoroacetate 80:20, centrifuge at 1860 g for
5 min, add the supernatant to a pre-washed (not otherwise described) Bond Elut CBA
(carboxylic acid, weak cation exchange) SPE cartridge. Wash the residue with 1 mL
MeOH:10 mM pH 7 sodium acetate containing 10 mM sodium trifluoroacetate 80:20 and
add the supernatant to the SPE cartridge. Wash the SPE cartridge with 4 mL MeOH,
elute with 5 mL MeOH:trifluoroacetic acid 95:5. Evaporate the eluate to dryness under
Imidocarb
317
reduced pressure at 50◦ , reconstitute the residue with 500 µL mobile phase, vortex for
15 s, sonicate for 3 min, centrifuge at 1860 g for 5 min, filter (0.45 µm), inject a 50 µL
aliquot. (Use silanized glassware.)
HPLC VARIABLES
Column: 100 × 4.6 3 µm Spherisorb S3W-C18
Mobile phase: Gradient. A was MeCN:10 mM pH 7 sodium acetate containing 10 mM
sodium trifluoroacetate 15:85. B was MeCN:10 mM pH 2 sodium acetate containing
10 mM trifluoroacetic acid and 10 mM tetramethylammonium chloride 10:90. A:B 100:0
for 5 min, to 0:100 (step gradient), maintain at 0:100 for 15 min, return to initial
conditions (step gradient), re-equilibrate for 10 min.
Flow rate: 1
Injection volume: 50
Detector: UV 260
CHROMATOGRAM
Retention time: 14
Limit of detection: 1–2 ng/g
KEY WORDS
cow; kidney; SPE
REFERENCE
Tarbin, J.A.; Shearer, G. High-performance liquid chromatographic determination of imidocarb in cattle
kidney with cation-exchange clean-up, J.Chromatogr., 1992, 577, 376–381.
ANNOTATED BIBLIOGRAPHY
Coldham, N.G.; Moore, A.S.; Sivapathasundaram, S.; Sauer, M.J. Imidocarb depletion from cattle liver
and mechanism of retention in isolated bovine hepatocytes, Analyst, 1994, 119, 2549–2552. [LOD
74 ng/g]
Coldham, N.G.; Moore, A.S.; Dave, M.; Graham, P.J.; Sivapathasundaram, S.; Lake, B.G.; Sauer, M.J.
Imidocarb residues in edible bovine tissues and in vitro assessment of imidocarb metabolism and
cytotoxicity, Drug Metab.Dispos., 1995, 23, 501–505.
318
Iobenguane
Iobenguane
Molecular formula: C8 H10 IN3
H
I
N
NH2
NH
Molecular weight: 275.09
CAS Registry No: 80663-95-2
Merck Index: 13, 5028
SAMPLE
Matrix: blood, urine
Sample preparation: Inject a 200 µL aliquot of serum or urine onto column A and
elute to waste with mobile phase A; after 5 min, backflush the contents of column A
onto column B with mobile phase B, monitor the effluent from column B. After 15 min,
re-equilibrate column A with mobile phase A for 5 min. After 5 determinations, flush
column A with MeOH.
HPLC VARIABLES
Column: A 30 × 4 5 µm LiChrosorb C8; B 250 × 4 5 µm LiChrosorb C8
Mobile phase: A 60 mM pH 6.0 phosphate buffer; B MeCN:60 mM pH 6.0 phosphate
buffer containing 1% sodium heptanesulfonate 35:65
Flow rate: 1
Injection volume: 200
Detector: UV 232 (urine), UV 254 (serum)
CHROMATOGRAM
Retention time: 14
Limit of detection: 50 ng/mL (S/N = 3)
OTHER SUBSTANCES
Extracted: metoclopramide (13), sulfamethoxazole/trimethoprim (?) (11)
Noninterfering: acetaminophen, diazepam, dimenhydrinate, etoposide, prolonium
iodide
KEY WORDS
column-switching; serum
REFERENCE
Schwabe, D.; Rohrbach, E.; Köhl, U. Determination of m-iodobenzylguanidine in serum and urine by
high-performance liquid chromatography, J.Chromatogr., 1989, 487, 177–182.
SAMPLE
Matrix: blood
Sample preparation: Plasma. Condition a 1 mL 100 mg Bakerbond cyano SPE cartridge with 1 mL MeOH and 2 mL water. Add 500 µL plasma to the SPE cartridge,
wash with 2 mL water, wash with 2 mL MeOH, elute with 1 mL 100 mM HCl in MeOH.
Evaporate the eluate to dryness under a stream of nitrogen at 60◦ , reconstitute the
residue with 200 µL mobile phase, vortex for 15 s, centrifuge at 9 500 g for 2 min,
inject a 100 µL aliquot. Whole blood. Condition a 1 mL 100 mg Bakerbond cyano SPE
cartridge with 1 mL MeOH and 2 mL water, retaining 0.5 mL water above the sorbent.
Place a 3 mL Bakerbond SPE filtration column above the SPE cartridge. Mix 500 µL
whole blood with 1.5 mL water, add to the filtration column, wash with 1 mL water,
remove the filtration column. Wash the SPE cartridge with 1 mL water, wash with 2 mL
MeOH, elute with 1 mL 100 mM HCl in MeOH. Evaporate the eluate to dryness under
a stream of nitrogen at 60◦ , reconstitute the residue with 200 µL mobile phase, vortex
for 15 s, centrifuge at 9 500 g for 2 min, inject a 100 µL aliquot.
Iobenguane
319
HPLC VARIABLES
Column: 300 × 3.9 10 µm µBondapak C18
Mobile phase: MeCN:25 mM pH 4.0 ammonium phosphate buffer 20:80
Flow rate: 1
Injection volume: 100
Detector: UV 254
CHROMATOGRAM
Limit of detection: 75 ng/mL
Limit of quantitation: 100 ng/mL
KEY WORDS
plasma; SPE; whole blood
REFERENCE
Wafelman, A.R.; Konings, M.C.P.; Rosing, H.; Hoefnagel, C.A.; Taal, B.G.; Maes, R.A.A.; Beijnen, J.H.
High-performance liquid chromatographic determination of metaiodobenzylguanidine in whole blood
and plasma of cancer patients, J.Pharm.Biomed.Anal., 1994, 12, 1173–1179.
ANNOTATED BIBLIOGRAPHY
Sparidans, R.W.; Taal, B.G.; Beijnen, J.H. Bioanalysis of m-iodobenzylguanidine in plasma by highperformance liquid chromatography after derivatization with benzoin, J.Chromatogr.B, 1999, 730,
193–199. [SPE; fluorescence detection; LOQ 2 ng/mL]
Wafelman, A.R.; Nortier, Y.L.M.; Rosing, H.; Underberg, W.J.M.; Maes, R.A.A.; Beijnen, J.H. Highperformance liquid chromatographic determination of m-iodobenzylguanidine in urine of cancer
patients, J.Chromatogr., 1993, 622, 71–77. [SPE; LOQ 200 ng/mL]
320
Iodixanol
Iodixanol
OH
Molecular
formula: C35 H44 I6 N6 O15
Molecular weight: 1550.18
CAS Registry No: 92339-11-2
Merck Index: 13, 5045
H
HO
OH
HO
H
N
H
O
I
O
I
N
I
N
O
I
H3C
OH
N
OH
I
N
N
O
OH
O
H
I
OH
OH
O
CH3
SAMPLE
Matrix: blood
Sample preparation: Dialyze 110 µL plasma against 175 µL water in the recipient
channel using a Cuprophane cellulose dialysis membrane (molecular weight cut-off 15
000), pump 4 mL water in a pulsed fashion through the recipient channel, pass the
water through column A, elute the contents of column A onto column B using the mobile
phase, monitor the effluent from column B. (Purge the system with 0.01% Triton X-100
in water.) (ASTED XL system)
HPLC VARIABLES
Column: A 5 × 1.6 10 µm Hypersil ODS; B 15 × 3.2 7 µm Brownlee RP-18 Newguard +
250 × 4.6 5 µm Brownlee OD-5A, Spheri-5, RP-18
Mobile phase: MeCN:water 9:91
Flow rate: 1
Detector: UV 244
CHROMATOGRAM
Retention time: 7 (exo isomer), 9 (endo isomer)
Limit of detection: 19–130 ng/mL
Limit of quantitation: 52–340 ng/mL
KEY WORDS
dialysis; monkey; human; plasma; rat
REFERENCE
Jacobsen, P.B. On-line dialysis and quantitative high-performance liquid chromatography analysis of
iodixanol in human, rat and monkey plasma, J.Chromatogr.B, 2000, 749, 135–142.
SAMPLE
Matrix: urine
Sample preparation: Dilute urine 1:20 with water, inject an aliquot.
HPLC VARIABLES
Column: 250 × 4.6 5 µm C18 (Becton Dickinson)
Mobile phase: Gradient. MeCN:water 8:92 for 5 min, to 16:84 (step gradient), maintain
at 16:84 for 10 min, return to initial conditions (step gradient), re-equilibrate for 5 min.
Detector: UV 250
CHROMATOGRAM
Retention time: 11.6 (exo), 13.8 (endo)
Limit of detection: 640 ng/mL
Iodixanol
321
REFERENCE
Kerr, S.W.; Wolyniec, W.W.; Filipovic, Z.; Nodop, S.G.; Braza, F.; Winquist, R.J.; Noonan, T.C. Repeated
measurement of intestinal permeability as an assessment of colitis severity in HLA-B27 transgenic
rats, J.Pharmacol.Exp.Ther., 1999, 291, 903–910.
ANNOTATED BIBLIOGRAPHY
Molander, P.; Theodorsen, M.; Lundanes, E.; Soerenssen, D.M.; Greibrokk, T. Temperature effects on
packed-capillary liquid chromatography of the X-ray contrast agent iodixanol, J.Chromatogr.Sci.,
2000, 38, 157–161.
Nomura, H.; Teshima, E.; Hakusui, H. Simple isocratic high-performance liquid chromatographic method
for measurement of iodixanol in human plasma, J.Chromatogr., 1991, 572, 333–338. [size exclusion
chromatography]
322
Iopanoic acid
Iopanoic acid
Molecular formula: C11 H12 I3 NO2
Molecular weight: 570.93
CAS Registry No: 96-83-3
Merck Index: 13, 5074
I
CH3
I
I
COOH
NH2
SAMPLE
Matrix: blood
Sample preparation: Mix 50 µL plasma with 50 µL 4 mM IS in MeOH, centrifuge at
14 000 rpm for 4 min, inject a 1–5 µL aliquot of the supernatant.
HPLC VARIABLES
Guard column: Present but not specified
Column: 150 × 4.6 Hisep shielded hydrophobic phase
Mobile phase: Gradient. MeOH:50 mM pH 3.4 phosphate buffer from 12:88 to 70:30
over 2 min, maintain at 70:30 for 30 min, return to initial conditions over 5 min.
Flow rate: 1.5
Injection volume: 1–5
Detector: UV 231, UV 254
CHROMATOGRAM
Retention time: 7.1
Internal standard: 2,4,6-triiodobenzoic acid (23.5)
Limit of detection: 6.25 ng
KEY WORDS
dog; plasma
REFERENCE
Andeejani, A.M.; Hughes, H.; Feuchuk, D.M.; Aboul-Enein, H.Y. Rapid assay of iopanoic acid in dog
plasma using a Hisep column, Biomed.Chromatogr., 1994, 8, 26–28.
SAMPLE
Matrix: blood, tissue
Sample preparation: Blood. Shake 500 µL whole blood with 5 mL MeOH for 30 s,
centrifuge at 2000 rpm for 2 min, inject a 20 µL aliquot of the supernatant. Liver.
Shake 1 g liver homogenate with 10 mL MeOH for 30 s, centrifuge at 2000 rpm for
2 min, inject a 20 µL aliquot of the supernatant.
HPLC VARIABLES
Column: 300 × 4 10 µm LiChrosorb RP-18
Mobile phase: MeOH:water 60:40 containing 5 mM tetrabutylammonium phosphate
Flow rate: 2
Injection volume: 20
Detector: UV 232
CHROMATOGRAM
Retention time: 7.15
Limit of detection: 1 µg/mL
OTHER SUBSTANCES
Extracted: diatrizoic acid (UV 242) (1.30), iothalamic acid (UV 242) (1.30), ioxaglic acid
(UV 242) (1.33)
Iopanoic acid
323
KEY WORDS
whole blood; liver
REFERENCE
Crowley, R.; Kacprzak, J. The determination of commonly used iodinated contrast media in postmortem
samples using HPLC and TLC, J.Anal.Toxicol., 1986, 10, 53–55.
324
Iopromide
Iopromide
CH3
O
Molecular formula: C18 H24 I3 N3 O8
Molecular weight: 791.11
CAS Registry No: 73334-07-3
Merck Index: 13, 5078
O
H3CO
OH
I
I
H
N
N
H
OH
N
I
OH
OH
O
SAMPLE
Matrix: blood
Sample preparation: Mix plasma with 4 vol 5% perchloric acid, centrifuge, inject a
20 µL aliquot of the supernatant.
HPLC VARIABLES
Column: 250 × 4 LiChrosorb C18
Mobile phase: MeCN:water 4:96 adjusted to pH 2.5 with phosphoric acid
Flow rate: 1.5
Injection volume: 20
Detector: UV 254
CHROMATOGRAM
Retention time: 11, 13 (isomers)
Limit of detection: 500 ng/mL
OTHER SUBSTANCES
Extracted: iohexol (4.5, 5.5 (isomers))
KEY WORDS
plasma
REFERENCE
Gaspari, F.; Perico, N.; Ruggenenti, P.; Mosconi, L.; Amuchastegui, C.S.; Guerini, E.; Daina, E.; Remuzzi,
G. Plasma clearance of nonradioactive iohexol as a measure of glomerular filtration rate, J.Am.Soc.
Nephrol., 1995, 6, 257–263.
SAMPLE
Matrix: water
Sample preparation: Condition a 3 mL 200 mg LiChrolut EN SPE cartridge with
water, 9 mL MeOH, and 9 mL water adjusted to pH 3.5 with nitric acid. Condition a
3 mL 250 mg LiChrolut Envi-Carb SPE cartridge (Supelco) with water, 9 mL MeOH,
and 9 mL water adjusted to pH 2 with nitric acid. Adjust pH of 500–1000 mL water to
3.5 with nitric acid, pass through the EN cartridge at 200 mL/h. Adjust the pH of the
eluate to 2 with nitric acid and pass through the Envi-Carb SPE cartridge at 300 mL/h.
Dry cartridges under vacuum for 1 min. Elute the EN cartridge with 6 mL MeOH. Elute
the Envi-Carb cartridge with 8 mL MeCN:water 50:50 containing a trace of ammonium
acetate in the reverse direction. Combine the eluates and evaporate to dryness under a
stream of nitrogen, reconstitute the residue with 500 µL mobile phase A, inject a 10 µL
aliquot.
HPLC VARIABLES
Column: 150 × 2 3 µm Luna C18(2)
Column temperature: 45
Iopromide
325
Mobile phase: Gradient. A was 0.05% trifluoroacetic acid in water. B was 0.05%
trifluoroacetic acid in MeOH. A:B from 100:0 to 95:5 over 10 min, to 75:25 over 15 min,
return to initial conditions over 2 min, re-equilibrate for 5 min.
Flow rate: 0.25
Injection volume: 10
Detector: MS, Micromass Quattro-LC, electrospray, positive ion mode, drying gas nitrogen, nebulizing gas nitrogen, collision gas argon, m/z 792–573; UV 242
CHROMATOGRAM
Retention time: 21.5, 22.5 (isomers)
Limit of detection: ca. 50 pg/mL
OTHER SUBSTANCES
Extracted: diatrizoate (m/z 615–361) (11), iohexol (m/z 822–803) (16), iotrolan (m/z 814)
(18)
KEY WORDS
SPE
REFERENCE
Putschew, A.; Schittko, S.; Jekel, M. Quantification of triiodinated benzene derivatives and X-ray contrast media in water samples by liquid chromatography-electrospray tandem mass spectrometry,
J.Chromatogr.A, 2001, 930, 127–134.
ANNOTATED BIBLIOGRAPHY
Putschew, A.; Wischnack, S.; Jekel, M. Occurrence of triiodinated X-ray contrast agents in the aquatic
environment, Sci.Total Environ., 2000, 255, 129–134. [iopromide; diatrizoate; iotrolan; iotroxin acid;
iotroxin]
Sacher, F.; Lange, F.T.; Brauch, H.-J.; Blankenhorn, I. Pharmaceuticals in groundwaters. Analytical
methods and results of a monitoring program in Baden-Württemberg, Germany, J.Chromatogr.A, 2001,
938, 199–210. [amidotrizoic acid; amoxicillin; anhydro-erythromycin; atenolol; betaxolol; bezafibrate;
bisoprolol; carbamazepine; chloramphenicol; clarithromycin; clenbuterol; clofibric acid; cloxacillin;
cyclophosphamide; dapsone; diazepam; diclofenac; dicloxacillin; dimethylaminophenazone; erythromycin; etofibrate; fenofibrate; fenoprofen; furazolidone; gemfibrozil; ibuprofen; ifosfamide; indomethacin; iomeprol; iopamidol; iopromide; ketoprofen; metoprolol; metronidazole; nafcillin; naproxen;
oleandomycin; oxacillin; penicillin G; penicillin V; pentoxifylline; phenazone; pindolol; propranolol;
propyphenazone; ronidazole; roxithromycin; albuterol; simvastatin; sotalol; spiramycin; sulfadiazine;
sulfadimidine; sulfamerazine; sulfamethoxazole; terbutaline; trimethoprim; tylosin; virginiamycin;
sulfamethazine]
Vanderford, B.J.; Pearson, R.A.; Rexing, D.J.; Snyder, S.A. Analysis of endocrine disruptors, pharmaceuticals, and personal care products in water using liquid chromatography/tandem mass spectrometry, Anal.Chem., 2003, 75, 6265–6274. [hydrocodone; trimethoprim; acetaminophen; caffeine;
erythromycin; sulfamethoxazole; fluoxetine; pentoxifylline; meprobamate; phenytoin; carbamazepine;
DEET; diazepam; oxybenzone; progesterone; iopromide; naproxen; ibuprofen; diclofenac; triclosan;
gemfibrozil; ethinyl estradiol; estradiol; testosterone; SPE]
326
Ioversol
Ioversol
H
O
Molecular formula: C18 H24 I3 N3 O9
Molecular weight: 807.11
CAS Registry No: 87771-40-2
Merck Index: 13, 5085
O
HO
HO
OH
N
OH
I
I
H
N
N
I
OH
OH
O
SAMPLE
Matrix: blood, CSF, tissue, urine
Sample preparation: Blood. Mix plasma or serum with 1 vol of MeCN:EtOH:water
60:38.4:1.6 or 20% trifluoroacetic acid at 4◦ , let stand overnight at 4◦ , centrifuge at 13
000 g at 4◦ for 10 min, inject an aliquot of the supernatant. Brain. Homogenize (Janke
& Kunkel Ikawerk homogenizer) with an equal volume of saline at 20 000 rpm at room
temperature for 3 min, centrifuge at 12 000 g at 4◦ for 30 min, filter (Centrisart 1)
while centrifuging at 4◦ , inject an aliquot of the ultrafiltrate. CSF. Dilute 50–200-fold
with water, inject an aliquot. Urine. Dilute with 4 vol of water. Mix with 1 vol of
MeCN:EtOH:water 60:38.4:1.6 at 4◦ , let stand overnight at 4◦ , centrifuge at 13 000 g
at 4◦ for 10 min, inject an aliquot of the supernatant. Alternatively, filter (Centrisart 1
with 5000 Da cut-off) while centrifuging at 4◦ , inject an aliquot of the ultrafiltrate.
HPLC VARIABLES
Column: 250 × 4.6 Spheri-5 RP-18
Mobile phase: Gradient. MeCN:water from 1:99 to 30:70 over 20 min
Flow rate: 1
Injection volume: 10
Detector: UV 220–280
CHROMATOGRAM
Retention time: 9.9
OTHER SUBSTANCES
Extracted: iohexol (11.1 (endo), 11.3 (exo))
Noninterfering: diatrizoate, iothalamate
KEY WORDS
brain; plasma; serum; ultrafiltrate
REFERENCE
Jacobsen, P.B. High performance liquid chromatography with multiwavelength detection: a technique for identification of iodinated x-ray contrast agents in human body fluids and brain tissue,
Am.J.Neuroradiol., 1992, 13, 1521–1525.
Ipratropium bromide
Ipratropium bromide
Molecular formula: C20 H30 BrNO3
Molecular weight: 412.37
CAS Registry No: 22254-24-6
Merck Index: 13, 5092
327
CH3
H3C
N+
CH3
Br−
O
OH
O
SAMPLE
Matrix: blood
Sample preparation: Inject 100 µL plasma onto column A and elute to waste with
mobile phase A; after 10 min, backflush the contents of column A onto column B with
mobile phase B; after 2 min, remove column A from the circuit, elute column B with
mobile phase B, monitor the effluent from column B, re-equilibrate column A with
mobile phase A for 5 min.
HPLC VARIABLES
Column: A 25 × 4 25 µm LiChrospher 60 XDS (SO3 /diol); B 250 × 4 5 µm LiChrospher
60 RP-Select B
Column temperature: 25 ± 0.1
Mobile phase: A MeOH:2 mM lithium perchlorate adjusted to pH 3.0 with 1 M perchloric acid 3:97; B MeCN:50 mM pH 3.0 phosphate buffer containing 0.5 mM sodium
butanesulfonate 20:80
Flow rate: 1
Injection volume: 100
Detector: UV 220
OTHER SUBSTANCES
Extracted: atropine (16)
KEY WORDS
column-switching; plasma
REFERENCE
Chiap, P.; Rbeida, O.; Christiaens, B.; Hubert, P.; Lubda, D.; Boos, K.-S.; Crommen, J. Use of a novel
cation-exchange restricted-access material for automated sample clean-up prior to the determination
of basic drugs in plasma by liquid chromatography, J.Chromatogr.A, 2002, 975, 145–155.
328
Ipriflavone
Ipriflavone
O
CH3
Molecular formula: C18 H16 O3
Molecular weight: 280.32
CAS Registry No: 35212-22-7
Merck Index: 13, 5093
H3C
O
O
SAMPLE
Matrix: blood, tissue, urine
Sample preparation: Homogenize (Ultra-Turrax T25) tissue with 4 vol of water. Vortex
50 µL plasma, urine, or tissue homogenate with 125 µL 1 µg/mL IS in MeCN, centrifuge
at 14 000 g for 1 min, inject a 50 µL aliquot of the supernatant.
HPLC VARIABLES
Column: 250 × 4.6 10 µm LiChrosorb RP-18
Mobile phase: MeCN:MeOH:50 mM pH 3 acetate buffer 35:25:40
Flow rate: 1.5
Injection volume: 50
Detector: UV 254
CHROMATOGRAM
Retention time: 12
Internal standard: testosterone (6)
Limit of detection: 20 ng/mL (plasma), 50 ng/mL (tissue), 100 ng/mL (urine)
KEY WORDS
brain; fat; heart; intestine; kidney; liver; lung; muscle; plasma; rat; spleen; stomach
REFERENCE
Kim, S.H.; Lee, J.S.; Lee, M.G. Determination of a isoflavone derivative, ipriflavone, and its metabolites,
M1 and M5, in rat plasma, urine, and tissue homogenate by high-performance liquid chromatography,
Res.Commun.Mol.Pathol.Pharmacol., 1997, 98, 313–324.
329
Isoflupredone
Isoflupredone
O
OH
CH3
Molecular formula: C21 H27 FO5
Molecular weight: 378.43
CAS Registry No: 338-95-4, 338-98-7 (21-acetate)
Merck Index: 13, 5192
OH
CH3
HO
F
H
H
O
SAMPLE
Matrix: blood, synovial fluid
Sample preparation: Mix 500 µL plasma or synovial fluid with 4 mL pH 4 potassium
phosphate buffer and 5 mL dichloromethane, centrifuge at 3000 g. Evaporate the organic
layer to dryness, reconstitute the residue with 1 mL mobile phase, inject a 70 µL aliquot.
HPLC VARIABLES
Column: 250 × 4.6 CR-J01 C18 (Shimadzu)
Mobile phase: MeCN:50 mM pH 3.5 potassium phosphate buffer (ratio not given)
Flow rate: 0.5
Injection volume: 70
Detector: UV 240
CHROMATOGRAM
Limit of quantitation: 10 ng/mL
KEY WORDS
horse; plasma; for isoflupredone and isoflupredone acetate
REFERENCE
Lillich, J.D.; Bertone, A.L.; Schmall, L.M.; Ruggles, A.J.; Sams, R.A. Plasma, urine, and synovial fluid disposition of methylprednisolone acetate and isoflupredone acetate after intra-articular administration
in horses, Am.J.Vet.Res., 1996, 57, 187–192.
330
Isopropamide iodide
Isopropamide iodide
H3C
H3C
Molecular formula: C23 H33 IN2 O
Molecular weight: 480.42
CAS Registry No: 71-81-8
Merck Index: 13, 5222
CH3
I−
N+
NH2
CH3
CH3
O
SAMPLE
Matrix: blood
Sample preparation: Condition a 3 mL diol SPE cartridge (Baker) with 2 mL MeOH
and 2 mL water. Add 1 mL plasma to the SPE cartridge, wash with 2 mL water, wash
with 2 mL MeOH, elute with 3 mL 60 mM KBr in MeOH. Evaporate the eluate to
dryness under a stream of nitrogen at 60◦ , reconstitute the residue with 200 µL mobile
phase, inject an aliquot. (Prepare 60 mM KBr in MeOH by sonicating for 45 min.)
HPLC VARIABLES
Column: 250 × 4 LiChrosorb RP18
Mobile phase: MeOH:water 55:45 containing 4.325 g/L sodium octanesulfonate and
2 mL/L N, N-dimethyloctylamine, adjusted to pH 3.0 with phosphoric acid
Flow rate: 1
Detector: UV 220
CHROMATOGRAM
Retention time: 5
OTHER SUBSTANCES
Extracted: clidinium bromide (9.5), mepenzolate bromide (8)
KEY WORDS
plasma; SPE
REFERENCE
Russ-Kirschenbaum, R.; Koziol, T.; Woolf, E. Solid phase extraction of quaternary ammonium compounds
on diol columns: Application to the HPLC determination of CK-1649 in plasma, J.Liq.Chromatogr.,
1989, 12, 3051–3059.
SAMPLE
Matrix: formulations
Sample preparation: Sonicate a tablet in 35 mL MeOH:water 45:55 and 10 mL
40 µg/mL IS in MeOH:water 45:55, make up to 50 mL with MeOH:water 45:55, centrifuge at 3020 g for 10 min, inject an aliquot of the supernatant. Mix 1 mL oral solution
with 10 mL 40 µg/mL IS in MeOH:water 45:55, make up to 50 mL with MeOH:water
45:55, inject an aliquot.
HPLC VARIABLES
Column: 150 × 4.1 5 µm Rsil C18 (RSL, Belgium)
Column temperature: 25
Mobile phase: MeOH:water 55:45 containing 20 mM sodium 1-octanesulfonate and
10 mM N,N-dimethyloctylamine, adjusted to pH 3.0 with orthophosphoric acid
Flow rate: 1
Injection volume: 10
Detector: UV 220
Isopropamide iodide
331
CHROMATOGRAM
Retention time: 4
Internal standard: fenpiverinium bromide (6)
OTHER SUBSTANCES
Simultaneous: chlorpheniramine (10), glycopyrrolate (10), mepenzolate bromide (6),
methyl paraben (2.5), pentienate bromide (7), phenylpropanolamine (3), tiemonium
iodide (5)
Noninterfering: cinnarizine, haloperidol
KEY WORDS
tablets; oral solutions; stability-indicating
REFERENCE
De Schutter, J.A.; Van den Bossche, W.; De Moerloose, P. Separation and determination of isopropamide
iodide in pharmaceutical formulations by reversed-phase ion-pair high-performance liquid chromatography, J.Chromatogr., 1986, 366, 321–328.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 150 × 4.1 5 µm PRP-1 (Hamilton)
Mobile phase: Gradient. MeCN:50 mM ammonium acetate from 10:90 to 50:50 over
30 min.
Flow rate: 0.5
Injection volume: 20
Detector: MS, Finnigan MAT TSQ-70, thermospray, discharge-off mode, vaporizer 90◦ ,
ion-source 250◦ , repeller 50–100 V, make-up flow MeCN:50 mM ammonium acetate
10:90 at 1 mL/min, collision gas pressure 0.5 Pa, collision energy 20 eV, m/z 353–238
CHROMATOGRAM
Limit of detection: 50 pg
OTHER SUBSTANCES
Simultaneous: antrenyl, clidinium, mepenzolate (14.5), methylbenacyzine, neostigmine, pipenzolate (15.5), propantheline (22), valethamate
REFERENCE
van der Hoeven, R.A.M.; Reeuwijk, H.J.E.M.; Tjaden, U.R.; van der Greef, J. Analysis of quaternary
ammonium drugs by thermospray liquid chromatography-mass spectrometry using a resin-based
stationary phase, J.Chromatogr.A, 1996, 741, 75–84.
332
Itopride
Itopride
H
O
O
N
N
Molecular formula: C20 H26 N2 O4
Molecular weight: 358.43
CAS Registry No: 122898-67-3
H3CO
OCH3
SAMPLE
Matrix: blood, urine
Sample preparation: Inject 200 µL serum or urine onto column A and elute to waste
with mobile phase A. After 4 min, backflush the contents of column A onto column B
with mobile phase B; after 1 min, remove column A from the circuit, elute column B
with mobile phase B, monitor the effluent from column B. Re-equilibrate column A with
mobile phase A.
HPLC VARIABLES
Column: A 10 × 4 25–40 µm Nucleosil CN (Macherey-Nagel); B 10 × 4.6 5 µm TSKgel
ODS-80TM + 150 × 4.6 5 µm TSKgel ODS-80TM (Tosoh)
Mobile phase: A 100 mM pH 7.0 phosphate buffer; B MeCN:50 mM pH 5.5 phosphate
buffer 20:80
Flow rate: 1
Injection volume: 200
Detector: F ex 308 em 344
CHROMATOGRAM
Retention time: 13
Limit of quantitation: 5 ng/mL (serum), 20 ng/mL (urine)
OTHER SUBSTANCES
Extracted: metabolites
KEY WORDS
column-switching; pharmacokinetics; serum
REFERENCE
Takahara, E.; Fukuoka, H.; Takagi, T.; Nagata, O.; Kato, H. Simultaneous determination of a new
gastrointestinal prokinetic agent (HSR-803) and its metabolites in human serum and urine by highperformance liquid chromatography using automated column-switching, J.Chromatogr., 1992, 576,
174–178.
Kinetin
H
O
N
N
Molecular formula: C10 H9 N5 O
Molecular weight: 215.21
CAS Registry No: 525-79-1
Merck Index: 13, 5329
H
N
N
N
SAMPLE
Matrix: solutions
Sample preparation: Inject an aliquot of a solution in 200 mM HCl.
HPLC VARIABLES
Guard column: present but not specified
Column: 150 × 3.3 Vydac
Mobile phase: MeOH:buffer 4:96 (The buffer was 12.5 mM citric acid containing 25 mM
sodium acetate, 30 mM NaOH, and 10 mM acetic acid.)
Detector: E, ESA Coulochem II, 650 mV; UV 260
CHROMATOGRAM
Retention time: 8
REFERENCE
Barciszewski, J.; Siboska, G.E.; Pedersen, B.O.; Clark, B.F.C.; Rattan, S.I.S. Evidence for the presence
of kinetin in DNA and cell extracts, FEBS Lett., 1996, 393, 197–200.
HPLC Methods for Recently Approved Pharmaceuticals. by George Lunn
Copyright 2005 John Wiley & Sons, Ltd. ISBN: 0-471-66941-5
333
Lafutidine
H
O
Molecular formula: C22 H29 N3 O4 S
O
Molecular weight: 431.56
CAS Registry No: 118288-08-7
Merck Index: 13, 5362
N
S
O
O
N
N
SAMPLE
Matrix: blood
Sample preparation: Mix 1 mL plasma with 500 µL 1 M NaOH, extract with 3 mL
ethyl acetate. Evaporate the extract to dryness under reduced pressure, reconstitute
the residue with 100 µL 100 mM HCl, wash with 1 mL ethyl acetate. Add 750 µL
100 mM NaOH to the aqueous layer, extract with 3 mL ethyl acetate containing 20 ng
IS. Evaporate the organic layer to dryness, reconstitute the residue with 200 µL mobile
phase, inject an aliquot.
HPLC VARIABLES
Guard column: Guard-Pak
Column: Cosmosil 5C18-AR
Column temperature: 40
Mobile phase: MeCN:10 mM pH 5.9 phosphate buffer 20:80
Flow rate: 1
Detector: UV 230
CHROMATOGRAM
Internal standard: 4-amino-3-nitroanisole
KEY WORDS
plasma
REFERENCE
Itoh, H.; Naito, T.; Takeyama, M. Lafutidine changes levels of somatostatin, calcitonin gene-related
peptide, and secretin in human plasma, Biol.Pharm.Bull., 2002, 25, 379–382.
334
HPLC Methods for Recently Approved Pharmaceuticals. by George Lunn
Copyright 2005 John Wiley & Sons, Ltd. ISBN: 0-471-66941-5
335
Lamivudine
Lamivudine
NH2
N
Molecular formula: C8 H11 N3 O3 S
Molecular weight: 229.26
CAS Registry No: 134678-17-4
Merck Index: 13, 5367
HO
O
N
O
S
SAMPLE
Matrix: blood
Sample preparation: Condition a 1 mL 100 mg Bond Elut-C SPE cartridge with 1 mL
MeOH and 1 mL 100 mM pH 7.0 ammonium acetate buffer. Heat plasma at 58◦ for 1 h
to inactivate HIV. Vortex 800 µL plasma with 300 µL 2 µg/mL hexobarbital in 25 mM
pH 7.0 ammonium acetate buffer for 30 s and centrifuge at 18 000 g for 5 min. Add 1 mL
of the supernatant to the SPE cartridge, wash with 1 mL 100 mM pH 7.0 ammonium
acetate buffer, suck dry for 1 min, elute with 800 µL MeOH. Evaporate the eluate to
dryness under a stream of nitrogen at 40◦ and reconstitute the residue with 100 µL
mobile phase. Vortex for 30 s, centrifuge at 18 000 g for 3 min, and inject an 80 µL
aliquot.
HPLC VARIABLES
Guard column: 20 × 3.9 5 µm Polarity dC18 (Waters)
Column: 150 × 3.9 5 µm Polarity dC18 (Waters)
Column temperature: 40
Mobile phase: Gradient. A was 10 mM pH 6.5 ammonium acetate buffer. B was 10 mM
pH 6.5 ammonium acetate buffer:MeCN:MeOH 20:50:30. A:B 96:4 for 15 min, to 36:64
over 15 min, maintain at 36:64 for 3 min, re-equilibrate at initial conditions for 7 min.
Flow rate: 1.1
Injection volume: 80
Detector: UV 269 for 11 min, UV 250 for 3 min, UV 271 for 10 min, UV 230 for 9 min
CHROMATOGRAM
Retention time: 8.6
Internal standard: hexobarbital (30.6)
Limit of quantitation: 10.0 ng/mL
OTHER SUBSTANCES
Extracted: abacavir (25.1), didanosine (13.6), nevirapine (27.3), stavudine (15.7), zalcitabine (5.9), zidovudine (23.8)
Noninterfering: tenofovir
KEY WORDS
plasma; SPE
REFERENCE
Rezk, N.L.; Tidwell, R.R.; Kashuba, A.D.M. Simultaneous determination of six HIV nucleoside analogue
reverse transcriptase inhibitors and nevirapine by liquid chromatography with ultraviolet absorbance
detection, J.Chromatogr.B, 2003, 791, 137–147.
SAMPLE
Matrix: blood
Sample preparation: Condition a 100 mg Dual Zone C18 SPE cartridge (Diazem) with
2 mL MeOH and 2 mL water. Dilute 500 µL serum with 1 mL water, add to the SPE
cartridge, wash with 500 µL water, elute with 1 mL MeOH. Evaporate the eluate to
336
Lamivudine
dryness with vortexing under reduced pressure at 40◦ and reconstitute the residue with
300 µL MeOH, inject a 10 µL aliquot.
HPLC VARIABLES
Column: two 150 × 4.6 3 µm Luna C18 columns in series
Column temperature: 60
Mobile phase: Gradient. MeCN:water from 5:95 to 45:55 over 20 min.
Flow rate: 0.85
Injection volume: 10
Detector: UV 250
CHROMATOGRAM
Retention time: 9.5
Limit of detection: 260 ng/mL
OTHER SUBSTANCES
Extracted: abacavir (17, LOD 75 ng/mL), didanosine (10.5, LOD 120 ng/mL), stavudine
(11.5, LOD 40 ng/mL), zalcitabine (7.5, LOD 440 ng/mL), zidovudine (16, LOD 30 ng/mL)
KEY WORDS
serum; SPE
REFERENCE
Simon, V.A.; Thiam, M.D.; Lipford, L.C. Determination of serum levels of thirteen human immunodeficiency virus-suppressing drugs by high-performance liquid chromatography, J.Chromatogr.A, 2001,
913, 447–453.
SAMPLE
Matrix: blood
Sample preparation: Vortex 100 µL serum with 20 µL 20% trichloroacetic acid for
10 s, centrifuge at 16 000 g for 5 min, inject an aliquot of the supernatant.
HPLC VARIABLES
Column: 250 × 4.6 5 µm Hypersil C18
Mobile phase: MeOH: buffer 11.7:88.3 (The buffer was 43 mM orthophosphoric acid
containing 10 mM triethylammonium acetate, adjusted to pH 7.0 with 5 M KOH.)
Flow rate: 1
Injection volume: 100
Detector: UV 280
CHROMATOGRAM
Retention time: 9.5
Limit of detection: 5 ng/mL
Limit of quantitation: 20 ng/mL
KEY WORDS
pharmacokinetics; serum
REFERENCE
Zhou, X.-J.; Sommadossi, J.-P. Rapid quantitation of (−)-2′ -deoxy-3′ -thiacytidine in human serum by
high-performance liquid chromatography with ultraviolet detection, J.Chromatogr.B, 1997, 691,
417–424.
ANNOTATED BIBLIOGRAPHY
Aboul-Enein, H.Y.; Hefnawy, M.M. High throughput analysis of lamivudine in pharmaceutical preparations using monolithic silica HPLC column, Anal.Lett., 2003, 36, 2527–2538. [LOD 12.5 ng/mL]
Lamivudine
337
Aymard, G.; Legrand, M.; Trichereau, N.; Diquet, B. Determination of twelve antiretroviral agents
in human plasma sample using reversed-phase high-performance liquid chromatography,
J.Chromatogr.B, 2000, 744, 227–240. [column-switching; LOQ 20 ng/mL for lamivudine; amprenavir;
efavirenz; indinavir; nelfinavir; ritonavir; saquinavir; abacavir; didanosine; lamivudine; stavudine;
nevirapine; zidovudine]
Fan, B.; Bartlett, M.G.; Stewart, J.T. Determination of lamivudine/stavudine/efavirenz in human serum
using liquid chromatography/electrospray tandem mass spectrometry with ionization polarity switch,
Biomed.Chromatogr., 2002, 16, 383–389. [SPE; LOQ 1.1 ng/mL for lamivudine]
Fan, B.; Stewart, J.T. Determination of zidovudine/lamivudine/nevirapine in human plasma using ionpair HPLC, J.Pharm.Biomed.Anal., 2002, 28, 903–908. [SPE; LOQ 59 ng/mL for lamivudine]
Gibbs, J.E.; Rashid, T.; Thomas, S.A. Effect of transport inhibitors and additional anti-HIV drugs on the
movement of lamivudine (3TC) across the guinea pig brain barriers, J.Pharmacol.Exp.Ther., 2003,
306, 1035–1041.
Harker, A.J.; Evans, G.L.; Hawley, A.E.; Morris, D.M. High-performance liquid chromatographic assay
for 2′ -deoxy-3′ -thiacytidine in human serum, J.Chromatogr.B, 1994, 657, 227–232. [SPE; carbovir is
internal standard]
Hoetelmans, R.M.; Profijt, M.; Mennhorst, P.L.; Mulder, J.W.; Beijnen, J.H. Quantitative determination
of (−)-2′ -deoxy-3′ -thiacytidine (lamivudine) in human plasma, saliva and cerebrospinal fluid by highperformance liquid chromatography with ultraviolet detection, J.Chromatogr.B, 1998, 713, 387–394.
[SPE; LOQ 10 ng/mL]
Hsyu, P.-H.; Lloyd, T.L. Automated high-performance liquid chromatographic analysis of (−)- 2′ -deoxy-3′ thiacytidine in biological fluids using the automated sequential trace enrichment of dialysate systems,
J.Chromatogr.B, 1994, 655, 253–259. [dialysate; serum; urine; column-switching]
Kakuda, T.N.; Page, L.M.; Anderson, P.L.; Henry, K.; Schacker, T.W.; Rhame, F.S.; Acosta, E.P.;
Brundage, R.C.; Fletcher, C.V. Pharmacological basis for concentration-controlled therapy with
zidovudine, lamivudine, and indinavir, Antimicrob.Agents Chemother., 2001, 45, 236–242. [SPE;
no HPLC of indinavir]
Kenney, K.B.; Wring, S.A.; Carr, R.M.; Wells, G.N.; Dunn, J.A. Simultaneous determination of
zidovudine and lamivudine in human serum using HPLC with tandem mass spectrometry,
J.Pharm.Biomed.Anal., 2000, 22, 967–983. [ultrafiltration; LOQ 2.5 ng/mL]
Morris, D.; Selinger, K. Determination of 2′ -deoxy-3′ -thiacytidine (3TC) in human urine by liquid chromatography: direct injection with column switching, J.Pharm.Biomed.Anal., 1994, 12, 255–264. [LOQ
500 ng/mL]
Moyer, T.P.; Temesgen, Z.; Enger, R.; Estes, L.; Charlson, J.; Oliver, L.; Wight, A. Drug monitoring of
antiretroviral therapy for HIV-1 infection: method validation and results of a pilot study, Clin.Chem.,
1999, 45, 1465–1476. [SPE; LOQ 100 ng/mL for lamivudine; didanosine; lamivudine; stavudine;
zalcitabine; zidovudine; delavirdine; nevirapine; indinavir; nelfinavir; ritonavir; saquinavir]
Ozkan, S.A.; Uslu, B. Rapid HPLC assay for lamivudine in pharmaceuticals and human serum,
J.Liq.Chromatogr.Rel.Technol., 2002, 25, 1447–1456. [LOQ 14 ng/mL; deflazacort is internal standard]
Pereira, A.S.; Kenney, K.B.; Cohen, M.S.; Hall, J.E.; Eron, J.J.; Tidwell, R.R.; Dunn, J.A. Simultaneous
determination of lamivudine and zidovudine concentrations in human seminal plasma using highperformance liquid chromatography and tandem mass spectrometry, J.Chromatogr.B, 2000, 742,
173–183. [ultrafiltration; LOQ 5 ng/mL]
Rajagopalan, P.; Boudinot, F.D.; Chu, C.K.; Tennant, B.C.; Baldwin, B.H.; Schinazi, R.F. Pharmacokinetics of (−)-2′ -3′ -dideoxy-3′ -thiacytidine in woodchucks, Antimicrob.Agents Chemother., 1996, 40,
642–645. [LOQ 250 ng/mL]
Rezk, N.L.; Tidwell, R.R.; Kashuba, A.D.M. Simultaneous determination of six HIV nucleoside analogue
reverse transcriptase inhibitors and nevirapine by liquid chromatography with ultraviolet absorbance
detection, J.Chromatogr.B, 2003, 791, 137–147. [plasma; LOQ 10 ng/mL; zalcitabine; lamivudine;
didanosine; stavudine; zidovudine; abacavir; hexobarbital is internal standard; SPE]
Rodriguez, J.F.; Rodriguez, J.L.; Santana, J.; Garcı́a, H.; Rosario, O. Simultaneous quantitation of intracellular zidovudine and lamivudine triphosphates in human immunodeficiency virus-infected individuals, Antimicrob.Agents Chemother., 2000, 44, 3097–3100. [LC-MS]
Santos-Magalhaes, N.S.; Pontes, A.; Cavalcante, R.M.; Costa, R.M.R.; Rangel, F.A.; Guimaraes, M.I.V.;
de Carvalho, J.N.L.; de Souza, S.D.F.; de Oliveira, H.M.; Esteves, I.L.C.; Ramalho, M.S.; Vieira, S.L.A.;
Alves, A.J. Bioequivalence of two lamivudine tablet formulations, Arzneimittelforschung, 2001, 51,
310–314.
Simon, V.A.; Thiam, M.D.; Lipford, L.C. Determination of serum levels of thirteen human immunodeficiency virus-suppressing drugs by high-performance liquid chromatography, J.Chromatogr.A, 2001,
338
Lamivudine
913, 447–453. [SPE; LOD 260 ng/mL for lamivudine; zalcitabine; lamivudine; stavudine; didanosine;
zidovudine; nevirapine; abacavir; indinavir; delavirdine; nelfinavir; saquinavir; ritonavir; efavirenz]
Solas, C.; Li, Y.-F.; Xie, M.-Y.; Sommadossi, J.-P.; Zhou, X.-J. Intracellular nucleotides of (−)-2′ ,3′ -deoxy3′ -thiacytidine in peripheral blood mononuclear cells of a patient infected with human immunodeficiency virus, Antimicrob.Agents Chemother., 1998, 42, 2989–2995.
Uslu, B.; Ozkan, S.A. Determination of lamivudine and zidovudine in binary mixtures using first
derivative spectrophotometric, first derivative of the ratio-spectra and high-performance liquid
chromatography-UV methods, Anal.Chim.Acta, 2002, 466, 175–185. [LOQ 25 ng/mL for lamivudine;
finasteride is internal standard]
Wang, L.H.; Chittick, G.E.; McDowell, J.A. Single-dose pharmacokinetics and safety of abacavir
(1592U89), zidovudine, and lamivudine administered alone and in combination in adults with human
immunodeficiency virus infection, Antimicrob.Agents Chemother., 1999, 43, 1708–1715.
Williams, L.D.; Von Tungeln, L.S.; Beland, F.A.; Doerge, D.R. Liquid chromatographic-mass spectrometric determination of the metabolism and disposition of the anti-retroviral nucleoside analogs zidovudine
and lamivudine in C57BL/6N and B6C3F1 mice, J.Chromatogr.B, 2003, 798, 55–62. [SPE; LC-MS]
Zheng, J.J.; Wu, S.T.; Emm, T.A. High-performance liquid chromatographic assay for the determination
of 2′ -deoxy-3′ -thiacytidine (lamivudine) in human plasma, J.Chromatogr.B, 2001, 761, 195–201. [SPE;
LOQ 10 ng/mL]
339
Latanoprost
Latanoprost
HO
O
O
Molecular formula: C26 H40 O5
Molecular weight: 432.59
CAS Registry No: 130209-82-4
Merck Index: 13, 5391
HO
CH3
CH3
OH
SAMPLE
Matrix: aqueous humor, tissue
Sample preparation: Homogenize (Polytron) rabbit eye tissue with 3 mL EtOH, centrifuge, evaporate the supernatant under a stream of nitrogen, reconstitute with EtOH,
inject an aliquot. Acidify aqueous humor to pH 3.4 with 1 M formic acid, extract with
3 mL ethyl acetate. Evaporate the ethyl acetate to dryness under a stream of nitrogen,
reconstitute with EtOH, inject an aliquot.
HPLC VARIABLES
Column: 5 µm Nucleosil C18
Mobile phase: Gradient. MeCN:water:acetic acid 35:65:0.1 for 11 min, to 46:54:0.1
over 1 min, maintain at 46:54:0.1 for 6 min, return to initial conditions over 3 min,
re-equilibrate for about 10 min. Alternatively, MeCN:water:acetic acid 25:75:0.1 for
15 min, to 40:60:0.1 over 10 min, return to initial conditions over 5 min, re-equilibrate
for 5 min.
Flow rate: 1
Detector: Radioactivity (3 H)
CHROMATOGRAM
Retention time: 22
OTHER SUBSTANCES
Extracted: metabolites
KEY WORDS
ciliary body; cornea; eye; pharmacokinetics; rabbit
REFERENCE
Sjöquist, B.; Basu, S.; Byding, P.; Bergh, K.; Stjernschantz, J. The pharmacokinetics of a new antiglaucoma drug, latanoprost, in the rabbit, Drug Metab.Dispos., 1998, 26, 745–754.
SAMPLE
Matrix: blood, feces, urine
Sample preparation: Blood, urine. Condition a Sep-Pak C18 with 10 mL EtOH and
10 mL water. Centrifuge plasma or urine at 800 g for 10 min, acidify to pH 3.5–4.0
with 1 M HCl, add to the SPE cartridge, wash with 10 mL water, wash with 10 mL
petroleum ether, elute with 10 mL methyl formate. Evaporate the eluate to dryness
under a stream of nitrogen, reconstitute the residue with EtOH, inject an aliquot.
Homogenize feces with 3 volumes of EtOH, centrifuge at 800 g for 15 min, evaporate
2 mL of the supernatant to dryness, reconstitute with 200 µL EtOH, add 1.8 mL water,
acidify to pH 3.5–4.0 with 1 M HCl, extract twice with 4 mL portions of ethyl acetate.
Evaporate the organic layer to dryness under a stream of nitrogen, reconstitute the
residue with EtOH, inject an aliquot.
HPLC VARIABLES
Guard column: 30 × 4 30–40 µm Presorb RP-18
340
Latanoprost
Column: 125 × 4.6 5 µm Nucleosil C18
Mobile phase: Gradient. MeCN:water:acetic acid 20:80:0.1 for 20 min, to 40:60:0.1 over
20 min, return to initial conditions over 5 min, re-equilibrate for 10 min.
Flow rate: 1
Detector: Radioactivity (3 H)
CHROMATOGRAM
Retention time: 20
OTHER SUBSTANCES
Extracted: metabolites
KEY WORDS
monkey; pharmacokinetics; plasma
REFERENCE
Sjöquist, B.; Tajallaei, S.; Stjernschantz, J. Pharmacokinetics of latanoprost in the cynomolgus monkey.
1st communication: single intravenous, oral or topical administration on the eye, Arzneimittelforschung,
1999, 49, 225–233.
341
Leflunomide
Leflunomide
F3C
Molecular formula: C12 H9 F3 N2 O2
Molecular weight: 270.21
CAS Registry No: 75706-12-6
Merck Index: 13, 5451
O
CH3
N
H
O
N
SAMPLE
Matrix: blood
Sample preparation: Mix 75 µL plasma with 200 µL 50 ng/mL IS in MeCN, centrifuge,
inject.
HPLC VARIABLES
Column: 150 × 4.6 Zorbax Eclipse XDB-C8
Mobile phase: Gradient. MeCN:10 mM ammonium formate containing 0.1% formic acid
0:100 for 3 min, to 90:10 over 17 min, return to initial conditions over 5 min.
Flow rate: 1
Detector: MS, PE Sciex API 2000 triple quadrupole, 50 µL/min entered the detector,
negative ionization, ionspray interface 150◦ , ionspray 4.5 kV, orifice 30 eV, nebulizer
gas nitrogen, m/z 269–82; UV 254
CHROMATOGRAM
Retention time: 16.5
Internal standard: indomethacin
OTHER SUBSTANCES
Extracted: metabolite A771726 (82) (12.7), 3-methylleflunomide (16.5)
KEY WORDS
human; plasma; rat
REFERENCE
Kalgutkar, A.S.; Nguyen, H.T.; Vaz, A.D.N.; Doan, A.; Dalvie, D.K.; McLeod, D.G.; Murray, J.C. In vitro
metabolism studies on the isoxazole ring scission in the anti-inflammatory agent leflunomide to its
active α-cyanoenol metabolite A771726: Mechanistic similarities with the cytochrome P450-catalyzed
dehydration of aldoximes, Drug Metab.Dispos., 2003, 31, 1240–1250.
SAMPLE
Matrix: blood
Sample preparation: Mix 250 µL plasma, 500 µL 100 mM pH 5 sodium acetate buffer,
and 100 µL 25 µg/mL IS in water, add 10 mL ethyl acetate, shake for 15 min, centrifuge
at 4000 rpm for 5 min. Evaporate the organic layer to dryness under a stream of
nitrogen at room temperature, reconstitute the residue with 200 µL mobile phase,
vortex for 1 min, inject a 50 µL aliquot.
HPLC VARIABLES
Guard column: C18
Column: 125 × 3 Nucleosil 100-5C18
Column temperature: 21
Mobile phase: MeCN:water:formic acid 40:59.8:0.2
Flow rate: 0.5
Injection volume: 50
Detector: UV 261
342
Leflunomide
CHROMATOGRAM
Retention time: 16.2
Internal standard: warfarin (12.2)
Limit of quantitation: 50 ng/mL
OTHER SUBSTANCES
Extracted: metabolite A771726 (8.2)
KEY WORDS
plasma
REFERENCE
Schmidt, A.; Schwind, B.; Gillich, M.; Brune, K.; Hinz, B. Simultaneous determination of leflunomide
and its active metabolite, A77 1726, in human plasma by high-performance liquid chromatography,
Biomed.Chromatogr., 2003, 17, 276–281.
Lercanidipine
Lercanidipine
Molecular formula: C36 H41 N3 O6
Molecular weight: 611.73
CAS Registry No: 100427-26-7
Merck Index: 13, 5465
343
H
H3C
N
CH3O
CH3
O
O
O H3C
N
CH3
CH3
NO2
SAMPLE
Matrix: blood
Sample preparation: Mix 1 mL plasma with 25 µL 660 ng/mL IS in MeOH and 50 µL
100 mM NaOH, add 4.5 mL hexane:isopropanol 99:1, vortex for 2 min, centrifuge at
2000 g for 10 min. Evaporate the organic layer to dryness under reduced pressure at
25◦ , reconstitute the residue with 50 µL mobile phase, vortex for 20 s, inject a 20 µL
aliquot. (Carry out all manipulations under yellow light.)
HPLC VARIABLES
Guard column: 4 × 4 5 µm 100 RP-18
Column: 250 × 4.6 10 µm Chiralpak AD
Column temperature: 30 ± 1
Mobile phase: Hexane:EtOH:diethylamine 95:5:0.1
Flow rate: 1.3
Injection volume: 20
Detector: MS, Micromass Quatro Micro LC triple quadrupole, electrospray interface,
positive ion mode, capillary voltage 3.5 kV, source 100◦ , desolvation 150◦ , nebulizing
gas nitrogen at 365 L/h, collision gas argon at 3.5 µbar, cone 40 V, collision energy
38.0 eV, m/z 612.40–100.10 (The make-up liquid was EtOH:10 mM ammonium acetate
95:5 pumped at 0.25 mL/min, which mixed with the column effluent. A splitter was used
so that 0.2 mL/min entered the detector.)
CHROMATOGRAM
Retention time: 5.98 (S), 6.50 (R)
Internal standard: amiodarone (collision energy 30.0 eV) (m/z 646.30–100.30) (3.47)
Limit of quantitation: 25 pg/mL
KEY WORDS
chiral; pharmacokinetics; plasma
REFERENCE
Jabor, V.A.P.; Coelho, E.B.; Ifa, D.R.; Bonato, P.S.; dos Santos, N.A.G.; Lanchote, V.L. Enantioselective
determination of lercanidipine in human plasma for pharmacokinetic studies by normal-phase liquid
chromatography-tandem mass spectrometry, J.Chromatogr.B, 2003, 796, 429–437.
SAMPLE
Matrix: formulations
Sample preparation: Sonicate tablet in 5 mL EtOH, make up to 10 mL with mobile
phase, centrifuge at 2700 g for 10 min. Dilute a 500 µL aliquot of the supernatant to
10 mL with mobile phase, inject an aliquot.
HPLC VARIABLES
Guard column: 30 × 4.6 Bondapak C18
Column: 150 × 3.9 5 µm Symmetry C18
Column temperature: 25 ± 1
Mobile phase: MeCN:10 mM pH 4.0 phosphate buffer 45:55
344
Lercanidipine
Flow rate: 1
Injection volume: 20
Detector: UV 356; E, glassy carbon working electrode 1000 mV, Ag/AgCl reference
electrode, platinum rod auxiliary electrode
CHROMATOGRAM
Retention time: 5
Limit of detection: 930 nM (UV), 750 nM (E)
Limit of quantitation: 1.2 µM (UV), 3.2 µM (E)
KEY WORDS
tablets
REFERENCE
Alvarez-Lueje, A.; Pujol, S.; Squella, J.A.; Núñz-Vergara, L.J. A selective HPLC method for determination of lercanidipine in tablets, J.Pharm.Biomed.Anal., 2003, 31, 1–9.
ANNOTATED BIBLIOGRAPHY
Boatto, G.; Nieddu, M.; Faedda, M.V.; De Caprariis, P. Enantiomeric separation by HPLC of 1,4dihydropyridines with vancomycin as chiral selector, Chirality, 2003, 15, 494–497. [isradipine;
nimodipine; nisoldipine; felodipine; lercanidipine; amlodipine]
Calleri, E.; De Lorenzi, E.; Siluk, D.; Markuszewski, M.; Kaliszan, R.; Massolini, G. Column liquid chromatography riboflavin binding protein-chiral stationary phase: Investigation of retention mechanism,
Chromatographia, 2002, 55, 651–658. [fenfluramine; dichloroisoproterenol; propranolol; oxprenolol;
alprenolol; acebutolol; ketoprofen; indoprofen; warfarin; lorazepam; oxazepam; isradipine; nimodipine;
amlodipine; nicardipine; lercanidipine; gallopamil; verapamil; bepridil]
Garzotti, M.; Hamdan, M. Supercritical fluid chromatography coupled to electrospray mass spectrometry: a powerful tool for the analysis of chiral mixtures, J.Chromatogr.B, 2002, 770, 53–61.
[alprenolol; lercanidipine; disopyramide; propafenone; tropicamide; atenolol; ofloxacin; albuterol;
econazole; miconazole; homatropine; nicardipine; sulpiride; verapamil; pindolol; flecainide; atropine;
clenbuterol; sulconazole; ketoconazole]
Letrozole
Letrozole
345
N
N
N
Molecular formula: C17 H11 N5
Molecular weight: 285.30
CAS Registry No: 112809-51-5
Merck Index: 13, 5469
NC
CN
SAMPLE
Matrix: blood, urine
Sample preparation: Condition a 1 mL 100 mg Bond Elut C8 SPE cartridge with
1 mL MeOH and 1 mL 100 mM HCl. Dilute urine with an equal volume of water.
Mix 1 mL plasma or diluted urine with 100 µL IS in water and 1 mL 100 mM HCl,
add to the SPE cartridge, wash with 2 mL 10 mM pH 7 phosphate buffer, wash with
500 µL MeCN:10 mM pH 7 phosphate buffer 20:80, elute with 2 mL MeCN:10 mM pH
7 phosphate buffer 40:60, inject a 100 µL aliquot of the eluate.
HPLC VARIABLES
Guard column: 20 × 4.6 5 µm Supelguard LC
Column: 250 × 4.6 5 µm ODS Hypersil C18
Mobile phase: MeCN:10 mM pH 7 phosphate buffer 30:70 (The buffer was 1.36 g
potassium dihydrogen phosphate and 1.42 g disodium hydrogen phosphate in 1 L water.)
Flow rate: 1.5
Injection volume: 100
Detector: F ex 230 em 295
CHROMATOGRAM
Retention time: 11.3
Internal standard: CGP 47 645 (1-[bis(4-cyanophenyl)fluoromethyl]-1,2,4-triazole)
(16.7)
Limit of quantitation: 1.40 nM (plasma), 2.80 nM (urine)
OTHER SUBSTANCES
Extracted: metabolite CGP 44 645 (12.3)
KEY WORDS
plasma; SPE
REFERENCE
Marfil, F.; Pineau, V.; Sioufi, A.; Godbillon, J. High-performance liquid chromatography of the aromatase
inhibitor, letrozole, and its metabolite in biological fluids with automated liquid-solid extraction and
fluorescence detection, J.Chromatogr.B, 1996, 683, 251–258.
346
Levetiracetam
Levetiracetam
Molecular formula: C8 H14 N2 O2
Molecular weight: 170.21
CAS Registry No: 102767-28-2
Merck Index: 13, 5482
CH3
O
NH2
N
O
SAMPLE
Matrix: blood
Sample preparation: Condition a 100 mg C18 SPE cartridge (Baker) with 2 mL MeOH
and 2 mL water. Add 200 µL serum and 100 µL 20 µg/mL IS in MeOH:water 2:98 to
the SPE cartridge, wash with 750 µL water, dry under vacuum for 3 min, elute with
1 mL MeOH:water 1:5, inject a 50 µL aliquot of the eluate.
HPLC VARIABLES
Column: 150 × 4.6 Spherisorb 3ODS2
Mobile phase: Gradient. MeCN:water from 6:94 to 20:80 over 5 min, to 40:60 over 1 min,
maintain at 40:60 for 4 min, return to initial conditions over 1 min, re-equilibrate for
4 min.
Flow rate: 1
Injection volume: 150
Detector: UV 205
CHROMATOGRAM
Retention time: 6.4
Internal standard: G025 (α-methyl-5,5-dimethyl-2-oxo-1-pyrrolidine acetamide) (7.8)
Limit of quantitation: 360 ng/mL
OTHER SUBSTANCES
Noninterfering: carbamazepine, carbamazepine dihydrodiol, carbamazepine epoxide, N-desmethylsuximide, ethosuximide, lamotrigine, loreclezole, monohydroxycarbamazepine, phenobarbital, phenytoin, primidone, valproic acid, vigabatrin
KEY WORDS
comparison with GC; pharmacokinetics; serum; SPE
REFERENCE
Vermeij, T.A.C.; Edelbroek, P.M. High-performance liquid chromatographic and megabore gas-liquid
chromatographic determination of levetiracetam (ucb L059) in human serum after solid-phase extraction, J.Chromatogr.B, 1994, 662, 134–139.
SAMPLE
Matrix: blood, saliva
Sample preparation: Vortex 100 µL serum or saliva with 100 µL IS solution and
25 µL 5 M NaOH for 10 s, add 1 mL dichloromethane, vortex for 1 min, centrifuge at
3000 rpm for 5 min. Evaporate the organic layer to dryness, reconstitute the residue
with 200 µL mobile phase, inject a 50 µL aliquot.
HPLC VARIABLES
Guard column: PRP-1 (Burdick & Jackson)
Column: 100 × 8 4 µm Nova-pak C18 Radial-Pak
Mobile phase: Gradient. A was MeCN:buffer 12:88. B was MeCN:buffer 45:55. A:B
100:0 for 7 min, to A:B 0:100 (step gradient), maintain at 0:100 for 5 min, return to
initial conditions (step gradient), re-equilibrate for 5 min. (Prepare A by dissolving
Levetiracetam
347
2.28 g dipotassium hydrogen phosphate trihydrate and 5.44 g potassium dihydrogen
phosphate in 880 mL water and then adding 120 mL MeCN. Prepare B by dissolving
2.28 g dipotassium hydrogen phosphate trihydrate and 5.44 g potassium dihydrogen
phosphate in 550 mL water and then adding 450 mL MeCN.)
Flow rate: 1
Injection volume: 50
Detector: UV 208
CHROMATOGRAM
Internal standard: ucb 17025
Limit of quantitation: ≤1.3 µg/mL
KEY WORDS
serum
REFERENCE
Grim, S.A.; Ryan, M.; Miles, M.V.; Tang, P.H.; Strawsburg, R.H.; deGrauw, T.J.; Fakhoury, T.A.; Baumann, R.J. Correlation of levetiracetam concentrations between serum and saliva, Ther.Drug Monit.,
2003, 25, 61–66.
ANNOTATED BIBLIOGRAPHY
Ratnaraj, N.; Doheny, H.C.; Patsalos, P.N. A micromethod for the determination of the new antiepileptic
drug levetiracetam (ucb L059) in serum or plasma by high performance liquid chromatography,
Ther.Drug Monit., 1996, 18, 154–157. [LOQ 5 µM]
348
Levonordefrin
Levonordefrin
Molecular formula: C9 H13 NO3
Molecular weight: 183.20
CAS Registry No: 829-74-3
Merck Index: 13, 6725
OH
HO
HO
CH3
NH2
SAMPLE
Matrix: blood
Sample preparation: Prepare an SPE column by packing 50–100 mesh Bio-Rex 70
(Na+ ) cation exchange resin (Bio-Rad) into a 40 × 10 polypropylene column and washing
three times with 1 M HCl, three times with 1 M NaOH, once with 1 M pH 6.5 sodium
acetate, and once with 0.01% disodium EDTA. Mix 2 mL plasma with 5 mL 0.1%
disodium EDTA, 500 µL 1 M pH 6.5 sodium acetate, and 4 ng IS, add to the SPE
column, wash with 10 mL water, elute with 1 mL 700 mM sulfuric acid and 3.5 mL
2 M ammonium sulfate containing 0.1% disodium EDTA. Adjust the pH of the eluate to
8.6 with 3 mL 1 M pH 8.6 Tris-HCl buffer containing 2% disodium EDTA, add 100 mg
activated alumina, shake for 3 min. Centrifuge, discard the liquid, wash the alumina
three times with 10 mL distilled water. Centrifuge to remove excess water, elute with
200 µL 300 mM perchloric acid, inject a 50–100 µL aliquot of the eluate.
HPLC VARIABLES
Column: 150 × 4.6 5 µm Spherisorb ODS2
Mobile phase: 100 mM pH 5.0 sodium dihydrogen phosphate containing 2 mM sodium
heptanesulfonate and 0.001% disodium EDTA
Flow rate: 1.5
Injection volume: 50–100
Detector: E, Bioanalytical Systems LC-4A, glassy carbon electrode +0.50 V, Ag/AgCl
reference electrode
CHROMATOGRAM
Retention time: 12
Internal standard: 3,4-dihydroxybenzylamine (20)
Limit of detection: 50 pg/mL
OTHER SUBSTANCES
Extracted: epinephrine (14), norepinephrine (6)
KEY WORDS
human; plasma; rabbit; SPE
REFERENCE
Jackman, G.P.; Oddie, C.J.; Skews, H.; Bobik, A. High-performance liquid chromatographic determination of plasma catecholamines during α-methyldopa therapy, J.Chromatogr., 1984, 308, 301–305.
349
Levosimendan
Levosimendan
H
NC
N
NC
Molecular formula: C14 H12 N6 O
Molecular weight: 280.28
CAS Registry No: 141505-33-1
Merck Index: 13, 5491
N N
N
O
H
H3C
SAMPLE
Matrix: blood
Sample preparation: Mix 500 µL plasma with 25 µL 1.5 µg/mL IS in 50 mM pH 7.2
phosphate buffer. Using a Cuprophan cellulose membrane (cut-off 15 kDa) dialyze a
370 µL aliquot against 18 mL 40 mM pH 4.0 ammonium acetate buffer. Pump the buffer
through a 650 µL recipient channel over 18 min and allow it to flow through column
A. At the end of this time, backflush the contents of column A onto column B with the
mobile phase, monitor the effluent from column B. (ASTED system. After use, flush the
donor side with 10 mL 0.05% Triton X-100 in 0.86% NaCl solution. Flush the recipient
side with 4 mL 40 mM pH 4.0 ammonium acetate buffer. Regenerate column A with
1 mL 40 mM pH 4.0 ammonium acetate buffer.)
HPLC VARIABLES
Column: A 5.8 × 4.6 10 µm Hypersil ODS; B 250 × 4 10 µm LiChrosorb RP-18
Mobile phase: MeOH:THF:32 mM sodium dihydrogen phosphate 65:1:45, adjusted to
apparent pH 3.5 with phosphoric acid
Flow rate: 1
Detector: UV 380
CHROMATOGRAM
Retention time: 6.4
Internal standard: OR-1097 ([[4-(1,4,5,6-tetrahydro-6-oxo-3-pyridazinyl)phenyl]hydrazono]propanedinitrile) (5.4)
Limit of detection: 5 ng/mL (S/N = 3)
OTHER SUBSTANCES
Interfering: carbamazepine
KEY WORDS
ASTED; dialysate; plasma
REFERENCE
Karlsson, M.; Korkolainen, T.; Wikberg, T. Automated analysis of levosimendan in human plasma by
on-line dialysis and liquid chromatography, Biomed.Chromatogr., 1997, 11, 54–58.
SAMPLE
Matrix: blood
Sample preparation: Mix 200 (rat) or 500 (dog) µL plasma with 200 µL 50 mM pH
9 phosphate buffer, add 200 µL 1 M HCl, add 4 mL ethyl acetate:n-hexane 50:50,
vortex for 2 min, centrifuge at 3500 g for 5 min. Evaporate 3 mL of the organic layer to
dryness under a stream of nitrogen at 40◦ , reconstitute the residue with 200 µL 0.5%
pH 9 triethylammonium acetate buffer, inject a 60 µL aliquot. Prepare human plasma
samples in the same fashion, but use 1 mL plasma and twice the volumes of reagents.
Alternatively filter (Ultrafree-MC 10000) 400 µL plasma while centrifuging at 1200 g
for 30 min, inject a 40 µL aliquot of the ultrafiltrate.
350
Levosimendan
HPLC VARIABLES
Guard column: Guard-Pak CN
Column: 250 × 4.6 5 µm Cyclobond I
Column temperature: 30
Mobile phase: MeOH:buffer 30:70-33:67 (Prepare the buffer by dissolving 10 mL triethylamine in 2 L and adjusting pH to 6.0 with acetic acid.)
Flow rate: 1
Injection volume: 40–60
Detector: UV 380
CHROMATOGRAM
Retention time: 19 (+), 21 (−)
Limit of quantitation: 10 ng/mL
KEY WORDS
chiral; dog; human; plasma; rat; ultrafiltrate
REFERENCE
Wikberg, T.; Korkolainen, T.; Karlsson, M. Enantiomeric bioanalysis of simendan and levosimendan by
chiral high-performance liquid chromatography, Chirality, 1996, 8, 511–517.
351
Lidamidine
Lidamidine
CH3
H
H
H
N
N
N
CH3
Molecular formula: C11 H16 N4 O
Molecular weight: 220.27
CAS Registry No: 66871-56-5, 65009-35-0
(HCl)
Merck Index: 13, 5502
O
CH3
NH
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 × 4 ODS
Mobile phase: MeOH:water:ammonium carbonate 60:40:0.02
Detector: UV 254
REFERENCE
Zalipsky, J.J.; Won, C.M.; Patel, D.M. Analytical-physical profile of lidamidine hydrochloride (WHR1142A), a novel antidiarrheal agent, Arzneimittelforschung, 1978, 28, 1441–1447.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 × 2.1 Zorbax Sil
Mobile phase: MeOH:dichloromethane:ammonium hydroxide 50:50:0.1
Detector: UV 254
REFERENCE
Zalipsky, J.J.; Won, C.M.; Patel, D.M. Analytical-physical profile of lidamidine hydrochloride (WHR1142A), a novel antidiarrheal agent, Arzneimittelforschung, 1978, 28, 1441–1447.
352
Lincomycin
Lincomycin
CH3
H3C
CH3
N
HO
Molecular formula: C18 H34 N2 O6 S
Molecular weight: 406.54
CAS Registry No: 154-21-2, 7179-49-9
(HCl monohydrate)
Merck Index: 13, 5522
NH
O
OH
O
OH
S
OH
CH3
SAMPLE
Matrix: blood
Sample preparation: Mix 500 µL serum with 500 µL MeCN, let stand for 20 min,
centrifuge at 10 000 g for 5 min, inject a 10 µL aliquot of the supernatant.
HPLC VARIABLES
Guard column: LichroCart 4-4 RP-18
Column: 125 × 4 5 µm Supersphere RP-18
Column temperature: 35
Mobile phase: MeCN:0.02% trifluoroacetic acid 40:60
Flow rate: 0.75
Injection volume: 10
Detector: MS, ThermoQuest Finnigan LCQ ion trap, APCI positive mode, vaporizer 450◦ ,
capillary 200◦ , capillary 9 V, discharge current 5 µA, sheath gas 0.6 L/min, auxiliary
gas 3 L/min, m/z 405
CHROMATOGRAM
Retention time: 2.2
OTHER SUBSTANCES
Extracted: clindamycin (m/z 425) (3.7)
KEY WORDS
lincomycin is IS in original paper; serum
REFERENCE
Martens-Lobenhoffer, J.; Banditt, P. Sensitive and specific determination of clindamycin in human
serum and bone tissue applying liquid chromatography-atmospheric pressure chemical ionization-mass
spectrometry, J.Chromatogr.B, 2001, 755, 143–149.
SAMPLE
Matrix: blood
Sample preparation: Vortex 250 µL plasma with 50 µL water for 15 s, add 50 µL 20%
trichloroacetic acid in water, vortex for 15 s, centrifuge at 6800 g for 10 min, inject a
20 µL aliquot of the supernatant.
HPLC VARIABLES
Guard column: 25 × 3 5 µm Hypersil RP-18
Column: 100 × 3 5 µm Hypersil RP-18
Mobile phase: Gradient. MeCN:10 mM ammonium acetate from 30:70 to 70:30 over
0.5 min, maintain at 70:30 for 7.5 min, return to initial conditions over 0.5 min, reequilibrate for 9.5 min.
Injection volume: 20
Detector: MS, ThermoQuest Finnigan MAT, ESI, first 3 min and last 8 min of run
diverted to waste, spray 3.5 kV, capillary 5 V, octapole 1 offset – 2 V, lens – 16 V,
Lincomycin
353
octapole 2 offset – 5 V, RF amplitude 400 V, collision energy 1.2 V, m/z 407.3–389.1–
359.2–126.3
CHROMATOGRAM
Retention time: 4.0
Limit of detection: 1.3 ng/mL (for clindamycin)
Limit of quantitation: 50 ng/mL (for clindamycin)
OTHER SUBSTANCES
Extracted: clindamycin (m/z 425.3–389.2–377.2–126.3) (7.4)
KEY WORDS
dog; lincomycin is IS in original; plasma
REFERENCE
Cherlet, M.; Croubels, S.; De Backer, P. Determination of clindamycin in animal plasma by highperformance liquid chromatography combined with electrospray ionization mass spectrometry, J.Mass
Spectrom., 2002, 37, 848–853.
354
Lindane
Lindane
Cl
Cl
Cl
Cl
Molecular formula: C6 H6 Cl6
Molecular weight: 290.83
CAS Registry No: 58-89-9
Merck Index: 13, 5523
Cl
Cl
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 300 × 3.9 10 µm µBondapak C18
Mobile phase: MeCN:water 60:40
Flow rate: 2.5
Detector: UV 254
OTHER SUBSTANCES
Simultaneous: hexachlorobenzene
REFERENCE
Gopalaswamy, U.V.; Aiyar, A.S. Biotransformation of lindane in the rat, Bull.Environ.Contam.Toxicol.,
1984, 32, 148–156.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 600 × 0.2 5 µm ODS (Phenomenex)
Mobile phase: Carbon dioxide
Detector: UV 280
CHROMATOGRAM
Retention time: 13
OTHER SUBSTANCES
Simultaneous: carbofuran (8), chlorpyrifos (9.9), naphthol (10.1)
KEY WORDS
SFC; UV restrictor at 100◦ ; pressure from 100 to 300 atm at 4 atm/min
REFERENCE
Pyo, D.; Kim, H.; Li, W.; Lee, M.L. Supercritical fluid chromatographic detection by use of a parallel flow
restrictor, J.Liq.Chromatogr.Rel.Technol., 1997, 20, 3389–3399.
355
Linezolid
Linezolid
O
N
O
Molecular formula: C16 H20 FN3 O4
Molecular weight: 337.35
CAS Registry No: 165800-03-3
Merck Index: 13, 5526
F
N
O
H
N
H
CH3
O
SAMPLE
Matrix: blood
Sample preparation: Condition a 1 mL 100 mg C2 SPE cartridge (Varian) with two
1 mL portions of MeCN and two 1 mL portions of water. Mix 50 µL plasma with 25 µL
water and 1 mL 100 ng/mL IS in water, add to the SPE cartridge, wash with 1 mL
MeCN:water 5:95, elute with 500 µL MeOH. Evaporate the eluate to dryness under a
stream of nitrogen at 40◦ , reconstitute the residue with 200 µL mobile phase, inject a
100 µL aliquot.
HPLC VARIABLES
Column: 150 × 4.6 5 µm Zorbax RXC8
Mobile phase: MeCN:water 20:80
Flow rate: 1
Injection volume: 100
Detector: UV 251
CHROMATOGRAM
Retention time: 11
Internal standard: PNU-101145 ((S)-N-[[3-[3-fluoro-4-[4-[(1-hydroxycyclopropyl)carbonyl]-1-piperazinyl]phenyl]- 2-oxo-5-oxazolidinyl]methyl]-acetamide) (9)
Limit of detection: 10 ng/mL
KEY WORDS
dog; mouse; plasma; rabbit; rat; SPE
REFERENCE
Peng, G.W.; Stryd, R.P.; Murata, S.; Igarashi, M.; Chiba, K.; Aoyama, H.; Aoyama, M.; Zenki, T.;
Ozawa, N. Determination of linezolid in plasma by reversed-phase high-performance liquid
chromatography, J.Pharm.Biomed.Anal., 1999, 20, 65–73.
SAMPLE
Matrix: blood
Sample preparation: Condition a Sep-Pak C18 with 1 mL MeCN and 1 mL water. Mix
500 µL plasma with 50 µL 10 µg/mL IS in MeCN, add to the SPE cartridge, wash with
1 mL water, elute with 300 µL MeCN. Evaporate the eluate to dryness under a stream
of nitrogen at 50◦ , reconstitute the residue with 100 µL MeCN, inject a 10 µL aliquot.
HPLC VARIABLES
Column: 150 × 4.6 5 µm Shim-Pack CLC-CN C18
Mobile phase: MeCN:20 mM ammonium acetate 80:20
Flow rate: 1
Injection volume: 10
Detector: MS, Finnigan LCQ, APCI, corona discharge 4.5 kV, vaporization 430◦ , capillary 150◦ 32 V, positive mode, m/z 296.2
356
Linezolid
CHROMATOGRAM
Retention time: 3.44
Internal standard: N-carbobenzoxy-3-fluoro-4-morpholinylaniline (m/z 223.2) (2.91)
Limit of detection: 50 ng/mL
Limit of quantitation: 100 ng/mL
KEY WORDS
plasma; SPE
REFERENCE
Phillips, O.A.; Abdel-Hamid, M.E.; Al-Hassawi, N.A. Determination of linezolid in human plasma by
LC-MS-MS, Analyst, 2001, 126, 609–614.
ANNOTATED BIBLIOGRAPHY
Ehrlich, M.; Trittler, R.; Daschner, F.D.; Kümmerer, K. A new and rapid method for monitoring the new
oxazolidinone antibiotic linezolid in serum and urine by high performance liquid chromatographyintegrated sample preparation, J.Chromatogr.B, 2001, 755, 373–377. [column-switching]
Gentry-Nielsen, M.J.; Olsen, K.M.; Preheim, L.C. Pharmacodynamic activity and efficacy of linezolid in a
rat model of pneumococcal pneumonia, Antimicrob.Agents Chemother., 2002, 46, 1345–1351. [linezolid;
rat; ceftriaxone; cephalexin is internal standard]
Gross, M.; Bürli, R.; Jones, P.; Garcia, M.; Batiste, B.; Kaizerman, J.; Moser, H.; Jiang, V.; Hoch, U.;
Duan, J.-X.; Tanaka, R.; Johnson, K.W. Pharmacology of novel heteroaromatic polycycle antibacterials,
Antimicrob.Agents Chemother., 2003, 47, 3448–3457. [LC-MS; linezolid; vancomycin]
Johnson, R.A.; Haan, D.E.; James, C.A.; Hopkins, N.K. Determination of linezolid, PNU-100766, in
human plasma and urine using high-performance liquid chromatography with ultraviolet detection
(Abstract 2487), Pharm.Res., 1997, 14, S374.
Sisson, T.L.; Jungbluth, G.L.; Hopkins, N.K. A pharmacokinetic evaluation of concomitant administration of linezolid and aztreonam, J.Clin.Pharmacol., 1999, 39, 1277–1282. [SPE]
Welshman, I.R.; Sisson, T.A.; Jungbluth, G.L.; Stalker, D.J.; Hopkins, N.K. Linezolid absolute bioavailability and the effect of food on oral bioavailability, Biopharm.Drug Dispos., 2001, 22, 91–97.
357
Liothyronine
Liothyronine
Molecular formula: C15 H12 I3 NO4
Molecular weight: 650.97
CAS Registry No: 6893-02-3
Merck Index: 13, 5532
O
HO
I
I
OH
NH2
O
I
SAMPLE
Matrix: blood, urine
Sample preparation: Condition a diol SPE cartridge with 3 mL MeOH and 3 mL water.
Serum. Vortex 40 µL serum with 50 µL MeCN, dilute with 200 µL water, centrifuge at
3500 rpm for 15 min, remove organic solvents under a stream of nitrogen at 45◦ , add
to the SPE cartridge, elute with 3 mL MeOH. Evaporate the eluate to dryness under a
stream of nitrogen at 45◦ , reconstitute the residue with 200 µL 1 µg/mL IS in water,
inject a 20 µL aliquot. Urine. Add 100 µL urine to the SPE cartridge, elute with 3 mL
MeOH. Evaporate the eluate to dryness under a stream of nitrogen at 45◦ , reconstitute
the residue with 200 µL 1 µg/mL IS in water, inject a 20 µL aliquot.
HPLC VARIABLES
Column: 150 × 4.0 5 µm Inertsil ODS-3
Mobile phase: MeOH:2% acetic acid 65:35
Flow rate: 1
Injection volume: 20
Detector: UV 240
CHROMATOGRAM
Retention time: 3
Internal standard: anthraquinone (10.4)
Limit of detection: 1 ng
OTHER SUBSTANCES
Extracted: levothyroxine (LOD 2 ng) (4.7)
KEY WORDS
serum; SPE
REFERENCE
Samanidou, V.F.; Gika, H.G.; Papadoyannis, I.N. Rapid HPLC analysis of thyroid gland
hormones tri-iodothyronine (T3) and thyroxine (T4) in human biological fluids after SPE,
J.Liq.Chromatogr.Rel.Technol., 2000, 23, 681–692.
358
Lomerizine
Lomerizine
F
Molecular formula: C27 H30 F2 N2 O3
Molecular weight: 468.53
CAS Registry No: 101477-55-8,
101477-54-7 (di HCl)
Merck Index: 13, 5584
OCH3
H3CO
N
F
OCH3
N
SAMPLE
Matrix: blood, tissue
Sample preparation: Plasma. Mix 100 µL plasma with 10 µL 1 µg/mL IS in MeOH
and 90 µL MeOH, add 200 µL 2 M NaOH, shake for 5 min, vortex with 750 µL n-hexane,
centrifuge at 1500 g for 5 min, repeat the extraction of the aqueous layer. Evaporate the
combined extracts to dryness under a stream of nitrogen, reconstitute the residue with
2 mL MeOH:1 M HCl 90:10, add to a 5 × 5 44–88 µm SP-Toyopearl SPE column, wash
with 1 mL MeOH:water 90:10, elute with 1 mL MeOH:1 M ammonia solution 90:10.
Evaporate the eluate to dryness under a stream of nitrogen, reconstitute the residue
with 50 µL MeCN:20 mM pH 5.5 phosphate buffer 70:30, inject a 5–20 µL aliquot.
Tissue. Homogenize brain tissue with 320 mM sucrose, centrifuge at 1300 g for 3 min.
remove the supernatant and centrifuge it at 17 000 g for 10 min to give the P2 fraction.
Mix 400 µL of the P2 fraction with 20 µL 1 µg/mL IS in MeOH, add 800 µL 2 M NaOH,
shake for 5 min, vortex with 3 mL n-hexane, centrifuge at 1500 g for 5 min, repeat the
extraction of the aqueous layer. Evaporate the combined extracts to dryness under a
stream of nitrogen, reconstitute the residue with 2 mL MeOH:1 M HCl 90:10, add to a
5 × 5 44–88 µm SP-Toyopearl SPE column, wash with 1 mL MeOH:water 90:10, elute
with 1 mL MeOH:1 M ammonia solution 90:10. Evaporate the eluate to dryness under a
stream of nitrogen, reconstitute the residue with 50 µL MeCN:20 mM pH 5.5 phosphate
buffer 70:30, inject a 5–20 µL aliquot.
HPLC VARIABLES
Column: 200 × 2.1 5 µm Inertsil C8
Mobile phase: MeCN:20 mM pH 5.5 potassium phosphate buffer 69:31
Flow rate: 0.2
Injection volume: 5–20
Detector: UV 210
CHROMATOGRAM
Retention time: 7.7
Internal standard: cinnarizine (8.6)
Limit of detection: 100 pg
OTHER SUBSTANCES
Extracted: flunarizine (9.5)
KEY WORDS
brain; plasma; rat; SPE
REFERENCE
Waki, H.; Ando, S. Column liquid chromatography of calcium channel blockers, J.Chromatogr., 1989,
494, 408–412.
359
Lopinavir
Lopinavir
H3C
CH3
H
Molecular formula: C37 H48 N4 O5
Molecular weight: 628.80
CAS Registry No: 192725-17-0
Merck Index: 13, 5594
N
N
O
N
O
O
N
OH
H
CH3
O
H3C
H
SAMPLE
Matrix: blood
Sample preparation: Mix 250 µL plasma with 50 µL MeOH, add 100 µL 2 µg/mL IS
in MeOH, add 250 µL 1 M NaOH, add 3 mL hexane:ethyl acetate 50:50, shake at high
speed for 25 min, centrifuge at 3000 g for 15 min. Evaporate the organic layer to dryness
under a stream of air, reconstitute the residue with 1 mL initial mobile phase, inject a
20 µL aliquot.
HPLC VARIABLES
Guard column: 10 × 2.1 Symmetry Shield
Column: 30 × 2.1 3.5 µm Symmetry C18
Mobile phase: Gradient. MeCN:5 mM pH 3.25 acetate buffer from 25:75 to 80:20 over
4 min using a nonlinear gradient (not specified).
Flow rate: 0.35
Injection volume: 20
Detector: MS, PE Sciex API 3000, turbo ionspray source, column effluent split 1:1 before
entering source
CHROMATOGRAM
Retention time: 3.1
Internal standard: Abbott A-86093 (3.2)
Limit of detection: 750 pg/mL
Limit of quantitation: 16.3 ng/mL
OTHER SUBSTANCES
Extracted: amprenavir (2.7, LOQ 16.3 ng/mL, LOD 380 pg/mL), indinavir (2.0, LOQ
16.3 ng/mL, LOD 1.5 ng/mL), nelfinavir (2.5, LOQ 16.3 ng/mL, LOD 330 pg/mL), ritonavir (2.9, LOQ 51.2 ng/mL, LOD 650 pg/mL), saquinavir (2.4, LOQ 16.3 ng/mL, LOD
780 pg/mL)
KEY WORDS
plasma
REFERENCE
Frerichs, V.A.; DiFrancesco, R.; Morse, G.D. Determination of protease inhibitors using liquid
chromatography-tandem mass spectrometry, J.Chromatogr.B, 2003, 787, 393–403.
SAMPLE
Matrix: blood
Sample preparation: Mix 1 mL plasma with 200 µL 10 µg/mL IS in water, add 200 µL
100 mM NaOH, mix, add 4 mL diethyl ether, shake for 5 min, centrifuge at 2500 rpm for
5 min. Evaporate the organic layer to dryness under a stream of nitrogen, reconstitute
the residue with 200 µL initial mobile phase, inject a 100 µL aliquot.
360
Lopinavir
HPLC VARIABLES
Column: 250 × 4.6 Stability RP18 (CIL, France)
Mobile phase: Gradient. MeCN:50 mM pH 5.65 phosphate buffer from 36:64 to 64:36
over 25 min, to 80:20 (step gradient), maintain at 80:20 for 10 min, re-equilibrate at
initial conditions for 5 min.
Flow rate: 1.5
Injection volume: 100
Detector: UV 240 for 5 min, UV 215 for 22 min, UV 260 for rest of run
CHROMATOGRAM
Retention time: 18.9
Internal standard: JR051012 (Janssen Cilag) (28.2)
Limit of quantitation: 50 ng/mL
OTHER SUBSTANCES
Extracted: amprenavir (11.2), efavirenz (19.9), indinavir (8.5), nelfinavir (24.1), nevirapine (3.3), ritonavir (17.6), saquinavir (16.7)
Noninterfering: acetaminophen, amineptine, amphotericin B, aspirin, bromazepam,
buspirone, citalopram, clobazam, diazepam, didanosine, fluconazole, flunitrazepam,
fluvoxamine, hydroxyitraconazole, isoniazid, itraconazole, lamivudine, loprazolam,
lorazepam, metronidazole, minalcipram, nordiazepam, omeprazole, paroxetine,
pyrimethamine, rifampin, sertraline, stavudine, sulfadiazine, trimethoprim,
venlafaxine, zalcitabine, zidovudine, zolpidem, zopiclone
KEY WORDS
plasma
REFERENCE
Titier, K.; Lagrange, F.; Péhourcq, F.; Edno-Mcheik, L.; Moore, N.; Molimard, M. High-performance liquid chromatographic method for the simultaneous determination of the six HIV-protease inhibitors
and two non-nucleoside reverse transcriptase inhibitors in human plasma, Ther.Drug Monit., 2002,
24, 417–424.
ANNOTATED BIBLIOGRAPHY
Droste, J.A.H.; Verweij-van Wissen, C.P.W.G.M.; Burger, D.M. Simultaneous determination of the HIV
drugs indinavir, amprenavir, saquinavir, ritonavir, lopinavir, nelfinavir, the nelfinavir hydroxymetabolite M8, and nevirapine in human plasma by reversed-phase high-performance liquid chromatography,
Ther.Drug Monit., 2003, 25, 393–399. [indinavir; amprenavir; saquinavir; ritonavir; lopinavir; nelfinavir; nevirapine]
Faux, J.; Venisse, N.; Olivier, J.C.; Bouquet, S. Rapid high-performance liquid chromatography determination of lopinavir, a novel HIV-1 protease inhibitor, in human plasma, Chromatographia, 2001, 54,
469–473. [SPE; LOQ 187 ng/mL]
Faux, J.; Venisse, N.; Le Moal, G.; Dupuis, A.; Bouquet, S. Simultaneous determination of six HIV
protease inhibitors, one metabolite, and two non-nucleoside reverse transcriptase inhibitors in
human plasma by isocratic reversed-phase liquid chromatography after solid-phase extraction,
Chromatographia, 2003, 58, 421–426. [SPE; amprenavir; indinavir; lopinavir; nelfinavir; ritonavir;
saquinavir; efavirenz; nevirapine; prazepam is internal standard]
Justesen, U.S.; Pedersen, C.; Klitgaard, N.A. Simultaneous quantitative determination of the HIV protease inhibitors indinavir, amprenavir, ritonavir, lopinavir, saquinavir, nelfinavir and the nelfinavir
active metabolite M8 in plasma by liquid chromatography, J.Chromatogr.B, 2003, 783, 491–500.
[indinavir; amprenavir; ritonavir; lopinavir; saquinavir; nelfinavir; LOQ 25 ng/mL]
Keil, K.; Frerichs, V.A.; DiFrancesco, R.; Morse, G. Reverse phase high-performance liquid chromatography method for the analysis of amprenavir, efavirenz, indinavir, lopinavir, nelfinavir and its active
metabolite (M8), ritonavir, and saquinavir in heparinized human plasma, Ther.Drug Monit., 2003, 25,
340–346. [LOQ 100 ng/mL]
Leibenguth, P.; Le Guellec, C.; Besnier, J.-M.; Bastides, F.; Macé, M.; Gaudet, M.-L.; Autret-Leca, E.;
Paintaud, G. Therapeutic drug monitoring of HIV protease inhibitors using high-performance liquid
Lopinavir
361
chromatography with ultraviolet or photodiode array detection, Ther.Drug Monit., 2001, 23, 679–688.
[indinavir; saquinavir; lopinavir; ritonavir; nelfinavir; amprenavir; carbamazepine is internal standard;
LOQ 50-100 ng/mL]
Marzolini, C.; Béguin, A.; Telenti, A.; Schreyer, A.; Buclin, T.; Biollaz, J.; Decosterd, L.A. Determination
of lopinavir and nevirapine by high-performance liquid chromatography after solid-phase extraction:
application for the assessment of their transplacental passage at delivery, J.Chromatogr.B, 2002, 774,
127–140. [LOQ 100 ng/mL; clozapine is internal standard]
Poirier, J.-M.; Robidou, P.; Jaillon, P. Simultaneous determination of the six HIV protease inhibitors
(amprenavir, indinavir, lopinavir, nelfinavir, ritonavir, and saquinavir) plus M8 nelfinavir metabolite
and the nonnucleoside reverse transcription inhibitor efavirenz in human plasma by solid-phase
extraction and column liquid chromatography, Ther.Drug Monit., 2002, 24, 302–309. [LOQ 25 ng/mL]
Ray, J.; Pang, E.; Carey, D. Simultaneous determination of indinavir, ritonavir and lopinavir (ABT 378)
in human plasma by high-performance liquid chromatography, J.Chromatogr.B, 2002, 775, 225–230.
[100 ng/mL]
Rentsch, K.M. Sensitive and specific determination of eight antiretroviral agents in plasma by
high-performance liquid chromatography-mass spectrometry, J.Chromatogr.B, 2003, 788, 339–350.
[SPE; amprenavir; efavirenz; indinavir; lopinavir; nelfinavir; nevirapine; ritonavir; saquinavir; LOQ
1–250 ng/mL]
Tribut, O.; Arvieux, C.; Michelet, C.; Chapplain, J.-M.; Allain, H.; Bentué-Ferrer, D. Simultaneous quantitative assay of six HIV protease inhibitors, one metabolite, and two non-nucleoside reverse transcriptase inhibitors in human plasma by isocratic reversed-phase liquid chromatography, Ther.Drug Monit.,
2002, 24, 554–562. [nevirapine; efavirenz; indinavir; amprenavir; nelfinavir; ritonavir; lopinavir;
saquinavir; LOQ 25 ng/mL]
Turner, M.L.; Reed-Walker, K.; King, J.R.; Acosta, E.P. Simultaneous determination of nine antiretroviral compounds in human plasma using liquid chromatography, J.Chromatogr.B, 2003, 784, 331–341.
[indinavir; nelfinavir; saquinavir; ritonavir; amprenavir; delavirdine; efavirenz; lopinavir; metabolites;
LOQ 50 ng/mL]
Usami, Y.; Oki, T.; Nakai, M.; Sagisaka, M.; Kaneda, T. A simple HPLC method for simultaneous
determination of lopinavir, ritonavir and efavirenz, Chem.Pharm.Bull., 2003, 26, 715–718. [LOQ
21–60 ng/mL]
362
Loteprednol etabonate
Loteprednol etabonate
Molecular formula: C24 H31 ClO7
Molecular weight: 466.96
CAS Registry No: 82034-46-6
Merck Index: 13, 5605
O
CH3
HO
CH3
H
H
O
Cl
O
O
CH3
O
H
O
SAMPLE
Matrix: bile, blood, urine
Sample preparation: Vortex 100 µL blood with 200 µL MeCN:DMSO 95:5, cool at 0◦
for several min, centrifuge at 3000 rpm for 10 min, inject an aliquot of the supernatant.
(A similar procedure can also be used for bile and urine, see Bodor,N.; Wu,W.-M.;
Murakami,T.; Engel,S. Soft drugs 19. Pharmacokinetics, metabolism and excretion of a
novel soft corticosteroid, loteprednol etabonate, in rats. Pharm.Res. 1995, 12, 875–879.)
HPLC VARIABLES
Column: 75 × 3.9 4 µm Nova-Pak phenyl
Mobile phase: MeCN:water:acetic acid 45:54:1
Flow rate: 1
Injection volume: 20
Detector: UV 254
CHROMATOGRAM
Retention time: 5.12
Limit of detection: <100 ng/mL
KEY WORDS
rat; whole blood
REFERENCE
Bodor, N.; Murakami, T.; Wu, W.-M. Soft drugs 18. Oral and rectal delivery of loteprednol etabonate, a
novel soft corticosteroid, in rats – for safer treatment of gastrointestinal inflammation, Pharm.Res.,
1995, 12, 869–874.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 75 × 3.9 4 µm Nova-Pak phenyl
Mobile phase: MeCN:water:acetic acid 50:50:1
Flow rate: 1.5
Detector: UV 254
CHROMATOGRAM
Retention time: 2.2
Loteprednol etabonate
363
OTHER SUBSTANCES
Simultaneous: metabolites
REFERENCE
Bodor, N.; Drustrup, J.; Wu, W. Effect of cyclodextrins on the solubility and stability of a novel soft
corticosteroid, loteprednol etabonate, Pharmazie, 2000, 55, 206–209.
Marbofloxacin
H3C
N
O
N
N
CH3
N
Molecular formula: C17 H19 FN4 O4
Molecular weight: 362.35
CAS Registry No: 115550-35-1
Merck Index: 13, 5774
F
COOH
O
SAMPLE
Matrix: blood
Sample preparation: Mix 200 µL plasma with 800 µL 1.5 µg/mL IS in 100 mM pH 7.4
phosphate buffer, add 6 mL chloroform (Caution! Chloroform is a carcinogen!), shake at
200 oscillations/min for 30 min, centrifuge at 13 000 g for 6 min. Evaporate the organic
layer to dryness under a stream of nitrogen at 40◦ , reconstitute the residue with 200 µL
phosphate-buffered saline, inject a 10–80 µL aliquot.
HPLC VARIABLES
Guard column: Novapak C18 Guard-Pak
Column: 150 × 3.9 5 µm Novapak C18
Mobile phase: MeCN:buffer 20:80 (The buffer was 20 mM potassium dihydrogen phosphate, 6 mM phosphoric acid, and 12 mM tetraethylammonium bromide, adjusted to
pH 3.0 with 2 M NaOH.)
Flow rate: 1
Injection volume: 10–80
Detector: F ex 338 em 425
CHROMATOGRAM
Retention time: 2.20
Internal standard: enrofloxacin (3.30)
Limit of detection: 15 ng/mL
OTHER SUBSTANCES
Simultaneous: danofloxacin (2.80), difloxacin (4.52), orbifloxacin (3.09), sarafloxacin
(4.40)
Interfering: ciprofloxacin (2.28), norfloxacin (2.16)
KEY WORDS
plasma
REFERENCE
Garcia, M.A.; Solans, C.; Aramayona, J.J.; Rueda, S.; Bregante, M.A. Determination of marbofloxacin
in plasma samples by high-performance liquid chromatography using fluorescence detection,
J.Chromatogr. B, 1999, 729, 157–161.
SAMPLE
Matrix: tissue
Sample preparation: Condition a 6 mL 10 mm SDB-PRS or MPC-SD (mixed C8 and
cation-exchange) SPE disc (3M) with two 1 mL portions of MeCN:96% acetic acid
95:5. Mix 1 g minced kidney with 150 ng IS, 10 mL MeCN, and 2.5 g sodium sulfate,
homogenize (turrax) for 2 min, centrifuge at 3000 g for 5 min, filter the supernatant
through 2.5 g sodium sulfate and Whatman paper. Acidify the filtrate with 2.5 mL 96%
acetic acid and add to the SPE disc, dry under vacuum for 5 min, elute with four 1 mL
portions of MeOH:1 M ammonia 75:25. Evaporate the eluate to dryness under a stream
364
HPLC Methods for Recently Approved Pharmaceuticals. by George Lunn
Copyright 2005 John Wiley & Sons, Ltd. ISBN: 0-471-66941-5
Marbofloxacin
365
of nitrogen at 35◦ , reconstitute the residue with 300 µL dilute formic acid (pH 2.5), filter
(0.45 µm), inject a 50 µL aliquot.
HPLC VARIABLES
Column: 70 × 4 5 µm Nucleosil 100-5 C18
Column temperature: 25
Mobile phase: Gradient. MeCN:dilute formic acid (pH 2.5) from 2:98 to 70:30 over 5 min,
maintain at 70:30 for 1 min.
Flow rate: 1
Injection volume: 50
Detector: MS, Micromass Quattro triple-stage quadrupole, ESI, 100 µL/min of column
effluent entered the detector, capillary 3.2 kV, source 130◦ , desolvation 400◦ , desolvation
gas nitrogen 650 L/h, nebulizer gas nitrogen 75 L/h, collision gas argon, cone 30 V,
collision energy 15 eV m/z 363–320
CHROMATOGRAM
Retention time: 2.97
Internal standard: quinine (m/z 325–160) (2.74)
Limit of detection: <20 ng/g
OTHER SUBSTANCES
Extracted: cinoxacin (m/z 263–217) (3.91), ciprofloxacin (m/z 332–245) (3.06),
danofloxacin (m/z 358–96) (3.10), enoxacin (m/z 321–206) (2.97), enrofloxacin (m/z
360–245) (3.19), flumequine (m/z 262–202) (4.88), nalidixic acid (m/z 233–215) (4.78),
norfloxacin (m/z 320–233) (3.01), ofloxacin (m/z 362–261) (3.01), oxolinic acid (m/z
262–216) (4.18)
KEY WORDS
kidney; pig; SPE
REFERENCE
Van Vyncht, G.; Jànosi, A.; Bordin, G.; Toussaint, B.; Maghuin-Rogister, G.; De Pauw, E.;
Rodriguez, A.R. Multiresidue determination of (fluoro)quinolone antibiotics in swine kidney using
liquid chromatography-tandem mass spectrometry, J.Chromatogr.A, 2002, 952, 121–129.
ANNOTATED BIBLIOGRAPHY
Ballesteros, O.; Toro, I.; Sanz-Nebot, V.; Navalóan, A.; Vı́lchez, J.L.; Barbosa, J. Optimization of the
composition and pH of the mobile phase used for separation and determination of a series of
quinolone antibacterials regulated by the European Union, Chromatographia, 2002, 56, 413–421.
[ciprofloxacin; enrofloxacin; norfloxacin; marbofloxacin; danofloxacin; pipemidic acid; difloxacin;
sarafloxacin; flumequine; oxolinic acid; LOD 10 ng/mL; fluorescence detection]
Garcia, M.A.; Solans, C.; Calvo, A.; Royo, M.; Hernandez, E.; Rey, R.; Bregante, M.A. HPLC separation
and quantification of ofloxacin enantiomers in rabbit plasma. Application to pharmacokinetic studies,
Chromatographia, 2002, 56, 39–42. [marbofloxacin is internal standard; fluorescence detection]
Gigosos, P.G.; Revesado, P.R.; Cadahı́a, O.; Fente, C.A.; Vazquez, B.I.; Franco, C.M.; Cepeda, A.
Determination of quinolones in animal tissues and eggs by high-performance liquid chromatography
with photodiode-array detection, J.Chromatogr.A, 2000, 871, 31–36. [enrofloxacin; ciprofloxacin;
difloxacin; norfloxacin; marbofloxacin; SPE; LOD 4 ng; UV 280]
Hernández-Arteseros, J.A.; Boronat, I.; Compaño, R.; Prat, M.D. Liquid chromatographic separation
of fluoroquinolone antibacterials used as veterinary drugs, Chromatographia, 2000, 52, 295–300.
[danofloxacin; sarafloxacin; difloxacin; marbofloxacin; enrofloxacin; ciprofloxacin; fluorescence
detection]
Johnson, G.; Westwood, S.; Houghton, R. A generic approach for the HPLC separation of quinolone
antibiotics, Chromatographia, 2002, 55, S127–S131. [nalidixic acid; oxolinic acid; sarafloxacin;
difloxacin; pipemidic acid; flumequine; ofloxacin; marbofloxacin; enrofloxacin; ciprofloxacin;
danofloxacin]
366
Marbofloxacin
Schneider, M.; Thomas, V.; Boisrame, B.; Deleforge, J. Pharmacokinetics of marbofloxacin in dogs after
oral and parenteral administration, J.Vet.Pharmacol.Ther., 1996, 19, 56–61. [ofloxacin is internal
standard]
Toussaint, B.; Bordin, G.; Janosi, A.; Rodriguez, A.R. Validation of a liquid chromatography-tandem
mass spectrometry method for the simultaneous quantification of 11 (fluoro)quinolone antibiotics in
swine kidney, J.Chromatogr.A, 2002, 976, 195–206. [danofloxacin; cinoxacin; ciprofloxacin; enoxacin;
enrofloxacin; flumequine; marbofloxacin; nalidixic acid; norfloxacin; ofloxacin; oxolinic acid; SPE]
Yorke, J.C.; Froc, P. Quantitation of nine quinolones in chicken tissues by high-performance liquid
chromatography with fluorescence detection, J.Chromatogr.A, 2000, 882, 63–77. [ciprofloxacin;
danofloxacin; enrofloxacin; nalidixic acid; oxolinic acid; sarafloxacin; difloxacin; flumequine;
marbofloxacin; LOQ 7.5–150 ng/g]
Masoprocol
Masoprocol
CH3
HO
Molecular formula: C18 H22 O4
Molecular weight: 302.36
CAS Registry No: 500-38-9, 27686-84-6 (meso)
Merck Index: 13, 6726
HO
367
OH
OH
CH3
SAMPLE
Matrix: blood
Sample preparation: Condition a Bond Elut C18 SPE cartridge with three 3 mL
portions of MeOH and three 3 mL portions of water. Mix 500 µL serum with 2.5 µg IS,
add to the SPE cartridge, wash with 3 mL water, elute with 300 µL 100 mM HCl in
MeOH, inject a 100 µL aliquot.
HPLC VARIABLES
Column: 250 × 4.6 5 µm Adsorbosphere C18
Mobile phase: Gradient MeCN:water:trifluoroacetic acid from 30:70:0.1 to 70:30:0.1
over 15 min, maintain at 70:30:0.1 for 15 min.
Flow rate: 1.5
Injection volume: 100
Detector: UV (wavelength not specified)
CHROMATOGRAM
Retention time: 8
Internal standard: tetra-O-methylnordihydroguaiaretic acid (Prepare IS by dissolving
1 equiv masoprocol and 6 equiv potassium carbonate in acetone, add 4 equiv dimethyl
sulfate (Caution! Dimethyl sulfate is highly toxic, particularly upon inhalation, and
a carcinogen! Perform the reaction in a chemical fume hood!), reflux for 8 h, add 6
equiv 1 M HCl, evaporate the acetone under reduced pressure, extract three times with
100 mL portions of ethyl acetate. Combine the organic layers, wash three times with
100 mL portions of 100 mM HCl, wash three times with 100 mL portions of 1 M NaCl,
evaporate the organic layer to dryness, recrystallize the product from dichloromethane.)
(20)
Limit of detection: 500 ng/mL
KEY WORDS
mouse; pharmacokinetics; plasma; SPE
REFERENCE
Lambert, J.D.; Meyers, R.O.; Timmermann, B.N.; Dorr, R.T. Pharmacokinetic analysis by highperformance liquid chromatography of intravenous nordihydroguaiaretic acid in the mouse,
J.Chromatogr.B, 2001, 754, 85–90.
368
Maxacalcitol
Maxacalcitol
H3C
CH3
O
H
CH3
OH
CH3
Molecular formula: C29 H42 O4
Molecular weight: 418.61
CAS Registry No: 103909-75-7
Merck Index: 13, 5783
H
CH2
HO
OH
SAMPLE
Matrix: blood
Sample preparation: Condition a 3 mL 200 mg Bond Elut C18 SPE cartridge with
3 mL MeOH:water 80:20, 2 mL water, and 2 mL 1 M HCl. Condition a 1 mL 100 mg
Bond Elut NH2 SPE cartridge with 1 mL hexane:isopropanol 70:30 and 1 mL hexane:isopropanol 90:10. Mix 20 µL 16 ng/mL IS in EtOH with 1 mL serum, add 200 µL
1 M HCl, mix, add to the C18 SPE cartridge, wash with 2 mL water, wash with
3 mL MeOH:water 50:50, elute with 4 mL MeOH:water 80:20. Evaporate the eluate to dryness under a stream of nitrogen at 60◦ , reconstitute the residue with
100 µL hexane:isopropanol 90:10, add to the NH2 SPE cartridge, wash with 1 mL
hexane:isopropanol 90:10, elute with 2 mL hexane:isopropanol 70:30. Evaporate the
eluate to dryness, reconstitute the residue with 50 µL mobile phase, inject a 20 µL
aliquot.
HPLC VARIABLES
Column: 250 × 2 5 µm Capcell Pak C18 UG120
Mobile phase: MeOH:10 mM ammonium acetate 90:10
Flow rate: 0.2
Injection volume: 20
Detector: MS, Finnigan TSQ-700 triple quadrupole, electrospray 4.5 kV, positive ion,
tube lens 120 V, collision gas argon 2.0 mtorr, collision energy -20 eV, (m/z 436–297)
CHROMATOGRAM
Retention time: 4.5
Internal standard: ED-94 ((5Z,7E,20S)-20α-(2-hydroxy-2-methylpropoxy)-9,10-secopregna-5,7,10(19)-trien-1α,3β-diol) (m/z 422–297) (4.5)
Limit of quantitation: 20 pg/mL
KEY WORDS1
serum; SPE
REFERENCE
Ishigai, M.; Asoh, Y.; Kumaki, K. Determination of 22-oxacalcitriol, a new analog of 1α,25dihydroxyvitamin D3 , in human serum by liquid chromatography-mass spectrometry, J.Chromatogr.B,
1998, 706, 261–267.
369
Medetomidine
Medetomidine
Molecular formula: C13 H16 N2
Molecular weight: 200.28
CAS Registry No: 86347-14-0
Merck Index: 13, 5811
CH3
H3C
CH3
N
N
H
SAMPLE
Matrix: blood
Sample preparation: Condition a Sep-Pak C18 SPE cartridge with water, MeOH, and
100 mM ammonium acetate. Add 5 mL plasma to the SPE cartridge, wash with 100 mM
ammonium acetate, elute with MeOH:100 mM ammonium acetate 75:25. Evaporate the
eluate to dryness under reduced pressure, reconstitute the residue in 200 µL mobile
phase, inject a 50 µL aliquot.
HPLC VARIABLES
Column: 150 × 4.6 5 µm Hitachi gel #3056
Mobile phase: MeOH:100 mM ammonium acetate 65:35
Flow rate: 1
Injection volume: 50
Detector: MS, Hitachi M-1000, APCI interface, drift voltage 21 V, nebulizer 260◦ , vaporizer 399◦ , multiplier voltage 1500 VF, m/z 201
CHROMATOGRAM
Retention time: 7.5
Internal standard: detomidine (m/z 187) (6.5)
Limit of quantitation: 1–2 ng/mL
OTHER SUBSTANCES
Extracted: atipamazole (m/z 213) (8.5), midazolam (m/z 326) (10.5)
KEY WORDS
pharmacokinetics; pig; plasma; SPE
REFERENCE
Kanazawa, H.; Nishimura, R.; Sasaki, N.; Takeuchi, A.; Takai, N.; Nagata, Y.; Matsushima, Y. Determination of medetomidine, atipamazole and midazolam by liquid chromatography-mass spectrometry,
Biomed.Chromatogr., 1995, 9, 188–191.
SAMPLE
Matrix: blood
Sample preparation: Mix 200 µL 250 mM NaOH with 500 µL plasma, add 6 mL
dichloromethane, mix gently for 10 min, centrifuge at 1700 g for 10 min. Evaporate
4 mL of the lower organic layer to dryness under a stream of nitrogen, reconstitute the
residue with 100 µL 50 mM pH 3.2 phosphate buffer, vortex for 1.5 min, centrifuge at
1700 g for 10 min, inject a 50 µL aliquot.
HPLC VARIABLES
Guard column: 20 × 4.6 5 µm Supelguard LC-DP
Column: 250 × 4.6 5 µm Supelcosil LC-DP
Mobile phase: MeCN:50 mM phosphate buffer:triethylamine 27:73:0.05, adjusted to pH
3.2
Flow rate: 1
Injection volume: 50
370
Medetomidine
Detector: UV 215
CHROMATOGRAM
Retention time: 14.6
Limit of detection: 5 ng/mL
OTHER SUBSTANCES
Extracted: atipamezole (16.2)
KEY WORDS
pharmacokinetics; plasma; reindeer
REFERENCE
Ranheim, B.; Horsberg, T.E.; Nymoen, U.; Soli, N.E.; Tyler, N.J.; Arnemo, J.M. Reversal of
medetomidine-induced sedation in reindeer (Rangifer tarandus tarandus) with atipamezole increases
the medetomidine concentration in plasma, J.Vet.Pharmacol.Ther., 1997, 20, 350–354.
ANNOTATED BIBLIOGRAPHY
Kanazawa, H.; Nagata, Y.; Matsushima, Y.; Takai, N.; Uchiyama, H.; Nishimura, R.; Takeuchi, A. Liquid
chromatography-mass spectrometry for the determination of medetomidine and other anaesthetics in
plasma, J.Chromatogr., 1993, 631, 215–220. [SPE; flumazenil; butorphanol; atropine; ketamine;
xylazine; medetomidine; atipamezole; midazolam; dog; LOD 0.5–2.5 ng/mL]
Meglutol
371
Meglutol
Molecular formula: C6 H10 O5
HO
HOOC
CH3
COOH
Molecular weight: 162.14
CAS Registry No: 503-49-1
Merck Index: 13, 5831
SAMPLE
Matrix: blood
Sample preparation: Condition a 30 × 10 column of DEAE-cellulose (Serva) with 10
vol of 100 mM pH 7.0 sodium perchlorate and 15 vol of water. Mix 6 mL plasma with
6 mL MeCN, centrifuge at 4000 g for 10 min, suspend the pellet in 6 mL MeCN:water
50:50, centrifuge at 4000 g for 10 min. Combine the supernatants, adjust the pH of a
10 mL aliquot to 7.0 with 100 mM NaOH, add to the DEAE-cellulose column, wash with
10 mL water, elute with 10 mL 100 mM perchloric acid. Freeze-dry the eluate overnight
at 0◦ , reconstitute the residue with 500 µL mobile phase, centrifuge at 12 000 g for
2 min, inject a 100 µL aliquot.
HPLC VARIABLES
Guard column: 50 × 4 micro-Guard-NH2
Column: 300 × 7.8 Aminex HPX-87 cation-exchange (Bio-Rad)
Mobile phase: 6.5 mM sulfuric acid
Flow rate: 0.5
Injection volume: 100
Detector: UV 210
CHROMATOGRAM
Retention time: 8
OTHER SUBSTANCES
Extracted: adipic acid (12), citric acid (5), 2-oxoglutaric acid (6.4)
KEY WORDS
plasma; SPE
REFERENCE
Lippe, G.; Trevisan, R.; Nosadini, R.; Fabris, R.; Deana, R. 3-Hydroxy-3-methylglutaric, adipic, and
2-oxoglutaric acids measured by HPLC in the plasma from diabetic patients, Clin.Biochem., 1987, 20,
275–279.
372
Melatonin
Melatonin
Molecular formula: C13 H16 N2 O2
Molecular weight: 232.28
CAS Registry No: 73-31-4
Merck Index: 13, 5838
H
N
O
H3CO
N
CH3
H
SAMPLE
Matrix: blood, CSF
Sample preparation: For total melatonin, mix 300 µL 60 mM trichloroacetic acid with
1 mL plasma or CSF, let stand at 0◦ for 10 min, centrifuge at 5000 g for 10 min, adjust
the pH of the supernatant to 7.4 with 20 µL 1 M NaOH, add to a 1 mL Chem-Elut 1001
SPE cartridge (without preconditioning), let stand for 3–5 min, elute with two 4 mL
portions of dichloromethane. Evaporate the eluate to dryness at 37◦ , reconstitute the
residue with 60 µL mobile phase, inject a 40 µL aliquot. For free melatonin add 1 mL
plasma or CSF directly to the SPE cartridge.
HPLC VARIABLES
Column: 150 × 4.6 3 µm Extrasil ODS-2 (EsseCi, Italy)
Mobile phase: MeCN:75 mM sodium acetate 28:72 (pH 5.0)
Flow rate: 1
Injection volume: 40
Detector: F ex 275 em 345; E, Chromsystem EC 41000, 0.9 V
CHROMATOGRAM
Retention time: 5
Limit of quantitation: 0.5 pg/mL (F)
KEY WORDS
plasma; SPE
REFERENCE
Rizzo, V.; Porta, C.; Moroni, M.; Scoglio, E.; Moratti, R. Determination of free and total (free plus proteinbound) melatonin in plasma and cerebrospinal fluid by high-performance liquid chromatography with
fluorescence detection, J.Chromatogr.B, 2002, 774, 17–24.
SAMPLE
Matrix: formulations
Sample preparation: Powder tablets and weigh out an amount equivalent to 100 mg
melatonin, add 100 mL 50 mM pH 3.0 phosphate buffer, sonicate for 10 min, centrifuge
at 3000 rpm for 10 min, filter (0.20 µm), inject an aliquot.
HPLC VARIABLES
Guard column: 50 × 4.6 5 µm Hypersil ODS
Column: 250 × 4.6 5 µm Hypersil ODS
Mobile phase: MeCN:MeOH:15 mM pH 2.43 phosphate buffer 14:19:67 containing
0.25% triethylamine (pH 3.0)
Flow rate: 1
Injection volume: 20
Detector: UV 223
CHROMATOGRAM
Retention time: 6.0
Internal standard: adenosine (2.2)
Melatonin
373
Limit of detection: 65 ng/mL
Limit of quantitation: 125 ng/mL
KEY WORDS
tablets
REFERENCE
Raggi, M.A.; Bugamelli, F.; Pucci, V. Determination of melatonin in galenic preparations by LC and
voltammetry, J.Pharm.Biomed.Anal., 2002, 29, 283–289.
ANNOTATED BIBLIOGRAPHY
Andrisano, V.; Bertucci, C.; Battaglia, A.; Cavrini, V. Photostability of drugs: photodegradation of melatonin and its determination in commercial formulations, J.Pharm.Biomed.Anal., 2000, 23, 15–23.
[post-column photochemical derivatization]
Azekawa, T.; Sano, A.; Aoi, K.; Sei, H.; Morita, Y. Concurrent on-line sampling of melatonin in pineal
microdialysates from conscious rat and its analysis by high-performance liquid chromatography with
electrochemical detection, J.Chromatogr., 1990, 530, 47–55.
Bechgaard, E.; Lindhardt, K.; Martinsen, L. High-performance liquid chromatographic analysis of melatonin in human plasma and rabbit serum with on-line column enrichment, J.Chromatogr.B, 1998, 712,
177–181. [column-switching; LOD <1 ng/mL]
Chanut, E.; Nguyen-Legros, J.; Versaux-Botteri, C.; Trouvin, J.H.; Launay, J.M. Determination of melatonin in rat pineal, plasma and retina by high- performance liquid chromatography with electrochemical
detection, J.Chromatogr.B, 1998, 709, 11–18. [LOD 15 pg/mL]
Chin, J.R. Determination of six indolic compounds, including melatonin, in rat pineal using highperformance liquid chromatography with serial fluorimetric-electrochemical detection, J.Chromatogr.,
1990, 528, 111–121.
Dubbels, R.; Reiter, R.J.; Klenke, E.; Goebel, A.; Schnakenberg, E.; Ehlers, C.; Schiwara, H.W.;
Schloot, W. Melatonin in edible plants identified by radioimmunoassay and by high performance
liquid chromatography-mass spectrometry, J.Pineal Res., 1995, 18, 28–31.
Eriksson, K.; Ostin, A.; Levin, J.-O. Quantification of melatonin in human saliva by liquid
chromatography-tandem mass spectrometry using stable isotope dilution, J.Chromatogr.B, 2003,
794, 115–123. [SPE; LOD 1 pg/mL]
Goldman, M.E.; Hamm, H.; Erickson, C.K. Determination of melatonin by high-performance liquid
chromatography with electrochemical detection, J.Chromatogr., 1980, 190, 217–220.
Hall, F.; Tengerdy, C.; Morita, M.; Pautler, E. Determination of bovine retinal melatonin with HPLC-EC,
Curr.Eye Res., 1985, 4, 847–850.
Hamase, K.; Tomita, T.; Kiyomizu, A.; Zaitsu, K. Determination of pineal melatonin by precolumn derivatization reversed-phase high-performance liquid chromatography and its application to the study of
circadian rhythm in rats and mice, Anal.Biochem., 2000, 279, 106–110.
Harumi, T.; Akutsu, H.; Matsushima, S. Simultaneous determination of serotonin, N-acetylserotonin
and melatonin in the pineal gland of the juvenile golden hamster by high-performance liquid chromatography with electrochemical detection, J.Chromatogr.B, 1996, 675, 152–156.
Härtter, S.; Morita, S.; Bodin, K.; Ursing, C.; Tybring, G.; Bertilsson, L. Determination of exogenous
melatonin and its 6-hydroxy metabolite in human plasma by liquid chromatography-mass spectrometry, Ther.Drug Monit., 2001, 23, 282–286. [LC-MS; LOD <2 ng/mL]
Hernandez, G.; Abreu, P.; Alonso, R.; Calzadilla, C.H. Determination of pineal melatonin by highperformance liquid chromatography with electrochemical detection: application for rhythm studies
and tissue explants, J.Pineal Res., 1990, 8, 11–19.
Iinuma, F.; Hamase, K.; Matsubayashi, S.; Takahashi, M.; Watanabe, M.; Zaitsu, K. Sensitive determination of melatonin by precolumn derivatization and reversed-phase high-performance liquid
chromatography, J.Chromatogr.A, 1999, 835, 67–72. [derivatization; LOD 500 amole; rat pineal
gland]
Itoh, M.T.; Hattori, A.; Sumi, Y. Hydroxyindole-O-methyltransferase activity assay using highperformance liquid chromatography with fluorometric detection: determination of melatonin
enzymatically formed from N-acetylserotonin and S-adenosyl-L-methionine, J.Chromatogr.B, 1997,
692, 217–221.
374
Melatonin
Kulczykowska, E.; Iuvone, P.M. Highly sensitive and specific assay of plasma melatonin using highperformance liquid chromatography with fluorescence detection preceded by solid-phase extraction,
J.Chromatogr.Sci., 1998, 36, 175–178. [fish; LOD 3 pg/mL]
Mills, M.H.; King, M.G.; Keats, N.G.; McDonald, R.A. Melatonin determination in human urine by highperformance liquid chromatography with fluorescence detection, J.Chromatogr., 1986, 377, 350–355.
Naylor, S.; Johnson, K.L.; Williamson, B.L.; Klarskov, K.; Gleich, G.J. Structural characterization of
contaminants in commercial preparations of melatonin by on-line HPLC-electrospray ionizationtandem mass spectrometry, Adv.Exp.Med.Biol., 1999, 467, 769–777.
Peniston-Bird, J.F.; Di, W.L.; Street, C.A.; Kadva, A.; Stalteri, M.A.; Silman, R.E. HPLC assay of melatonin in plasma with fluorescence detection, Clin.Chem., 1993, 39, 2242–2247.
Presits, P.; Molnár-Perl, I. HPLC of tryptophan and its metabolites using simultaneously UV, native
fluorescence and pre-column fluorescence derivatization, Chromatographia, 2003, 57, S87–S92. [UV
detection; fluorescence detection; niacin; niacinamide; melatonin]
Sagara, Y.; Okatani, Y.; Yamanaka, S.; Kiriyama, T. Determination of plasma 5-hydroxytryptophan, 5hydroxytryptamine, 5-hydroxyindoleacetic acid, tryptophan and melatonin by high-performance liquid
chromatography with electrochemical detection, J.Chromatogr., 1988, 431, 170–176.
Torano, J.S.; van Rijn-Bikker, P.; Merkus, P.; Guchelaar, H.-J. Quantitative determination of melatonin
in human plasma and cerebrospinal fluid with high-performance liquid chromatography and fluorescence detection, Biomed.Chromatogr., 2000, 14, 306–310. [fluorescence detection; 5-fluorotryptamine
is internal standard; LOD 8 pg/mL]
Vitale, A.A.; Ferrari, C.C.; Aldana, H.; Affanni, J.M. Highly sensitive method for the determination of
melatonin by normal-phase high-performance liquid chromatography with fluorometric detection,
J.Chromatogr.B, 1996, 681, 381–384.
Wakabayashi, H.; Shimada, K.; Aizawa, Y. Determination of serotonin and melatonin in rat pineal
gland by high-performance liquid chromatography with ultraviolet and fluorometric dual detection,
Chem.Pharm.Bull., 1985, 33, 3875–3880.
Wakabayashi, H.; Shimada, K.; Aizawa, Y. Variation of melatonin and serotonin content in rat pineal
gland with sex and oestrous phase difference determined by high-performance liquid chromatography
with fluorimetric detection, J.Chromatogr., 1986, 381, 21–28.
Yamada, K.; Sakamoto, Y.; Satoh, T. A simple method for the measurement of pineal melatonin with a
high performance liquid chromatography-UV detection, Res.Commun.Chem.Pathol.Pharmacol., 1984,
46, 283–287.
Melengestrol acetate
Melengestrol acetate
O
CH3
Molecular formula: C25 H32 O4
Molecular weight: 396.52
CAS Registry No: 2919-66-6, 5633-18-1 (melengestrol only)
Merck Index: 13, 5840
CH3
CH3
OCOCH3
CH2
H
H
375
H
O
CH3
SAMPLE
Matrix: tissue
Sample preparation: Condition a 3 mL 500 mg CN SPE cartridge with 3 mL ethyl
acetate and 5 mL hexane. Cut about 10 g partially thawed fat into small pieces, place
in a glass funnel with the outlet covered by glass wool, heat the fat in a microwave oven
at high power for 2 or 3 min, collect the rendered fat in a beaker. Add 5 mL MeCN to
2 g liquid fat, heat at 60◦ , shake for 5 min on a reciprocating shaker at high speed, cool
in an ice bath for 5 min, centrifuge at 3000 g for 5 min, decant the acetonitrile extract,
repeat the extraction procedure, combine the extracts. Add 3 mL hexane to the combined
extracts, vortex for 30 s, centrifuge at 1800 g for 5 min, discard the hexane, repeat the
hexane wash. Dry the MeCN layer under a stream of nitrogen at 60◦ , dissolve the residue
in 4 mL hexane, add 1 mL 100 mM NaOH, add 500 µL 1 M magnesium chloride, shake
for 30 s, heat at 60◦ for 15 min, centrifuge at 1800 g for 5 min. Remove the hexane, add
4 mL hexane to the residue, shake for 30 s, centrifuge at 1800 g for 5 min. Combine the
hexane extracts and evaporate to dryness under a stream of nitrogen at 60◦ (to remove
any water), reconstitute with 1 mL hexane. Add the sample to the SPE cartridge, wash
with 5 mL hexane, wash with 6 mL ethyl acetate:hexane 5:95, elute with three 1 mL
and one 500 µL portions of ethyl acetate:hexane 20:80. Evaporate the eluate to dryness
under a stream of nitrogen at 60◦ , reconstitute the residue with 1 mL MeCN:water
70:30, add 10 µL 10% HCl, shake, filter (0.22 µm), inject a 40 µL aliquot.
HPLC VARIABLES
Guard column: 20 × 4.6 5 µm C18 end-capped with 20% carbon load
Column: 250 × 4.6 5 µm C18 end-capped with 20% carbon load
Mobile phase: MeCN:water 70:30
Flow rate: 1
Injection volume: 40
Detector: UV 291
CHROMATOGRAM
Retention time: 9.32
Limit of detection: 3 ppb
Limit of quantitation: 10 ppb
OTHER SUBSTANCES
Extracted: chlormadinone (8.57), megestrol (8.92)
KEY WORDS
cow; fat; pig; SPE
REFERENCE
Andresen, M.T.; Fesser, A.C.E. Liquid chromatographic determination of progestogens in animal fat,
J.AOAC Int., 1996, 79, 1037–1042.
SAMPLE
Matrix: tissue
376
Melengestrol acetate
Sample preparation: Condition a 100 mg C18 SPE cartridge (Varian) with MeOH and
10 mM pH 8.5 Tris buffer. Fill a 22 mL vessel (from bottom to top) with 5 g alumina
(dried at 120◦ for 48 h before use), 6 g anhydrous sodium sulfate, and 2 g melted
(microwave) kidney fat. There is filter paper between the alumina and sodium sulfate.
Pass hexane down through the layers at 60◦ at 1500 psi followed by MeCN at 50◦ at
1500 psi. Store the MeCN at −20◦ for 30 min to precipitate fat, filter through a plug
of glass wool, evaporate to dryness under a stream of nitrogen at 40◦ , reconstitute the
residue with 2 mL MeOH, mix with 5 mL water, add to the SPE cartridge, wash with
2 mL 20 mM pH 8.5 Tris buffer, wash with 2 mL MeOH:water 40:60, elute with 2 mL
MeOH. Evaporate the eluate to dryness under a stream of nitrogen at 40◦ , reconstitute
the residue with 75 µL MeCN, add 75 µL 0.5% formic acid, mix, inject a 75 µL aliquot.
(The extraction is done in a Dionex ASE system (advanced solvent extraction).)
HPLC VARIABLES
Column: 150 × 3 5 µm Symmetry (Waters)
Column temperature: 40
Mobile phase: Gradient. A:B from 45:55 to 100:0 over 12 min. A was MeCN:water:formic
acid 10:90:0.5. B was MeCN:water:formic acid 90:10:0.5.
Flow rate: 0.4
Injection volume: 75
Detector: MS, Micromass Quattro Ultima, positive electrospray, capillary 2.5 kV, cone
40 V, source 120◦ , cone gas 189 L/h, desolvation gas 652 L/h, m/z 397.2–279.3–279.3
CHROMATOGRAM
Retention time: 8.8
Limit of detection: <2 ng/g
OTHER SUBSTANCES
Extracted: chloromadinone acetate (m/z 405.2–309.2–345.3) (8.6), chlorotestosterone
acetate (m/z 365.2–305.2–323.3) (12.2), delmadinone acetate (m/z 403.2–205.1–181.1)
(7.8), flurogestone acetate (m/z 407.2–267.4–225.4) (4.5), medroxyprogesterone acetate
(m/z 387.2–327.3–285.3) (8.8), megestrol acetate (m/z 385.2–267.3–325.3) (8.4)
KEY WORDS
fat; kidney; SPE
REFERENCE
Hooijerink, H.; van Bennekom, E.O.; Nielen, M.W.F. Screening for gestagens in kidney fat using
accelerated solvent extraction and liquid chromatography electrospray tandem mass spectrometry,
Anal.Chim.Acta, 2003, 483, 51–59.
ANNOTATED BIBLIOGRAPHY
Campbell, H.M.; Sauvé, F. Liquid chromatographic determination of melengestrol acetate in feeds,
J.AOAC Int., 1993, 76, 1163–1167.
Chichila, T.M.; Edlund, P.O.; Henion, J.D.; Epstein, R.L. Determination of melengestrol acetate in
bovine tissues by automated coupled-column normal-phase high-performance liquid chromatography,
J.Chromatogr., 1989, 488, 389–406. [column-switching]
Huopalahti, R.P.; Henion, J.D. Application of supercritical fluid extraction and high performance liquid
chromatography/mass spectrometry for the determination of some anabolic agents directly from bovine
tissue samples, J.Liq.Chromatogr.Rel.Technol., 1996, 19, 69–87. [UV detection; LC-MS; cow; muscle;
liver; LOD 100 ppb; SFE; dexamethasone; trenbolone; triamcinolone acetonide; zeranol; diethylstilbestrol; medroxyprogesterone; melengestrol acetate]
Joos, P.E.; Van Ryckeghem, M. Liquid chromatography-tandem mass spectrometry of some anabolic
steroids, Anal.Chem., 1999, 71, 4701–4710. [taleranol; estriol; trenbolone; boldenone; fluoxymesterone;
nortestosterone; nandrolone; methylboldenone; zeranol; estrone; ethinyl estradiol; diethylstilbestrol;
Melengestrol acetate
377
hexestrol; estradiol; dienestrol; testosterone; estranediol; acetoxyprogesterone; delmadinone;
norgestrel; methyltestosterone; methandriol; medroxyprogesterone; progesterone; megestrol;
chlormadinone; melengestrol; norethandrolone; chlortestosterone; stanozolol; hydroxyprogesterone;
caproxyprogesterone]
Parks, O.W.; Shadwell, R.J.; Lightfield, A.R.; Maxwell, R.J. Determination of melengestrol acetate in
supercritical fluid-solid phase extracts of bovine fat tissue by HPLC-UV and GC-MS, J.Chromatogr.Sci.,
1996, 34, 353–357.
Roybal, J.E. High pressure liquid chromatographic determination of melengestrol acetate in dry feed
supplements, J.Assoc.Off.Anal.Chem., 1981, 64, 661–664.
Weigand, J.L.; Dille, D.S. Determination of melengestrol acetate in feedstuffs with liquid chromatographic preparatory column cleanup and quantitative analysis, J.Assoc.Off.Anal.Chem., 1988, 71,
707–709.
378
Memantine
Memantine
Molecular formula: C12 H21 N
Molecular weight: 179.30
CAS Registry No: 19982-08-2
Merck Index: 13, 5851
NH2
H3C
CH3
SAMPLE
Matrix: blood
Sample preparation: Mix 1 mL plasma with 50 µL 1 µg/mL IS in 10 mM HCl, 1 mL
1 M NaOH, and 4 mL n-heptane:isoamyl alcohol 98.5:1.5 on a rocking platform at
low speed for 10 min, centrifuge at 1500 g for 10 min. Add the organic supernatant
to 150 µL 100 mM HCl, mix at moderate speed for 10 min, centrifuge at 1500 g for
10 min. Evaporate the aqueous layer to dryness under reduced pressure at 45◦ for 1 h,
reconstitute the residue with 50 µL 1 M pH 10.3 carbonate buffer, add 25 µL 1% dansyl
chloride in MeCN, vortex, let stand at room temperature for 45 min. Evaporate to
dryness under reduced pressure at 45◦ for 30 min, reconstitute the residue with 125 µL
MeCN:water 75:25, vortex briefly, centrifuge for 3–5 min, inject a 40 µL aliquot of the
supernatant.
HPLC VARIABLES
Column: 250 × 4.6 5 µm Supelcosil LC-18
Mobile phase: MeCN:buffer 73:27 (Prepare mobile phase by mixing 730 mL MeCN,
270 mL 25 mM potassium dihydrogen phosphate, 500 µL orthophosphoric acid, and
600 µL n-butylamine.)
Flow rate: 1.8
Injection volume: 40
Detector: F ex 235 em 470
CHROMATOGRAM
Retention time: 12.1
Internal standard: amantadine (7.6)
Limit of quantitation: 3 ng/mL (S/N 20)
OTHER SUBSTANCES
Extracted: amoxapine (13.72), m-chlorophenylpiperazine (8.21), clovoxamine (8.45),
desipramine (14.54), desmethylcitalopram (6.68), desmethylclomipramine (20.05),
desmethyldoxepin (10.55), desmethylmaprotiline (9.56), desmethylmianserin (12.87),
desmethylsertraline (15.41), desmethyltrimipramine (19.06), fenfluramine (10.02),
fluoxetine (12.94), fluvoxamine (8.36), trans-10-hydroxynortriptyline (5.48), cis-10hydroxynortriptyline (6.18), maprotiline (15.67), norclozapine (8.35), norfenfluramine
(5.32), norfluoxetine (8.50), nortriptyline (17.45), paroxetine (10.15), propranolol (7.11),
protriptyline (14.59), rimantadine (13.48), sertraline (31.70)
Noninterfering: amitriptyline, bupropion, citalopram, clomipramine, clozapine, doxepin, haloperidol, 2-hydroxydesipramine, 2-hydroxyimipramine, imipramine, loxapine,
moclobemide, olanzapine, risperidone
KEY WORDS
derivatization; plasma
REFERENCE
Suckow, R.F.; Zhang, M.F.; Collins, E.D.; Fischman, M.W.; Cooper, T.B. Sensitive and selective liquid
chromatographic assay of memantine in plasma with fluorescence detection after pre-column
derivatization, J.Chromatogr.B, 1999, 729, 217–224.
Memantine
379
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 100 × 4.6 5 µm Prodigy ODS(3) (Phenomenex)
Mobile phase: Gradient. MeOH:water:formic acid from 50:50:0.1 to 70:30:0.1 over 3 min,
maintain at 70:30:0.1 for 2 min, return to initial conditions over 0.5 min, re-equilibrate
for 3.5 min.
Flow rate: 0.8
Injection volume: 100
Detector: MS, Agilent HP-1100 MSD, drying gas 30 L/min at 300◦ , nebulizer pressure
30 psi, capillary entrance 3500 V, capillary exit 70 V, m/z 180
CHROMATOGRAM
Retention time: 3.6
Limit of quantitation: 100 pM (water), 500 pM (PBS)
KEY WORDS
use silanized glassware
REFERENCE
Koeberle, M.J.; Hughes, P.M.; Wilson, C.G.; Skellern, G.G. Development of a liquid chromatographymass spectrometric method for measuring the binding of memantine to different melanins, J.Chromatogr.B, 2003, 787, 313–322.
ANNOTATED BIBLIOGRAPHY
Duh, T.-H.; Wu, H.-L.; Kou, H.-S.; Lu, C.-Y. (2-Naphthoxy)acetyl chloride, a simple fluorescent reagent,
J.Chromatogr.A, 2003, 987, 205–209. [derivatization; amantadine; memantine]
380
Menthol
Menthol
CH3
Molecular formula: C10 H20 O
Molecular weight: 156.26
CAS Registry No: 89-78-1
Merck Index: 13, 5861
OH
H3C
CH3
SAMPLE
Matrix: blood
Sample preparation: Heat 5 µL serum, 45 µL water, and 200 mM NaOH in
EtOH:water 95:5 at 100◦ for 15 min, cool, add 1 mL water, add 3 mL hexane, vortex
for 2 min, centrifuge at 3000 rpm for 5 min. Remove the organic layer and evaporate
it to dryness under a stream of nitrogen, reconstitute the residue in 20 µL acetone,
add 50 µL 5 mM 4-dimethylaminopyridine in acetone, mix well, add 100 µL 20 mM
3-(2-phthalimidyl)benzoyl azide in acetone, mix well, heat at 125◦ in a stoppered vial for
30 min, cool in a water bath, inject a 10 µL aliquot. (Omit 4-dimethylaminopyridine for
cholesterol determinations. Synthesis of 3-(2-phthalimidyl)benzoyl azide is as follows.
Mix 680 mg m-aminobenzoic acid in 50 mL diethyl ether with 670 mg o-phthalaldehyde
in 100 mL diethyl ether, mix, stir at room temperature for 2 days, filter to obtain 3-(2phthalimidyl)benzoic acid as a white solid. Dissolve 500 mg 3-(2-phthalimidyl)benzoic
acid in 15 mL DMF, add 560 mg diphenylphosphoryl azide in 4 mL DMF, add 200 mg
triethylamine, stir at 0◦ for 2 h, add 60 mL 5% sodium bicarbonate in water, add 100 mL
diethyl ether, shake. Wash the organic layer twice with 100 mL portions of cold water.
Collect the solid at the interface and combine it with the residue obtained when the
organic layer is evaporated under reduced pressure. Take up the crude product in
acetone, add water to precipitate pure product, repeat this procedure twice to obtain
3-(2-phthalimidyl)benzoyl azide as white needles (mp 123–126◦ ).)
HPLC VARIABLES
Column: 100 × 6 ERC-ODS-1161 (Erma, Tokyo)
Mobile phase: MeCN:water 70:30
Flow rate: 1
Injection volume: 10
Detector: F ex 302 em 440
CHROMATOGRAM
Retention time: 12
Limit of detection: 100–400 fmol
OTHER SUBSTANCES
Extracted: decanol (21), nonanol (14), octanol (10),
KEY WORDS
derivatization; serum; serum extraction only validated for cholesterol
REFERENCE
Tsuruta, Y.; Date, Y.; Kohashi, K. (2-Phthalimidyl)benzoylazides as fluorescence labeling reagents for
alcohols in high-performance liquid chromatography, Anal.Sci., 1991, 7, 411–414.
381
Mepenzolate bromide
Mepenzolate bromide
H3C
CH3
N+
O
Br−
Molecular formula: C21 H26 BrNO3
Molecular weight: 420.35
CAS Registry No: 76-90-4
Merck Index: 13, 5873
O
OH
SAMPLE
Matrix: blood
Sample preparation: Condition a 3 mL diol SPE cartridge (Baker) with 2 mL MeOH
and 2 mL water. Add 1 mL plasma to the SPE cartridge, wash with 2 mL water, wash
with 2 mL MeOH, elute with 3 mL 60 mM KBr in MeOH. Evaporate the eluate to
dryness under a stream of nitrogen at 60◦ , reconstitute the residue with 200 µL mobile
phase, inject an aliquot. (Prepare 60 mM KBr in MeOH by sonicating for 45 min.)
HPLC VARIABLES
Column: 250 × 4 LiChrosorb RP18
Mobile phase: MeOH:water 55:45 containing 4.325 g/L sodium octanesulfonate and
2 mL/L N,N-dimethyloctylamine, adjusted to pH 3.0 with phosphoric acid
Flow rate: 1
Detector: UV 220
CHROMATOGRAM
Retention time: 8
OTHER SUBSTANCES
Extracted: clidinium bromide (9.5), isopropamide iodide (5)
KEY WORDS
plasma; SPE
REFERENCE
Russ-Kirschenbaum, R.; Koziol, T.; Woolf, E. Solid phase extraction of quaternary ammonium compounds
on diol columns: Application to the HPLC determination of CK-1649 in plasma, J.Liq.Chromatogr.,
1989, 12, 3051–3059.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 150 × 4.1 5 µm PRP-1 (Hamilton)
Mobile phase: Gradient. MeCN:50 mM ammonium acetate from 10:90 to 50:50 over
30 min.
Flow rate: 0.5
Injection volume: 20
Detector: MS, Finnigan MAT TSQ-70, thermospray, vaporizer 90◦ , ion-source 250◦ ,
repeller 50–100 V, make up flow 1 mL/min of MeCN:50 mM ammonium acetate 10:90,
collision gas pressure 0.5 Pa, 35 eV, m/z 340
CHROMATOGRAM
Retention time: 14
Limit of detection: 50 pg
382
Mepenzolate bromide
OTHER SUBSTANCES
Simultaneous: antrenyl (m/z 348), clidinium (m/z 352) (15), isopropamide (m/z 353),
pipenzolate (m/z 354) (15), propantheline (m/z 368) (22), valethanate (m/z 306)
REFERENCE
van der Hoeven, R.A.M.; Reeuwijk, H.J.E.M.; Tjaden, U.R.; van der Greef, J. Analysis of quaternary
ammonium drugs by thermospray liquid chromatography-mass spectrometry using a resin-based
stationary phase, J.Chromatogr.A, 1996, 741, 75–84.
383
Mepixanox
Mepixanox
Molecular formula: C20 H21 NO3
Molecular weight: 323.38
CAS Registry No: 17854-59-0
Merck Index: 13, 5885
O
O
OCH3
N
SAMPLE
Matrix: blood
Sample preparation: Add 1 mL plasma or serum and 4 mL 900 ng/mL IS in water to a
previously mixed Toxi Tube A (Analytical Systems), rotate for 5 min, centrifuge at 3000 g
for 5 min. Evaporate the organic layer to dryness under a stream of nitrogen, reconstitute
the residue with 200 µL MeCN:diethylamine 100:0.004, inject an aliquot. (Toxi Tube A
contains 3.5% sodium carbonate, 3.5% sodium bicarbonate, 18% dichloromethane, and
17% dichloroethane, buffered at pH 9.0.)
HPLC VARIABLES
Column: 250 × 4 5 µm Si 60 (Merck)
Mobile phase: MeCN:isopropanol:diethylamine 50:50:0.004
Flow rate: 1
Injection volume: 50
Detector: UV 237
CHROMATOGRAM
Retention time: 6.1
Internal standard: chlorpromazine (8.6)
Limit of detection: 1 ng/mL
Limit of quantitation: 5 ng/mL
OTHER SUBSTANCES
Simultaneous: amitriptyline (12.2), chlordiazepoxide (5.4), dimefline (6.4), dipyrone
(9.1), doxapram (4.0), fluphenazine (2.7), imipramine (15.4), levomepromazine (4.0),
nikethamide (5.5), promazine (12.7), promethazine (8.1), thioridazine (12.0)
KEY WORDS
normal phase; pharmacokinetics; plasma; serum
REFERENCE
Grossi, G.; Lippi, A.; Battistoni, R.; Martelli, E.; Sturani, C. High-performance liquid chromatographic
determination of mepixanthone in serum, J.Chromatogr., 1984, 309, 214–218.
384
Mequinol
Mequinol
OH
CH3
Molecular formula: C7 H8 O2
O
Molecular weight: 124.14
CAS Registry No: 150-76-5
SAMPLE
Matrix: blood
Sample preparation: Mix 100 µL serum with 1 mL 20 µg/mL IS in mobile phase,
inject a 20 µL aliquot.
HPLC VARIABLES
Guard column: present but not specified
Column: 150 × 4.6 5 µm Pinkerton ISRP (Regis)
Mobile phase: MeOH:100 mM pH 6.8 potassium phosphate buffer 2.5:97.5
Flow rate: 1.2
Injection volume: 20
Detector: UV 280
CHROMATOGRAM
Retention time: 5
Internal standard: 4-ethoxyphenol (8)
Limit of detection: 2 µg/mL
OTHER SUBSTANCES
Extracted: acetaminophen (2.9), caffeine (3.8), metoclopramide (3.8), salicylic acid (3.2),
theophylline (4)
KEY WORDS
serum
REFERENCE
Dawson, C.M.; Belcher, H.J.C.R.; Rainbow, S.J.; Tickner, T.R. Measurement of 4-hydroxyanisole in
serum by direct injection high-performance liquid chromatography, J.Chromatogr., 1990, 534, 267–270.
385
Methenamine
Methenamine
Molecular formula: C6 H12 N4
N
N
N
N
Molecular weight: 140.19
CAS Registry No: 100-97-0
Merck Index: 13, 5994
SAMPLE
Matrix: air
Sample preparation: Pull air through a coated filter at 150 mL/min for 20 min,
shake filter with 3 mL MeCN for 1 min, filter (Millex SR) the solution, inject a 10 µL
aliquot. (Prepare filter as follows. Mix 300 mg 2,4-dinitrophenylhydrazine hydrochloride, 500 µL 85% phosphoric acid, 1.5 mL glycerol:EtOH 20:80, and 9 mL MeCN. Dip
glass fiber filter in this solution for a few seconds, let dry on glass surface at room
temperature for 2 h, store in a desiccator over saturated NaCl. Before use, recrystallize
2,4-dinitrophenylhydrazine hydrochloride twice from 4 M HCl. Methenamine is converted to formaldehyde on the filter and the formaldehyde 2,4-dinitrophenylhydrazone
is detected by HPLC.)
HPLC VARIABLES
Column: 100 × 5 10 µm Waters Radial Pak A C18
Mobile phase: MeOH:water 40:60
Flow rate: 0.8
Injection volume: 10
Detector: UV 365
CHROMATOGRAM
Retention time: k′ 2.2
Limit of quantitation: 500 ng/mL
KEY WORDS
derivatization
REFERENCE
Levin, J.-O.; Fängmark, I. High-performance liquid chromatographic determination of hexamethylenetetramine in air, Analyst, 1988, 113, 511–513.
386
Methoprene
Methoprene
Molecular formula: C19 H34 O3
Molecular weight: 310.47
CAS Registry No: 40596-69-8
Merck Index: 13, 6013
CH3
H3CO
H3C
CH3
CH3
O
CH3
O
CH3
SAMPLE
Matrix: blood, urine
Sample preparation: Condition a 3 mL 500 mg Sep-Pak C18 SPE cartridge with 3 mL
MeCN and 3 mL water. Vortex 500 µL plasma or urine with 100 µL 1 M pH 5.0 acetic
acid for 30 s, centrifuge at 1000 g for 5 min, add the supernatant to the SPE cartridge,
wash with 3 mL water, elute with 2 mL MeOH, elute with 2 mL MeCN. Evaporate the
combined eluates to 500 µL under a stream of nitrogen, inject a 10 µL aliquot.
HPLC VARIABLES
Guard column: 20 × 4 5 µm Supelco
Column: 3000 × 3.9 10 µm µBondapak C18
Mobile phase: Gradient. MeCN:water adjusted to pH 4.0 with 1 M acetic acid 55:45
to 60:40 over 10 min, to 80:20 over 3 min, return to initial conditions over 5 min,
re-equilibrate for 2 min.
Flow rate: 0.6 for 9 min, to 1 over 1 min, maintain at 1 for 8 min
Injection volume: 10
Detector: UV 254
CHROMATOGRAM
Retention time: 18.4
Limit of detection: 100 ng/mL (S/N 3)
Limit of quantitation: 150 ng/mL
OTHER SUBSTANCES
Extracted: methoprene acid (12.3), permethrin (UV 210) (17.6), m-phenoxybenzoic acid
(UV 210) (7.9), m-phenoxybenzyl alcohol (UV 210) (7.3)
KEY WORDS
plasma; rat; SPE
REFERENCE
Abu-Qare, A.W.; Abou-Donia, M.B. A solid phase extraction reversed-phase HPLC method for the
simultaneous determination of methoprene, permethrin and selected metabolites in rat plasma and
urine, Biomed.Chromatogr., 2001, 15, 464–470.
Methoxychlor
Methoxychlor
Molecular formula: C16 H15 Cl3 O2
Molecular weight: 345.65
CAS Registry No: 72-43-5
Merck Index: 13, 6020
H3CO
387
OCH3
CCl3
SAMPLE
Matrix: blood, urine
Sample preparation: Add 1 mL urine or whole blood and 3 mL water to a Toxi-Tube A,
mix by gentle inversion for 5 min, centrifuge at 1500 g for 5 min. Evaporate the organic
layer to dryness under a stream of nitrogen at 40◦ , reconstitute the residue with 50 µL
MeCN:water 50:50, vortex, inject a 10 (urine) or 30 (blood) µL aliquot.
HPLC VARIABLES
Guard column: 20 mm long Symmetry C18 (Waters)
Column: 250 × 4.6 5 µm Symmetry C8 (Waters)
Column temperature: 30
Mobile phase: Gradient. MeCN:buffer 15:85 for 6.5 min, 35:65 for 18.5 min (step gradient), 80:20 for 3 min (step gradient), re-equilibrate at initial conditions for 7 min. (The
buffer was 6 g sodium dihydrogen phosphate in 1 L water, adjusted to pH 3.8 with 10%
phosphoric acid.)
Flow rate: 1 for 6.5 min, to 1.5 over 18.5 min, maintain at 1.5 until re-equilibration is
complete
Injection volume: 10–30
Detector: UV 200
CHROMATOGRAM
Retention time: 27.368
OTHER SUBSTANCES
Extracted: acamprosate (abs 200.5) (2.97), acebutolol (abs 233.4) (10.233), acefylline
heptaminol (abs 207.5) (3.628), acenocoumarol (abs 204) (20.052), acepromazine (abs
249.9) (10.763), aceprometazine (abs 240.5) (14.225), acetaminophen (abs 200.5) (5.592),
acetazolamide (abs 265.3) (6.927), acetiamin (abs 200.5) (11.78), acetorphan (abs 200.5)
(22.495), acetylleucine (abs 200.5) (9.825), acetylmethionine (abs 200.5) (5.03), 6acetylmorphine (abs 208.7) (7.32), actinoquinol (abs 202.8) (4.637), acyclovir (abs 200.5)
(3.073), adenosine (abs 206.4) (2.697), adrafinil (abs 200.5) (13.833), albendazole (abs
218.1) (17.777), aldicarb (abs 200.5) (15.143), alfuzosin (abs 244) (10.37), alimemazine
(abs 253.5) (15.257), alminoprofen (abs 201.7) (18.695), almitrine (abs 200.5) (25.905),
alprazolam (abs 220.5) (16.972), altazide (abs 226.3) (16.368), altretamine (abs 229.9)
(17.833), ambemonium (abs 200.5) (10.543), amfepramone (abs 200.5) (8.688), amiloride
(abs 214.6) (3.608), amineptine (abs 200.5) (14.032), amiodarone (abs 204) (21.915),
amisulpride (abs 225.2) (8.923), amitriptyline (abs 206.4) (15.878), amlodipine (abs
200.5) (15.093), amobarbital (abs 200.5) (16.617), amoxapine (abs 211.l) (14.187), amoxicillin (abs 200.5) (3.067), amphetamine (abs 200.5) (3.71), amphotericin B (abs 346.2)
(15.718), ampicillin (abs 200.5) (3.827), anethole (abs 205.2) (23.902), apomorphine (abs
206.4) (8.962), aprindine (abs 200.5) (16.967), arecoline (abs 207.5) (3.1), aspartame
(abs 200.5) (9.8), astemizole (abs 200.5) (13.16), atenolol (abs 200.5) (3.637), atrazine
(abs 221.6) (18.177), atropine (abs 200.5) (10.388), bacampicillin (abs 200.5) (14.687),
bamethan (abs 200.5) (5.91), bamifylline (abs 207.5) (10.288), barbital (abs 200.5)
(10.445), benazepril (abs 205.2) (17.003), bendroflumethiazide (abs 208.7) (18.632), benfluorex (abs 200.5) (16.378), bentazone (abs 224) (13.615), benzbromarone (abs 204)
(26.075), benzene (abs 209.9) (19.465), benzophenone (abs 200.5) (16.967), benzoylecgonine (abs 200.5) (9.678), benzydamine (abs 215.8) (14.955), bepridil (abs 202.8) (19.503),
388
Methoxychlor
betahistine (abs 200.5) (3.155), betamethasone (abs 200.5) (13.277), betaxolol (abs 200.5)
(13.405), bezafibrate (abs 200.5) (18.268), BHT (abs 200.5) (22.015), bidesethylchloroquine (abs 219.3) (3.867), biperiden (abs 200.5) (14.847), biscoumacetate (abs 208.7)
(19.172), bisoprolol (abs 200.5) (12.283), boldine (abs 218.1) (8.068), bromadiolone (abs
202.8) (25.363), bromazepam (abs 232.2) (14.732), bromocriptine (abs 200.5) (16.652),
brompheniramine (abs 200.5) (13.935), buclizide (abs 200.5) (22.752), buflomedil (abs
201.7) (11.3), bumetanide (abs 200.5) (19.055), buprenorphine (abs 212.2) (14.035), buspirone (abs 236.9) (12.523), butobarbital (abs 200.5) (14.858), N-butylscopolammonium
bromide (abs 200.5) (12.232), butylparaben (abs 200.5) (20.332), cafedrine (abs 206.4)
(10.235), caffeic acid (abs 321.2) (8.963), caffeine (abs 205.2) (6.647), canrenoate (abs
287.8) (16.52), captodiamine (abs 200.5) (20.15), captopril (abs 200.5) (9.652), carbamazepine (abs 213.4) (15.763), carbaryl (abs 220.5) (17.968), carbimazole (abs 200.5)
(11.138), carbinoxamine (abs 200.5) (12.81), carbutamide (abs 200.5) (14.547), carbutamine (abs 200.5) (14.475), carnitine (abs 287.8) (16.058), carpipramine (abs 200.5)
(16.18), carteolol (abs 214.6) (5,935), cefadroxil (abs 200.5) (3.188), cefatrizine (abs
200.5) (3.808), cefazolin (abs 200.5) (8.423), cefixime (abs 287.8) (4.823), cefpodoxime (abs
200.5) (18.945), ceftriaxone (abs 245.2) (5.34), cefuroxine (abs 278.3) (16.293, 16.552),
celiprolol (abs 232.2) (11.493), cephalexin (abs 200.5) (3.875, 4.792), cetirizine (abs
200.5) (15.683), chlorambucil (abs 201.7) (22.387), chloramphenicol (abs 200.5) (14.105),
chlordiazepoxide (abs 244) (15.223), chlorhexidine (abs 200.5) (13.532), chloridazone
(abs 227.5) (12.293), chlormadinone (abs 283.1) (24.11), chlormezanone (abs 200.5)
(15.493), chlorobenzoic acid (abs 200.5) (16.143), chlorophacinone (abs 200.5) (20.978),
4-chlorophenylbiguanide (abs 200.5) (10.068), chloropicrin (abs 202.8) (19.433), chloroquine (abs 221.6) (5.442), chlorpheniramine (abs 200.5) (12.925), chlorpromazine (abs
254.7) (16.035), chlorpropamide (abs 200.5) (17.657), chlortoluron (abs 209.9) (17.573),
cibenzoline (abs 200.5) (13.143), cicletanine (abs 200.5) (13.808), cilazapril (abs 200.5)
(14.367), cimetidine (abs 200.5) (3.602), cinchonine (abs 202.8) (10.198), cinnarizine
(abs 200.5) (19.258), ciprofibrate (abs 200.5) (21.22), ciprofloxacin (abs 278.3) (9.102),
cisapride (abs 214.6) (14.627), clenbuterol (abs 211.1) (10.802), clidinium (abs 200.5)
(13.27), clindamycin (abs 200.5) (11.962), clobazam (abs 229.9) (19.19), clobenzorex (abs
200.5) (13.912), clocinizine (abs 200.5) (20.432), clofibrate (abs 200.5) (18.267), clofibride (abs 200.5) (21.067), clomethiazole (abs 249.9) (15.958), clomipramine (abs 200.5)
(16.442), clonazepam (abs 200.5) (17.417), clonidine (abs 200.5) (6.128), clorazepate
(abs 227.5) (18.44), clotiazepam (abs 211.1) (21.652), cloxacillin (abs 200.5) (15.702),
cocaine (abs 200.5) (11.92), codeine (abs 212.2) (4.975), codethyline (abs 211.1) (8.735),
colchicine (abs 244) (13.118), colchicoside (abs 244) (5.32), colchicosine (abs 200.5)
(5.078), cortivazol (abs 207.5) (12.002), cotinine (abs 200.5) (4.7), coumachlor (abs 204)
(22.153), coumafen (abs 205.2) (20.355), coumatetralyl (abs 264.1) (4.993), crimidine
(abs 251.1) (3.703), cyamemazine (abs 270) (14.993), cyclandelate (abs 200.5) (26.383),
cycloguanil (abs 200.5) (10,793), cyproheptadine (abs 224) (15.015), cyromazine (abs
214.6) (3.283), dacarbazine (abs 323.5) (3.6), dapsone (abs 200.5) (12.583), desethylatrazine (abs 213.4) (12.583), desipramine (abs 200.5) (14.87), desoxy-2-phenobarbital
(abs 200.5) (11.047), dexamethasone (abs 241.7) (13.127), dextromethorphan (abs 200.5)
(13.312), dextromoramide (abs 200.5) (15.835), dextropropoxyphene (abs 200.5) (15.82),
di-syston (abs 200.5) (26.795), diamorphine (abs 206.4) (11.152), diaveridine (abs 200.5)
(7.022), diazepam (abs 200.5) (20.327), diazinon (abs 200.5) (25.763), dibekacin (abs
200.5) (14.053), dichlorprop (abs 200.5) (16.947), diclofenac (abs 200.5) (22.115), diethylstilbestrol (abs 200.5) (20.882), difemerine (abs 200.5) (13.222), digitoxin (abs 219.3)
(18.725), digoxin (abs 220.5) (13.852), dihydralazine (abs 219.3) (2.81), dihydrocodeine
(abs 208.7) (4.7), diltiazem (abs 200.5) (13.992), dimerazole (abs 200.5) (8.007), dimethyl
phthalate (abs 200.5) (17.04), dimetridazole (abs 320) (9.955, 10.118), dinoseb (abs
213.4) (23.652), diphacinone (abs 200.5) (19.63), dipheniramine (abs 200.5) (14.092),
diprophylline (abs 206.4) (3.625), dipropyline (abs 207.5) (16.305), dipyridamole (abs
285.5) (13.197), disopyramide (abs 200.5) (11.445), disulfiram (abs 216.9) (25.13), diuron
(abs 211.1) (18.503), domperidone (abs 207.5) (12.355), dosulepin (abs 200.5) (14.943),
doxepin (abs 206.4) (14.095), doxorubicin (abs 232.2) (12.057), doxylamine (abs 200.5)
(11.147), droperidol (abs 202.8) (21.18), dropropizine (abs 200.5) (7.243), econazole (abs
200.5) (20.137), EDDP (abs 200.5) (14.655), embutramide (abs 200.5) (17.365), emetine
(abs 202.8) (9.385), enalapril (abs 211.1) (3.432), enoxacin (abs 268.9) (7.672), ephedrine
Methoxychlor
389
(abs 206.4) (5.655), epinephrine (abs 200.5) (2.87), esculin (abs 202.8) (5.277), eserine (abs 204) (8.253), estazolam (abs 221.6) (16.495), β-estradiol (abs 200.5) (18.202),
estriol (abs 200.5) (13.142), ethosuximide (abs 200.5) (10.485), ethyl paraben (abs 200.5)
(16.19), etodolac (abs 225.2) (21.503), famotidine (abs 202.8) (3.487), felodipine (abs
200.5) (24.423), fenbufen (abs 200.5) (19.292), fenfluramine (abs 207.5) (13.055), fenofibrate (abs 200.5) (18.262), fenoprofen (abs 200.5) (21.16), fenoverine (abs 200.5) (15.57),
fenozolone (abs 220.5) (12.913), fenproporex (abs 200.5) (19.263), fentanyl (abs 255.8)
(14.202), flavone (abs 201.7) (20.768), flecaine (abs 204) (14.417), floctafenine (abs 209.9)
(17.177), flubendazole (abs 211.1) (16.745), fluconazole (abs 200.5) (11.398), flucytosine
(abs 200.5) (3.052), flunarizine (abs 200.5) (19.317), flunindione (abs 222.8) (17.892),
flunitrazepam (abs 200.5) (18.558), fluorouracil (abs 204) (3.433), fluoxetine (abs 200.5)
(16.185), flupenthixol (abs 228.7) (17.358), fluphenazine (abs 259.4) (17.357), flurbiprofen
(abs 200.5) (21.337), flutamide (abs 200.5) (22.248), fluvoxamine (abs 200.5) (15.347), folic
acid (abs 200.5) (3.583), fumaric acid (abs 206.4) (2.985), furaltadone (abs 347.4) (8,927),
furazolidone (abs 347.4) (12.24), furosemide (abs 234.6) (15.17), fusidic acid (abs 200.5)
(24.86), gentamicin (abs 200.5) (14.037), glibenclamide (abs 200.5) (21.953), gliclazide
(abs 200.5) (20.5), glipizide (abs 200.5) (17.603), glutathione (abs 200.5) (2.835), griseofulvin (abs 292.6) (18.392), guaiacol (abs 200.5) (13.787), guaifenesin (abs 200.5) (11.435),
guanfacine (abs 200.5) (11.387), halofantrine (abs 258.2) (22.993), haloperidol (abs 200.5)
(14.415), harpagoside (abs 279.5) (13.48), hematoporphyrin (abs 347.4) (18.66), histidine
(abs 211.1) (2.613), hydrochlorothiazide (abs 226.3) (9.397), hydrocortisone (abs 242.9)
(17.735), 4-hydroxybutyrate (abs 207.5) (2.772), 3-hydroxytyramine (abs 200.5) (3.002),
hydroxyzine (abs 200.5) (15.267), hyoscyamine (abs 200.5) (9.657), ibuprofen (abs 200.5)
(23.815), imidazole (abs 205.2) (2.74), imipramine (abs 200.5) (15.113), inchonidine (abs
202.8) (10.480), indomethacin (abs 201.7) (21.748), indoramine (abs 200.5) (12.533),
iodoform (abs 200.5) (22.05), isoquercitrin (abs 205.2) (10.582), isosorbide (abs 200.5)
(18.48), isothipendyl (abs 248.8) (13.467), isradipine (abs 200.5) (22.352), josamycine (abs
231.1) (16.763), ketamine (abs 202.8) (9.637), ketoconazole (abs 202.8) (15.738), ketoprofen (abs 200.5) (19.628), lacidipine (abs 239.3) (27.235), lactic acid (abs 200.5) (3.227),
lanzoprazole (abs 200.5) (16.613), levamisole (abs 213.4) (6.97), levodopa (abs 200.5)
(3.575), levomepromazine (abs 251.1) (15.842), levopenbutolol (abs 200.5) (15.928), lidocaine (abs 200.5) (9.922), linuron (abs 209.9) (21.253), lisuride (abs 209.9) (4.53), lobeline
(abs 200.5) (14.625), loflazepate (abs 228.7) (21.027), lomustine (abs 229.9) (22.982),
loprazolam (abs 200.5) (13.387), loratadine (abs 200.5) (22.943), lorazepam (abs 228.7)
(17.175), loxapine (abs 209.9) (14.553), LSD (abs 200.5) (12.003), lufenuron (abs 209.9)
(27.658), maprotiline (abs 200.5) (15.508), MCPA (abs 200.5) (15.877), MDA (abs 200.5)
(8.058), MDMA (abs 200.5) (9.058), MDPA (abs 200.5) (11.185), mebendazole (abs 209.9)
(16.077), mebezonium (abs 225.2) (3.4), meclozine (abs 200.5) (19.955), mecoprop (abs
200.5) (17.705), medazepam (abs 200.5) (15.83), medifoxamine (abs 200.5) (13.833),
medroxyprogesterone (abs 241.7) (24.203), mefenorex (abs 207.5) (11.897), mefloquine
(abs 222.8) (16.597), megestrol (abs 290.2) (23.815), melatonin (abs 200.5) (12.923),
melphalan (abs 201.7) (12.93), meperidine (abs 200.5) (11.77), mesalamine (abs 205.2)
(4.737), metamitron (abs 200.5) (11.853), metaproterenol (abs 200.5) (4.15), metformin
(abs 233.4) (2.803), methacycline (abs 242.9) (11.493), methadone (abs 200.5) (15.753),
methamphetamine (abs 206.4) (8.433), methionine (abs 200.5) (2.988), methyclothiazide
(abs 226.3) (15.36), methyldopa (abs 200.5) (2.96), 3-methyldopamine (abs 200.5) (3.23),
4-methyldopamine (abs 200.5) (4.185), methylhydroxyprogesterone (abs 242.9) (21.852),
methylparaben (abs 200.5) (14.04), methylprednisolone (abs 245.2) (18.887), metoclopramide (abs 213.4) (9.915), metoprolol (abs 200.5) (10.722), metronidazole (abs 320)
(6.778), mexiletine (abs 200.5) (11.468), mianserin (abs 200.5) (13.787), miconazole (abs
202.8) (21.53), midazolam (abs 200.5) (14.873), minaprin (abs 204) (11.225), minocycline
(abs 200.5) (22.637), minoxidil (abs 231.1) (9.76), moclobemide (abs 200.5) (10.218),
molsidomine (abs 312.8) (10.007), monodesbutylhalofantrine (abs 258.2) (20.45), monodesethylchloroquine (abs 219.3) (4.675), moroxydine (abs 236.9) (2.873), morphine (abs
211.1) (3.315), N-acetyl-l-tyrosine ethyl ester (abs 200.5) (11.997), nadolol (abs 200.5)
(6.77), nadoxolol (abs 211.1) (12.445), naftidrofuryl (abs 225.2) (15.832), nalidixic acid
(abs 258.2) (16.008), nalorphine (abs 211.1) (4.76), naloxone (abs 200.5) (14.028), naltrexone (abs 205.2) (6.087), nefopam (abs 200.5) (12.653), nemonapride (abs 212.2)
(14.985), neopynamine (abs 209.9) (3.233), neostigmine (abs 200.5) (4.782), netilmicin
(abs 206.4) (12.082), niacin (abs 209.9) (3.163), niacinamide (abs 214.6) (3.61), niaprazine
390
Methoxychlor
(abs 200.5) (11.092), nicardipine (abs 205.2) (15.528), nicergoline (abs 202.8) (15.452),
niclosamide (abs 205.2) (23.92), nifedipine (abs 236.9) (19.485), niflumic acid (abs 200.5)
(21.968), nifuroxazide (abs 200.5) (13.43), nikethamide (abs 200.5) (10.623), nitrazepam
(abs 200.5) (16.927), nitrendipine (abs 236.9) (22.087), nitrofural (abs 260.6) (10.323),
nizatidine (abs 316.4) (3.302), noramidopyrine (abs 200.5) (9.157), norclomipramine
(abs 200.5) (16.617), norephedrine (abs 205.2) (5.015), norepinephrine (abs 200.5) (2.8),
norethisterone (abs 240.5) (24.038), norgestrel (abs 241.7) (21.565), norpropoxyphene
(abs 200.5) (15.478), nortriptyline (abs 206.4) (15.603), noscapine (abs 213.4) (12.827),
ofloxacin (abs 295) (8.648), omeprazole (abs 200.5) (14.065), opiclone (abs 200.5) (10.372),
opipramol (abs 255.8) (14.163), oxacillin (abs 200.5) (14.76), oxatomide (abs 205.2)
(15.797), oxazepam (abs 228.7) (16.745), oxprenolol (abs 200.5) (12.017), pancuronium
(abs 200.5) (3.027), papaverine (abs 251.1) (12.12), paraminosalicylic acid (abs 207.5)
(5.875), parbendazole (abs 207.5) (18.15), parconazole (abs 202.8) (15.5), paroxetine (abs
200.5) (15.275), pefloxacin (abs 278.3) (8.942), penfluridol (abs 200.5) (20.183), pentaerythritol (abs 200.5) (23.082), pentazocine (abs 200.5) (12.522), pentobarbital (abs
200.5) (16.437), pentoxifylline (abs 206.4) (11.477), pentoxyverine (abs 200.5) (15.582),
perindopril (abs 206.4) (13.698), perphenazine (abs 255.8) (15.96), phenindione (abs
226.3) (18.062), phenobarbital (abs 200.5) (13.993), phenol (abs 200.5) (13.422), phenoxyethanol (abs 200.5) (13.375), phenylbutazone (abs 200.5) (24.098), phenytoin (abs
200.5) (16.288), phloroglucinol (abs 202.8) (4.172), pholcodine (abs 211.1) (2.687), phorate (abs 200.5) (26.477), phosdrin (abs 215.8) (13.615), phthalic acid (abs 201.7) (6.59),
pilocarpine (abs 214.6) (4.622), pimaricin (abs 303.3) (13.637), pimozide (abs 205.2)
(17.192), pinaverium (abs 213.4) (21.337), pindolol (abs 215.8) (8.568), pipamperone (abs
200.5) (10.918), piperacillin (abs 200.5) (12.758), piperine (abs 341.5) (20.373), piperonyl
butoxide (abs 202.8) (27.627), pipothiazine (abs 262.9) (14.695), piracetam (abs 200.5)
(3.295), piretanide (abs 200.5) (17.8), pirisudanol (abs 209.9) (3.462), piroxicam (abs
200.5) (16.57), pivampicillin (abs 200.5) (15.813), pizotifene (abs 200.5) (15.197), pralidoxime (abs 295) (2.863), prazepam (abs 200.5) (23.36), prazosin (abs 246.4) (10.608),
prednisolone (abs 246.4) (14.113), prednisone (abs 241.7) (14.178), prifinium (abs 200.5)
(15.622), primidone (abs 200.5) (11.13), pristinamycin (abs 227.5) (17.235), procaine (abs
292.6) (5.218), progesterone (abs 242.9) (23.835), proguanil (abs 200.5) (13.61), promethazine (abs 251.1) (14.482), prometrine (abs 221.6) (21.89), propafenone (abs 211.1)
(15.128), propazine (abs 221.6) (20.433), propericyazine (abs 270) (14.168), propoxur
(abs 200.5) (17.293), propranolol (abs 213.4) (13.06), propylparaben (abs 200.5) (18.34),
proscillaridine a (abs 200.5) (15.585), protopine (abs 205.2) (11.707), psilocybin (abs
220.5) (3.343), pyrazinamide (abs 208.7) (3.823), pyridostigmine (abs 200.5) (3.228),
pyrimethamine (abs 208.7) (12.497), pyroglutamic acid (abs 200.5) (3.008), quercetin
(abs 202.8) (14.168), quinapril (abs 200.5) (16.782), quinidine (abs 208.7) (11.025),
quinine (abs 208) (11.253), quinupramine (abs 200.5) (15.168), ramipril (abs 206.4)
(15.678), ranitidine (abs 228.7) (3.74), reserpine (abs 218.1) (16.433), resorcinol (abs
200) (8.027), rifabutin (abs 208.7) (17.583), rifampin (abs 236.9) (16.167), rifamycin (abs
225.2) (20.85), rilmenidine (abs 200.5) (9.8), ronidazole (abs 308) (8.265), rosmarinic acid
(abs 200.5) (10.637), rosoxacin (abs 271.2) (13.467), roxithromycin (abs 200.5) (15.833),
rutine (abs 204) (10.1), saccharin (abs 200.5) (5.907), salicylic acid (abs 202.8) (12.12),
scopolamine (abs 200.5) (7.39), secnidazole (abs 318.8) (9.668), secobarbital (abs 200.5)
(17.42), selegiline (abs 207.5) (10.712), simazine (abs 220.5) (15.752), sisomicin (abs
200.5) (14.032), sotalol (abs 200.5) (3.842), spironolactone (abs 239.3) (20.68), strychnine
(abs 207.5) (9.202), sulbutiamine (abs 200.5) (12.97), sulfachloropyrazine (abs 271.2)
(14.763), sulfadiazine (abs 200.5) (8.375), sulfadimethoxine (abs 200.5) (14.735), sulfadoxine (abs 200.5) (13.345), sulfaguanidine (abs 200.5) (3.795), sulfamethoxazole (abs
200.5) (13.445), sulfamethoxypyridazine (abs 200.5) (11.217), sulfanilamide (abs 200.5)
(5.047), sulfathiazole (abs 200.5) (9.022), sulindac (abs 200.5) (16.627), sulpiride (abs
212.2) (3.858), sultopride (abs 212.2) (13.012), synephrine (abs 200.5) (3.02), tamoxifen
(abs 200.5) (20.652), temazepam (abs 200.5) (18.562), tenoxicam (abs 200.5) (12.733),
terbutaline (abs 200.5) (3.683), terfenadine (abs 200.5) (19.118), terpine (abs 312.8)
(13.868), tetracaine (abs 312.8) (13.69), tetracycline (abs 274.8) (9.888), tetramisole
(abs 214.6) (6.97), tetrazepam (abs 226.3) (22.378), theobromine (abs 204) (3.79), theodrenaline (abs 201.7) (3.682), theophylline (abs 202.8) (4.877), thiamphenicol (abs
200.5) (7), thiopental (abs 285.5) (19.202), thioproperazine (abs 265.3) (15.212), thioridazine (abs 262.9) (17.168), tianeptine (abs 206.4) (14.877), tiapride (abs 213.4) (5.468),
Methoxychlor
391
tiaprofenic acid (abs 200.5) (17.653), ticlopidine (abs 200.5) (13.813), tiemonium (abs
200.5) (11.795), timolol (abs 296.1) (10.295), tinidazole (abs 317.6) (10.563), tioclomarol
(abs 200.5) (22.525), tobramycin (abs 200.5) (13.393), tofisopam (abs 204) (18.508),
toloxatone (abs 204) (14.107), toluene (abs 215.8) (21.88), trandolapril (abs 206.4)
(16.993), trazodone (abs 211.1) (12.683), triamterene (abs 215.8) (8.705), triazolam
(abs 220.5) (17.353), trichlorocarbanilide (abs 264.1) (25.573), trichloromethiazide (abs
225.2) (14.907), trifluoperazine (abs 258.2) (17.747), trifluperidol (abs 200.5) (21.638),
trihexyphenidyl (abs 200.5) (15.298), trimebutine (abs 213.4) (14.588), trimetazidine
(abs 206.4) (6.06), trimethoprim (abs 205.2) (8.282), trimipramine (abs 200.5) (15.943),
triprolidine (abs 200.5) (13.133), tritoqualine (abs 214.6) (20.968), tropatepine (abs
200.5) (15.425), vanillin (abs 231.1) (11.687), verapamil (abs 201.7) (15.365), veratrine
(abs 220.5) (13.647), veratrole (abs 201.7) (16.377), viloxazine (abs 200.5) (10.993), vinblastine (abs 200.5) (8.37), vincamine (abs 221.6) (12.08), vincristine (abs 220.5) (13.765),
vinylbital (abs 200.5) (16.583), virginiamycin (abs 227.5) (17.21), vitamin B1 (abs 200.5)
(2.597), vitamin B2 (abs 267.7) (7.182), vitamin B5 (abs 200.5) (3.772), vitamin B6 (abs
200.5) (2.895), vitamin B12 (abs 207.5) (3.777), vitamin C (abs 249.9) (2.928), vitamin H (abs 200.5) (8.89), warfarin (abs 205.2) (20.358), xanthydrol (abs 209.9) (18.857),
xipamide (abs 218.1) (18.823), xylene (abs 218.1) (23.985), yohimbine (abs 220.5) (11.51),
zipeprol (abs 205.2) (13.45), zolpidem (abs 208.7) (11.882), zuclopenthixol (abs 206.4)
(16.325) [abs = wavelength of maximum absorbance]
KEY WORDS
whole blood
REFERENCE
Gaillard, Y.; Pépin, G. Use of high-performance liquid chromatography with photodiode-array UV detection for the creation of a 600-compound library. Application to forensic toxicology, J.Chromatogr.A,
1997, 763, 149–163.
SAMPLE
Matrix: cell cultures
Sample preparation: Add 20% MeCN to cell culture, let stand at room temperature
for 15 min, centrifuge at 600 g for 5 min, inject a 400 µL aliquot of the supernatant.
HPLC VARIABLES
Column: 150 × 4.6 5 µm Zorbax SB-C8
Mobile phase: Gradient. MeCN:water:acetic acid 40:60:1 for 2 min, to 90:10:1 over
8 min, maintain at 90:10:1 for 15 min
Injection volume: 400
Detector: UV 254
CHROMATOGRAM
Retention time: 12
OTHER SUBSTANCES
Extracted: metabolites
REFERENCE
Charles, G.D.; Bartels, M.J.; Gennings, C.; Zacharewski, T.R.; Freshour, N.L.; Gollapudi, B.B.; Carney, E.W. Incorporation of S-9 activation into an ER-α transactivation assay, Repro.Toxicol., 2000, 14,
207–216.
392
Methyltestosterone
Methyltestosterone
CH3 OH
CH3
CH3
H
Molecular formula: C20 H30 O2
Molecular weight: 302.45
CAS Registry No: 58-18-4
Merck Index: 13, 6148
H
H
O
SAMPLE
Matrix: blood
Sample preparation: Condition a 1 mL 100 mg Bond Elut C1 SPE cartridge with 2
column vol of MeCN and 2 column vol of water. Mix 500 µL serum with 25 µL 1 µg/mL
IS in MeCN, add 500 µL water, vortex for 5 s, add to the SPE cartridge, wash with
3 column vol of water, wash with 1 column vol of MeCN:water 20:80, air dry at high
vacuum for 10 min. Elute with 1 mL hexane:chloroform 50:50 (Caution! Chloroform
is a carcinogen!), evaporate the eluate to dryness under a stream of nitrogen at 45◦ ,
reconstitute with 50 µL MeCN, vortex while adding 200 µL water, inject a 50 µL aliquot.
HPLC VARIABLES
Guard column: 15 × 4.6 7 µm silica (Brownlee)
Column: 220 × 4.6 5 µm silica (Brownlee)
Column temperature: 50
Mobile phase: MeCN:50 mM pH 2.5 sodium phosphate buffer 21:79
Flow rate: 1
Injection volume: 50
Detector: UV 247
CHROMATOGRAM
Retention time: 5.8
Internal standard: testosterone propionate (9.1)
Limit of detection: 2 ng/mL
KEY WORDS
serum: SPE
REFERENCE
Lampert, B.L.; Stewart, J.T. Determination of anabolic steroids and zeranol in human serum by isocratic
reverse phase HPLC on silica, J.Liq.Chromatogr., 1989, 12, 3231–3249.
393
Metrizamide
Metrizamide
HO
O
H
OH
Molecular formula: C18 H22 I3 N3 O8
OH
OH
Molecular weight: 789.10
CAS Registry No: 31112-62-6
Merck Index: 13, 6176
N
O
H
O
H3C
I
I
N
CH3
N
I
O
CH3
H
SAMPLE
Matrix: blood
Sample preparation: Mix 1 mL plasma with 50 µL 1.5 mg/mL IS and 200 µL 20% zinc
sulfate in water, add 200 µL saturated aqueous barium hydroxide, mix, heat on a steam
bath for 2 min, cool, centrifuge, inject a 25 µL aliquot.
HPLC VARIABLES
Guard column: 60 × 4 Corasil C18
Column: 250 × 4.6 Partisil 10 ODS
Mobile phase: MeOH:water:acetic acid 5:94.5:0.5
Flow rate: 2
Injection volume: 25
Detector: UV 244
CHROMATOGRAM
Retention time: 3.9
Internal standard: 3-dimethylaminomethyleneimino-2,4,6-triiodobenzoic acid (11.2)
Limit of detection: 720 ng/mL
Limit of quantitation: 2.5 µg/mL
KEY WORDS
pharmacokinetics; plasma
REFERENCE
Kullberg, M.P.; Biddlecome, C.E.; Ross, R.W.; Edelson, J. High-performance liquid chromatographic
determination of metrizamide in plasma, J.Chromatogr., 1979, 168, 533–537.
394
Metyrosine
Metyrosine
Molecular formula: C10 H13 NO3
Molecular weight: 195.21
CAS Registry No: 672-87-7
Merck Index: 13, 6183
O
H2N
OH
CH3
HO
SAMPLE
Matrix: blood
Sample preparation: Condition a 1 mL Bond Elut SCX SPE cartridge with 2 mL
MeOH and 2 mL 100 mM HCl. Vortex 1 mL serum with 20 µL 100 µg/mL IS in MeOH
and 1 mL water for 2 min, add to the SPE cartridge, wash with 2 mL water, air dry for
3 min, elute with four 250 µL portions of buffer, inject a 100 µL aliquot. (The buffer was
1 M dipotassium hydrogen phosphate adjusted to pH 5.0 with 1 M phosphoric acid.)
HPLC VARIABLES
Column: 250 × 4.6 5 µm Spherisorb ODS2
Mobile phase: MeCN:MeOH:buffer 5:2.5:92.5 (Prepare mobile phase by dissolving 30 mg
sodium 1-heptanesulfonate hydrate, 1.379 g sodium dihydrogen phosphate monohydrate, 2.5 mL MeOH, and 5 mL MeCN in 100 mL water. Adjust pH to 3.0 with 100 mM
phosphoric acid.)
Flow rate: 1
Injection volume: 100
Detector: F ex 282 em 370
CHROMATOGRAM
Retention time: 10.4
Internal standard: dopamine hydrochloride (8.9)
Limit of detection: 1 µg/mL
OTHER SUBSTANCES
Extracted: methyldopa (LOD 100 ng/mL) (6.2)
KEY WORDS
cow; serum; SPE
REFERENCE
Hefnawy, M.M.; Stewart, J.T. Determination of metyrosine and its metabolite in serum by reversed
phase high performance liquid chromatography using solid phase extraction and fluorescence detection,
J.Liq.Chromatogr.Rel.Technol., 1997, 20, 3009–3016.
SAMPLE
Matrix: solutions
Sample preparation: Prepare metyrosine methyl esters by treatment of racemic metyrosine with absolute MeOH and concentrated sulfuric acid (J.Am.Chem.Soc. 1978, 100,
6536). Dissolve 5 mg of the racemic metyrosine methyl esters in 5 mL MeCN:water
50:50 containing 0.5% v/v triethylamine. Heat a 50 µL aliquot of this solution at 45◦ for
15 min, add 10 µL 0.5% w/v 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl isothiocyanate in
MeCN, heat at 45◦ for 2 h, add 10 µL 0.5% v/v hydrazine in MeCN (Caution! Hydrazine
is carcinogenic and toxic!), heat at 45◦ for 15 min. Evaporate the mixture under a
stream of nitrogen, reconstitute the residue with 250 µL mobile phase, inject a 100 µL
aliquot. (Prepare 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl isothiocyanate solution and
hydrazine solution fresh each day.)
Metyrosine
395
HPLC VARIABLES
Column: 250 × 4.6 5 µm Spherisorb ODS
Mobile phase: MeCN:water 35:65 containing 0.1% triethylamine, adjusted to pH 4.0
with trifluoroacetic acid
Flow rate: 1
Injection volume: 100
Detector: UV 250
CHROMATOGRAM
Retention time: 7.32 (L), 8.73 (D)
KEY WORDS
chiral; derivatization
REFERENCE
Hefnawy, M.M.; Stewart, J.T. HPLC separation of metyrosine enantiomers as methyl esters derivatized
with 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl isothiocyanate, J.Liq.Chromatogr.Rel.Technol., 1998,
21, 381–389.
396
Micafungin
Micafungin
HO
HO
Molecular formula: C56 H71 N9 O23 S
OH
O
O
N
N
H
H3C
H2N
Molecular weight: 1270.29
CAS Registry No: 235114-32-6,
208538-73-2 (Na salt)
Merck Index: 13, 6199
O
OH
O
S
O
O
H
N
N
O
HOH
O H N
HO
H H
CH3
O
H N
N
O
HO
O
N
OH
H
O
O
HO
OH
H3C
SAMPLE
Matrix: blood
Sample preparation: Acidify plasma, add MeCN, centrifuge, dilute the supernatant
with phosphate buffer, inject a 75 µL aliquot.
HPLC VARIABLES
Column: 150 × 4.6 5 µm TSK-GEL ODS80TM
Column temperature: 50
Mobile phase: MeCN:20 mM potassium dihydrogen phosphate 41:59
Flow rate: 1
Injection volume: 75
Detector: F ex 273 em 464
CHROMATOGRAM
Retention time: 12
Limit of detection: 10 ng/mL
Limit of quantitation: 100 ng/mL
KEY WORDS
pharmacokinetics; plasma; rabbit
REFERENCE
Groll, A.H.; Mickiene, D.; Petraitis, V.; Petraitiene, R.; Ibrahim, K.H.; Piscitelli, S.C.; Bekersky, I.;
Walsh, T.J. Compartmental pharmacokinetics and tissue distribution of the antifungal echinocandin
lipopeptide micafungin (FK463) in rabbits, Antimicrob.Agents Chemother., 2001, 45, 3322–3327.
SAMPLE
Matrix: tissue
Sample preparation: Homogenize (Ultra-Turrax 10N head) tissue with 4 vol of ice-cold
pH 7.4 phosphate-buffered saline at 0◦ twice for 30 s each time, let stand at 4◦ C for
30 min, centrifuge at 2000 g for 10 min, add to a C8 SPE cartridge (Varian), wash and
elute using MeCN/50 mM pH 4.0 ammonium acetate mixtures (unspecified). Evaporate
the eluate to dryness under a stream of nitrogen at 40◦ , reconstitute the residue with
MeOH:50 mM pH 4.0 ammonium acetate 50:50, inject a 75 µL aliquot.
HPLC VARIABLES
Column: 150 × 4.6 5 µm Alltech Inertsil C8
Mobile phase: MeCN:20 mM pH 4.0 ammonium acetate 45:55
Flow rate: 0.75
Micafungin
397
Injection volume: 75
Detector: UV 273
CHROMATOGRAM
Retention time: 8
Limit of detection: 10 ng/mL
Limit of quantitation: 50 ng/mL
KEY WORDS
brain; kidney; liver; lung; plasma; SPE; spleen
REFERENCE
Groll, A.H.; Mickiene, D.; Petraitis, V.; Petraitiene, R.; Ibrahim, K.H.; Piscitelli, S.C.; Bekersky, I.;
Walsh, T.J. Compartmental pharmacokinetics and tissue distribution of the antifungal echinocandin
lipopeptide micafungin (FK463) in rabbits, Antimicrob.Agents Chemother., 2001, 45, 3322–3327.
398
Milnacipran
Milnacipran
Molecular formula: C15 H22 N2 O
H2N
Molecular weight: 246.35
CAS Registry No: 92623-85-3
Merck Index: 13, 6216
CH3
N
O
CH3
SAMPLE
Matrix: blood
Sample preparation: Vortex 1 mL whole blood with 3 mL water and 20 µL 100 µg/mL
IS in MeOH for 1 min, add to a Toxi Tube A, shake gently for 5 min, centrifuge at
2800 rpm for 10 min. Evaporate the organic layer to dryness, reconstitute the residue
with 100 µL MeOH, inject a 20 µL aliquot.
HPLC VARIABLES
Column: 250 × 4.6 5 µm Symmetry (Waters)
Column temperature: 30
Mobile phase: MeCN:MeOH:100 mM ammonium acetate 30:30:40
Flow rate: 1
Injection volume: 20
Detector: UV 220
CHROMATOGRAM
Retention time: 4.64
Internal standard: tetrazepam (21.7)
Limit of quantitation: 10 ng/mL
OTHER SUBSTANCES
Extracted: bromazepam (5.7), nordiazepam (10.4)
KEY WORDS
whole blood
REFERENCE
Rop, P.P.; Sournac, M.H.; Burle, J.; Fornaris, M.; Coiffait, P.E. Blood concentration of milnacipran in a
case of a fatal automobile accident, J.Anal.Toxicol., 2002, 26, 123–126.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 × 4.6 5 µm Satisfaction RP 18 AB (CIL, France)
Column temperature: 45
Mobile phase: MeCN:buffer 35:65 (The buffer was 25 mM potassium dihydrogen phosphate and 10 mM triethylamine adjusted to pH 4.80 with phosphoric acid.)
Flow rate: 1
Injection volume: 20
Detector: UV 225
CHROMATOGRAM
Retention time: 4.26
Milnacipran
399
OTHER SUBSTANCES
Simultaneous: citalopram (7.66), desmethylcitalopram (6.98), desmethylmirtazapine
(3.63), desmethylvenlafaxine (3.16), fluoxetine (17.94), fluvoxamine (12.44), mirtazapine
(5.14), norfluoxetine (15.22), paroxetine (10.24), sertraline (20.00), venlafaxine (4.74)
REFERENCE
Dallet, P.; Labat, L.; Richard, M.; Langlois, M.H.; Dubost, J.P. A reversed-phase HPLC method development for the separation of new antidepressants, J.Liq.Chromatogr.Rel.Technol., 2002, 25, 101–111.
ANNOTATED BIBLIOGRAPHY
Duverneuil, C.; de la Grandmaison, G.L.; De Mazancourt, P.; Alvarez, J.-C. A high-performance liquid
chromatography method with photodiode-array UV detection for therapeutic drug monitoring of the
nontricyclic antidepressant drugs, Ther.Drug Monit., 2003, 25, 565–573. [LOD 2.5–10 ng/mL; plasma;
fluoxetine; norfluoxetine; sertraline; paroxetine; citalopram; fluvoxamine; moclobemide; mirtazapine;
milnacipran; toloxatone; venlafaxine; viloxazine]
Titier, K.; Castaing, N.; Scotto-Gomez, E.; Pehourcq, F.; Moore, N.; Molimard, M. High-performance liquid chromatographic method with diode array detection for identification and quantification of the eight
new antidepressants and five of their active metabolites in plasma after overdose, Ther.Drug Monit.,
2003, 25, 581–587. [LOQ 25–100 ng/mL; fluvoxamine; paroxetine; sertraline; fluoxetine; citalopram;
mirtazapine; milnacipran; venlafaxine; norfluoxetine]
400
Mirtazapine
Mirtazapine
Molecular formula: C17 H19 N3
Molecular weight: 265.35
CAS Registry No: 61337-67-5
Merck Index: 13, 6230
N
N
N
CH3
SAMPLE
Matrix: blood
Sample preparation: Mix 1 mL whole blood with 20 µL 1 µg/mL dibenzepin in
MeOH:water 50:50, add 300 µL pH 11 Tris buffer, mix, add 500 µL butyl acetate,
vortex for 2 min, centrifuge. Preserve the aqueous layer (A). Remove the organic layer
and add it to 75 µL 10 mM ammonium acetate buffer containing 0.1% formic acid (pH
3.2), evaporate (?). Add 75 µL MeCN, sonicate for 5 min, centrifuge at 5000 rpm for
5 min, keep the extract as B. Add 20 µL 1 µg/mL enalapril in MeOH:water 50:50 and
120 mg NaCl to the aqueous layer (A), mix, add 500 µL pH 3 phosphate buffer, add
600 µL 8.5% phosphoric acid, add 5 mL dichloromethane:isopropanol 95:5, shake at
250 cycles/min in a bench-top shaker for 30 min, centrifuge at 5000 rpm for 5 min.
Remove the lower organic layer and evaporate it to dryness under a stream of air at
45◦ . Reconstitute the residue with 150 µL initial mobile phase, sonicate, centrifuge,
combine with extract B, inject a 30 µL aliquot. (Sample preparation from Gergov,M.;
Robson,J.N.; Ojanperä,I.; Heinonen,O.P.; Vuori,E. Simultaneous screening and quantitation of 18 antihistamine drugs in blood by liquid chromatography ionspray tandem
mass spectrometry. Forensic Sci.Inter. 2001, 121, 108–115.)
HPLC VARIABLES
Guard column: 40 mm long 4 µm Purospher RP-18 LiChro Cart 4-4
Column: 100 × 2.1 4 µm Genesis C18 (Jones Chromatography)
Column temperature: 35
Mobile phase: Gradient. MeCN:buffer from 20:80 to 100:0 over 10 min, maintain at
0:100 for 3 min, re-equilibrate at initial conditions for 5 min. (The buffer was 10 mM
ammonium acetate containing 0.1% formic acid (pH 3.2).)
Flow rate: 0.2
Injection volume: 30
Detector: MS, PE Sciex API 365 triple-stage quadrupole LC-MS-MS, PE Sciex Turbo Ion
Spray interface, positive ion mode, needle voltage 5.2 kV, nebulizer gas air at 60 psi,
curtain gas nitrogen at 40 psi, collision cell gas nitrogen at 40 psi, turbo ionspray heater
375◦ , heater gas flow 7 L/min
CHROMATOGRAM
Retention time: 4.4
Internal standard: dibenzepin, enalapril
Limit of detection: <20 ng/mL
OTHER SUBSTANCES
Extracted: acebutolol (3.8, LOD 0.1 µg/mL), acrivastine (5.7, LOD <0.02 µg/mL),
alprazolam (6.1, LOD <0.02 µg/mL), alprenolol (5.4, LOD 0.01 µg/mL), amantadine
(3.4, LOD 0.1 µg/mL), amiloride (2.0, LOD 0.1 µg/mL), aminophenazone (2.8, LOD
<5 µg/mL), amiodarone (10.2, LOD 0.05 µg/mL), amitriptyline (6.6, LOD <0.02 µg/mL),
astemizole (5.8, LOD <0.02 µg/mL), atenolol (1.7, LOD 0.30 µg/mL), azacyclonol (5.1,
LOD 0.02 µg/mL), benzhexol (6.6, LOD <0.02 µg/mL), benzoylecgonine (3.3, LOD
0.01 µg/mL), betaxolol (5.5, LOD 0.01 µg/mL), biperidine (6.2, LOD <0.02 µg/mL),
bisoprolol (5.0, LOD <0.02 µg/mL), brompheniramine (5.3, LOD 0.002 µg/mL),
bupivacaine (5.1, LOD <0.02 µg/mL), buprenorphine (5.9, LOD 0.01 µg/mL),
buspirone (5.1, LOD 0.002 µg/mL), caffeine (2.8, LOD 1 µg/mL), carbamazepine
Mirtazapine
401
(6.1, LOD <0.02 µg/mL), carbinoxamine (5.1, LOD 0.002 µg/mL), carisoprodol
(6.7, LOD <5 µg/mL), carvedilol (6.2, LOD <0.02 µg/mL), celiprolol (4.3, LOD
0.05 µg/mL), cetirizine (6.3, LOD 0.05 µg/mL), chlorcyclizine (6.6, LOD <0.02 µg/mL),
chlordiazepoxide (5.7, LOD <0.02 µg/mL), chlormezanone (5.8, LOD <5 µg/mL),
chloroquine (2.7, LOD 0.02 µg/mL), chlorpheniramine (5.1, LOD 0.002 µg/mL),
chlorpromazine (7.0, LOD 0.02 µg/mL), chlorpropamide (6.7, LOD <5 µg/mL),
chlorprothixene (7.0, LOD <0.02 µg/mL), cinnarizine (7.9, LOD <0.02 µg/mL),
citalopram (5.7, LOD <0.02 µg/mL), clemastine (7.7, LOD 0.02 µg/mL), clobazam
(7.3, LOD <0.02 µg/mL), clobutinol (5.3, LOD 0.02 µg/mL), clomethiazole (6.2,
LOD 0.5 µg/mL), clomipramine (7.1, LOD <0.02 µg/mL), clonazepam (6.6, LOD
<0.02 µg/mL), clonidine (2.8, LOD 0.1 µg/mL), clozapine (5.6, LOD <0.02 µg/mL),
cocaine (4.6, LOD <0.02 µg/mL), codeine (2.5, LOD 0.1 µg/mL), coumatetralyl
(8.4, LOD 0.05 µg/mL), cyclizine (5.8, LOD <0.02 µg/mL), dextropropoxyphene
(6.6, LOD 0.05 µg/mL), demoxepam (5.8, LOD 0.02 µg/mL), dextromethorphan
(5.5, LOD <0.02 µg/mL), diazepam (8.1, LOD 0.02 µg/mL), diltiazem (5.8,
LOD <0.02 µg/mL), diphenhydramine (5.7, LOD <0.02 µg/mL), dipyridamole (5.4,
LOD 0.005 µg/mL), disopyramine (4.4, LOD <0.02 µg/mL), dixyrazine (6.8, LOD
0.005 µg/mL), doxapram (4.8, LOD <0.02 µg/mL), doxepin (5.9, LOD <0.02 µg/mL),
ebastine (9.6, LOD 0.005 µg/mL), embutramide (6.7, LOD 0.005 µg/mL), ergotamine
(5.5, LOD 0.005 µg/mL), ethenzamide (5.0, LOD 0.05 µg/mL), ethylmorphine (3.2,
LOD 0.05 µg/mL), ethylparathion (9.7, LOD <5 µg/mL), etodroxizine (6.4, LOD
<0.02 µg/mL), felodipine (9.6, LOD 0.02 µg/mL), fenazepam (7.5, LOD <0.02 µg/mL),
fenfluramine (5.3, LOD <0.02 µg/mL), fenkamfamine (5.1, LOD <0.02 µg/mL), fentanyl
(5.5, LOD <0.02 µg/mL), fexofenadine (6.3, LOD <0.02 µg/mL), flecainide (5.9, LOD
<0.02 µg/mL), fluconazole (4.0, LOD 0.1 µg/mL), flumazenil (5.2, LOD <0.02 µg/mL),
flunitrazepam (7.1, LOD 0.002 µg/mL), fluoxetine (6.8, LOD 0.1 µg/mL), flupentixol
(7.5, LOD 0.18 µg/mL), fluvoxamine (6.3, LOD 0.02 µg/mL), glibenclamide (8.5, LOD
<0.02 µg/mL), glipizide (6.8, LOD <0.05 µg/mL), haloperidol (6.1, LOD <0.02 µg/mL),
histapyrrodine (6.3, LOD 0.02 µg/mL), hydrocodone (3.0, LOD 0.05 µg/mL),
hydroxychloroquine (2.4, LOD <0.3 µg/mL), hydroxyzine (6.3, LOD <0.02 µg/mL),
imipramine (6.4, LOD 0.05 µg/mL), indomethacin (8.6, LOD 0.05 µg/mL), isoniazid (2.2,
LOD 3 µg/mL), isradipine (8.6, LOD 0.05 µg/mL), ketamine (3.6, LOD <0.05 µg/mL),
ketobemidone (3.3, LOD <0.05 µg/mL), ketoprofen (7.3, LOD 0.1 µg/mL), ketorolac (6.2,
LOD 0.05 µg/mL), labetalol (4.9, LOD 0.05 µg/mL), lamotrigine (4.0, LOD 0.1 µg/mL),
levocabastine (5.8, LOD 0.01 µg/mL), levomepromazine (6.5, LOD 0.02 µg/mL), lidocaine
(3.7, LOD <0.05 µg/mL), loratadine (9.3, LOD 0.002 µg/mL), lorazepam (6.6, LOD
0.02 µg/mL), lormetazepam (7.4, LOD <0.02 µg/mL), LSD (4.7, LOD <0.02 µg/mL),
malathion (8.9, LOD 10 µg/mL), maprotiline (6.4, LOD <0.02 µg/mL), MDMA (3.3, LOD
0.02 µg/mL), meclozine (8.5, LOD <0.02 µg/mL), medazepam (6.3, LOD <0.02 µg/mL),
meloxicam (7.1, LOD 0.01 µg/mL), melperone (5.0, LOD <0.02 µg/mL), meperidine
(4.7, LOD <0.02 µg/mL), mepivacaine (3.7, LOD <0.02 µg/mL), meprobamate (4.9,
LOD 0.1 µg/mL), mesoridazine (5.4, LOD <0.02 µg/mL), methamphetamine (3.3,
LOD 0.05 µg/mL), methadone (6.7, LOD <0.02 µg/mL), methylparathion (8.6, LOD
10 µg/mL), methylphenidate (4.2, LOD <0.02 µg/mL), metoclopramide (3.8, LOD
<0.02 µg/mL), metoprolol (4.1, LOD 0.02 µg/mL), metronidazole (2.6, LOD 1 µg/mL),
mexiletine (4.4, LOD 0.05 µg/mL), mianserin (5.7, LOD <0.02 µg/mL), midazolam
(5.9, LOD <0.02 µg/mL), mizolastine (5.5, LOD 0.01 µg/mL), moclobemide (3.7,
LOD 0.05 µg/mL), molindone (4.0, LOD <0.02 µg/mL), monoacetylmorphine (2.7,
LOD 0.1 µg/mL), morphine (2.0, LOD 0.1 µg/mL), nicotine (2.2, LOD 0.05 µg/mL),
nifedipine (7.5, LOD 0.02 µg/mL), nikethamide (3.6, LOD <0.02 µg/mL), nitrazepam
(6.5, LOD <0.02 µg/mL), nizatidine (1.7, LOD 1 µg/mL), nomifensine (4.6, LOD
<0.02 µg/mL), nortriptyline (6.4, LOD <0.02 µg/mL), norverapamil (6.2, LOD 1 µg/mL),
noscapine (5.0, LOD <0.02 µg/mL), olanzapine (3.0, LOD 0.05 µg/mL), ondansetron
(4.6, LOD <0.02 µg/mL), orphenadrine (6.1, LOD <0.02 µg/mL), oxazepam (6.3, LOD
<0.02 µg/mL), oxcarbazepine (5.3, LOD 0.02 µg/mL), oxprenolol (4.7, LOD 0.02 µg/mL),
oxycodone (2.8, LOD 0.05 µg/mL), papaverine (4.8, LOD <0.02 µg/mL), acetaminophen
(2.5, LOD <5 µg/mL), paroxetine (6.2, LOD 0.02 µg/mL), pemoline (3.3, LOD
0.05 µg/mL), pentazocine (5.0, LOD <0.02 µg/mL), pentifylline (7.3, LOD <5 µg/mL),
pentoxyverine (6.6, LOD <0.02 µg/mL), perphenazine (6.9, LOD 0.002 µg/mL),
phenazone (3.9, LOD 0.05 µg/mL), phencyclidine (5.3, LOD 0.05 µg/mL), pheniramine
402
Mirtazapine
(4.1, LOD 0.02 µg/mL), phenylbutazone (9.0, LOD <5 µg/mL), phenylpropanolamine
(2.5, LOD 0.3 µg/mL), phenytoin (6.1, LOD 0.05 µg/mL), pindolol (3.3, LOD 0.05 µg/mL),
piroxicam (6.6, LOD 0.02 µg/mL), pitofenone (5.4, LOD <0.02 µg/mL), pizotifen (6.5,
LOD <0.02 µg/mL), practolol (1.8, LOD 0.1 µg/mL), prazosin (4.1, LOD 0.05 µg/mL),
prilocaine (3.8, LOD <0.02 µg/mL), primidone (4.0, LOD <5 µg/mL), procainamide
(2.2, LOD 0.05 µg/mL), prochlorperazine (7.5, LOD 0.02 µg/mL), promazine (6.2,
LOD <0.02 µg/mL), promethazine (6.0, LOD 0.05 µg/mL), propafenone (6.3, LOD
<0.02 µg/mL), propranolol (5.4, LOD 0.02 µg/mL), propyphenazone (6.6, LOD
0.50 µg/mL), pseudoephedrine (2.6, LOD 1 µg/mL), quinine (4.2, LOD 0.02 µg/mL),
ranitidine (1.8, LOD 0.1 µg/mL), risperidone (4.9, LOD <0.02 µg/mL), rocurone
(3.8, LOD 0.1 µg/mL), ropivacaine (4.6, LOD <0.02 µg/mL), salicylamide (4.2, LOD
<5 µg/mL), selegiline (4.1, LOD 0.05 µg/mL), sertindole (7.2, LOD <0.02 µg/mL),
sertraline (6.8, LOD 0.02 µg/mL), sulindac (6.5, LOD 0.02 µg/mL), simazine (6.0, LOD
0.1 µg/mL), sincocaine (6.5, LOD <0.02 µg/mL), sisapride (5.9, LOD <0.02 µg/mL),
sotalol (2.1, LOD 0.1 µg/mL), strychnine (5.3, LOD 0.05 µg/mL), sulpiride (1.9, LOD
0.1 µg/mL), sulthiame (4.1, LOD 0.05 µg/mL), temazepam (7.2, LOD <0.02 µg/mL),
terbutaline (2.3, LOD 0.1 µg/mL), terfenadine (8.1, LOD 0.002 µg/mL), terodiline
(6.7, LOD <0.02 µg/mL), tetracaine (5.7, LOD <0.02 µg/mL), dronabinol (12.3,
LOD 0.05 µg/mL), tetrahydrozoline (3.6, LOD 0.1 µg/mL), theobromine (2.3, LOD
<5 µg/mL), theophylline (2.4, LOD <5 µg/mL), thioridazine (7.5, LOD 0.02 µg/mL),
timolol (3.8, LOD 0.05 µg/mL), thiothixene (6.7, LOD 0.02 µg/mL), tolbutamide (7.1,
LOD <5 µg/mL), toremifene (8.7, LOD 0.02 µg/mL), tramadol (4.2, LOD 0.02 µg/mL),
trazodone (5.2, LOD <0.02 µg/mL), triamterene (3.2, LOD 0.1 µg/mL), triazolam (6.7,
LOD 0.002 µg/mL), trimeprazine (6.4, LOD <0.02 µg/mL), trimethoprim (3.1, LOD
0.05 µg/mL), trimipramine (6.7, LOD <0.02 µg/mL), venlafaxine (4.9, LOD 0.02 µg/mL),
verapamil (6.5, LOD <0.02 µg/mL), warfarin (7.9, LOD <0.02 µg/mL), yohimbine (4.5,
LOD <0.02 µg/mL), zolpidem (4.7, LOD <0.02 µg/mL), zopiclone (4.0, LOD 0.1 µg/mL)
KEY WORDS
whole blood
REFERENCE
Gergov, M.; Ojanperä, I.; Vuori, E. Simultaneous screening for 238 drugs in blood by
liquid chromatography-ionspray tandem mass spectrometry with multiple-reaction monitoring,
J.Chromatogr.B, 2003, 795, 41–53.
SAMPLE
Matrix: blood
Sample preparation: Condition a 3 mL 130 mg Bond Elut Certify SPE cartridge with
3 mL MeOH, 3 mL water, and 1 mL 100 mM pH 6.0 potassium phosphate buffer. Vortex
1 mL plasma, 2 mL 100 mM pH 6.0 potassium phosphate buffer, and 100 µL 1 µg/mL
IS in EtOH, add to the SPE cartridge, wash with 3 mL water, wash with 1 mL 1 M
acetic acid, wash with 3 mL MeOH, draw air through the column for 5 min, elute with
3 mL dichloromethane:isopropanol:ammonium hydroxide 78:20:2. Evaporate the eluate
to dryness under a stream of air at 55◦ , reconstitute the residue with 120 µL mobile
phase, inject a 80 µL aliquot.
HPLC VARIABLES
Guard column: 25 × 4.6 10 µm Chiralpak AD
Column: 50 × 4.6 10 µm Chiralpak AD
Mobile phase: Hexane:EtOH:isopropanol 98:1:1
Flow rate: 1.5
Injection volume: 80
Detector: UV 290
CHROMATOGRAM
Retention time: 12.5 (+), 15.0 (−)
Mirtazapine
403
Internal standard: imipramine (3.8)
Limit of quantitation: 10 ng/mL
KEY WORDS
chiral; plasma; SPE
REFERENCE
Dodd, S.; Burrows, G.D.; Norman, T.R. Chiral determination of mirtazapine in human blood plasma by
high-performance liquid chromatography, J.Chromatogr.B, 2000, 748, 439–443.
SAMPLE
Matrix: formulations
Sample preparation: Extract a 300 mg amount powdered tablets with 100 mL
MeCN:water 50:50, inject a 10 µL aliquot.
HPLC VARIABLES
Column: 250 × 4.6 5 µm Hypersil ODS
Column temperature: 40
Mobile phase: MeCN:MeOH:THF:buffer 14.875:12.67:7.455:65 (The buffer was 18 g/L
tetramethylammonium hydroxide pentahydrate in water adjusted to pH 7.4 with 85%
phosphoric acid.)
Flow rate: 1.5
Injection volume: 10
Detector: UV 210
CHROMATOGRAM
Limit of detection: 0.01–0.04%
Limit of quantitation: 0.02–0.12%
OTHER SUBSTANCES
Simultaneous: impurities
KEY WORDS
comparison with capillary electrophoresis; tablets
REFERENCE
Wynia, G.S.; Windhorst, G.; Post, P.C.; Maris, F.A. Development and validation of a capillary electrophoresis method within a pharmaceutical quality control environment and comparison with
high-performance liquid chromatography, J.Chromatogr.A, 1997, 773, 339–350.
ANNOTATED BIBLIOGRAPHY
Dallet, P.; Labat, L.; Richard, M.; Langlois, M.H.; Dubost, J.P. A reversed-phase HPLC method development for the separation of new antidepressants, J.Liq.Chromatogr.Rel.Technol., 2002, 25, 101–111.
[fluvoxamine; fluoxetine; sertraline; paroxetine; citalopram; venlafaxine; milnacipran; mirtazapine]
Duverneuil, C.; de la Grandmaison, G.L.; De Mazancourt, P.; Alvarez, J.-C. A high-performance liquid
chromatography method with photodiode-array UV detection for therapeutic drug monitoring of the
nontricyclic antidepressant drugs, Ther.Drug Monit., 2003, 25, 565–573. [LOD 2.5–10 ng/mL; plasma;
fluoxetine; norfluoxetine; sertraline; paroxetine; citalopram; fluvoxamine; moclobemide; mirtazapine;
milnacipran; toloxatone; venlafaxine; viloxazine]
Frahnert, C.; Rao, M.L.; Grasmäder, K. Analysis of eighteen antidepressants, four atypical antipsychotics and active metabolites in serum by liquid chromatography: a simple tool for therapeutic
drug monitoring, J.Chromatogr.B, 2003, 794, 35–47. [SPE; sulpiride; moclobemide; amisulpride;
venlafaxine; normirtazapine; melperone; reboxetine; zolpidem; nordoxepin; diazepam; risperidone;
benperidol; normaprotiline; dibenzepine; opipramol; fluvoxamine; quetiapine; desipramine; citalopram;
norfluoxetine; norclozapine; nortriptyline; haloperidol; paroxetine; maprotiline; mirtazapine; fluoxetine; doxepin; norclomipramine; imipramine; trifluperidol; olanzapine; trimipramine; amitriptyline;
404
Mirtazapine
ziprasidone; promethazine; mianserin; clomipramine; clozapine; fluphenazine; nefazodone; sertraline;
chlorprothixene; thioridazine; pimozide; LOQ 5–10 ng/mL]
Labat, L.; Dallet, P.; Kummer, E.; Dubost, J.P. Spectrophotometric, spectrofluorimetric, HPLC and CZE
determination of mirtazapine in pharmaceutical tablets, J.Pharm.Biomed.Anal., 2002, 28, 365–371.
[pirenzepine is internal standard]
Maris, F.A.; Dingler, E.; Niehues, S. High-performance liquid chromatographic assay with fluorescence
detection for the routine monitoring of the antidepressant mirtazapine and its demethyl metabolite in
human plasma, J.Chromatogr.B, 1999, 721, 309–316. [LOQ 0.5 ng/mL]
Moody, J.D.; Freeman, J.P.; Fu, P.P.; Cerniglia, C.E. Biotransformation of mirtazapine by Cunninghamella elegans, Drug Metab.Dispos., 2002, 30, 1274–1279.
Morgan, P.E.; Tapper, J.; Spencer, E.P. Measurement of total mirtazapine and normirtazapine in
plasma/serum by liquid chromatography with fluorescence detection, J.Chromatogr.B, 2003, 798,
211–215. [LOQ 1 ng/mL; imipramine is internal standard]
Ptácek, P.; Klı́ma, J.; Macek, J. Determination of mirtazapine in human plasma by liquid chromatography, J.Chromatogr.B, 2003, 794, 323–328. [fluorescence detection; LOQ 1.5 ng/mL; zolpidem is
internal standard]
Romiguieres, T.; Pehourcq, F.; Matoga, M.; Begaud, B.; Jarry, C. Determination of mirtazapine and its
demethyl metabolite in plasma by high-performance liquid chromatography with ultraviolet detection; Application to management of acute intoxication, J.Chromatogr.B, 2002, 775, 163–168. [LOQ
20 ng/mL; opipramol is internal standard]
Thieme, D.; Sachs, H. Improved screening capabilities in forensic toxicology by application of liquid chromatography-tandem mass spectrometry, Anal.Chim.Acta, 2003, 492, 171–186. [alprazolam;
dothiepin; piritramide; cocaine; lorazepam; lormetazepam; clonazepam; flunitrazepam; bromazepam;
midazolam; flurazepam; nitrazepam; temazepam; medazepam; nordazepam; diazepam; methylclonazepam; triazolam; oxazepam; haloperidol; benperidol; sulpiride; amisulpride; mirtazapine; citalopram; olanzapine; paroxetine; fluoxetine; sertraline; zopiclone; zolpidem; risperidone; quetiapine;
fentanyl; pipamperone; meperidine; buprenorphine; propoxyphene; pentazocine; phenazocine; EDDP;
tilidine; methadone; morphine; codeine; dihydrocodeine; acetylmorphine; amphetamine; ephedrine;
norephedrine; pseudoephedrine; methylephedrine; amphetaminil; benzphetamine; methylphenidate;
nikethamide; amfeparone; clobenzorex; atropine; scopolamine; ajmaline; aconitine; colchicine; strychnine; metoprolol; acebutolol; propranolol; sotalol; atenolol; bisoprolol; amiloride; triamterene; warfarin;
brodifacoum; coumatetralyl; phenprocoumon; methaqualone; clomethiazole; acetaminophen; methoxamine; vecuronium; neostigmine; LSD]
Titier, K.; Castaing, N.; Scotto-Gomez, E.; Pehourcq, F.; Moore, N.; Molimard, M. High-performance liquid chromatographic method with diode array detection for identification and quantification of the eight
new antidepressants and five of their active metabolites in plasma after overdose, Ther.Drug Monit.,
2003, 25, 581–587. [LOQ 25–100 ng/mL; fluvoxamine; paroxetine; sertraline; fluoxetine; citalopram;
mirtazapine; milnacipran; venlafaxine; norfluoxetine]
405
Misoprostol
Misoprostol
H3C
Molecular formula: C22 H38 O5
Molecular weight: 382.53
CAS Registry No: 59122-46-2
Merck Index: 13, 6232
O
O
OH
OCH3
CH3
HO
SAMPLE
Matrix: blood
Sample preparation: Mix 200 µL plasma with 200 µL 100 ppm 2-naphthoic acid
and 300 µL MeCN, shake well, centrifuge for 10 min, inject a 20 µL aliquot of the
supernatant.
HPLC VARIABLES
Column: 250 × 4.6 5 µm Zorbax C8
Mobile phase: MeCN:MeOH:20 mM pH 3.0 potassium phosphate buffer 30:24:46
Flow rate: 1.7
Injection volume: 20
Detector: UV 210; Radioactivity (3 H)
CHROMATOGRAM
Retention time: 18.5 (diastereomers not resolved)
Internal standard: 2-naphthoic acid (6)
Limit of quantitation: LOQ 1 µg (UV), 12 pg (radioactivity detector)
OTHER SUBSTANCES
Extracted: iloprost (LOQ 500 ng (UV), 42 pg (radioactivity detector)) (14.7, 15.9 (diastereomers))
KEY WORDS
plasma; rat
REFERENCE
Womack, I.M.; Lee, A.S.; Kamath, B.; Agrawal, K.C.; Kishore, V. A high performance liquid radiochromatographic assay for the simultaneous analysis of iloprost and misoprostol, Prostaglandins, 1996,
52, 249–259.
406
Mizolastine
Mizolastine
F
O
Molecular formula: C24 H25 FN6 O
Molecular weight: 432.49
CAS Registry No: 108612-45-9
Merck Index: 13, 6242
H
N
N
N
N
N
N
CH3
SAMPLE
Matrix: blood
Sample preparation: Mix 1 mL whole blood with 20 µL 1 µg/mL dibenzepin in
MeOH:water 50:50, add 300 µL pH 11 Tris buffer, mix, add 500 µL butyl acetate,
vortex for 2 min, centrifuge. Preserve the aqueous layer (A). Remove the organic layer
and add it to 75 µL 10 mM ammonium acetate buffer containing 0.1% formic acid (pH
3.2), evaporate (?). Add 75 µL MeCN, sonicate for 5 min, centrifuge at 5000 rpm for
5 min, keep the extract as B. Add 20 µL 1 µg/mL enalapril in MeOH:water 50:50 and
120 mg NaCl to the aqueous layer (A), mix, add 500 µL pH 3 phosphate buffer, add
600 µL 8.5% phosphoric acid, add 5 mL dichloromethane:isopropanol 95:5, shake at
250 cycles/min in a bench-top shaker for 30 min, centrifuge at 5000 rpm for 5 min.
Remove the lower organic layer and evaporate it to dryness under a stream of air at
45◦ . Reconstitute the residue with 150 µL initial mobile phase, sonicate, centrifuge,
combine with extract B, inject a 30 µL aliquot. (Sample preparation from Gergov,M.;
Robson,J.N.; Ojanperä,I.; Heinonen,O.P.; Vuori,E. Simultaneous screening and quantitation of 18 antihistamine drugs in blood by liquid chromatography ionspray tandem
mass spectrometry. Forensic Sci.Inter. 2001, 121, 108–115.)
HPLC VARIABLES
Guard column: 40 mm long 4 µm Purospher RP-18 LiChro Cart 4-4
Column: 100 × 2.1 4 µm Genesis C18 (Jones Chromatography)
Column temperature: 35
Mobile phase: Gradient. MeCN:buffer from 20:80 to 100:0 over 10 min, maintain at
0:100 for 3 min, re-equilibrate at initial conditions for 5 min. (The buffer was 10 mM
ammonium acetate containing 0.1% formic acid (pH 3.2).)
Flow rate: 0.2
Injection volume: 30
Detector: MS, PE Sciex API 365 triple-stage quadrupole LC-MS-MS, PE Sciex Turbo Ion
Spray interface, positive ion mode, needle voltage 5.2 kV, nebulizer gas air at 60 psi,
curtain gas nitrogen at 40 psi, collision cell gas nitrogen at 40 psi, turbo ionspray heater
375◦ , heater gas flow 7 L/min
CHROMATOGRAM
Retention time: 5.5
Internal standard: dibenzepin, enalapril
Limit of detection: 10 ng/mL
OTHER SUBSTANCES
Extracted: acebutolol (3.8, LOD 0.1 µg/mL), acrivastine (5.7, LOD <0.02 µg/mL),
alprazolam (6.1, LOD <0.02 µg/mL), alprenolol (5.4, LOD 0.01 µg/mL), amantadine
(3.4, LOD 0.1 µg/mL), amiloride (2.0, LOD 0.1 µg/mL), aminophenazone (2.8, LOD
<5 µg/mL), amiodarone (10.2, LOD 0.05 µg/mL), amitriptyline (6.6, LOD <0.02 µg/mL),
astemizole (5.8, LOD <0.02 µg/mL), atenolol (1.7, LOD 0.30 µg/mL), azacyclonol (5.1,
LOD 0.02 µg/mL), benzhexol (6.6, LOD <0.02 µg/mL), benzoylecgonine (3.3, LOD
0.01 µg/mL), betaxolol (5.5, LOD 0.01 µg/mL), biperidine (6.2, LOD <0.02 µg/mL),
bisoprolol (5.0, LOD <0.02 µg/mL), brompheniramine (5.3, LOD 0.002 µg/mL),
bupivacaine (5.1, LOD <0.02 µg/mL), buprenorphine (5.9, LOD 0.01 µg/mL),
buspirone (5.1, LOD 0.002 µg/mL), caffeine (2.8, LOD 1 µg/mL), carbamazepine
Mizolastine
407
(6.1, LOD <0.02 µg/mL), carbinoxamine (5.1, LOD 0.002 µg/mL), carisoprodol
(6.7, LOD <5 µg/mL), carvedilol (6.2, LOD <0.02 µg/mL), celiprolol (4.3, LOD
0.05 µg/mL), cetirizine (6.3, LOD 0.05 µg/mL), chlorcyclizine (6.6, LOD <0.02 µg/mL),
chlordiazepoxide (5.7, LOD <0.02 µg/mL), chlormezanone (5.8, LOD <5 µg/mL),
chloroquine (2.7, LOD 0.02 µg/mL), chlorpheniramine (5.1, LOD 0.002 µg/mL),
chlorpromazine (7.0, LOD 0.02 µg/mL), chlorpropamide (6.7, LOD <5 µg/mL),
chlorprothixene (7.0, LOD <0.02 µg/mL), cinnarizine (7.9, LOD <0.02 µg/mL),
citalopram (5.7, LOD <0.02 µg/mL), clemastine (7.7, LOD 0.02 µg/mL), clobazam
(7.3, LOD <0.02 µg/mL), clobutinol (5.3, LOD 0.02 µg/mL), clomethiazole (6.2,
LOD 0.5 µg/mL), clomipramine (7.1, LOD <0.02 µg/mL), clonazepam (6.6, LOD
<0.02 µg/mL), clonidine (2.8, LOD 0.1 µg/mL), clozapine (5.6, LOD <0.02 µg/mL),
cocaine (4.6, LOD <0.02 µg/mL), codeine (2.5, LOD 0.1 µg/mL), coumatetralyl
(8.4, LOD 0.05 µg/mL), cyclizine (5.8, LOD <0.02 µg/mL), dextropropoxyphene
(6.6, LOD 0.05 µg/mL), demoxepam (5.8, LOD 0.02 µg/mL), dextromethorphan
(5.5, LOD <0.02 µg/mL), diazepam (8.1, LOD 0.02 µg/mL), diltiazem (5.8,
LOD <0.02 µg/mL), diphenhydramine (5.7, LOD <0.02 µg/mL), dipyridamole (5.4,
LOD 0.005 µg/mL), disopyramine (4.4, LOD <0.02 µg/mL), dixyrazine (6.8, LOD
0.005 µg/mL), doxapram (4.8, LOD <0.02 µg/mL), doxepin (5.9, LOD <0.02 µg/mL),
ebastine (9.6, LOD 0.005 µg/mL), embutramide (6.7, LOD 0.005 µg/mL), ergotamine
(5.5, LOD 0.005 µg/mL), ethenzamide (5.0, LOD 0.05 µg/mL), ethylmorphine (3.2,
LOD 0.05 µg/mL), ethylparathion (9.7, LOD <5 µg/mL), etodroxizine (6.4, LOD
<0.02 µg/mL), felodipine (9.6, LOD 0.02 µg/mL), fenazepam (7.5, LOD <0.02 µg/mL),
fenfluramine (5.3, LOD <0.02 µg/mL), fenkamfamine (5.1, LOD <0.02 µg/mL), fentanyl
(5.5, LOD <0.02 µg/mL), fexofenadine (6.3, LOD <0.02 µg/mL), flecainide (5.9, LOD
<0.02 µg/mL), fluconazole (4.0, LOD 0.1 µg/mL), flumazenil (5.2, LOD <0.02 µg/mL),
flunitrazepam (7.1, LOD 0.002 µg/mL), fluoxetine (6.8, LOD 0.1 µg/mL), flupentixol
(7.5, LOD 0.18 µg/mL), fluvoxamine (6.3, LOD 0.02 µg/mL), glibenclamide (8.5, LOD
<0.02 µg/mL), glipizide (6.8, LOD <0.05 µg/mL), haloperidol (6.1, LOD <0.02 µg/mL),
histapyrrodine (6.3, LOD 0.02 µg/mL), hydrocodone (3.0, LOD 0.05 µg/mL),
hydroxychloroquine (2.4, LOD <0.3 µg/mL), hydroxyzine (6.3, LOD <0.02 µg/mL),
imipramine (6.4, LOD 0.05 µg/mL), indomethacin (8.6, LOD 0.05 µg/mL), isoniazid (2.2,
LOD 3 µg/mL), isradipine (8.6, LOD 0.05 µg/mL), ketamine (3.6, LOD <0.05 µg/mL),
ketobemidone (3.3, LOD <0.05 µg/mL), ketoprofen (7.3, LOD 0.1 µg/mL), ketorolac (6.2,
LOD 0.05 µg/mL), labetalol (4.9, LOD 0.05 µg/mL), lamotrigine (4.0, LOD 0.1 µg/mL),
levocabastine (5.8, LOD 0.01 µg/mL), levomepromazine (6.5, LOD 0.02 µg/mL), lidocaine
(3.7, LOD <0.05 µg/mL), loratadine (9.3, LOD 0.002 µg/mL), lorazepam (6.6, LOD
0.02 µg/mL), lormetazepam (7.4, LOD <0.02 µg/mL), LSD (4.7, LOD <0.02 µg/mL),
malathion (8.9, LOD 10 µg/mL), maprotiline (6.4, LOD <0.02 µg/mL), MDMA (3.3, LOD
0.02 µg/mL), meclozine (8.5, LOD <0.02 µg/mL), medazepam (6.3, LOD <0.02 µg/mL),
meloxicam (7.1, LOD 0.01 µg/mL), melperone (5.0, LOD <0.02 µg/mL), meperidine
(4.7, LOD <0.02 µg/mL), mepivacaine (3.7, LOD <0.02 µg/mL), meprobamate (4.9,
LOD 0.1 µg/mL), mesoridazine (5.4, LOD <0.02 µg/mL), methamphetamine (3.3,
LOD 0.05 µg/mL), methadone (6.7, LOD <0.02 µg/mL), methylparathion (8.6, LOD
10 µg/mL), methylphenidate (4.2, LOD <0.02 µg/mL), metoclopramide (3.8, LOD
<0.02 µg/mL), metoprolol (4.1, LOD 0.02 µg/mL), metronidazole (2.6, LOD 1 µg/mL),
mexiletine (4.4, LOD 0.05 µg/mL), mianserin (5.7, LOD <0.02 µg/mL), midazolam
(5.9, LOD <0.02 µg/mL), mirtazapine (4.4, LOD <0.02 µg/mL), moclobemide (3.7,
LOD 0.05 µg/mL), molindone (4.0, LOD <0.02 µg/mL), monoacetylmorphine (2.7,
LOD 0.1 µg/mL), morphine (2.0, LOD 0.1 µg/mL), nicotine (2.2, LOD 0.05 µg/mL),
nifedipine (7.5, LOD 0.02 µg/mL), nikethamide (3.6, LOD <0.02 µg/mL), nitrazepam
(6.5, LOD <0.02 µg/mL), nizatidine (1.7, LOD 1 µg/mL), nomifensine (4.6, LOD
<0.02 µg/mL), nortriptyline (6.4, LOD <0.02 µg/mL), norverapamil (6.2, LOD 1 µg/mL),
noscapine (5.0, LOD <0.02 µg/mL), olanzapine (3.0, LOD 0.05 µg/mL), ondansetron
(4.6, LOD <0.02 µg/mL), orphenadrine (6.1, LOD <0.02 µg/mL), oxazepam (6.3, LOD
<0.02 µg/mL), oxcarbazepine (5.3, LOD 0.02 µg/mL), oxprenolol (4.7, LOD 0.02 µg/mL),
oxycodone (2.8, LOD 0.05 µg/mL), papaverine (4.8, LOD <0.02 µg/mL), acetaminophen
(2.5, LOD <5 µg/mL), paroxetine (6.2, LOD 0.02 µg/mL), pemoline (3.3, LOD
0.05 µg/mL), pentazocine (5.0, LOD <0.02 µg/mL), pentifylline (7.3, LOD <5 µg/mL),
pentoxyverine (6.6, LOD <0.02 µg/mL), perphenazine (6.9, LOD 0.002 µg/mL),
phenazone (3.9, LOD 0.05 µg/mL), phencyclidine (5.3, LOD 0.05 µg/mL), pheniramine
408
Mizolastine
(4.1, LOD 0.02 µg/mL), phenylbutazone (9.0, LOD <5 µg/mL), phenylpropanolamine
(2.5, LOD 0.3 µg/mL), phenytoin (6.1, LOD 0.05 µg/mL), pindolol (3.3, LOD 0.05 µg/mL),
piroxicam (6.6, LOD 0.02 µg/mL), pitofenone (5.4, LOD <0.02 µg/mL), pizotifen (6.5,
LOD <0.02 µg/mL), practolol (1.8, LOD 0.1 µg/mL), prazosin (4.1, LOD 0.05 µg/mL),
prilocaine (3.8, LOD <0.02 µg/mL), primidone (4.0, LOD <5 µg/mL), procainamide
(2.2, LOD 0.05 µg/mL), prochlorperazine (7.5, LOD 0.02 µg/mL), promazine (6.2,
LOD <0.02 µg/mL), promethazine (6.0, LOD 0.05 µg/mL), propafenone (6.3, LOD
<0.02 µg/mL), propranolol (5.4, LOD 0.02 µg/mL), propyphenazone (6.6, LOD
0.50 µg/mL), pseudoephedrine (2.6, LOD 1 µg/mL), quinine (4.2, LOD 0.02 µg/mL),
ranitidine (1.8, LOD 0.1 µg/mL), risperidone (4.9, LOD <0.02 µg/mL), rocurone
(3.8, LOD 0.1 µg/mL), ropivacaine (4.6, LOD <0.02 µg/mL), salicylamide (4.2, LOD
<5 µg/mL), selegiline (4.1, LOD 0.05 µg/mL), sertindole (7.2, LOD <0.02 µg/mL),
sertraline (6.8, LOD 0.02 µg/mL), sulindac (6.5, LOD 0.02 µg/mL), simazine (6.0, LOD
0.1 µg/mL), sincocaine (6.5, LOD <0.02 µg/mL), sisapride (5.9, LOD <0.02 µg/mL),
sotalol (2.1, LOD 0.1 µg/mL), strychnine (5.3, LOD 0.05 µg/mL), sulpiride (1.9, LOD
0.1 µg/mL), sulthiame (4.1, LOD 0.05 µg/mL), temazepam (7.2, LOD <0.02 µg/mL),
terbutaline (2.3, LOD 0.1 µg/mL), terfenadine (8.1, LOD 0.002 µg/mL), terodiline
(6.7, LOD <0.02 µg/mL), tetracaine (5.7, LOD <0.02 µg/mL), dronabinol (12.3,
LOD 0.05 µg/mL), tetrahydrozoline (3.6, LOD 0.1 µg/mL), theobromine (2.3, LOD
<5 µg/mL), theophylline (2.4, LOD <5 µg/mL), thioridazine (7.5, LOD 0.02 µg/mL),
timolol (3.8, LOD 0.05 µg/mL), thiothixene (6.7, LOD 0.02 µg/mL), tolbutamide (7.1,
LOD <5 µg/mL), toremifene (8.7, LOD 0.02 µg/mL), tramadol (4.2, LOD 0.02 µg/mL),
trazodone (5.2, LOD <0.02 µg/mL), triamterene (3.2, LOD 0.1 µg/mL), triazolam (6.7,
LOD 0.002 µg/mL), trimeprazine (6.4, LOD <0.02 µg/mL), trimethoprim (3.1, LOD
0.05 µg/mL), trimipramine (6.7, LOD <0.02 µg/mL), venlafaxine (4.9, LOD 0.02 µg/mL),
verapamil (6.5, LOD <0.02 µg/mL), warfarin (7.9, LOD <0.02 µg/mL), yohimbine (4.5,
LOD <0.02 µg/mL), zolpidem (4.7, LOD <0.02 µg/mL), zopiclone (4.0, LOD 0.1 µg/mL)
KEY WORDS
whole blood
REFERENCE
Gergov, M.; Ojanperä, I.; Vuori, E. Simultaneous screening for 238 drugs in blood by
liquid chromatography-ionspray tandem mass spectrometry with multiple-reaction monitoring,
J.Chromatogr.B, 2003, 795, 41–53.
SAMPLE
Matrix: blood
Sample preparation: Mix 1 g plasma with 20 µL 15 µg/mL IS in MeOH, add 200 µL
MeCN, vortex, centrifuge at 11 000 g for 4 min, inject a 200 µL aliquot onto column
A and elute to waste with mobile phase A; after 2 min, backflush the contents of
column A onto column B with mobile phase B; after 1.5 min, remove column A from
the circuit, elute column B with mobile phase B, monitor the effluent from column B.
Backflush column A with MeCN:water 50:50, MeCN, MeOH:water 50:50, re-equilibrate
with mobile phase A.
HPLC VARIABLES
Column: A 75 × 2.1 30–40 µm Perisorb C18; B 20 × 4.6 40 µm Pelliguard C18
(Supelchem) + 150 × 4.6 5 µm Hypersil BDS C8
Mobile phase: A MeCN:water 10:90; B MeCN:25 mM pH 4.5 phosphate buffer 40:60
Flow rate: 1
Injection volume: 200
Detector: UV 285
CHROMATOGRAM
Retention time: 6.6
Internal standard: SL 86.0116 (chloro analogue of mizolastine) (8.2)
Limit of quantitation: 2.5 ng/mL
Mizolastine
409
KEY WORDS
column-switching; comparison with liquid–liquid extraction and SPE; plasma
REFERENCE
Ascalone, V.; Guinebault, P.; Rouchouse, A. Determination of mizolastine, a new antihistaminic drug,
in human plasma by liquid-liquid extraction and column-switching techniques in combination with
high-performance liquid chromatography, J.Chromatogr., 1993, 619, 275–284.
SAMPLE
Matrix: blood
Sample preparation: Mix 1 g plasma with 20 µL 15 µg/mL IS in MeOH, add 500 µL
700 mM pH 9 borate buffer, add 7 mL diethyl ether, shake on a tumble extractor at
40 rpm for 10 min, centrifuge at 2000 g at 5◦ for 5 min. Remove the organic layer and
add it to 25 mM pH 2.6 phosphate buffer, shake on a tumble extractor at 20 rpm for
10 min. Discard the organic layer, remove traces of ether from the aqueous layer with a
stream of nitrogen at 40◦ , inject a 150 µL aliquot. (Prepare 700 mM pH 9 borate buffer
by dissolving 6.18 g boric acid and 7.46 g KCl in 100 mL water and adjusting to pH 9
with ca. 50 mL 1 M NaOH.)
HPLC VARIABLES
Guard column: 20 × 4.6 40 µm Pelliguard C18 (Supelchem)
Column: 150 × 4.6 5 µm Hypersil BDS C8
Mobile phase: MeCN:25 mM pH 2.5 phosphate buffer:triethylamine 35:65:0.1
Flow rate: 1
Injection volume: 150
Detector: UV 285
CHROMATOGRAM
Retention time: 3.6
Internal standard: SL 86.0116 (chloro analogue of mizolastine) (4.6)
Limit of quantitation: 0.5 ng/mL
KEY WORDS
comparison with column-switching and SPE; liquid–liquid extraction; plasma
REFERENCE
Ascalone, V.; Guinebault, P.; Rouchouse, A. Determination of mizolastine, a new antihistaminic drug,
in human plasma by liquid-liquid extraction and column-switching techniques in combination with
high-performance liquid chromatography, J.Chromatogr., 1993, 619, 275–284.
SAMPLE
Matrix: blood
Sample preparation: Condition a 100 mg 40 µm Bakerbond CN SPE cartridge with
1 mL MeOH saturated with ammonium dihydrogen phosphate and 2 mL water. Mix
1 mL plasma with 500 µL 200 ng/mL IS in MeCN:water, mix, add a 1.4 mL aliquot
to the SPE cartridge, wash twice with 2 mL portions of water, wash twice with 1 mL
portions of MeOH:water 30:70, dry, elute with 600 µL MeOH saturated with ammonium
dihydrogen phosphate, mix the eluate (ca. 300 µL) with 400 µL 50 mM pH 4.6 phosphate
buffer, inject a 650 µL aliquot.
HPLC VARIABLES
Column: 250 × 4.6 5 µm Ultrabase C8
Mobile phase: MeCN:50 mM pH 4.6 phosphate buffer:triethylamine 45:55:0.1
Flow rate: 1
Injection volume: 650
Detector: UV 285
410
Mizolastine
CHROMATOGRAM
Retention time: 8
Internal standard: SL 86.0116 (chloro analogue of mizolastine) (10)
Limit of quantitation: 1 ng/mL
KEY WORDS
comparison with column-switching and liquid–liquid extraction; plasma; SPE
REFERENCE
Ascalone, V.; Guinebault, P.; Rouchouse, A. Determination of mizolastine, a new antihistaminic drug,
in human plasma by liquid-liquid extraction and column-switching techniques in combination with
high-performance liquid chromatography, J.Chromatogr., 1993, 619, 275–284.
Moexipril
Moexipril
H3C
O
O
Molecular formula: C27 H34 N2 O7
N
Molecular weight: 498.57
CAS Registry No: 103775-10-6,
82586-52-5 (HCl)
Merck Index: 13, 6250
H
HOOC
CH3
411
OCH3
N
OCH3
O
SAMPLE
Matrix: formulations
Sample preparation: Inject an aliquot of a 50 µg/mL solution in water.
HPLC VARIABLES
Column: 5 µm Ultrasphere-ODS
Mobile phase: MeCN:THF:50 mM pH 2 ammonium phosphate 35:10:55
Flow rate: 1
Detector: UV 220
OTHER SUBSTANCES
Simultaneous: degradants
KEY WORDS
lyophilized powder
REFERENCE
Strickley, R.G.; Visor, G.C.; Lin, L.H.; Gu, L. An unexpected pH effect on the stability of moexipril
lyophilized powder, Pharm.Res., 1989, 6, 971–975.
412
Mofezolac
Mofezolac
O
N
COOH
Molecular formula: C19 H17 NO5
Molecular weight: 339.34
CAS Registry No: 78967-07-4
Merck Index: 13, 6254
H3CO
OCH3
SAMPLE
Matrix: blood, urine
Sample preparation: Condition a Sep-Pak C18 SPE cartridge with 5 mL MeOH and
5 mL water. Plasma. Add 4 mL ice-cold MeCN to 500 µL plasma, vortex for 30 s, place on
ice for 30 min, centrifuge refrigerated at 2000 g for 15 min. Evaporate the supernatant
to dryness under a stream of nitrogen, reconstitute the residue with 2 mL water, add to
the SPE cartridge, wash with 3 mL water, elute with 4 mL MeOH. Evaporate the eluate
to dryness under a stream of nitrogen, reconstitute the residue with 100 µL MeOH,
inject a 20 µL aliquot. Urine. Mix 1 mL urine with 1 mL 10 mM pH 7.0 phosphate
buffer, add to the SPE cartridge, wash with 3 mL water, elute with 4 mL MeOH.
Evaporate the eluate to dryness under a stream of nitrogen, reconstitute the residue
with 200 µL MeOH:water 40:60, inject a 20 µL aliquot. (10 mM pH 7.0 Phosphate
buffer was 35.81 g disodium hydrogen phosphate dodecahydrate and 13.61 g potassium
dihydrogen phosphate in 1 L water, pH adjusted to 7.0 with NaOH or phosphoric acid.)
HPLC VARIABLES
Column: 150 × 6 5 µm Shimadzu Shim-pack CLC ODS
Column temperature: 35
Mobile phase: Gradient. MeOH:20 mM pH 6.4 potassium dihydrogen phosphate buffer
from 0:100 to 100:0 over 33 min.
Flow rate: 1.5
Injection volume: 20
Detector: UV 254
CHROMATOGRAM
Retention time: 23.9
Limit of detection: 10 ng/mL (plasma), 100 ng/mL (urine)
OTHER SUBSTANCES
Extracted: metabolites
KEY WORDS
pharmacokinetics; plasma; SPE
REFERENCE
Marunaka, T.; Maniwa, M. High-performance of liquid chromatographic determination of [3,4-di(4-methoxyphenyl)-5-isoxazolyl]acetic acid and its metabolites in human plasma and urine,
J.Chromatogr., 1987, 422, 227–233.
413
Mometasone furoate
Mometasone furoate
Cl
HO
Molecular formula: C27 H30 Cl2 O6
Molecular weight: 521.44
CAS Registry No: 83919-23-7
Merck Index: 13, 6264
O
O
CH3
O
O
CH3
Cl
H
CH3
H
O
SAMPLE
Matrix: blood
Sample preparation: Mix 400 µL plasma with IS, extract with 8 mL nchlorobutane:ethyl acetate 95:5. Evaporate the organic layer to dryness, reconstitute
the residue with 35 µL MeOH, inject an 8 µL aliquot.
HPLC VARIABLES
Column: 33 × 4.6 3 µm LC-18-DB
Mobile phase: MeOH:25 mM ammonium acetate 80:20
Flow rate: 1
Injection volume: 8
Detector: MS, PE Sciex API-III, positive ion daughter, m/z 355.0
CHROMATOGRAM
Retention time: 24
Internal standard: betamethasone 17,21-dipropionate (m/z 279.3)
Limit of quantitation: 49.7 pg/mL
OTHER SUBSTANCES
Extracted: mometasone (15), metabolites
KEY WORDS
pharmacokinetics; plasma
REFERENCE
Affrime, M.B.; Cuss, F.; Padhi, D.; Wirth, M.; Pai, S.; Clement, R.P.; Lim, J.; Kantesaria, B.; Alton, K.;
Cayen, M.N. Bioavailability and metabolism of mometasone furoate following administration by
metered-dose and dry-powder inhalers in healthy human volunteers, J.Clin.Pharmacol., 2000, 40,
1227–1236.
SAMPLE
Matrix: blood, simulated gastric fluid, simulated intestinal fluid, simulated lung fluid
Sample preparation: Mix 500 µL plasma, simulated gastric fluid, simulated intestinal fluid, or simulated lung fluid with 500 µL 10 µg/mL IS in EtOH, add 4 mL
dichloromethane, mix on a roller mixer for 30 min, centrifuge at 2500 rpm for 15 min.
Evaporate the organic layer to dryness under a stream of nitrogen at 37◦ , reconstitute
the residue with 250 µL mobile phase, centrifuge at 15 000 rpm for 3 min, inject a 50 µL
aliquot of the supernatant.
HPLC VARIABLES
Column: 150 × 4.6 5 µm Ultrasphere C8
Mobile phase: MeOH:water 59:41
Flow rate: 1.5
Injection volume: 50
Detector: UV 248
414
Mometasone furoate
CHROMATOGRAM
Retention time: 17
Internal standard: dexamethasone 21-acetate (7)
Limit of detection: 50 ng/mL
Limit of quantitation: 200 ng/mL
OTHER SUBSTANCES
Extracted: degradants
Simultaneous: beclomethasone dipropionate (33.9), budesonide (12.3), fluticasone propionate (16.3), hydrocortisone (3.2), prednisone (2.7)
KEY WORDS
plasma
REFERENCE
Teng, X.W.; Foe, K.; Brown, K.F.; Cutler, D.J.; Davies, N.M. High-performance liquid chromatographic
analysis of mometasone furoate and its degradation products. Application to in vitro degradation
studies, J.Pharm.Biomed.Anal., 2001, 26, 313–319.
ANNOTATED BIBLIOGRAPHY
Teng, X.W.; Cutler, D.J.; Davies, N.M. Mometasone furoate degradation and metabolism in human
biological fluids and tissues, Biopharm.Drug Dispos., 2003, 24, 321–333. [plasma; urine; microsomal
incubations; lung tissue]
415
Monensin
Monensin
Molecular formula: C36 H62 O11
Molecular weight: 670.87
CAS Registry No: 17090-79-8
Merck Index: 13, 6270
H3C
HO
CH3
H3C
H3CO
H3C
O
H
CH3
COOH
O
H
H
O
H
O
CH3
H
CH3
O
CH3
HO
OH
SAMPLE
Matrix: blood
Sample preparation: Vortex 100 µL MeOH:water 80:20 and 400 µL trichloroacetic
acid solution with 500 µL plasma, centrifuge at 4000 rpm for 4 min, filter (Spin-X) the
supernatant, inject a 30 µL aliquot of the filtrate. (Prepare trichloroacetic acid solution
as follows. Dissolve 85 g trichloroacetic acid in 15 mL water. Store this solution in the
refrigerator. Dilute 150 µL of this solution with 100 mL acetone.)
HPLC VARIABLES
Guard column: C18
Column: 250 × 4.6 5 µm Supelco Discovery C18
Mobile phase: MeOH:10 mM ammonium acetate 85:15
Flow rate: 0.8 for 15 min, 1 for 10 min
Injection volume: 30
Detector: MS, PE Sciex API 100 single quadrupole, turbo ionspray, turbo probe 150◦ , air
6 L/min, 50 µL/min flowed into detector, m/z 693.7
CHROMATOGRAM
Retention time: 15
Limit of detection: 4 ng/mL
Limit of quantitation: 8 ng/mL
OTHER SUBSTANCES
Extracted: lasalocid (10), narasin (23), salinomycin (20)
KEY WORDS
chicken; plasma
REFERENCE
Hormazábal, V.; Yndestad, M. Determination of amprolium, ethopabate, lasalocid, monensin, narasin,
and salinomycin in chicken tissues, plasma, and egg using liquid chromatography-mass spectrometry,
J.Liq.Chromatogr.Rel.Technol., 2000, 23, 1585–1598.
416
Morantel
Morantel
H3C
N
S
Molecular formula: C12 H16 N2 S
Molecular weight: 220.34
CAS Registry No: 20574-50-9,
26155-31-7 (tartrate)
Merck Index: 13, 6289
N
CH3
SAMPLE
Matrix: abomasal fluid, blood, feces, intestinal fluid, ruminal fluid
Sample preparation: Add 100 µL 50 µg/mL IS to 5 mL plasma, abomasal fluid, intestinal fluid, ruminal fluid or 5 g feces, mix, add 10 mL 200 mM ammonium hydroxide,
add 30 mL chloroform (Caution! Chloroform is a carcinogen!), shake mechanically for
30 min, centrifuge at 2000 g for 15 min, filter (Whatman 1 PS) the chloroform layer. Add
3 mL 1 M sulfuric acid to the filtrate, shake for 15 min, centrifuge at 2000 g for 5 min.
Remove a 1 mL aliquot, neutralize with 2 drops concentrated ammonium hydroxide,
inject a 50 µL aliquot.
HPLC VARIABLES
Column: 10 µm Bondex C18 (Phenomenex)
Mobile phase: MeCN:pH 3.5 ammonium acetate buffer 28:72
Flow rate: 1.3
Injection volume: 50
Detector: UV 318
CHROMATOGRAM
Retention time: 5.64
Internal standard: pyrantel tartrate (3.97)
Limit of detection: 25 ng/mL (plasma, ruminal fluid), 20 ng/mL (abomasal fluid, intestinal fluid), 50 ng/g (feces)
KEY WORDS
cow; plasma
REFERENCE
Lanusse, C.E.; Gascon, L.H.; Ranjan, S.; Prichard, R.K. Morantel tartrate release from a longacting intraruminal device in cattle: pharmacokinetics and gastrointestinal distribution,
J.Vet.Pharmacol.Ther., 1992, 15, 117–123.
Mosapride
Mosapride
417
H3C
O
F
Molecular formula: C21 H25 ClFN3 O3
Molecular weight: 421.90
CAS Registry No: 112885-41-3,
112885-42-4 (citrate)
Merck Index: 13, 6306
O
N
H
N
NH2
Cl
O
SAMPLE
Matrix: blood
Sample preparation: Condition a Sep-Pak C18 SPE cartridge with two 3 mL portions
of MeOH, two 3 mL portions of water, and two 2 mL portions of 20 mM pH 7.0 phosphate
buffer. Mix 50 µL 100 µg/mL IS in water, 2 mL pH 7.0 sodium phosphate buffer, and
1 mL plasma, add to the SPE cartridge, wash with two 3 mL portions of water, elute
with 2 mL MeOH, evaporate the eluate to dryness under reduced pressure at 50◦ ,
dissolve the residue in 400 µL MeOH:0.05% acetic acid 45:55, centrifuge at 5000 rpm
for 10 min, inject a 180 µL aliquot.
HPLC VARIABLES
Guard column: 20 × 9 µBondapak C18
Column: 150 × 4 5 µm Shim-pack CLC-ODS (Shimadzu)
Mobile phase: MeOH:0.05% acetic acid from 20:80 to 38.7:61.3 over 7 min, to 90:10 in
2 min, maintain at 90:10 for 4 min, from 90:10 to 20:80 in 0.5 min, maintain at 20:80
for 4.5 min.
Flow rate: 1.3
Injection volume: 180
Detector: F ex 314 em 354
CHROMATOGRAM
Retention time: 13
Internal standard: AD-9675 ((+)-4-amino-5-chloro-2-ethoxy-N-[[4-propyl-2-morpholinyl]methyl]benzamide citrate) (10.5)
OTHER SUBSTANCES
Extracted: metabolites
KEY WORDS
plasma; rat; SPE
REFERENCE
Yokoyama, I.; Mizuki, Y.; Yamaguchi, T.; Fujii, T. Simultaneous enantiomeric determination of a gastroprokinetic agent mosapride citrate and its metabolite in plasma using α1 -acid glycoprotein HPLC
column, J.Pharm.Biomed.Anal., 1997, 15, 1527–1535.
SAMPLE
Matrix: formulations
Sample preparation: Let powdered 50 mg tablet stand in 25 mL MeOH for 6 h with
occasional sonication, filter (0.45 µm). Dilute the filtrate to 10 µg/mL with mobile phase.
HPLC VARIABLES
Guard column: 20 mm long Pelliguard LC-18 (Supelco)
Column: 150 × 4.6 5 µm RP C-18
418
Mosapride
Column temperature: 40
Mobile phase: MeOH:20 mM pH 4.0 potassium phosphate buffer 70:30
Flow rate: 1.1
Injection volume: 20
Detector: UV 274
CHROMATOGRAM
Retention time: 6.1
Internal standard: risperidone (4.1)
KEY WORDS
tablets
REFERENCE
Krishnaiah, Y.S.R.; Murthy, T.K.; Sankar, D.G.; Satyanarayana, V. The determination of mosapride
citrate in bulk drug samples and pharmaceutical dosage forms using HPLC, Anal.Sci., 2002, 18,
1269–1271.
SAMPLE
Matrix: solutions
Sample preparation: Inject a 100 µL aliquot of a solution in 200 µL 20 mM disodium
hydrogen phosphate:20 mM citric acid 65:35.
HPLC VARIABLES
Guard column: 10 × 3 Chiral AGP
Column: 100 × 4 10 µm Chiral AGP (Chrom Tech)
Mobile phase: Gradient. A was MeOH. B was 20 mM disodium hydrogen phosphate. C
was 20 mM citric acid. A:B:C for 0:65:35 for 4 min, to 5:60:35 over 6 min, maintain at
5:60:35 for 3 min, to 15:48:37 over 1 min, to 25:40:35 over 11 min, to 0:65:35 over 1 min,
maintain at 0:65:35 for 4 min. (The pH of the mobile phase was 4.4 between 4 and 6 min
and 5.0 between 20 and 22 min.)
Flow rate: 1
Injection volume: 100
Detector: F ex 314 em 354
CHROMATOGRAM
Retention time: 20.8 (R), 21.8 (S)
Internal standard: AD-9675 ((+)-4-amino-5-chloro-2-ethoxy-N-[[4-propyl-2-morpholinyl]methyl]benzamide citrate) (9.5)
KEY WORDS
chiral
REFERENCE
Yokoyama, I.; Mizuki, Y.; Yamaguchi, T.; Fujii, T. Simultaneous enantiomeric determination of a gastroprokinetic agent mosapride citrate and its metabolite in plasma using α1 -acid glycoprotein HPLC
column, J.Pharm.Biomed.Anal., 1997, 15, 1527–1535.
ANNOTATED BIBLIOGRAPHY
Itoh, H.; Nagano, T.; Takeyama, M. Effects of mosapride citrate on human plasma levels of motilin,
gastrin, somatostatin, and secretin, Biol.Pharm.Bull., 2001, 24, 1072–1075. [LOQ 10 ng/mL]
Karlsson, A.; Aspegren, A. The use of mobile phase pH and column temperature to reverse the retention
order of enantiomers on Chiral-AGP, Chromatographia, 1998, 47, 189–196.
Mosapride
419
Krishnaiah, Y.S.R.; Murthy, T.K.; Sankar, D.C.; Satyanarayana, V. A validated RP-HPLC method for the
determination of mosapride citrate in bulk drug samples and pharmaceutical formulations, Pharmazie,
2002, 57, 814–816.
Kumar, Y.R.; Babu, J.M.; Sarma, M.S.P.; Seshidhar, B.; Reddy, S.S.; Reddy, G.S.; Vyas, K. Application
of LC-MS/MS for the identification of a polar impurity in mosapride, a gastroprokinetic drug,
J.Pharm.Biomed.Anal., 2003, 32, 361–368.
420
Moxifloxacin
Moxifloxacin
Molecular formula: C21 H24 FN3 O4
Molecular weight: 401.43
CAS Registry No: 151096-09-2
Merck Index: 13, 6316
H
OCH3
N
H
N
N
H
F
COOH
O
SAMPLE
Matrix: blood
Sample preparation: Condition a 1 mL 30 mg OASIS HLB SPE cartridge with 1 mL
MeOH and 1 mL water. Mix 1 mL plasma with 100 µL 500 ng/mL IS in water,
add to the SPE cartridge, wash with two 1 mL portions of water, elute with 1 mL
MeOH:trifluoroacetic acid 99.9:0.1. Evaporate the eluate to dryness under reduced
pressure, reconstitute the residue with 100 µL mobile phase, inject a 10 µL aliquot.
HPLC VARIABLES
Column: 100 × 4.6 5 µm Hypersil C18
Mobile phase: MeCN:0.1% formic acid 40:60
Flow rate: 1
Injection volume: 10
Detector: MS, PE Sciex API 3000, TurboIonSpray, positive ion mode, source 400◦ ,
ionspray 3500 V, declustering 65 V, focusing 300 V, collision energy 20 eV, collision gas
nitrogen, column effluent split 1:5 before entering the detector, m/z 402–384
CHROMATOGRAM
Retention time: 2.75
Internal standard: lomefloxacin (m/z 352–308) (2.32)
Limit of detection: 50 pg/mL
Limit of quantitation: 1 ng/mL
KEY WORDS
plasma; SPE
REFERENCE
Vishwanathan, K.; Bartlett, M.G.; Stewart, J.T. Determination of moxifloxacin in human plasma by
liquid chromatography electrospray ionization tandem mass spectrometry, J.Pharm.Biomed.Anal.,
2002, 30, 961–968.
SAMPLE
Matrix: blood, tissue
Sample preparation: Condition a 1 mL 30 mg OASIS HLB SPE cartridge with 1 mL
MeOH and 1 mL water. Homogenize (Ultra Turrax) 250 mg lung tissue with 2.5 mL
PBS, centrifuge at 3080 g for 15 min. Mix 1.2 mL plasma or lung homogenate with
300 µL 10 µg/mL IS in water, add a 1.5 mL aliquot to the SPE cartridge, wash with
1 mL water, dry with air, elute with 1 mL MeOH:trifluoroacetic acid 99.9:0.1. Evaporate
the eluate to dryness under a stream of nitrogen at 50◦ , reconstitute the residue with
500 µL mobile phase, inject a 50 µL aliquot.
HPLC VARIABLES
Column: 150 × 4.6 5 µm Supelcosil ABZ+
Mobile phase: MeCN:buffer 18:82 (The buffer was 10 mM potassium dihydrogen phosphate adjusted to pH 4 with orthophosphoric acid.)
Flow rate: 1.25
Moxifloxacin
421
Injection volume: 50
Detector: UV 296
CHROMATOGRAM
Retention time: 6.2
Internal standard: enrofloxacin (3.5)
Limit of detection: 6.5 ng/mL (plasma), 50 ng/g (lung)
Limit of quantitation: 30 ng/mL (plasma), 400 ng/g (lung)
KEY WORDS
lung; plasma; SPE
REFERENCE
Lemoine, T.; Breilh, D.; Ducint, D.; Dubrez, J.; Jougon, J.; Velly, J.F.; Saux, M.C. Determination of moxifloxacin (BAY 12–8039) in plasma and lung tissue by high-performance liquid chromatography with
ultraviolet detection using a fully automated extraction method with a new polymeric cartridge,
J.Chromatogr.B, 2000, 742, 247–254.
SAMPLE
Matrix: blood, vitreous humor
Sample preparation: Serum. Rotate 400 µL serum with 3.2 mL dichloromethane at
20 rpm for 10 min, shake for 1 min. Shake the organic layer with 200 µL 20 mM pH 2.0
orthophosphoric acid for 1 min, centrifuge at 2000 g at 10◦ for 10 min, inject a 20 µL
aliquot of the aqueous layer. Vitreous humor. Centrifuge vitreous humor briefly, inject
a 20 µL aliquot.
HPLC VARIABLES
Column: 75 × 4.6 3 µm Ultrasphere C18
Mobile phase: MeCN:buffer 12:88 (serum) or 10:90 (vitreous humor) (The buffer was
5 mM tetrabutylammonium bromide adjusted to pH 1.8 with phosphoric acid.)
Flow rate: 1.5
Injection volume: 20
Detector: UV 296
CHROMATOGRAM
Limit of detection: 10 ng/mL
KEY WORDS
rabbit; serum
REFERENCE
Bronner, S.; Jehl, F.; Peter, J.-D.; Ploy, M.-C.; Renault, C.; Arvis, P.; Monteil, H.; Prevost, G. Moxifloxacin efficacy and vitreous penetration in a rabbit model of Staphylococcus aureus endophthalmitis
and effect on gene expression of leucotoxins and virulence regulator factors, Antimicrob.Agents
Chemother., 2003, 47, 1621–1629.
ANNOTATED BIBLIOGRAPHY
Ba, B.B.; Etienne, R.; Ducint, D.; Quentin, C.; Saux, M.-C. Determination of moxifloxacin in growth
media by high-performance liquid chromatography, J.Chromatogr.B, 2001, 754, 107–112. [fluorescence
detection; LOQ 50 ng/mL]
Djabarouti, S.; Boselli, E.; Allaouchiche, B.; Ba, B.; Nguyen, A.T.; Gordien, J.B.; Bernadou, J.M.; Saux,
M.C.; Breilh, D. Determination of levofloxacin in plasma, bronchoalveolar lavage and bone tissues by
high-performance liquid chromatography with ultraviolet detection using a fully automated extraction
method, J.Chromatogr.B, 2004, 799, 165–172. [SPE; moxifloxacin is internal standard]
422
Moxifloxacin
Liang, H.; Kays, M.B.; Sowinski, K.M. Separation of levofloxacin, ciprofloxacin, gatifloxacin, moxifloxacin,
trovafloxacin and cinoxacin by high-performance liquid chromatography: application to levofloxacin
determination in human plasma, J.Chromatogr.B, 2002, 772, 53–63.
Paillard, D.; Grellet, J.; Dubois, V.; Saux, M.-C.; Quentin, C. Discrepancy between uptake and intracellular activity of moxifloxacin in a Staphylococcus aureus-human THP-1 monocytic cell model,
Antimicrob.Agents Chemother., 2002, 46, 288–293. [column-switching; fluorescence detection; LOD
1 ng/mL]
Stass, H.; Dalhoff, A. Determination of BAY 12–8039, a new 8-methoxyquinolone, in human body fluids
by high-performance liquid chromatography with fluorescence detection using on-column focusing,
J.Chromatogr.B, 1997, 702, 163–174.
Tobin, C.M.; Sunderland, J.; White, L.O.; MacGowan, A.P.; Reeves, D.S. An isocratic high performance
liquid chromatography (HPLC) assay for moxifloxacin, a new 8-methoxyquinolone, J.Antimicrob.Chemother., 1998, 42, 278–279.
Wallace, A.W.; Victory, J.M.; Amsden, G.W. Lack of bioequivalence of gatifloxacin when coadministered with calcium-fortified orange juice in healthy volunteers, J.Clin.Pharmacol., 2003, 43, 92–96.
[moxifloxacin is internal standard]
Xuan, D.; Zhong, M.; Mattoes, H.; Bui, K.Q.; McNabb, J.; Nicolau, D.P.; Quintiliani, R.; Nightingale, C.H.
Streptococcus pneumoniae response to repeated moxifloxacin or levofloxacin exposure in a rabbit tissue
cage model, Antimicrob.Agents Chemother., 2001, 45, 794–799. [serum; fluorescence detection; LOQ
100 ng/mL; ciprofloxacin is IS]
423
Moxonidine
Moxonidine
OCH3 H
Molecular formula: C9 H12 ClN5 O
Molecular weight: 241.68
CAS Registry No: 75438-57-2
Merck Index: 13, 6318
H3C
N
N
N
N
Cl
N
H
SAMPLE
Matrix: blood, urine
Sample preparation: Plasma. Condition a 10 mm 6 mL SPE disk (3M Empore) with
MeOH and water. Mix 1 mL plasma with 1 mL water at 0◦ , add 10 ng IS, add to the SPE
disk, wash with MeOH:water 15:85, elute with MeOH:water:trifluoroacetic acid 95:5:3.
Evaporate the eluate to dryness under reduced pressure at 45◦ , reconstitute the residue
with 150 µL 100 mM ammonium acetate, inject a 125 µL aliquot. Urine. Condition a
1 mL Bakerbond carboxylic acid SPE cartridge with MeOH and water. Mix 1 mL urine
with 1 mL water, add 100 ng IS, vortex, add to the SPE cartridge, wash with 1 mL
water, wash with 1 mL MeOH:water 50:50, elute with MeOH:water:trifluoroacetic acid
95:5:3. Evaporate the eluate to dryness under reduced pressure at 45◦ , reconstitute the
residue with 150 µL 100 mM ammonium acetate, inject a 60 µL aliquot.
HPLC VARIABLES
Column: 250 × 4.6 5 µm Supelcosil C18 LC-ABZ
Mobile phase: Gradient. MeCN:25 mM pH 5 ammonium acetate 0:100 for 2 min, to 4:96
over 20 min, maintain at 4:96 for 4 min, return to initial conditions over 1 min.
Flow rate: 1
Injection volume: 60–125
Detector: MS, PE Sciex API-III Plus, APCI, heated nebulizer, 25% of column effluent
entered the detector, m/z 242–44
CHROMATOGRAM
Retention time: 27
Internal standard: clonidine (m/z 230–213)
Limit of quantitation: 50 pg/mL
OTHER SUBSTANCES
Extracted: metabolites
KEY WORDS
plasma; SPE
REFERENCE
He, M.M.; Abraham, T.L.; Lindsay, T.J.; Schaefer, H.C.; Pouliquen, I.J.; Payne, C.; Czeskis, B.; Shipley, L.A.; Oliver, S.D.; Mitchell, M.I. Metabolism and disposition of the antihypertensive agent moxonidine in humans, Drug Metab.Dispos., 2003, 31, 334–342.
Nadifloxacin
HO
CH3
N
N
Molecular formula: C19 H21 FN2 O4
Molecular weight: 360.38
CAS Registry No: 124858-35-1
Merck Index: 13, 6371
F
COOH
O
SAMPLE
Matrix: blood
Sample preparation: Shake 1 mL plasma, 10 µL 100 ng/mL IS in MeOH, 1 mL 25 mM
pH 6.86 phosphate buffer, and 5 mL chloroform for 10 min (Caution! Chloroform is a
carcinogen!), centrifuge at 1800 g for 10 min. Remove a 3.5 mL aliquot of the lower
chloroform layer and evaporate it to dryness under a stream of air at 40◦ , reconstitute
the residue with 120 µL MeOH, inject a 40 µL aliquot.
HPLC VARIABLES
Column: 150 × 4.6 5 µm TSK-gel ODS-80TM
Mobile phase: MeCN:THF:25 mM pH 5.5 ammonium phosphate buffer 35:3:65
Flow rate: 1
Injection volume: 40
Detector: UV 295
CHROMATOGRAM
Retention time: 6.8
Internal standard: OPC-7258 (9-fluoro-6,7-dihydro-5-methyl-8-morpholinyl-1-oxo-1H,
5H-benzo[i,j]quinolizine-2-carboxylic acid) (11.3)
Limit of detection: 1 ng/mL
Limit of quantitation: 5 ng/mL
KEY WORDS
human; pharmacokinetics; plasma; rat
REFERENCE
Koike, M.; Akiyama, H.; Shimizu, T. High-performance liquid chromatographic procedure for the determination of rat plasma concentrations of a new antibacterial agent, (±)-9-fluoro-6,7-dihydro-8(4-hydroxy-1-piperidyl)-5-methyl-1-oxo-1H,5H- benzo[i, j]quinolizine-2-carboxylic acid, for topical use,
J.Chromatogr., 1990, 526, 235–239.
424
HPLC Methods for Recently Approved Pharmaceuticals. by George Lunn
Copyright 2005 John Wiley & Sons, Ltd. ISBN: 0-471-66941-5
425
Naftopidil
Naftopidil
O
N
OH
N
Molecular formula: C24 H28 N2 O3
Molecular weight: 392.49
CAS Registry No: 57149-07-2
Merck Index: 13, 6382
H3CO
SAMPLE
Matrix: blood
Sample preparation: Mix 1 mL plasma, 10 µL 5 µg/mL IS in MeOH, 500 µL 1 M
pH 9.2 dipotassium hydrogen phosphate buffer, and 8 mL diethyl ether, rotate for
10 min. Add 5 mL of the organic layer to 300 µL 50 mM sulfuric acid, rotate for 10 min,
centrifuge at 1000 g, discard the organic layer, remove traces of ether from the aqueous
layer with a stream of nitrogen for 30 s. Remove a 250 µL aliquot of the aqueous layer
and add it to 50 µL 1 M pH 9.2 dipotassium hydrogen phosphate buffer, mix, inject a
100 µL aliquot.
HPLC VARIABLES
Column: 250 × 4.6 5 µm RP-LiChrosorb Select B (Hibar)
Mobile phase: MeCN:MeOH:buffer 22.5:22.5:55 (The buffer was 20 mM potassium
dihydrogen phosphate adjusted to pH 1.8 with phosphoric acid.)
Flow rate: 0.8
Injection volume: 100
Detector: F ex 215 em 320
CHROMATOGRAM
Retention time: 9.3
Internal standard: carvedilol (7.7)
Limit of detection: 1 ng/mL
Limit of quantitation: 2 ng/mL
OTHER SUBSTANCES
Extracted: metabolites
KEY WORDS
pharmacokinetics; plasma
REFERENCE
Niebch, G.; Borbe, H.O.; Besenfelder, E. High-performance liquid chromatography of naftopidil, a novel
antihypertensive drug, and two metabolites in human plasma, J.Chromatogr., 1990, 534, 247–252.
SAMPLE
Matrix: tissue
Sample preparation: Mix 200 mg frozen (−80◦ ) tissue with 100 µL 1.16 µg/mL IS in
MeOH:water 50:50 and 200 µL water in a PTFE tube precooled in liquid nitrogen,
homogenize (Braun microdismembrator), pour the contents into a centrifuge tube, wash
in with 500 µL water, wash in with 500 µL acetone, vortex for 2 min, centrifuge at
4000 rpm at 4◦ for 20 min. Evaporate the supernatant to dryness under a stream of
nitrogen at 40◦ , reconstitute the residue with 1 mL 500 mM pH 3.5 potassium acetate
buffer, centrifuge, add the supernatant dropwise to a 1 mL 100 mg 40 µm Bond-Elut
cyano SPE cartridge, wash twice with 1 mL portions of water, elute with two 250 µL
aliquots of MeCN:100 mM pH 5 potassium acetate buffer 80:20, inject a 100 µL aliquot.
426
Naftopidil
HPLC VARIABLES
Guard column: 17 × 4 5 µm Spherisorb C6
Column: 150 × 4.6 5 µm Spherisorb C6
Mobile phase: MeCN:250 mM pH 4 potassium acetate buffer 65:35
Flow rate: 1
Injection volume: 100
Detector: F ex 285 em 360
CHROMATOGRAM
Retention time: 5.4
Internal standard: naftopidil (5.4)
Limit of quantitation: 10 pg/mg (S/N 10) (for carvedilol)
OTHER SUBSTANCES
Extracted: carvedilol (4.7)
KEY WORDS
heart; naftopidil is IS in original; SPE
REFERENCE
Behn, F.; Läer, S.; Scholz, H. Determination of carvedilol in human cardiac tissue by high-performance
liquid chromatography, J.Chromatogr.Sci., 2001, 39, 121–124.
427
Nandrolone
Nandrolone
CH3 OH
H
H
Molecular formula: C18 H26 O2
Molecular weight: 274.40
CAS Registry No: 434-22-0
Merck Index: 13, 6391
H
H
O
SAMPLE
Matrix: blood
Sample preparation: Condition a 1 mL 100 mg Bond Elut C1 SPE cartridge with 2
column vol of MeCN and 2 column vol of water. Mix 500 µL serum with 25 µL 5 µg/mL
IS in MeCN, add 500 µL water, vortex for 5 s, add to the SPE cartridge, wash with 2
column vol of water, wash with 1 column vol of MeCN:water 10:90, elute with 1 mL
MeCN:water 45:55, concentrate the eluate to 250 µL under a stream of nitrogen at 45◦ ,
inject a 50 µL aliquot.
HPLC VARIABLES
Guard column: 15 × 4.6 7 µm silica (Brownlee)
Column: 220 × 4.6 5 µm silica (Brownlee)
Column temperature: 60
Mobile phase: MeCN:100 mM pH 2.5 sodium phosphate buffer 15:85
Flow rate: 1
Injection volume: 50
Detector: UV 247
CHROMATOGRAM
Retention time: 8.2
Internal standard: spironolactone (7.8)
Limit of detection: 3 ng/mL
OTHER SUBSTANCES
Extracted: fluoxymesterone (5.4), methandrostenolone (9.3), methyltestosterone (9.6),
stanozolol (UV 230; LOD 7 ng/mL) (12.7), testosterone (8.6), zeranol (UV 263) (3.6)
KEY WORDS
serum: SPE
REFERENCE
Lampert, B.L.; Stewart, J.T. Determination of anabolic steroids and zeranol in human serum by isocratic
reverse phase HPLC on silica, J.Liq.Chromatogr., 1989, 12, 3231–3249.
SAMPLE
Matrix: blood, urine
Sample preparation: Condition a 3 mL 500 mg Baker C18 SPE cartridge with 2.5 mL
MeOH and 5 mL water. Serum. Mix 2 mL serum with 4 ng IS and 15 mL 150 mM pH
5 acetate buffer, sonicate for 5 min, add to the SPE cartridge, wash with 5 mL 150 mM
pH 5 acetate buffer, wash with 10 mL water, wash with 2.5 mL MeOH:water 40:60,
elute with 4 mL MeOH. Evaporate the eluate to dryness under a stream of nitrogen,
reconstitute the residue with 100 µL MeOH, inject a 5 µL aliquot. Urine. Heat 2 mL
urine, 10 ng IS, and 20 µL β-glucuronidase/aryl sulfatase (Helix pomatia) (Boehringer
Mannheim) at 37◦ for 12 h, cool, add 15 mL 150 mM pH 5 acetate buffer, add to the
SPE cartridge, wash with 5 mL 150 mM pH 5 acetate buffer, wash with 10 mL water,
wash with 2.5 mL MeOH:water 40:60, elute with 4 mL MeOH. Evaporate the eluate to
428
Nandrolone
dryness under a stream of nitrogen, reconstitute the residue with 100 µL MeOH, inject
a 5 µL aliquot.
HPLC VARIABLES
Column: 250 × 2 5 µm Kingsorb C18 (Phenomenex)
Mobile phase: MeCN:water 70:30 containing 2 mM ammonium acetate
Flow rate: 0.15
Injection volume: 5
Detector: MS, PE Sciex API-III Plus triple quadrupole, APCI, positive ion mode, nebulizer gas air at 400 kPa, curtain gas nitrogen at 0.6 L/min, auxiliary gas air 1.5 L/min,
collision gas argon, nebulizer 350◦ , discharge current 4 µA, orifice 90 V, m/z 275–83,
275–109
CHROMATOGRAM
Retention time: 5.6
Internal standard: d2 -17β-testosterone (m/z 291–99) (6.1)
Limit of quantitation: 100 pg/mL
OTHER SUBSTANCES
Extracted: 17α-nortestosterone (m/z 275–83, 275–109) (6.4), progesterone (m/z 315–97)
(11.4), 17α-testosterone (m/z 289–107, 289–109) (7.2), 17β-testosterone (m/z 289–107,
289–109) (6.1)
KEY WORDS
cow; serum; SPE
REFERENCE
Draisci, R.; Palleschi, L.; Ferretti, E.; Lucentini, L.; Cammarata, P. Quantitation of anabolic hormones
and their metabolites in bovine serum and urine by liquid chromatography-tandem mass spectrometry,
J.Chromatogr.A, 2000, 870, 511–522.
429
Narasin
Narasin
Molecular formula: C43 H72 O11
CH3
H3C
HOOC
Molecular weight: 765.02
CAS Registry No: 55134-13-9
Merck Index: 13, 6445
H
O
CH3
H
CH3
CH3
H3C
O
OH
CH3
OH
OH
CH3
H
O
O
O
CH3
H
O
CH3
CH3
SAMPLE
Matrix: blood
Sample preparation: Vortex 100 µL MeOH:water 80:20 and 400 µL trichloroacetic
acid solution with 500 µL plasma, centrifuge at 4000 rpm for 4 min, filter (Spin-X) the
supernatant, inject a 30 µL aliquot of the filtrate. (Prepare trichloroacetic acid solution
as follows. Dissolve 85 g trichloroacetic acid in 15 mL water. Store this solution in the
refrigerator. Dilute 150 µL of this solution with 100 mL acetone.)
HPLC VARIABLES
Guard column: C18
Column: 250 × 4.6 5 µm Supelco Discovery C18
Mobile phase: MeOH:10 mM ammonium acetate 85:15
Flow rate: 0.8 for 15 min, 1 for 10 min
Injection volume: 30
Detector: MS, PE Sciex API 100 single quadrupole, turbo ionspray, turbo probe 150◦ , air
6 L/min, 50 µL/min flowed into detector, m/z 693.7
CHROMATOGRAM
Retention time: 23
Limit of detection: 5 ng/mL
Limit of quantitation: 10 ng/mL
OTHER SUBSTANCES
Extracted: lasalocid (10), monensin (15), salinomycin (20)
KEY WORDS
chicken; plasma
REFERENCE
Hormazábal, V.; Yndestad, M. Determination of amprolium, ethopabate, lasalocid, monensin, narasin,
and salinomycin in chicken tissues, plasma, and egg using liquid chromatography-mass spectrometry,
J.Liq.Chromatogr.Rel.Technol., 2000, 23, 1585–1598.
430
Nartograstim
Nartograstim
Molecular formula: C850 H1344 N226 O245 S8
Molecular weight: 18905.67
CAS Registry No: 134088-74-7
Merck Index: 13, 4552
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: TSK gel ODS-120T (Tosoh)
Mobile phase: Gradient. MeCN:0.1% trifluoroacetic acid from 40:60 to 90:10 over 80 (?)
min.
Flow rate: 1
CHROMATOGRAM
Retention time: 39 (oxidized form), 42 (reduced form)
REFERENCE
Yamasaki, M.; Konishi, N.; Yamaguchi, K.; Itoh, S.; Yokoo, Y. Purification and characterization of recombinant human granulocyte colony-stimulating factor (rhG-CSF) derivatives: KW-2228 and other
derivatives, Biosci.Biotechnol.Biochem., 1998, 62, 1528–1534.
Nateglinide
Nateglinide
Molecular formula: C19 H27 NO3
Molecular weight: 317.42
CAS Registry No: 105816-04-4
Merck Index: 13, 6454
431
COOH
O
N
H
CH3
CH3
SAMPLE
Matrix: blood
Sample preparation: Mix 50 µL plasma with 10 µL 5 µg/mL IS in MeOH and 100 µL
MeCN for 10 min, centrifuge at 3000 g for 10 min. Evaporate the supernatant to dryness
under a stream of nitrogen at 50◦ , reconstitute the residue with 30 µL mobile phase,
inject a 20 µL aliquot.
HPLC VARIABLES
Guard column: present but not specified
Column: 250 × 3 5 µm ProntoSIL 120-5-C18 AQ (Bischoff)
Column temperature: 50
Mobile phase: MeCN:MeOH:buffer 30:8:70 (The buffer was 100 mM potassium hydrogen phosphate (sic) adjusted to pH 4.0 with 30% KOH.)
Flow rate: 1
Injection volume: 20
Detector: UV 210
CHROMATOGRAM
Retention time: 14.1
Internal standard: carbamazepine (6.7)
Limit of quantitation: 100 pg/mL
KEY WORDS
pharmacokinetics; plasma
REFERENCE
Bauer, S.; Störmer, E.; Kirchheiner, J.; Michael, C.; Brockmöller, J.; Roots, I. Rapid and simple method
for the analysis of nateglinide in human plasma using HPLC analysis with UV detection, J.Pharm.Biomed.Anal., 2003, 31, 551–555.
SAMPLE
Matrix: blood
Sample preparation: Mix 500 µL plasma with IS and 2 mL 50 mM pH 6.6 phosphate
buffer, add to a conditioned (unspecified) Sep-Pak SPE cartridge, wash with two 3 mL
portions of water, elute with 5 mL MeOH. Evaporate the eluate to dryness, reconstitute
the residue with 250 µL mobile phase, mix, centrifuge, inject a 150 µL aliquot.
HPLC VARIABLES
Column: 250 × 4.6 5 µm Supelcosil LC-ABZ
Mobile phase: MeCN:50 mM pH 6.27 phosphate buffer 35:65
Injection volume: 150
Detector: UV 210
CHROMATOGRAM
Internal standard: N-(trans-4-t-butylcyclohexylcarbonyl)-D-phenylalanine
Limit of quantitation: 50 ng/mL
432
Nateglinide
KEY WORDS
pharmacokinetics; plasma
REFERENCE
Weaver, M.L.; Orwig, B.A.; Rodriguez, L.C.; Graham, E.D.; Chin, J.A.; Shapiro, M.J.; McLeod, J.F.; Mangold, J.B. Pharmacokinetics and metabolism of nateglinide in humans, Drug Metab.Dispos., 2001, 29,
415–421.
ANNOTATED BIBLIOGRAPHY
Anderson, D.M.; Shelley, S.; Crick, N.; Buraglio, M. No effect of the novel antidiabetic agent nateglinide
on the pharmacokinetics and anticoagulant properties of warfarin in healthy volunteers, J.Clin.Pharmacol., 2002, 42, 1358–1365. [LOQ 50 ng/mL; SPE; column-switching]
Choudhury, S.; Hirschberg, Y.; Filipek, R.; Lasseter, K.; McLeod, J.F. Single-dose pharmacokinetics of
nateglinide in subjects with hepatic cirrhosis, J.Clin.Pharmacol., 2000, 40, 634–640. [SPE; LOQ
25.8 ng/mL]
Ono, I.; Matsuda, K.; Kanno, S. Determination of N-(trans-4-isopropylcyclohexylcarbonyl)-D-phenylalanine in human plasma by solid-phase extraction and column-switching high-performance liquid
chromatography with ultraviolet detection, J.Chromatogr.B, 1996, 678, 384–387. [LOQ 50 ng/mL]
Ono, I.; Matsuda, K.; Kanno, S. Determination of N-(trans-4-isopropylcyclohexanecarbonyl)-D-phenylalanine and its metabolites in human plasma and urine by column-switching high-performance liquid
chromatography with ultraviolet detection, J.Chromatogr.B, 1997, 692, 397–404.
Qi, M.; Wang, P.; Sun, Y.; Li, Y. Determination of the L-enantiomer of nateglinide in a bulk drug
substance by chiral reversed-phase liquid chromatography, J.Liq.Chromatogr.Rel.Technol., 2003, 26,
1839–1845. [LOD (L) 300 ng/mL (D) 400 ng/mL; LOQ (L) 800 ng/mL (D) 1000 ng/mL]
Nebivolol
Nebivolol
OH
O
H
N
433
OH
O
Molecular formula: C22 H25 F2 NO4
Molecular weight: 405.43
CAS Registry No: 99200-09-6
Merck Index: 13, 6459
F
F
SAMPLE
Matrix: solutions
Sample preparation: Inject a 20 µL aliquot of a 100 µg/mL solution in EtOH.
HPLC VARIABLES
Column: 150 × 4.6 5 µm Chiralpak AD-RH
Column temperature: 23 ± 1
Mobile phase: Isopropanol
Flow rate: 0.5
Injection volume: 20
Detector: UV 220
CHROMATOGRAM
Retention time: 13.17 (+), 17.45 (−)
KEY WORDS
chiral
REFERENCE
Aboul-Enein, H.Y.; Ali, I. HPLC enantiomeric resolution of nebivolol on normal and reversed amylose
based chiral phases, Pharmazie, 2001, 56, 214–216.
SAMPLE
Matrix: urine
Sample preparation: Mix 5 mL urine with 50 µL 10 µg/mL IS, add 500 µL 1 M pH
5.2 sodium acetate buffer, add 50 µL β-glucuronidase/aryl sulfatase (Helix pomatia)
(Boehringer Mannheim), heat at 50◦ for 1 h, cool, add 500 mg sodium hydrogen carbonate:potassium carbonate 2:1, add 1 mL t-butanol, add 5 mL MTBE, add 1 g sodium
sulfate, shake for 15 min, centrifuge for 10 min. Evaporate the organic layer to dryness
under reduced pressure at 50◦ , reconstitute the residue with 50 µL MeCN, inject a 5 µL
aliquot.
HPLC VARIABLES
Column: 55 × 4 3 µm Purospher STAR RP-18e (Merck)
Column temperature: 25
Mobile phase: Gradient. A MeCN. B was pH 3.5 ammonium acetate buffer (4 mM
ammonium acetate and 1% acetic acid). A:B from 0:100 to 80:20 over 4 min, re-equilibrate
at initial conditions for 3 min.
Flow rate: 1
Injection volume: 5
Detector: MS, PE Sciex API 2000 triple quadrupole, APCI, 10% of column effluent
entered the detector, positive ionization, interface 400◦ , ionization 5.9 kV, m/z 151.0
CHROMATOGRAM
Retention time: 3.82
Internal standard: bupranolol (216.1)
Limit of detection: 10 ng/mL
434
Nebivolol
OTHER SUBSTANCES
Extracted: acebutolol (m/z 116.2), alprenolol (m/z 116.3), atenolol (m/z 145.1), betaxolol
(m/z 116.1), befunolol(m/z 56.0), bisoprolol (m/z 116.3), bunitrolol (m/z 193.2) (2.97),
butofilolol (m/z 256.4), carazolol (m/z 116.3), carteolol (m/z 237.3), carvedilol (m/z 100.2)
(3.69), celiprolol (m/z 251.1), cloranolol (m/z 236.1), esmolol (m/z 145.1), indenolol (m/z
171.1), labetalol (m/z 91.1), levobunolol (m/z 236.3), mepindolol (m/z 116.2), metipranolol
(m/z 116.2), metoprolol (m/z 116.2), moprolol (m/z 116.2), nadolol (m/z 254.4), nifenalol
(m/z 165.2), oxprenolol (m/z 72.0), penbutolol (m/z 236.4), pindolol (m/z 116.2), propranolol (m/z 116.1), sotalol (m/z 133.2), talinolol (m/z 308.3) (3.83), timolol (m/z 261.2),
toliprolol (m/z 147.2)
REFERENCE
Thevis, M.; Opfermann, G.; Schänzer, W. High speed determination of beta-receptor blocking agents in
human urine by liquid chromatography/tandem mass spectrometry, Biomed.Chromatogr., 2001, 15,
393–402.
ANNOTATED BIBLIOGRAPHY
Ali, I.; Aboul-Enein, H.Y. Enantioseparation of some clinically used drugs by HPLC using cellulose
Tris (3,5-dichlorophenylcarbamate) chiral stationary phase, Biomed.Chromatogr., 2003, 17, 113–117.
[metoprolol; teratolol; tolamolol; nebivolol; econazole; miconazole; cromakalim; etodolac]
Woestenborghs, R.; Embrechts, L.; Heykants, J. HPLC-fluorescence method for the determination of
the new β1 -adrenoreceptor blocking agent nebivolol in human plasma, Methodol.Surv.Biochem.Anal.,
1988, 18, 215–216.
435
Nelfinavir
Nelfinavir
H
Molecular formula: C32 H45 N3 O4 S
Molecular weight: 567.79
CAS Registry No: 159989-64-7, 159989-65-8 (mesylate)
Merck Index: 13, 6471
CH3
O
S
O
CH3
CH3
CH3
N
HO
N
H
N
OH
H
H
SAMPLE
Matrix: blood
Sample preparation: Condition an Extrasep C18 SPE cartridge (Lida) with 2 mL
MeOH and 2 mL water. Dilute 500 µL serum with 500 µL water, add to the SPE
cartridge, wash with 500 µL water, elute with 1 mL MeOH. Evaporate the eluate to
dryness with vortexing under reduced pressure at 40◦ and reconstitute the residue with
300 µL MeOH, inject a 10 µL aliquot.
HPLC VARIABLES
Column: two 150 × 4.6 3 µm Luna C18 columns in series
Column temperature: 60
Mobile phase: Gradient. MeCN:4 mM sulfuric acid from 8:92 to 63:37 over 45 min,
maintain at 63:37 for 5 min.
Flow rate: 0.85
Injection volume: 10
Detector: UV 265 for 31 min then UV 240
CHROMATOGRAM
Retention time: 33.5
Limit of detection: 400 ng/mL
OTHER SUBSTANCES
Extracted: delavirdine (25.5, LOD 110 ng/mL), efavirenz (51, LOD 62 ng/mL), indinavir (24.5, LOD 210 ng/mL), nelfinavir (33.5, LOD 400 ng/mL), nevirapine (23.5, LOD
84 ng/mL), ritonavir (50.5, LOD 510 ng/mL), saquinavir (35, LOD 100 ng/mL)
KEY WORDS
SPE; serum
REFERENCE
Simon, V.A.; Thiam, M.D.; Lipford, L.C. Determination of serum levels of thirteen human immunodeficiency virus-suppressing drugs by high-performance liquid chromatography, J.Chromatogr.A, 2001,
913, 447–453.
SAMPLE
Matrix: blood
Sample preparation: Mix 250 µL plasma with 50 µL MeOH, add 100 µL 2 µg/mL IS
in MeOH, add 250 µL 1 M NaOH, add 3 mL hexane:ethyl acetate 50:50, shake at high
speed for 25 min, centrifuge at 3000 g for 15 min. Evaporate the organic layer to dryness
under a stream of air, reconstitute the residue with 1 mL initial mobile phase, inject a
20 µL aliquot.
HPLC VARIABLES
Guard column: 10 × 2.1 Symmetry Shield
436
Nelfinavir
Column: 30 × 2.1 3.5 µm Symmetry C18
Mobile phase: Gradient. MeCN:5 mM pH 3.25 acetate buffer from 25:75 to 80:20 over
4 min using a nonlinear gradient (not specified).
Flow rate: 0.35
Injection volume: 20
Detector: MS, PE Sciex API 3000, turbo ionspray source, column effluent split 1:1 before
entering source
CHROMATOGRAM
Retention time: 2.5
Internal standard: Abbott A-86093 (3.2)
Limit of detection: 330 pg/mL
Limit of quantitation: 16.3 ng/mL
OTHER SUBSTANCES
Extracted: amprenavir (2.7, LOQ 16.3 ng/mL, LOD 380 pg/mL), indinavir (2.0, LOQ
16.3 ng/mL, LOD 1.5 ng/mL), lopinavir (3.1, LOQ 16.3 ng/mL, LOD 750 pg/mL), ritonavir (2.9, LOQ 51.2 ng/mL, LOD 650 pg/mL), saquinavir (2.4, LOQ 16.3 ng/mL, LOD
780 pg/mL)
KEY WORDS
plasma
REFERENCE
Frerichs, V.A.; DiFrancesco, R.; Morse, G.D. Determination of protease inhibitors using liquid chromatography-tandem mass spectrometry, J.Chromatogr.B, 2003, 787, 393–403.
SAMPLE
Matrix: blood
Sample preparation: Mix 1 mL plasma with 200 µL 10 µg/mL IS in water, add 200 µL
100 mM NaOH, mix, add 4 mL diethyl ether, shake for 5 min, centrifuge at 2500 rpm for
5 min. Evaporate the organic layer to dryness under a stream of nitrogen, reconstitute
the residue with 200 µL initial mobile phase, inject a 100 µL aliquot.
HPLC VARIABLES
Column: 250 × 4.6 Stability RP18 (CIL, France)
Mobile phase: Gradient. MeCN:50 mM pH 5.65 phosphate buffer from 36:64 to 64:36
over 25 min, to 80:20 (step gradient), maintain at 80:20 for 10 min, re-equilibrate at
initial conditions for 5 min.
Flow rate: 1.5
Injection volume: 100
Detector: UV 240 for 5 min, UV 215 for 22 min, UV 260 for rest of run
CHROMATOGRAM
Retention time: 24.1
Internal standard: JR051012 (Janssen Cilag) (28.2)
Limit of quantitation: 50 ng/mL
OTHER SUBSTANCES
Extracted: amprenavir (11.2), efavirenz (19.9), indinavir (8.5), lopinavir (18.9), nevirapine (3.3), ritonavir (17.6), saquinavir (16.7)
Noninterfering: acetaminophen, amineptine, amphotericin B, aspirin, bromazepam,
buspirone, citalopram, clobazam, diazepam, didanosine, fluconazole, flunitrazepam, fluvoxamine, hydroxyitraconazole, isoniazid, itraconazole, lamivudine, loprazolam, lorazepam, metronidazole, minalcipram, nordiazepam, omeprazole, paroxetine, pyrimethamine, rifampin, sertraline, stavudine, sulfadiazine, trimethoprim, venlafaxine, zalcitabine, zidovudine, zolpidem, zopiclone
Nelfinavir
437
KEY WORDS
plasma
REFERENCE
Titier, K.; Lagrange, F.; Péhourcq, F.; Edno-Mcheik, L.; Moore, N.; Molimard, M. High-performance liquid chromatographic method for the simultaneous determination of the six HIV-protease inhibitors
and two non-nucleoside reverse transcriptase inhibitors in human plasma, Ther.Drug Monit., 2002,
24, 417–424.
SAMPLE
Matrix: blood
Sample preparation: Briefly vortex 250 µL 1 µg/mL IS in MeOH with 250 µL plasma,
add 500 µL 100 mM pH 10.5 ammonium hydroxide, vortex, add 2 mL MeCN:ethyl
acetate 10:90, vortex for 4 min, centrifuge at 2060 g, evaporate the organic layer to
dryness under a stream of nitrogen at 50◦ , reconstitute with 150 µL mobile phase,
vortex for 2 min, inject a 100 µL aliquot.
HPLC VARIABLES
Guard column: Nova-Pak C18
Column: 250 × 4.6 5 µm Symmetry C18 (Waters)
Mobile phase: MeCN:25 mM sodium dihydrogen phosphate adjusted to pH 3.4 with
phosphoric acid 42:58
Flow rate: 1.3
Injection volume: 100
Detector: UV 220
CHROMATOGRAM
Retention time: 8.2
Internal standard: 6,7-dimethyl-2,3-di(2-pyridyl)quinoxaline (9.9)
Limit of quantitation: 50 ng/mL
KEY WORDS
plasma
REFERENCE
Wu, E.Y.; Wilkinson, J.M.I.I.; Naret, D.G.; Daniels, V.L.; Williams, L.J.; Khalil, D.A.; Shetty, B.V. Highperformance liquid chromatographic method for the determination of nelfinavir, a novel HIV-1 protease
inhibitor, in human plasma, J.Chromatogr.B, 1997, 695, 373–380.
ANNOTATED BIBLIOGRAPHY
Aymard, G.; Legrand, M.; Trichereau, N.; Diquet, B. Determination of twelve antiretroviral agents in
human plasma sample using reversed-phase high-performance liquid chromatography, J.Chromatogr.B,
2000, 744, 227–240. [amprenavir; efavirenz; indinavir; nelfinavir; ritonavir; saquinavir; abacavir;
didanosine; lamivudine; stavudine; nevirapine; zidovudine; column-switching; SPE; LOQ 5–100 ng/mL]
Bouley, M.; Briere, C.; Padoin, C.; Petitjean, O.; Tod, M. Sensitive and rapid method for the simultaneous quantification of the HIV-protease inhibitors indinavir, nelfinavir, ritonavir, and saquinavir in
human plasma by reversed-phase liquid chromatography, Ther.Drug Monit., 2001, 23, 56–60. [LOQ
25–250 ng/mL]
Chi, J.; Jayewardene, A.L.; Stone, J.A.; Motoya, T.; Aweeka, F.T. Simultaneous determination of five
HIV protease inhibitors nelfinavir, indinavir, ritonavir, saquinavir and amprenavir in human plasma
by LC/MS/MS, J.Pharm.Biomed.Anal., 2002, 30, 675–684. [LOQ 5 ng/mL]
Cociglio, M.; Hillaire-Buys, D.; Peyrière, H.; Alric, R. Performance analysis of a rapid HPLC determination with the solvent demixing extraction of HIV antiproteases and efavirenz in plasma,
J.Chromatogr.Sci., 2003, 41, 80–86. [LOD 1–175 ng/mL]
Dailly, E.; Thomas, L.; Kergueris, M.F.; Jolliet, P.; Bourin, M. High-performance liquid chromatographic
assay to determine the plasma levels of HIV-protease inhibitors (amprenavir, indinavir, nelfinavir,
438
Nelfinavir
ritonavir and saquinavir) and the non-nucleoside reverse transcriptase inhibitor (nevirapine) after
liquid-liquid extraction, J.Chromatogr.B, 2001, 758, 129–135. [LOQ 50–400 ng/mL]
Droste, J.A.H.; Verweij-van Wissen, C.P.W.G.M.; Burger, D.M. Simultaneous determination of the HIV
drugs indinavir, amprenavir, saquinavir, ritonavir, lopinavir, nelfinavir, the nelfinavir hydroxymetabolite M8, and nevirapine in human plasma by reversed-phase high-performance liquid chromatography,
Ther.Drug Monit., 2003, 25, 393–399. [LOQ 50–70 ng/mL]
Faux, J.; Venisse, N.; Le Moal, G.; Dupuis, A.; Bouquet, S. Simultaneous determination of six HIV
protease inhibitors, one metabolite, and two non-nucleoside reverse transcriptase inhibitors in
human plasma by isocratic reversed-phase liquid chromatography after solid-phase extraction, Chromatographia, 2003, 58, 421–426. [LOQ 50 – 120 ng/mL; amprenavir; indinavir; lopinavir; nelfinavir;
ritonavir; saquinavir; efavirenz; nevirapine; prazepam is internal standard]
Hugen, P.W.H.; Verweij-van Wissen, C.P.W.G.M.; Burger, D.M.; Wuis, E.W.; Koopmans, P.P.; Hekster, Y.A. Simultaneous determination of the HIV-protease inhibitors indinavir, nelfinavir, saquinavir
and ritonavir in human plasma by reversed-phase high-performance liquid chromatography, J. Chromatogr.B, 1999, 727, 139–149. [LOQ 45 ng/mL]
Janoly, A.; Bleyzac, N.; Favetta, P.; Gagneu, M.C.; Bourhis, Y.; Coudray, S.; Oger, I.; Aulagner, G. Simple
and rapid high-performance liquid chromatographic method for nelfinavir, M8 nelfinavir metabolite,
ritonavir and saquinavir assay in plasma, J.Chromatogr.B, 2002, 780, 155–160. [LOD 200 ng/mL]
Justesen, U.S.; Pedersen, C.; Klitgaard, N.A. Simultaneous quantitative determination of the HIV protease inhibitors indinavir, amprenavir, ritonavir, lopinavir, saquinavir, nelfinavir and the nelfinavir
active metabolite M8 in plasma by liquid chromatography, J.Chromatogr.B, 2003, 783, 491–500. [LOQ
25 ng/mL]
Keil, K.; Frerichs, V.A.; DiFrancesco, R.; Morse, G. Reverse phase high-performance liquid chromatography method for the analysis of amprenavir, efavirenz, indinavir, lopinavir, nelfinavir and its active
metabolite (M8), ritonavir, and saquinavir in heparinized human plasma, Ther.Drug Monit., 2003, 25,
340–346. [LOQ 100–200 ng/mL]
Lamotte, C.; Peytavin, G.; Farinotti, R. Determination of nelfinavir, a potent HIV protease inhibitor,
and its active metabolite M8 in human plasma by high-performance liquid chromatography with
photodiode-array detection, J.Chromatogr.B, 1999, 735, 159–170. [LOQ 25 ng/mL]
Poirier, J.-M.; Radembino, N.; Robidou, P.; Jaillon, P. Simultaneous determination of the five HIVprotease inhibitors: Amprenavir, indinavir, nelfinavir, ritonavir, and saquinavir in human plasma
by solid-phase extraction and column liquid chromatography, Ther.Drug Monit., 2000, 22, 465–473.
[LOQ 25 ng/mL]
Poirier, J.-M.; Robidou, P.; Jaillon, P. Simultaneous determination of the six HIV protease inhibitors
(amprenavir, indinavir, lopinavir, nelfinavir, ritonavir, and saquinavir) plus M8 nelfinavir metabolite
and the nonnucleoside reverse transcription inhibitor efavirenz in human plasma by solid-phase
extraction and column liquid chromatography, Ther.Drug Monit., 2002, 24, 302–309. [LOQ 25 ng/mL]
Proust, V.; Toth, K.; Hulin, A.; Taburet, A.-M.; Gimenez, F.; Singlas, E. Simultaneous high-performance
liquid chromatographic determination of the antiretroviral agents amprenavir, nelfinavir, ritonavir,
saquinavir, delavirdine and efavirenz in human plasma, J.Chromatogr.B, 2000, 742, 453–458. [LOQ
50–150 ng/mL]
Remmel, R.P.; Kawle, S.P.; Weller, D.; Fletcher, C.V. Simultaneous HPLC assay for quantification of
indinavir, nelfinavir, ritonavir, and saquinavir in human plasma, Clin.Chem., 2000, 46, 73–81. [LOQ
20–50 ng/mL]
Rentsch, K.M. Sensitive and specific determination of eight antiretroviral agents in plasma by highperformance liquid chromatography-mass spectrometry, J.Chromatogr.B, 2003, 788, 339–350. [SPE;
LOQ 1–250 ng/mL; amprenavir; efavirenz; indinavir; lopinavir; nelfinavir; nevirapine; ritonavir;
saquinavir]
Sarasa-Nacenta, M.; López-Púa, Y.; Mallolas, J.; Blanco, J.L.; Gatell, J.M.; Carné, X. Simultaneous
determination of the HIV-protease inhibitors indinavir, amprenavir, ritonavir, saquinavir and nelfinavir in human plasma by reversed-phase high-performance liquid chromatography, J.Chromatogr.B,
2001, 757, 325–332. [SPE; LOQ 10–85 ng/mL]
Simon, V.A.; Thiam, M.D.; Lipford, L.C. Determination of serum levels of thirteen human immunodeficiency virus-suppressing drugs by high-performance liquid chromatography, J.Chromatogr.A, 2001,
913, 447–453. [SPE; LOD 30–440 ng/mL; zalcitabine; lamivudine; stavudine; didanosine; zidovudine;
nevirapine; abacavir; indinavir; delavirdine; nelfinavir; saquinavir; ritonavir; efavirenz]
Tribut, O.; Arvieux, C.; Michelet, C.; Chapplain, J.-M.; Allain, H.; Bentué-Ferrer, D. Simultaneous quantitative assay of six HIV protease inhibitors, one metabolite, and two non-nucleoside reverse transcriptase inhibitors in human plasma by isocratic reversed-phase liquid chromatography, Ther.Drug
Nelfinavir
439
Monit., 2002, 24, 554–562. [LOQ 25 ng/mL; nevirapine; efavirenz; indinavir; amprenavir; nelfinavir;
ritonavir; lopinavir; saquinavir]
Turner, M.L.; Reed-Walker, K.; King, J.R.; Acosta, E.P. Simultaneous determination of nine antiretroviral compounds in human plasma using liquid chromatography, J.Chromatogr.B, 2003, 784, 331–341.
[LOQ 50 ng/mL; indinavir; nelfinavir; saquinavir; ritonavir; amprenavir; delavirdine; efavirenz;
lopinavir]
van Heeswijk, R.P.G.; Hoetelmans, R.M.W.; Harms, R.; Meenhorst, P.L.; Mulder, J.W.; Lange, J.M.A.;
Beijnen, J.H. Simultaneous quantitative determination of the HIV protease inhibitors amprenavir,
indinavir, nelfinavir, ritonavir and saquinavir in human plasma by ion-pair high-performance liquid chromatography with ultraviolet detection, J.Chromatogr.B, 1998, 719, 159–168. [SPE; LOQ
25–50 ng/mL]
Villani, P.; Feroggio, M.; Gianelli, L.; Bartoli, A.; Montagna, M.; Maserati, R.; Regazzi, M.B. Antiretrovirals: simultaneous determination of five protease inhibitors and three nonnucleoside transcriptase
inhibitors in human plasma by a rapid high-performance liquid chromatography-mass spectrometry
assay, Ther.Drug Monit., 2001, 23, 380–388. [LOD 10–25 ng/mL; LOQ 20–40 ng/mL; saquinavir;
indinavir; ritonavir; nelfinavir; amprenavir; nevirapine; delavirdine; efavirenz]
Yamada, H.; Kotaki, H.; Nakamura, T.; Iwamoto, A. Simultaneous determination of the HIV protease
inhibitors indinavir, amprenavir, saquinavir, ritonavir and nelfinavir in human plasma by highperformance liquid chromatography, J.Chromatogr.B, 2001, 755, 85–89. [LOQ 50 ng/mL]
Zhang, K.E.; Wu, E.; Patick, A.K.; Kerr, B.; Zorbas, M.; Lankford, A.; Kobayashi, T.; Maeda, Y.; Shetty, B.;
Webber, S. Circulating metabolites of the human immunodeficiency virus protease inhibitor nelfinavir
in humans: Structural identification, levels in plasma, and antiviral activities, Antimicrob.Agents
Chemother., 2001, 45, 1086–1093. [LC-MS; reserpine is internal standard; LOQ 20 ng/mL]
440
Nequinate
Nequinate
Molecular formula: C22 H23 NO4
Molecular weight: 365.42
CAS Registry No: 13997-19-8
Merck Index: 13, 6498
H
O
N
H3C
OCH3
O
O
SAMPLE
Matrix: feed
Sample preparation: Slurry 100 g 100–200 mesh Dowex 50 W-X8 (H+ ) cation-exchange
resin with 500 mL 10% HCl, heat to boiling with continuous stirring, cool, discard liquid,
wash resin twice with 500 mL portions of water, wash with 250 mL MeOH, filter (Whatman No. 541 paper), rinse with 200 mL MeOH, air dry. Slurry 5 g resin with 50 mL 10%
HCl, pour into a chromatography column, wash with water until the effluent is neutral
to litmus, wash with 50 mL MeOH. Do not allow to go dry at any stage. Shake 20 g finely
ground feed with 100 mL 2% methanesulfonic acid in MeOH for 30 min, filter (Whatman
No. 541 paper). Add 25 mL filtrate to 100 mL 10% HCl and 100 mL dichloromethane,
shake for 1 min, extract the aqueous phase twice with 40 mL portions of dichloromethane.
Combine the organic layers and evaporate to dryness under reduced pressure at 40◦ ,
reconstitute the residue with 20–25 mL MeOH, add to the ion-exchange column, rinse
flask with 10 mL MeOH, add the rinse to the column, wash the column with 50 mL MeOH,
elute the column with 150 mL 2% methanesulfonic acid in MeOH. Mix the eluate with
300 mL 10% HCl and 130 mL dichloromethane, shake for 1 min, extract the aqueous layer
twice with 70 mL portions of dichloromethane. Combine the organic layers and evaporate
to dryness under reduced pressure at 40◦ , reconstitute the residue with 10 mL MeOH,
inject a 20 µL aliquot.
HPLC VARIABLES
Column: 300 × 3.9 µBondapak C18
Mobile phase: MeOH:water 75:25
Flow rate: 1.5
Injection volume: 20
Detector: UV 265
CHROMATOGRAM
Retention time: 8
Limit of quantitation: 1 µg/g
OTHER SUBSTANCES
Noninterfering: clopidol
KEY WORDS
SPE
REFERENCE
Merson, G.H.J.; Hill, L.A.; Johnson, S.F. Determination of methyl benzoquate in poultry feeding stuffs
using high-performance liquid chromatography, Analyst, 1985, 110, 761–764.
441
Neridronic acid
Neridronic acid
Molecular formula: C6 H17 NO7 P2
O
OH
HO
H2N
Molecular weight: 277.15
CAS Registry No: 79778-41-9
P
P
OH
OH
OH
O
SAMPLE
Matrix: formulations
Sample preparation: Dilute injections 100-fold, inject a 20 µL aliquot. Disintegrate
a 5 mg tablet in 100 mL water, sonicate for 5 min, centrifuge an aliquot at 3600 g for
4 min, inject a 20 µL aliquot of the supernatant.
HPLC VARIABLES
Column: 150 × 4.6 10 µm IC-PAK Anion HC (Waters)
Column temperature: 30
Mobile phase: 1.5 mM nitric acid containing 0.5 mM copper(II) nitrate (Prepare column
by pumping ILC Regenerant A (Waters) and 100 mM nitric acid for 30 min.)
Flow rate: 1
Injection volume: 20
Detector: UV 245
CHROMATOGRAM
Retention time: 1.5
OTHER SUBSTANCES
Simultaneous: alendronate (k′ 0.48), clodronate (k′ 26.4), etidronate (5.8 min), olpadronate (k′ 0.82), pamidronate (2 min)
KEY WORDS
complexation; injections; tablets
REFERENCE
Sparidans, R.W.; Den Hartigh, J.; Vermeij, P. High-performance ion-exchange chromatography with
in-line complexation of bisphosphonates and their quality control in pharmaceutical preparations,
J.Pharm.Biomed.Anal., 1995, 13, 1545–1550.
SAMPLE
Matrix: solutions
Sample preparation: Vortex 100 µL of an 80–500 µg/mL solution in water with 50 µL
EtOH, 40 µL pyridine, 10 µL triethylamine, and 2 µL phenylisothiocyanate, heat at
80◦ for 5 min, evaporate under nitrogen at 80◦ , reconstitute with 1 mL water, wash
twice with 2 mL portions of chloroform:1-pentanol 90:10 (Caution! Chloroform is a
carcinogen!), inject a 20 µL aliquot of the aqueous layer.
HPLC VARIABLES
Column: 100 × 3 5 µm Chromspher C18
Mobile phase: MeCN:20 mM pH 7–8 phosphate buffer containing 5 mM tetraethylammonium hydroxide and 0.5 mM etidronate (adsorption suppressor) 3:97
Flow rate: 0.4
Injection volume: 20
Detector: UV 240
OTHER SUBSTANCES
Simultaneous: alendronate, pamidronate
442
Neridronic acid
KEY WORDS
derivatization
REFERENCE
Sparidans, R.W.; Den Hartigh, J.; Beijnen, J.H.; Vermeij, P. Derivatization of pamidronate and other
amino(bis)phosphonates with different isothiocyanates prior to ion-pair liquid chromatography,
J.Chromatogr.A, 1997, 782, 211–217.
443
Nevirapine
Nevirapine
H
H3C
O
N
Molecular formula: C15 H14 N4 O
Molecular weight: 266.30
CAS Registry No: 129618-40-2
Merck Index: 13, 6514
N
N
N
SAMPLE
Matrix: blood
Sample preparation: Condition an Extrasep C18 SPE cartridge (Lida) with 2 mL
MeOH and 2 mL water. Dilute 500 µL serum with 500 µL water, add to the SPE
cartridge, wash with 500 µL water, elute with 1 mL MeOH. Evaporate the eluate to
dryness with vortexing under reduced pressure at 40◦ and reconstitute the residue with
300 µL MeOH, inject a 10 µL aliquot.
HPLC VARIABLES
Column: two 150 × 4.6 3 µm Luna C18 columns in series
Column temperature: 60
Mobile phase: Gradient. MeCN:4 mM sulfuric acid from 8:92 to 63:37 over 45 min,
maintain at 63:37 for 5 min.
Flow rate: 0.85
Injection volume: 10
Detector: UV 265 for 31 min then UV 240
CHROMATOGRAM
Retention time: 23.5
Limit of detection: 84 ng/mL
OTHER SUBSTANCES
Extracted: delavirdine (25.5, LOD 110 ng/mL), efavirenz (51, LOD 62 ng/mL), indinavir (24.5, LOD 210 ng/mL), nelfinavir (33.5, LOD 400 ng/mL), ritonavir (50.5, LOD
510 ng/mL), saquinavir (35, LOD 100 ng/mL)
KEY WORDS
SPE; serum
REFERENCE
Simon, V.A.; Thiam, M.D.; Lipford, L.C. Determination of serum levels of thirteen human immunodeficiency virus-suppressing drugs by high-performance liquid chromatography, J.Chromatogr.A, 2001,
913, 447–453.
SAMPLE
Matrix: blood
Sample preparation: Mix 1 mL plasma with 200 µL 10 µg/mL IS in water, add 200 µL
100 mM NaOH, mix, add 4 mL diethyl ether, shake for 5 min, centrifuge at 2500 rpm for
5 min. Evaporate the organic layer to dryness under a stream of nitrogen, reconstitute
the residue with 200 µL initial mobile phase, inject a 100 µL aliquot.
HPLC VARIABLES
Column: 250 × 4.6 Stability RP18 (CIL, France)
Mobile phase: Gradient. MeCN:50 mM pH 5.65 phosphate buffer from 36:64 to 64:36
over 25 min, to 80:20 (step gradient), maintain at 80:20 for 10 min, re-equilibrate at
initial conditions for 5 min.
Flow rate: 1.5
444
Nevirapine
Injection volume: 100
Detector: UV 240 for 5 min, UV 215 for 22 min, UV 260 for rest of run
CHROMATOGRAM
Retention time: 3.3
Internal standard: JR051012 (Janssen Cilag) (28.2)
Limit of quantitation: 100 ng/mL
OTHER SUBSTANCES
Extracted: amprenavir (11.2), efavirenz (19.9), indinavir (8.5), lopinavir (18.9), nelfinavir (24.1), ritonavir (17.6), saquinavir (16.7)
Noninterfering: acetaminophen, amineptine, amphotericin B, aspirin, bromazepam,
buspirone, citalopram, clobazam, diazepam, didanosine, fluconazole, flunitrazepam, fluvoxamine, hydroxyitraconazole, isoniazid, itraconazole, lamivudine, loprazolam, lorazepam, metronidazole, minalcipram, nordiazepam, omeprazole, paroxetine, pyrimethamine, rifampin, sertraline, stavudine, sulfadiazine, trimethoprim, venlafaxine, zalcitabine, zidovudine, zolpidem, zopiclone
KEY WORDS
plasma
REFERENCE
Titier, K.; Lagrange, F.; Péhourcq, F.; Edno-Mcheik, L.; Moore, N.; Molimard, M. High-performance liquid chromatographic method for the simultaneous determination of the six HIV-protease inhibitors
and two non-nucleoside reverse transcriptase inhibitors in human plasma, Ther.Drug Monit., 2002,
24, 417–424.
SAMPLE
Matrix: bulk
Sample preparation: Mix 24 mg drug substance with 4 mL MeCN and 80 mL mobile
phase, sonicate until all solid dissolves, cool, make up to 100 mL with mobile phase,
inject a 50 µL aliquot.
HPLC VARIABLES
Column: 150 × 4.6 5 µm Supelcosil LC-ABZ
Column temperature: 35
Mobile phase: MeCN:25 mM pH 5.0 ammonium dihydrogen phosphate buffer 20:80
Flow rate: 1
Injection volume: 50
Detector: UV 220
CHROMATOGRAM
Retention time: 7.44
Limit of detection: 0.001% (S/N 3)
Limit of quantitation: 0.003% (S/N 10)
OTHER SUBSTANCES
Simultaneous: impurities
KEY WORDS
robust; stability-indicating
REFERENCE
Li, Q.C.; Tougas, T.; Cohen, K.; Lee, R.; Meagan, P.; Corson, M.; Muchnick, T. Validation of a highperformance liquid chromatography method for the assay of and determination of related organic
impurities in nevirapine drug substance, J.Chromatogr.Sci., 2000, 38, 246–254.
Nevirapine
445
ANNOTATED BIBLIOGRAPHY
Aymard, G.; Legrand, M.; Trichereau, N.; Diquet, B. Determination of twelve antiretroviral agents
in human plasma sample using reversed-phase high-performance liquid chromatography,
J.Chromatogr.B, 2000, 744, 227–240. [amprenavir; efavirenz; indinavir; nelfinavir; ritonavir;
saquinavir; abacavir; didanosine; lamivudine; stavudine; nevirapine; zidovudine; column-switching;
SPE; LOQ 5–100 ng/mL]
Chi, J.; Jayewardene, A.L.; Stone, J.A.; Aweeka, F.T. An LC-MS-MS method for the determination of
nevirapine, a non-nucleoside reverse transcriptase inhibitor, in human plasma, J.Pharm.Biomed.Anal.,
2003, 31, 953–959. [LOQ 25 ng/mL]
Dailly, E.; Thomas, L.; Kergueris, M.F.; Jolliet, P.; Bourin, M. High-performance liquid chromatographic
assay to determine the plasma levels of HIV-protease inhibitors (amprenavir, indinavir, nelfinavir,
ritonavir and saquinavir) and the non-nucleoside reverse transcriptase inhibitor (nevirapine) after
liquid-liquid extraction, J.Chromatogr.B, 2001, 758, 129–135. [LOQ 50–400 ng/mL]
Droste, J.A.H.; Verweij-van Wissen, C.P.W.G.M.; Burger, D.M. Simultaneous determination of the
HIV drugs indinavir, amprenavir, saquinavir, ritonavir, lopinavir, nelfinavir, the nelfinavir
hydroxymetabolite M8, and nevirapine in human plasma by reversed-phase high-performance liquid
chromatography, Ther.Drug Monit., 2003, 25, 393–399. [LOQ 50–70 ng/mL]
Erickson, D.A.; Mather, G.; Trager, W.F.; Levy, R.H.; Keirns, J.J. Characterization of the in vitro
biotransformation of the HIV-1 reverse transcriptase inhibitor nevirapine by human hepatic
cytochromes P-450, Drug Metab.Dispos., 1999, 27, 1488–1495.
Fan, B.; Stewart, J.T. Determination of zidovudine/zalcitabine/nevirapine in human plasma by ion-pair
HPLC, J.Liq.Chromatogr.Rel.Technol., 2001, 24, 3017–3026. [SPE; LOQ 20–58 ng/mL; aprobarbital
is internal standard]
Fan, B.; Stewart, J.T. Determination of zidovudine/lamivudine/nevirapine in human plasma using ionpair HPLC, J.Pharm.Biomed.Anal., 2002, 28, 903–908. [SPE; LOQ 53–59 ng/mL]
Faux, J.; Venisse, N.; Le Moal, G.; Dupuis, A.; Bouquet, S. Simultaneous determination of six HIV
protease inhibitors, one metabolite, and two non-nucleoside reverse transcriptase inhibitors in
human plasma by isocratic reversed-phase liquid chromatography after solid-phase extraction,
Chromatographia, 2003, 58, 421–426. [LOQ 50 – 120 ng/mL; amprenavir; indinavir; lopinavir;
nelfinavir; ritonavir; saquinavir; efavirenz; nevirapine; prazepam is internal standard]
Hollanders, R.M.F.; van Ewijk-Beneken Kolmer, E.W.J.; Burger, D.M.; Wuis, E.W.; Koopmans, P.P.;
Hekster, Y.A. Determination of nevirapine, an HIV-1 non-nucleoside reverse transcriptase inhibitor,
in human plasma by reversed-phase high-performance liquid chromatography, J.Chromatogr.B, 2000,
744, 65–71. [LOD 50 ng/mL; LOQ 100 ng/mL]
Kishimoto, W.; Takano, J.; Senda, C.; Ishiguro, N.; Sakai, K.; Igarashi, T. Quantitative prediction of
in vivo drug interactions between nevirapine and antifungal agents from in vitro data in rats,
Biol.Pharm.Bull., 2000, 23, 1027–1032.
Lips, A.G.A.M.; Lameijer, W.; Fokkens, R.H.; Nibbering, N.M.M. Methodology for the development of
a drug library based upon collision-induced fragmentation for the identification of toxicologically
relevant drugs in plasma samples, J.Chromatogr.B, 2001, 759, 191–207. [plasma; LC-MS;
morphine; sotalol; acetaminophen; codeine; olanzapine; caffeine; amphetamine; lamotrigine; phenytoin;
nevirapine; phenobarbital; clozapine; midazolam; flurazepam; paroxetine; promethazine; flecainide;
haloperidol; oxazepam; phenprocoumon; lorazepam; naproxen; amitriptyline; zopiclone; sertraline;
zuclopenthixol; triazolam; flunitrazepam; saquinavir; pimozide; clomipramine; clobazam; valproic
acid; chlordiazepoxide; diazepam; ritonavir; ibuprofen; promazine is internal standard]
Lopez, R.M.; Pou, L.; Gomez, M.R.; Ruiz, I.; Monterde, J. Simple and rapid determination of nevirapine
in human serum by reversed-phase high-performance liquid chromatography, J.Chromatogr.B, 2001,
751, 371–376. [LOQ 100 ng/mL]
Marchei, E.; Valvo, L.; Pacifici, R.; Pellegrini, M.; Tossini, G.; Zuccaro, P. Simultaneous determination of
zidovudine and nevirapine in human plasma by RP-LC, J.Pharm.Biomed.Anal., 2002, 29, 1081–1088.
[SPE; LOD 25–50 ng/mL; LOQ 50–150 ng/mL]
Marzolini, C.; Béguin, A.; Telenti, A.; Schreyer, A.; Buclin, T.; Biollaz, J.; Decosterd, L.A. Determination
of lopinavir and nevirapine by high-performance liquid chromatography after solid-phase extraction:
application for the assessment of their transplacental passage at delivery, J.Chromatogr.B, 2002, 774,
127–140. [SPE; LOD 15–50 ng/mL; LOQ 60–100 ng/mL; clozapine is internal standard]
Moyer, T.P.; Temesgen, Z.; Enger, R.; Estes, L.; Charlson, J.; Oliver, L.; Wight, A. Drug monitoring of
antiretroviral therapy for HIV-1 infection: Method validation and results of a pilot study, Clin.Chem.,
1999, 45, 1465–1476. [serum; LOD 10–100 ng/mL; didanosine; lamivudine; stavudine; zalcitabine;
zidovudine; delavirdine; nevirapine; indinavir; nelfinavir; ritonavir; saquinavir]
446
Nevirapine
Palladino, D.E.; Hopkins, J.L.; Ingraham, R.H.; Warren, T.C.; Kapadia, S.R.; Van Moffaert, G.J.;
Grob, P.M.; Stevenson, J.M.; Cohen, K.A. High-performance liquid chromatography and photoaffinity
crosslinking to explore the binding environment of nevirapine to reverse transcriptase of human
immunodeficiency virus, J.Chromatogr.A, 1994, 676, 99–112.
Pav, J.W.; Rowland, L.S.; Korpalski, D.J.; Elvin, A.T. HPLC-UV method for the quantitation of
nevirapine (ViramuneTM ) in human plasma, serum, and milk following solid phase extraction (Abstract
4214), Pharm.Res., 1997, 14, S697.
Pav, J.W.; Rowland, L.S.; Korpalski, D.J. HPLC-UV method for the quantitation of nevirapine in
biological matrices following solid phase extraction, J.Pharm.Biomed.Anal., 1999, 20, 91–98. [SPE;
LOQ 25 ng/mL; plasma; serum; milk; CSF]
Rabin, L.; Hincenbergs, M.; Moreno, M.B.; Warren, S.; Linquist, V.; Datema, R.; Charpiot, B.; Seifert, J.;
Kaneshima, H.; McCune, J.M. Use of standardized SCID-hu Thy/Liv mouse model for preclinical
efficacy testing if anti-human immunodeficiency virus type I compounds, Antimicrob.Agents
Chemother., 1996, 40, 755–762. [plasma; LOD 8 ng/mL]
Rentsch, K.M. Sensitive and specific determination of eight antiretroviral agents in plasma by highperformance liquid chromatography-mass spectrometry, J.Chromatogr.B, 2003, 788, 339–350. [SPE;
LOQ 1–250 ng/mL; amprenavir; efavirenz; indinavir; lopinavir; nelfinavir; nevirapine; ritonavir;
saquinavir]
Rezk, N.L.; Tidwell, R.R.; Kashuba, A.D.M. Simple and rapid quantification of the non-nucleoside reverse
transcriptase inhibitors nevirapine, delavirdine, and efavirenz in human blood plasma using highperformance liquid chromatography with ultraviolet absorbance detection, J.Chromatogr.B, 2002, 774,
79–88. [LOQ 10–25 ng/mL; SPE; hexobarbital is internal standard]
Rezk, N.L.; Tidwell, R.R.; Kashuba, A.D.M. Simultaneous determination of six HIV nucleoside analogue
reverse transcriptase inhibitors and nevirapine by liquid chromatography with ultraviolet absorbance
detection, J.Chromatogr.B, 2003, 791, 137–147. [SPE; LOQ 10 ng/mL; zalcitabine; lamivudine;
didanosine; stavudine; zidovudine; abacavir; hexobarbital is internal standard]
Riska, P.; Lamson, M.; Macgregor, T.; Sabo, J.; Hattox, S.; Pav, J.; Keirns, J. Disposition and
biotransformation of the antiretroviral drug nevirapine in humans, Drug Metab.Dispos., 1999, 27,
895–901.
Riska, P.S.; Joseph, D.P.; Dinallo, R.M.; Davidson, W.C.; Keirns, J.J.; Hattox, S.E. Biotransformation
of nevirapine, a non-nucleoside HIV-1 reverse transcriptase inhibitor, in mice, rats, rabbits, dogs,
monkeys, and chimpanzees, Drug Metab.Dispos., 1999, 27, 1434–1447. [SPE]
Simon, V.A.; Thiam, M.D.; Lipford, L.C. Determination of serum levels of thirteen human
immunodeficiency virus-suppressing drugs by high-performance liquid chromatography,
J.Chromatogr.A, 2001, 913, 447–453. [SPE; LOD 30–440 ng/mL; zalcitabine; lamivudine; stavudine;
didanosine; zidovudine; nevirapine; abacavir; indinavir; delavirdine; nelfinavir; saquinavir; ritonavir;
efavirenz]
Störmer, E.; von Moltke, L.L.; Perloff, M.D.; Greenblatt, D.J. Differential modulation of P-glycoprotein
expression and activity by non-nucleoside HIV-1 reverse transcriptase inhibitors in cell culture,
Pharm.Res., 2002, 19, 1038–1045. [verapamil; efavirenz; nevirapine; delavirdine]
Tribut, O.; Arvieux, C.; Michelet, C.; Chapplain, J.-M.; Allain, H.; Bentué-Ferrer, D. Simultaneous
quantitative assay of six HIV protease inhibitors, one metabolite, and two non-nucleoside reverse
transcriptase inhibitors in human plasma by isocratic reversed-phase liquid chromatography,
Ther.Drug Monit., 2002, 24, 554–562. [LOQ 25 ng/mL; nevirapine; efavirenz; indinavir; amprenavir;
nelfinavir; ritonavir; lopinavir; saquinavir]
van Heeswijk, R.P.; Hoetelmans, R.M.; Meenhorst, P.L.; Mulder, J.W.; Beijnen, J.H. Rapid
determination of nevirapine in human plasma by ion-pair reversed- phase high-performance liquid
chromatography with ultraviolet detection, J.Chromatogr.B, 1998, 713, 395–399. [LOQ 52 ng/mL]
Villani, P.; Feroggio, M.; Gianelli, L.; Bartoli, A.; Montagna, M.; Maserati, R.; Regazzi, M.B.
Antiretrovirals: simultaneous determination of five protease inhibitors and three nonnucleoside
transcriptase inhibitors in human plasma by a rapid high-performance liquid chromatography-mass
spectrometry assay, Ther.Drug Monit., 2001, 23, 380–388. [LOD 10–25 ng/mL; LOQ 20–40 ng/mL;
saquinavir; indinavir; ritonavir; nelfinavir; amprenavir; nevirapine; delavirdine; efavirenz]
447
Nicarbazin
Nicarbazin
NO2
O2N
O
Molecular formula: C19 H18 N6 O6
N
N
Molecular weight: 426.38
CAS Registry No: 330-95-0
Merck Index: 13, 6519
H
H
H3C
N
CH3
N
CH3
SAMPLE
Matrix: blood
Sample preparation: Vortex 100 µL plasma with 200 µL MeCN, centrifuge for 5 min,
inject a 60 µL aliquot of the supernatant.
HPLC VARIABLES
Guard column: 15 × 4.6 C18
Column: 250 × 4.6 5 µm Keystone ODS/H
Column temperature: 35
Mobile phase: MeCN:water 60:40
Flow rate: 1
Injection volume: 60
Detector: UV 347
CHROMATOGRAM
Retention time: 8.5 (dinitrocarbanilide)
Limit of detection: 27–35 ng/mL
KEY WORDS
chicken; duck; goose; plasma
REFERENCE
Primus, T.M.; Kohler, D.J.; Goodall, M.A.; Yoder, C.; Griffin, D.; Miller, L.; Johnston, J.J. Determination
of 4,4′ -dinitrocarbanilide (DNC), the active component of the antifertility agent nicarbazin, in chicken,
duck, and goose plasma, J.Agric.Food Chem., 2001, 49, 3589–3593.
448
Nilutamide
Nilutamide
H3C
Molecular formula: C12 H10 F3 N3 O4
H3C
Molecular weight: 317.22
CAS Registry No: 63612-50-0
Merck Index: 13, 6572
H
N
O
N
CF3
O
NO2
SAMPLE
Matrix: blood, urine
Sample preparation: Extract 1 mL plasma or urine with 2 mL chloroform (Caution!
Chloroform is a carcinogen!). Evaporate an aliquot of the organic layer to dryness,
reconstitute the residue with 1 mL mobile phase, inject a 10–20 µL aliquot.
HPLC VARIABLES
Column: 250 mm long 5 µm C18
Mobile phase: MeOH
Flow rate: 1.2
Injection volume: 10–20
Detector: UV 254
CHROMATOGRAM
Retention time: 4.5
Limit of detection: 50 ng/mL
KEY WORDS
plasma
REFERENCE
Pendyala, L.; Creaven, P.J.; Huben, R.; Tremblay, D.; Bertagna, C. Pharmacokinetics of Anandron in
patients with advanced carcinoma of the prostate, Cancer Chemother.Pharmacol., 1988, 22, 69–76.
449
Nipradilol
Nipradilol
Molecular formula: C15 H22 N2 O6
Molecular weight: 326.34
CAS Registry No: 81486-22-8
Merck Index: 13, 6590
CH3
H3C
N
H
O
OH
O
ONO2
SAMPLE
Matrix: solutions
Sample preparation: Add 50 µL L-menthoxyacetyl chloride to a solution of 5–10 mg
nipradilol in 500 µL dry pyridine, let stand at room temperature for 30 min, add 50 µL
water, evaporate to dryness under reduced pressure, reconstitute with 5 mL chloroform
(Caution! Chloroform is a carcinogen!), wash twice with 3 mL portions of 1 M HCl, wash
with 3 mL saturated sodium bicarbonate solution, dry over anhydrous sodium sulfate,
inject an aliquot.
HPLC VARIABLES
Column: 200 × 4 10 µm Partisil-10
Mobile phase: Hexane:ethyl acetate 100:20
Flow rate: 1.5
Detector: UV 275
CHROMATOGRAM
Retention time: 14 (2′ R,3S), 15 (2′ S,3R), 17 (2′ S,3S), 20 (2′ R,3R) [ 2′ hydroxy, 3 nitroxy]
KEY WORDS
chiral; derivatization; normal phase
REFERENCE
Yoneda, M.; Shiratsuchi, M.; Yoshimura, M.; Ohkawa, Y.; Muramatsu, T. Optical resolution and determination of absolute configuration of nipradilol, Chem.Pharm.Bull., 1985, 33, 2735–2742.
450
Nitazoxanide
Nitazoxanide
O
O2N
Molecular formula: C12 H9 N3 O5 S
Molecular weight: 307.29
CAS Registry No: 55981-09-4
Merck Index: 13, 6595
S
N
O
O
CH3
N
H
SAMPLE
Matrix: blood, urine
Sample preparation: Plasma. Vortex 500 µL plasma and IS with 1 mL MeCN, centrifuge, inject a 20 µL aliquot of the supernatant. Urine. Mix 500 µL urine with 500 µL
1% Helix Pomatia juice (type H2, Sigma) in 100 mM pH 5 acetate buffer, heat at 37◦
overnight, inject an aliquot.
HPLC VARIABLES
Column: 100 × 4.6 5 µm Nucleosil C18
Mobile phase: Gradient. MeCN:20 mM pH 2.5 phosphate buffer from10:90 to 35:65 over
8 min.
Injection volume: 20
Detector: UV 360
CHROMATOGRAM
Internal standard: nifuroxazide
Limit of quantitation: 20 ng/mL (plasma), 200 ng/mL (urine)
KEY WORDS
only desacetylnitazoxanide is detected in plasma and urine; plasma
REFERENCE
Stockis, A.; Deroubaix, X.; Lins, R.; Jeanbaptiste, B.; Calderon, P.; Rossignol, J.F. Pharmacokinetics of
nitazoxanide after single oral dose administration in 6 healthy volunteers, Int.J.Clin.Pharmacol.Ther.,
1996, 34, 349–351.
SAMPLE
Matrix: microsomal incubations, urine
Sample preparation: Urine. Mix 2 mL urine with 10 µL 100 U/mL β-glucuronidase,
5 µL arylsulfatase (Helix pomatia) (Boehringer Mannheim), and 200 µL 1 M pH 5.5
acetate buffer, heat at 37◦ for 3 h, add 200 µL 1 M formic acid, centrifuge, inject a
100 µL aliquot. Microsomal incubations. Mix 1 mL microsomal incubation with 100 µL
2 M formic acid, add 550 µL MeCN, mix, centrifuge, inject an aliquot. (This method is
also said to work for plasma and feces, but there are no details.) (Only the deacylated
compound tizanoxide is detected in biological fluids.)
HPLC VARIABLES
Column: Nucleosil 5C18
Mobile phase: Gradient. MeCN:10 mM formic acid from 10:90 to 90:10 over (?)
Injection volume: 100
Detector: MS, VG Quattro II
Nitazoxanide
451
KEY WORDS
dog; human; liver; monkey; rat
REFERENCE
Broekhuysen, J.; Stockis, A.; Lins, R.L.; De Graeve, J.; Rossignol, J.F. Nitazoxanide: pharmacokinetics
and metabolism in man, Int.J.Clin.Pharmacol.Ther., 2000, 38, 387–394.
452
Nitenpyram
Nitenpyram
Cl
N
H3C
N
Molecular formula: C11 H15 ClN4 O2
Molecular weight: 270.72
CAS Registry No: 150824-47-8
Merck Index: 13, 6596
NO2
N
H
CH3
SAMPLE
Matrix: fruit, vegetables
Sample preparation: Condition a 3 mL 500 mg Bond Elut PSA (weak anion-exchange)
SPE cartridge with 10 mL acetone and 10 mL acetone:hexane 50:50. Condition a 6 mL
1 g Mega Bond Elut silica SPE cartridge with 10 mL acetone and 10 mL acetone:hexane
30:70. Blend (Polytron) 20 g sample with 100 mL MeCN for 2 min, filter (paper).
Add 5 g NaCl to the filtrate and shake mechanically for 1 min. Evaporate 50 mL
of the MeCN layer to near dryness, take up in 2 mL acetone, wash out flask with
2 mL acetone. Combine the acetone solutions with 4 mL hexane, add to the PSA SPE
cartridge, elute with 5 mL acetone:hexane 50:50. Evaporate the eluates and dissolve
in 2 mL acetone:hexane 30:70, add to the silica SPE cartridge, wash with 10 mL
acetone:hexane 30:70. Discard all effluent from the silica SPE cartridge. Elute with
10 mL acetone:hexane 40:60 (for acetamiprid and imidacloprid) and 20 mL acetone (for
nitenpyram). Evaporate each eluate to dryness, reconstitute the residue with 2 mL
MeOH, inject a 10 µL aliquot.
HPLC VARIABLES
Column: 75 × 4.6 3 µm Cadenza CD-C18 (Imtakt, Kyoto)
Column temperature: 50
Mobile phase: Gradient. MeCN:50 mM potassium dihydrogen phosphate 3:97 for 3 min,
to 40:60 over 7 min, maintain at 40:60 for 5 min, to 100:0 over 5 min, maintain at 100:0
for 5 min, to 5:95 over 5 min, maintain at 5:95 for 5 min.
Flow rate: 0.8
Injection volume: 10
Detector: UV 270
CHROMATOGRAM
Retention time: 11
Limit of detection: 20 ng/g
OTHER SUBSTANCES
Extracted: acetamiprid (UV 245; LOD 10 ng/g) (15), imidacloprid (LOD 10 ng/g) (14)
KEY WORDS
cucumber; eggplant; grape; Japanese radish; potato; SPE; tomato
REFERENCE
Obana, H.; Okihashi, M.; Akutsu, K.; Kitagawa, Y.; Hori, S. Determination of acetamiprid, imidacloprid,
and nitenpyram residues in vegetables and fruits by high-performance liquid chromatography with
diode-array detection, J.Agric.Food Chem., 2002, 50, 4464–4467.
453
Nomegestrol
Nomegestrol
H3C
CH3
O
OH
Molecular formula: C21 H28 O3
H
Molecular weight: 328.45
CAS Registry No: 58691-88-6
H
H
H
O
CH3
SAMPLE
Matrix: blood
Sample preparation: Condition a Sep Pak C18 SPE cartridge with MeOH and water.
Add 1 mL plasma to the SPE cartridge, wash with 5 mL water, wash with 2 mL
MeOH:water 70:30, elute with 2 mL MeOH. Evaporate the eluate to dryness, reconstitute the residue with 150 µL EtOH, inject a 20 µL aliquot.
HPLC VARIABLES
Column: 250 × 4.6 5 µm Ultrasphere
Mobile phase: MeOH:water 70:30
Flow rate: 1.2
Injection volume: 20
Detector: UV 280
CHROMATOGRAM
Retention time: 13
KEY WORDS
plasma; SPE; for nomegestrol acetate
REFERENCE
Ezan, E.; Benech, H.; Bucourt, R.; Ardouin, T.; Tchernatinsky, C.; Thomas, J.L.; Paris, J.; Grognet, J.M.
Enzyme immunoassay for nomegestrol acetate in human plasma, J.Steroid Biochem.Mol.Biol., 1993,
46, 507–514.
454
Nonoxynol-9
Nonoxynol-9
CAS Registry No: 26027-38-3
Merck Index: 13, 6711
(OCH2CH2)nOH
n=9
H3C
SAMPLE
Matrix: blood
Sample preparation: Mix 1 mL whole blood with 1 g solid NaCl, grind (sic) for 5 min,
extract twice with 4 mL portions of benzene (Caution! Benzene is a carcinogen!),
centrifuge at 8000 rpm for 30 min. Combine the extracts and evaporate them to dryness
under a stream of nitrogen, dry under vacuum at 50◦ for 1 h, reconstitute the residue
with 100 µL MeOH, centrifuge, inject a 20 µL aliquot of the supernatant.
HPLC VARIABLES
Column: 250 × 4.6 TSK-Gel C18
Mobile phase: MeOH:water (ratio not given)
Injection volume: 20
Detector: UV 221
CHROMATOGRAM
Retention time: 7.3
Limit of quantitation: 50 ng/mL
KEY WORDS
whole blood; also, using normal phase with a silica column and n-hexane at UV 276 gives
numerous peaks for oligomers.
REFERENCE
Yang, J.; Zhao, Z. Quantitative analysis of nonoxynol-9 in the blood, Contraception, 1991, 43, 161–166.
455
Nystatin
Nystatin
Molecular formula: C47 H75 NO17 (A1 )
Molecular weight: 926.09 (A1 )
CAS Registry No: 1400-61-9,
34786-70-4 (A1 )
Merck Index: 13, 6770
OH
H3C
HO
O
O
CH3
OH
OH
OH
OH
OH
OH
O
COOH
H3C
O
O
OH
CH3
NH2
OH
SAMPLE
Matrix: blood, tissue
Sample preparation: Plasma. Mix 800 µL MeOH with 400 µL plasma, let stand at 4◦
for 30 min, centrifuge at 2000 g for 10 min, centrifuge the supernatant in a separate
tube at 10 000 g for 4 min. Filter (Durapore 0.22 µm) 400 µL of the supernatant
while centrifuging at 4000 g for 4 min, inject a 200 µL aliquot of the filtrate. Tissue.
Homogenize (Tissuemizer 10 N head) tissue with 2 vol of ice-cold MeOH at 0◦ , let stand
at 4◦ for 30 min, centrifuge at 2000 g for 10 min, centrifuge the supernatant in a separate
tube at 10 000 g for 4 min. Filter (Durapore 0.22 µm) 400 µL of the supernatant while
centrifuging at 4000 g for 4 min, inject a 200 µL aliquot of the filtrate.
HPLC VARIABLES
Guard column: 15 × 3.2 5 µm NewGuard RP-18
Column: 300 × 3.9 10 µm µBondapak C18
Column temperature: 30
Mobile phase: MeCN:MeOH:10 mM sodium phosphate buffer containing 1 mM EDTA
30:30:40, adjusted to pH 6.0 with 85% phosphoric acid
Flow rate: 2
Injection volume: 200
Detector: UV 305
CHROMATOGRAM
Retention time: 8.1, 10.0
Limit of quantitation: 50 ng/mL
OTHER SUBSTANCES
Noninterfering: acetaminophen, amikacin, amitriptyline, carbamazepine, ceftazidime,
cyclosporine, digoxin, disopyramide, ethosuximide, gentamicin, lidocaine, lithium,
methotrexate, phenobarbital, phenytoin, primidone, procainamide, propranolol, quinidine, salicylic acid, theophylline, tobramycin, valproic acid, vancomycin
KEY WORDS
human; pharmacokinetics; plasma; rabbit
REFERENCE
Groll, A.H.; Mickiene, D.; Werner, K.; Piscitelli, S.C.; Walsh, T.J. High-performance liquid chromatographic determination of liposomal nystatin in plasma and tissues for pharmacokinetic and tissue
distribution studies, J.Chromatogr.B, 1999, 735, 51–62.
Octocrylene
Molecular formula: C24 H27 NO2
Molecular weight: 361.48
CH3
CN
O
CH3
O
CAS Registry No: 6197-30-4
SAMPLE
Matrix: sunscreen
Sample preparation: Heat 2 g sunscreen in 40 mL MeOH and 250 µL 2 M sulfuric
acid at 60◦ for 5 min until a homogeneous solution develops, cool, make up to 50 mL
with MeOH, dilute a 1 mL aliquot to 5 mL with initial mobile phase, inject a 5–50 µL
aliquot.
HPLC VARIABLES
Column: 150 × 4.6 5 µm PLRP-S (Polymer Labs)
Column temperature: 25
Mobile phase: Gradient. MeCN:THF:buffer 10:10:80 for 2.5 min, to 45:45:10 over
22.5 min, maintain at 45:45:10 for 10 min, return to initial conditions over 5 min, reequilibrate for 5 min. (The buffer was 1.4 g/L citric acid monohydrate containing 6.8 g/L
tetrabutylammonium hydroxide, adjusted to pH 9.0 with concentrated ammonia.)
Flow rate: 0.8 for 2.5 min, to 0.6 over 2.5 min, maintain at 0.6 for 10 min, return to 0.8
over 5 min, maintain at 0.8 for 5 min
Injection volume: 5–50
Detector: UV 302
CHROMATOGRAM
Retention time: 29.13
Limit of detection: 50–500 ppm
OTHER SUBSTANCES
Simultaneous: benzophenone-3 (25.93 min, UV 288), benzophenone-4 (12.47 min, UV
241), 3-benzylidene-d,l-camphor (26.47 min, UV 293), benzylidene camphor sulfonic acid
(15.77 min, UV 297), butyl methoxydibenzoylmethane (29.71 min, UV 359), camphor
benzalkonium methosulfate (6.178 min, UV 288), 3-(4′ -ethylbenzylidene)-d,l-camphor
(27.30 min, UV 297), homosalate (30.32 min, UV 241), isoamyl p-methoxycinnamate
(26.93 min, UV 307), isopropyl dibenzoylmethane (29.86 min, UV 350), megasol
(29.17 min, UV 241), octyl dimethyl PABA (28.83 min, UV 312), octyl methoxycinnamate
(29.43 min, UV 307), octyl salicylate (30.03 min, UV 241), octyl triazone (33.72 min, UV
312), PEG−25 p-aminobenzoic acid (10.79 min, UV 307), phenylbenzimidazole sulfonic
acid (8.898 min, UV 302), terephthalydiene dicamphor sulfonic acid (13.18 min, UV
340), urocanic acid (2.468 min, UV 278)
REFERENCE
Rastogi, S.C.; Jensen, G.H. Identification of UV filters in sunscreen products by high-performance liquid
chromatography-diode-array detection, J.Chromatogr.A, 1998, 828, 311–316.
456
HPLC Methods for Recently Approved Pharmaceuticals. by George Lunn
Copyright 2005 John Wiley & Sons, Ltd. ISBN: 0-471-66941-5
Oleic acid
457
Oleic acid
Molecular formula: C18 H34 O2
Molecular weight: 282.46
H3C(CH2)6
(CH2)6COOH
CAS Registry No: 112-80-1
Merck Index: 13, 6898
SAMPLE
Matrix: blood
Sample preparation: Mix 10 µL plasma with 200 µL 40 µg/mL IS in EtOH and 90 µL
EtOH, add 100 µL 2 M KOH in EtOH:water 50:50, heat at 80◦ for 20 min, cool to room
temperature. Add 200 µL 20 mM 2-nitrophenylhydrazine in 300 mM HCl:EtOH 50:50,
add 200 µL 250 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride in
EtOH:pyridine 97:3, heat at 80◦ for 5 min. Cool the solution to room temperature, add
10% KOH in MeOH:water 50:50, heat again at 80◦ for 5 min, cool to room temperature.
Add 4 mL 33 mM pH 4.6 potassium phosphate buffer:500 mM HCl 70:10, extract with
4 mL hexane by shaking vigorously for 2 min. Centrifuge at 20 000 rpm for 10 min,
evaporate the upper layer under a stream of nitrogen, resuspend the residue in 400 µL
MeOH, inject a 50 µL aliquot.
HPLC VARIABLES
Guard column: 20 × 2 20–40 µm LiChroprep C18
Column: 250 × 6 YMC FA C8 (Hichrom) + Apex 3 µm ODS (Jones Chromatography) in
series
Column temperature: 45
Mobile phase: Gradient. A was MeCN. B was MeCN:MeOH:water 75:11:14. A:B 0:100
for 20 min, from 0:100 to 100:0 over 5 min, maintain at 100:0 for 10 min, return to
initial conditions over for 2 min, re-equilibrate.
Flow rate: 1.5
Injection volume: 50
Detector: UV 400
CHROMATOGRAM
Retention time: 20
Internal standard: margaric acid
OTHER SUBSTANCES
Extracted: arachidonic acid (14.5), docosanoic acid (38), eicosanoic acid (34), eicosatrienoic
acid (17), eicosapentanoic acid (12), lauric acid (10), linoleic acid (15.5), linolenic acid
(12.5), gamma-linolenic acid (13), myristic acid (13), palmitic acid (18.5), palmitoleic acid
(15), stearic acid (27), tetracosanoic acid (42)
KEY WORDS
derivatization; plasma
REFERENCE
Bailey, A.L.; Southon, S. Determination of total long-chain fatty acids in human plasma and lipoproteins, before and during copper-stimulated oxidation, by high-performance liquid chromatography,
Anal.Chem., 1998, 70, 415–419.
SAMPLE
Matrix: blood
Sample preparation: Vortex 5 µL serum, 5 µL EtOH, and 50 µL 4% pyridine in
EtOH containing 20 mM HCl for 10 s, add 25 µL 50 mM reagent in DMF, add 15 µL
458
Oleic acid
2 M 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in water, heat at 37◦ for 10 min,
centrifuge at 1000 g for 5 min, inject a 10 µL aliquot of the supernatant. (The reagent
was 6,7-dimethoxy-1-methyl-2(1H)-quinoxalinone-3-propionylcarboxylic acid hydrazide.
Synthesis is as follows. Stir 483 g veratrole in 1.45 L acetic acid at 15◦ , add 683 g concentrated nitric acid (s.g. 1.05) over 1 h keeping the temperature below 40◦ (cool if
necessary), add 2.127 L fuming nitric acid (s.g. 1.50) over 1 h keeping the temperature
below 30◦ , allow to stand for 2 h, pour into a large volume of cold water, filter, wash
the solid until it is free from acid, recrystallize from EtOH to give 4,5-dinitroveratrole
(mp 129.5–130.5◦ ) (J.Am.Chem.Soc. 1946, 68, 1536). Reflux 5 g 4,5-dinitroveratrole in
200 mL benzene (Caution! Benzene is a carcinogen!), add 100 g 60 mesh iron powder
and 20 mL concentrated HCl in small portions over 1 h, reflux for 4 h, add 10 mL
water, reflux for 2 h, cool, make alkaline with 2.5 M NaOH, extract several times with
200 mL portions of benzene. Combine the extracts and evaporate to dryness, add 10 mL
concentrated HCl, recrystallize from EtOH to give 1,2-diamino-4,5-dimethoxybenzene
hydrochloride as slightly pink needles (mp 240◦ d) (Anal.Chim.Acta 1982, 134, 39). Dissolve 2.5 mmol 1,2-diamino-4,5-dimethoxybenzene monohydrochloride and 2.4 mmol
α-ketoglutaric acid in 30 mL 500 mM HCl, heat in a boiling water bath for 2 h, cool
in ice, filter, wash the precipitate with water, dry under vacuum, recrystallize from
MeOH:water 90:10 to give 6,7-dimethoxy-2(1H)-quinoxalinone-3-propionylcarboxylic
acid as yellow needles (mp 240◦ ) (Chem.Pharm.Bull. 1985, 33, 3493). Treat 1.5 g
6,7-dimethoxy-2(1H)-quinoxalinone-3-propionylcarboxylic acid in 100 mL MeOH with
ethereal diazomethane, evaporate to dryness under reduced pressure, dissolve the
residue in 30 mL chloroform (Caution! Chloroform is a carcinogen!), chromatograph on
a 250 × 35 column of 70–230 mesh silica gel 60 (Merck) with hexane:ethyl acetate 50:50
to give methyl 6,7-dimethoxy-1-methyl-2(1H)-quinoxalinone-3-propionylcarboxylate as
colorless needles (mp 178–179◦ ). Dissolve 900 mg methyl 6,7-dimethoxy-1-methyl2(1H)-quinoxalinone-3-propionylcarboxylate in 100 mL 45% hydrazine hydrate in water,
heat at 100◦ for 1 h, recrystallize the precipitate from EtOH to give 6,7-dimethoxy-1methyl-2(1H)-quinoxalinone-3-propionylcarboxylic acid hydrazide as colorless needles
(mp 205–206◦ ) (Analyst 1990, 115, 1363).)
HPLC VARIABLES
Column: 250 × 4.6 10 µm YMC-Pack C8 (Yamamura, Kyoto)
Column temperature: 30 ± 0.2
Mobile phase: Gradient. MeCN:water 55:45 for 56 min, to 95:5 over 16 min, maintain
at 95:5 for 4 min, re-equilibrate at initial conditions for 4 min.
Flow rate: 1
Injection volume: 100
Detector: F ex 360 em 435
CHROMATOGRAM
Retention time: 62.5
Limit of detection: 2–7 fmol
OTHER SUBSTANCES
Extracted: arachidonic acid (41), dihomo-gamma-linolenic acid (51), docosahexaenoic
acid (41), eicosapentaenoic acid (20.5), lauric acid (12), linoleic acid (39), linolenic acid
(27), margaric acid (66), myristic acid (24), myristoleic acid (16), palmitic acid (49),
palmitoleic acid (30), stearic acid (72)
Noninterfering: adipic acid, alcohols, aldehydes, amines, amino acids, benzoic acid,
cinnamic acid, α-keto acids, lactic acid, malic acid, malonic acid, oxalic acid, phenols,
salicylic acid, succinic acid, sugars
KEY WORDS
derivatization; serum
Oleic acid
459
REFERENCE
Iwata, T.; Inoue, K.; Nakamura, M.; Yamaguchi, M. Simple and highly sensitive determination of free
fatty acids in human serum by high performance liquid chromatography with fluorescence detection,
Biomed.Chromatogr., 1992, 6, 120–123.
SAMPLE
Matrix: blood
Sample preparation: Mix 100 µL serum with 20 µL 5 mM IS in isopropanol, add
500 µL isopropanol:n-heptane:2 M phosphoric acid 40:10:1, mix, let stand at room temperature for 5–10 min, add 200 µL n-heptane, add 300 µL water, vortex thoroughly,
centrifuge at 1000 g for 5 min. Remove a 200 µL aliquot of the upper organic layer and
evaporate it to dryness under a stream of nitrogen, reconstitute the residue in 6 µL
reagent, 500 µL MeCN, and ca. 1 mg potassium bicarbonate, flush the tube with nitrogen, close the PTFE-lined cap tightly, heat at 85◦ with vigorous stirring for 45 min (weigh
vial before and after heating to check for leakage), cool, remove stir bar, centrifuge,
inject a 10–25 µL aliquot of the supernatant. (The reagent was 50 mM p-bromophenacyl
bromide in MeCN containing 5 mM 18-crown-6, store protected from light.)
HPLC VARIABLES
Guard column: 4 × 4 5 µm CN
Column: 25 × 4 3 µm Spherisorb C6
Column temperature: 30
Mobile phase: MeCN:water 77:23
Flow rate: 1.3
Injection volume: 10–25
Detector: UV 254
CHROMATOGRAM
Retention time: 13
Internal standard: heptadecanoic acid (margaric acid) (15)
Limit of detection: 800 nM
OTHER SUBSTANCES
Extracted: arachidonic acid (9.8), docosahexaenoic acid (9), eicosapentaenoic acid (8),
elaidic acid (14), lauric acid (6), linoleic acid (10.5), linolenic acid (8.5), myristic acid
(8.8), myristoleic acid (7), palmitic acid (12), palmitoleic acid (9.5), stearic acid (18)
KEY WORDS
derivatization; serum
REFERENCE
Puttmann, M.; Krug, H.; von Ochsenstein, E.; Kattermann, R. Fast HPLC determination of serum free
fatty acids in the picomole range, Clin.Chem., 1993, 39, 825–832.
SAMPLE
Matrix: blood
Sample preparation: Sonicate 10 µL serum, 44 µL MeOH, and 1 µL pyridine for 5 min,
add 25 µL 100 mM reagent in DMF, add 20 µL 400 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in MeOH, let stand at 25◦ for 2 h, centrifuge, inject an aliquot.
(The reagent was 2-(5-hydrazinocarbonyl-2-furyl)-5,6-dimethoxybenzothiazole, which is
synthesized as follows. Pass dry hydrogen chloride into a mixture of 12.6 g methyl
2-furoate, 4.5 g paraformaldehyde, and 3.4 g anhydrous zinc chloride in 50 mL dry
chloroform for 3 h while holding the reaction temperature at 30◦ (Caution! Chloroform is a carcinogen!). After cooling, pour the contents of the flask into 100 mL
460
Oleic acid
cold water, remove the chloroform layer, extract the aqueous layer with chloroform
(cf Coll.Czech.Chem.Commun. 1960, 25, 1058). Combine the chloroform layers, neutralize, dry over anhydrous calcium chloride, evaporate, distil to give 5-chloromethyl
furyl-2-carboxylic acid methyl ester (bp 108◦ /4 mm Hg). Reflux 10 g 5-chloromethyl
furyl-2-carboxylic acid methyl ester and 25 g silver carbonate in 100 mL THF:water
70:30 for 5 h, filter through Celite, concentrate the filtrate under reduced pressure,
chromatograph the product on silica gel with chloroform to give 5-hydroxymethyl
furyl-2-carboxylic acid methyl ester as a light yellow oil. Add a solution of 2.9 g
5-hydroxymethyl furyl-2-carboxylic acid methyl ester in 30 mL dichloromethane to
12 g pyridinium chlorochromate in 100 mL dichloromethane, stir at room temperature
for 4 h, evaporate to dryness under reduced pressure, chromatograph on silica with
dichloromethane to give 5-formyl furyl-2-carboxylic acid methyl ester as a light yellow powder. Add 10 mL concentrated nitric acid dropwise to 20 g 4-bromoveratrole
in 60 mL acetic acid while keeping the temperature at 10–30◦ with occasional cooling; when the addition is complete, pour the reaction mixture into ice water. Collect
the precipitate and dissolve it in 500 mL hot EtOH, add activated charcoal, filter, add 40 mL water to the filtrate to give 4,5-dimethoxy-2-nitrobromobenzene as
a light yellow crystalline solid (mp 121–122◦ ). Prepare sodium sulfide by melting
together 5 g sodium sulfide nonahydrate and 700 mg sulfur, add this mixture to 5 g
4,5-dimethoxy-2-nitrobromobenzene in 50 mL EtOH:water 95:5, reflux for 30 min,
pour into ice water, collect the solid, recrystallize from dichloromethane to give
di(4,5-dimethoxy-2-nitrophenyl)sulfide as yellow needles (mp 231–232◦ ). Add 15 mL
concentrated HCl dropwise to 1.5 g di(4,5-dimethoxy-2-nitrophenyl)sulfide and 4.5 g tin
powder stirred at 40–50◦ in 150 mL EtOH, reflux for 1 h, cool to room temperature,
filter, add 1.17 g 5-formyl furyl-2-carboxylic acid methyl ester to the filtrate, reflux for
1 h, cool, filter, chromatograph the solid on silica gel with dichloromethane, recrystallize from EtOH to give 5-(5′ ,6′ -dimethoxybenzothiazolyl)-N-furan-2-carboxylic acid
methyl ester as a yellow powder (mp 192–202◦ ). Add 2 mL hydrazine hydrate (Caution! Hydrazine hydrate is a carcinogen!) to 800 mg 5-(5′ ,6′ -dimethoxybenzothiazolyl)N-furan-2-carboxylic acid methyl ester in 20 mL EtOH, reflux for 30 min, collect
the solid, wash with MeOH, dry under vacuum over phosphorus pentoxide to give
2-(5-hydrazinocarbonyl-2-furyl)-5,6-dimethoxybenzothiazole as a light yellow solid (mp
226–228◦ ).)
HPLC VARIABLES
Column: 250 × 4.6 5 µm Wakosil-II 5C18 HG
Column temperature: 40
Mobile phase: Gradient. MeCN:water from 70:30 to 75:25 over 25 min, to 100:0 over
15 min, maintain at 100:0.
Flow rate: 1
Injection volume: 10
Detector: F ex 363 em 452
CHROMATOGRAM
Retention time: 35
Limit of detection: 50 fmol
OTHER SUBSTANCES
Extracted: alprostadil (51), arachidonic acid (24), dinoprost (41), dinoprostone (50),
lauric acid (10), linoleic acid (25), linolenic acid (18), margaric acid (38), myristic acid
(19), myristoleic acid (12), palmitic acid (32), palmitoleic acid (21), stearic acid (42)
KEY WORDS
derivatization; serum
REFERENCE
Saito, M.; Ushijima, T.; Sasamoto, K.; Ohkura, Y.; Ueno, K. 2-(5-Hydrazinocarbonyl-2-furyl)-5,6-dimethoxybenzothiazole as a precolumn fluorescence derivatization reagent for carboxylic acids in
Oleic acid
461
high-performance liquid chromatography and its application to the assay of fatty acids in human
serum, Anal.Sci., 1995, 11, 103–107.
SAMPLE
Matrix: blood
Sample preparation: Vortex 10 µL plasma, 200 µL 500 mM pH 6.5 phosphate buffer,
50 µL 20 µM IS in MeOH, and 2 mL n-heptane:chloroform 50:50 for 2 min (Caution! Chloroform is a carcinogen!), centrifuge at 1000 g for 10 min. Remove the lower organic layer
and evaporate it to dryness, reconstitute the residue in two 100 µL aliquots of acetone.
Evaporate the acetone solution to dryness, add 2–3 mg finely powdered potassium bicarbonate:sodium sulfate 50:50, add 50 µL 800 µM dibenzo-18-crown-6 in acetone, add 50 µL
2 mM 7-acetoxy-4-bromomethylcoumarin in acetone, heat at 50◦ in the dark for 30 min,
inject a 50 µL aliquot. (7-Acetoxy-4-bromomethylcoumarin is available from TCI America
or Tokyo Kasei, or it may be prepared as follows. Reflux 50 g 7-hydroxy-4-methylcoumarin
(β-methylumbelliferone) and 100 mL acetic anhydride for 1 h, cool, pour into 500 mL cold
water, filter, dry the solid, recrystallize from EtOH to give 4-methyl-7-acetoxycoumarin.
Reflux 10 g 4-methyl-7-acetoxycoumarin, 9 g N-bromosuccinimide, a little 2,2′ -(azobis(2methylpropionitrile) (α,α ′ -azobisisobutyronitrile, Eastman), and 100 mL carbon tetrachloride for 20 h, cool, evaporate under reduced pressure to remove the solvent, wash
the residue with water, filter, dry, recrystallize from ethyl acetate/cyclohexane to give
7-acetoxy-4-bromomethyl-coumarin (mp 184–185◦ ) (J.Chromatogr. 1982, 234, 121).)
HPLC VARIABLES
Column: 250 × 4 5 µm LiChrosorb RP-18
Column temperature: 40
Mobile phase: Gradient. MeCN:MeOH:water from 35:35:30 to 0:90:10 over 70 min
(convex gradient).
Flow rate: 1.2
Injection volume: 50
Detector: F ex 365 em 460 following post-column reaction. The column effluent mixed
with 200 mM NaOH in MeOH:water 80:20 pumped at 0.4 mL/min and the mixture
flowed through a 3.5 m ×0.5 mm ID stainless steel coil at 50◦ to the detector.
CHROMATOGRAM
Retention time: 51
Internal standard: margaric acid (57)
Limit of detection: 5 pmol
OTHER SUBSTANCES
Extracted: arachidonic acid (37), capric acid (17), caproic acid (6), caprylic acid (11),
heptanoic acid (9), lauric acid (26), linoleic acid (42), linolenic acid (35), myristic acid
(36), myristoleic acid (28), nonanoic acid (15), palmitic acid (50), palmitoleic acid (39),
stearic acid (66), tridecanoic acid (30), undecanoic acid (21)
KEY WORDS
derivatization; plasma; post-column reaction
REFERENCE
Tsuchiya, H.; Hayashi, T.; Sato, M.; Tatsumi, M.; Takagi, N. Simultaneous separation and sensitive
determination of free fatty acids in blood plasma by high-performance liquid chromatography,
J.Chromatogr., 1984, 309, 43–52.
SAMPLE
Matrix: blood
Sample preparation: Vortex 50 µL plasma, 10 µL IS solution, and 450 µL micelle
solution for 10 s, add 25 µL 28 mg/mL 9-bromomethylacridine in acetone, mix. Remove
a 50 µL aliquot and heat it to 60◦ for 6 min, inject the whole amount through a 2 µm
462
Oleic acid
stainless steel filter of 8 sq mm area onto column A, wash to waste with 400 µL mobile
phase A, backflush the contents of column A onto column B with the mobile phase
B, monitor the effluent from column B. After each injection, backflush the stainless
steel filter with 1 mL buffer. (The micelle solution was 25 mM Arkopal N-130 (a polyoxyethylene(13)nonylphenol, Hoechst Holland, Amsterdam) in 10 mM pH 7.0 phosphate
buffer containing 6 mM tetrakis(decyl)ammonium bromide. 9-Bromomethylacridine is
available from TCI America or Tokyo Kasei, or it may be prepared as follows. Heat
10 g diphenylamine, 10 mL glacial acetic acid, and 40 g anhydrous zinc chloride to
220◦ , evaporate excess acetic acid with stirring, heat at 220–230◦ for 6 h, digest with
hot 10% sulfuric acid, make strongly alkaline with 25% ammonia to dissolve the
zinc chloride. Extract the insoluble residue with toluene. Extract the organic layer
with 10% sulfuric acid, make the aqueous layer alkaline with aqueous ammonia. Collect the yellow precipitate that separates and recrystallize it twice from petroleum
ether to give 9-methyl acridine as pale yellow needles (Chromatographia 1989, 28,
267). Reflux 560 mg 9-methylacridine, 445 mg N-bromosuccinimide, and 10 mg benzoyl peroxide in 30 mL carbon tetrachloride for more than 2 h, cool, chromatograph
on silica gel with benzene:ethyl acetate 30:1 (Caution! Benzene is a carcinogen!) to
obtain 9-bromomethylacridine as yellow crystals (mp 147–151◦ ) (Anal.Lett. 1987, 20,
1581).)
HPLC VARIABLES
Column: A 10 × 2.1 40 µm Chromsep C18 (Chrompack); B 100 × 3 5 µm Chromspher
C18 (Chrompack)
Mobile phase: A 10 mM pH 7.0 phosphate buffer; B Gradient. MeOH:water 75:25 for
3 min, to 100:0 over 12 min (concave gradient).
Injection volume: 50
Detector: UV 254; F ex 362 em 418
CHROMATOGRAM
Retention time: 10
Internal standard: heptadecanoic acid (13)
Limit of detection: 300 nM
OTHER SUBSTANCES
Extracted: arachidonic acid (7), linoleic acid (9), linolenic acid (6), myristic acid (8),
palmitic acid (10.5), palmitoleic acid (7.5), stearic acid (13.5)
KEY WORDS
column-switching; derivatization; plasma
REFERENCE
van der Horst, F.A.L.; Post, M.H.; Holthuis, J.J.M.; Brinkman, U.A.T. Automated high-performance
liquid chromatographic determination of plasma free fatty acids using on-line derivatization with 9bromomethylacridine based on micellar phase-transfer catalysis, J.Chromatogr., 1990, 500, 443–452.
SAMPLE
Matrix: solutions
Sample preparation: Mix 100 µL of a solution in EtOH, EtOH/water, or water with
400 µL reagent solution and 200 µL 20 mM 2-nitrophenylhydrazine hydrochloride in
water, heat at 60◦ for 20 min, add 100 µL 15% KOH in MeOH:water 80:20, heat at 60◦
for 15 min, cool, inject a 1–2 µL aliquot. (Prepare the reagent by mixing equal volumes
of 3% pyridine in EtOH and 250 mM 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride in EtOH.)
HPLC VARIABLES
Column: 250 × 4.6 5 µm YMC-C8 (Yamamuta Chemical Research, Kyoto)
Column temperature: 50
Oleic acid
463
Mobile phase: MeOH:water 86:14 adjusted to pH 4.5 with 100 mM HCl
Flow rate: 1.2
Injection volume: 1–2
Detector: UV 230; UV 400
CHROMATOGRAM
Retention time: 10.7
Limit of detection: 2.5–5 pmol (UV 230), 10–15 pmol (UV 400)
OTHER SUBSTANCES
Simultaneous: capric acid (3), lauric acid (5), linoleic acid (8.5), linolenic acid (7),
myristic acid (6.5), palmitic acid (10), palmitoleic acid (7.5), stearic acid (14)
KEY WORDS
derivatization
REFERENCE
Miwa, H.; Hiyama, C.; Yamamoto, M. High-performance liquid chromatography of short- and long-chain
fatty acids as 2-nitrophenylhydrazides, J.Chromatogr., 1985, 321, 165–174.
ANNOTATED BIBLIOGRAPHY
Abushufa, R.; Reed, P.; Weinkove, C. Fatty acids in erythrocytes measured by isocratic HPLC, Clin.Chem.,
1994, 40, 1707–1712. [oleic acid; arachidonic acid; palmitoleic acid; linoleic acid; eicosatrienoic acid;
palmitic acid; stearic acid]
Akasaka, K.; Suzuki, T.; Ohrui, H.; Meguro, H.; Shindo, Y.; Takahashi, H. 9-Bromomethylacridine a
novel fluorescent labeling reagent of carboxylic group for HPLC, Anal.Lett., 1987, 20, 1581–1594.
[caprylic acid; capric acid; lauric acid; linolenic acid; arachidonic acid; palmitoleic acid; myristic acid;
linoleic acid; oleic acid; palmitic acid; stearic acid]
Akasaka, K.; Ohrui, H.; Meguro, H. Determination of carboxylic acids by high-performance liquid chromatography with 2-(2,3-anthracenedicarboximido)ethyl trifluoromethanesulfonate as highly sensitive
fluorescent labelling reagent, Analyst, 1993, 118, 765–768. [LOD 1.4–3.8 pmol; caprylic acid; caproic
acid; lauric acid; myristoleic acid; eicosapentaenoic acid; linolenic acid; myristic acid; docosahexaenoic
acid; palmitoleic acid; arachidonic acid; linoleic acid; eicosatrienoic acid; palmitic acid; oleic acid;
eicosadienoic acid; margaric acid; stearic acid; gondoic acid]
Baty, J.D.; Pazouki, S.; Dolphin, J. Analysis of fatty acids as their anthrylmethyl esters by highperformance liquid chromatography with fluorescence detection, J.Chromatogr., 1987, 395, 403–411.
[plasma; LOD 50 ng; lauric acid; linolenic acid; arachidonic acid; myristic acid; palmitoleic acid; linoleic
acid; palmitic acid; oleic acid; stearic acid; arachidic acid; behenic acid; lignoceric acid; margaric acid]
Borch, R.F. Separation of long chain fatty acids as phenacyl esters by high pressure liquid chromatography, Anal.Chem., 1975, 47, 2437–2439. [LOD 100 ng; lauric acid; myristoleic acid; linolenic acid;
myristic acid; palmitoleic acid; arachidonic acid; linoleic acid; pentadecanoic acid; linolelaidic acid;
eicosatrienoic acid; palmitic acid; oleic acid; vaccenic acid; petroselinic acid; elaidic acid; eicosadienoic
acid; heptadecanoic acid; stearic acid; eicosaenoic acid; nonadecanoic acid; arachidic acid; erucic acid;
heneicosanoic acid; behenic acid; nervonic acid; lignoceric acid]
Carrier, A.; Parent, J. Liquid chromatography-mass spectrometry determination of free fatty acids
in phospholipid-based formulations, J.Liq.Chromatogr.Rel.Technol., 2001, 24, 97–107. [stearic acid;
linoleic acid; linolenic acid; palmitic acid; oleic acid]
Cooper, M.J.; Anders, M.W. Determination of long chain fatty acids as 2-naphthacyl esters by high
pressure liquid chromatography and mass spectrometry, Anal.Chem., 1974, 46, 1849–1852. [oleic acid;
linolenic acid; linoleic acid; dihomolinolenic acid; arachidonic acid]
Czauderna, M.; Kowalczyk, J. Separation of some mono-, di- and tri-unsaturated fatty acids containing 18 carbon atoms by high-performance liquid chromatography and photodiode array detection,
J.Chromatogr.B, 2001, 760, 165–178. [derivatization; linoleic acid; linolenic acid; oleic acid; ricinoleic
acid; myristic acid; palmitic acid; vaccenic acid; stearic acid]
Eguchi, Y. Analysis of lipoprotein lipase activity using high-performance liquid chromatography,
Biomed.Chromatogr., 2002, 16, 500–503. [oleic acid; derivatization]
Ergan, F.; Andre, G. Simple high performance liquid chromatography methods for monitoring lipase
reactions, Lipids, 1989, 24, 76–78. [oleic; acid; linolenic acid; linoleic acid; palmitic acid; stearic acid]
464
Oleic acid
Fuse, T.; Kusu, F.; Takamura, K. Determination of higher fatty acids in oils by high-performance liquid
chromatography with electrochemical detection, J.Chromatogr.A, 1997, 764, 177–182. [linoleic acid;
oleic acid; palmitic acid; stearic acid; post-column reaction; derivatization; camellia oil; olive oil;
rapeseed oil; corn oil; soybean oil; LOD 20 pmol]
Ghiggeri, G.M.; Candiano, G.; Delfino, G.; Queirolo, C.; Ginevri, F.; Perfamo, F.; Gusmano, R. Separation
of the 9-anthryldiazomethane derivates of fatty acids by high-performance liquid chromatography on a
fatty acid analysis column. Application to albumin-bound fatty acid analysis, J.Chromatogr., 1986, 381,
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Oleic acid
465
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Oleic acid
467
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Olmesartan
Olmesartan
Molecular formula: C29 H30 N6 O6
Molecular weight: 558.58
H3C CH3
HO
O
O
O
N
O
N
H3C
CAS Registry No: 144689-63-4
Merck Index: 13, 6909
469
O
H
CH3
N N
N
N
SAMPLE
Matrix: microsomal incubations
Sample preparation: Mix 100 µL microsomal incubation with 100 µL MeCN, centrifuge at 10 000 g for 3 min, inject a 10 µL aliquot.
HPLC VARIABLES
Column: 150 × 6.9 YMC-Pack ODS-A-312 C18
Mobile phase: MeCN:water:PIC A 45:55:2.2
Flow rate: 1
Injection volume: 10
Detector: UV 254
CHROMATOGRAM
Retention time: 6
KEY WORDS
human; intestine; liver; rat
REFERENCE
Kobayashi, N.; Fujimori, I.; Watanabe, M.; Ikeda, T. Real-time monitoring of metabolic reactions by
microdialysis in combination with tandem mass spectrometry: hydrolysis of CS-866 in vitro in human
and rat plasma, livers, and small intestines, Anal.Biochem., 2000, 287, 272–278.
470
Olopatadine
Olopatadine
O
COOH
Molecular formula: C21 H23 NO3
Molecular weight: 337.41
CAS Registry No: 113806-05-6,
140462-76-6 (HCl)
Merck Index: 13, 6910
N
CH3
CH3
SAMPLE
Matrix: blood
Sample preparation: Condition a Bond Elut C18 SPE cartridge with 1 mL MeOH,
2 mL water, 2 mL 1% bovine serum albumin in water, and 5 mL water. Vortex 250 µL
plasma, 50 µL 200 ng/mL IS in water, and 250 µL water, add to the SPE cartridge,
wash with 2 mL water, elute with 1 mL MeOH. Evaporate the eluate to dryness under
a stream of nitrogen, reconstitute the residue with 50 µL mobile phase, filter (0.2 µm
PTFE), inject a 10 µL aliquot.
HPLC VARIABLES
Column: 50 × 2 5 µm Develosil ODS HG-5
Mobile phase: MeOH:10 mM acetic acid 45:55
Flow rate: 0.1
Injection volume: 10
Detector: MS, Micromass Quattro, electrospray, ion source 120◦ , capillary 4.0 kV,
counter current electrode 1.2 kV, cone 20 V, positive mode, collision gas argon 0.2 Pa,
collision energy 20 eV, m/z 338–165
CHROMATOGRAM
Retention time: 2.5
Internal standard: KF11796 ((11Z)-11-[3-(dimethylamino)propylidene]-6,11-dihydrodibenz[b,e]oxepin-2-propionic acid) (m/z 352–247–179) (4)
Limit of quantitation: 1 ng/mL
OTHER SUBSTANCES
Extracted: metabolites
KEY WORDS
pharmacokinetics; plasma; SPE
REFERENCE
Fujita, K.; Magara, H.; Kobayashi, H. Determination of olopatadine, a new antiallergic agent, and its
metabolites in human plasma by high-performance liquid chromatography with electrospray ionization
tandem mass spectrometry, J.Chromatogr.B, 1999, 731, 345–352.
SAMPLE
Matrix: microsomal incubations
Sample preparation: Mix 100 µL microsomal incubation with 100 µL ice-cold MeCN,
centrifuge at 14 020 g for 10 min, filter the supernatant, inject an aliquot of the filtrate.
HPLC VARIABLES
Column: 150 × 6 5 µm YMC-Pack AM312
Mobile phase: MeCN:0.1% trifluoroacetic acid 20:80
Flow rate: 1
Detector: Radioactivity (14 C)
Olopatadine
471
OTHER SUBSTANCES
Extracted: metabolites
KEY WORDS
human; liver
REFERENCE
Kajita, J.; Inano, K.; Fuse, E.; Kuwabara, T.; Kobayashi, H. Effects of olopatadine, a new antiallergic agent, on human liver microsomal cytochrome P450 activities, Drug Metab.Dispos., 2002, 30,
1504–1511.
472
Orbifloxacin
Orbifloxacin
Molecular formula: C19 H20 F3 N3 O3
Molecular weight: 395.38
CAS Registry No: 113617-63-3
CH3
H
N
H3C
F
N
N
F
COOH
F
O
SAMPLE
Matrix: blood
Sample preparation: Mix 200 µL plasma with 800 µL 1.5 µg/mL IS in 100 mM pH 7.4
phosphate buffer, add 6 mL chloroform (Caution! Chloroform is a carcinogen!), shake at
200 oscillations/min for 30 min, centrifuge at 13 000 g for 6 min. Evaporate the organic
layer to dryness under a stream of nitrogen at 40◦ , reconstitute the residue with 200 µL
phosphate-buffered saline, inject an aliquot.
HPLC VARIABLES
Guard column: Novapack C18 Guard-Pak
Column: 150 × 3.9 5 µm Novapack C18
Mobile phase: MeCN:buffer 20:80 (The buffer was 20 mM potassium dihydrogen phosphate containing 6 mM phosphoric acid and 12 mM tetraethylammonium bromide, pH
adjusted to 3.0 with 2 M NaOH.)
Flow rate: 1
Detector: F ex 338 em 425
CHROMATOGRAM
Retention time: 3.09
Internal standard: norfloxacin (2.16)
Limit of detection: 9 ng/mL (sic)
Limit of quantitation: 4 ng/mL
OTHER SUBSTANCES
Simultaneous: ciprofloxacin (2.28), danofloxacin (2.80), difloxacin (4.52), enrofloxacin
(3.30), marbofloxacin (2.20), sarafloxacin (4.40)
KEY WORDS
plasma; rabbit
REFERENCE
Garcı́a, M.A.; Solans, C.; Aramayona, J.J.; Rueda, S.; Bregante, M.A. Determination of orbifloxacin in
rabbit plasma by high-performance liquid chromatography with fluorescence detection, J.Chromatogr.
Sci., 1999, 37, 199–202.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 × 4 Develosil ODS-7
Mobile phase: MeOH:dioxane:100 mM pH 3.5 citrate buffer 12:5:84 (Caution! Dioxane
is a carcinogen!)
Flow rate: 1.2
Detector: UV 290
Orbifloxacin
473
CHROMATOGRAM
Retention time: 6
OTHER SUBSTANCES
Simultaneous: degradation products
KEY WORDS
photodegradation
REFERENCE
Morimura, T.; Kohno, K.; Nobuhara, Y.; Matsukura, H. Photoreaction and active oxygen generation by
photosensitization of a new antibacterial fluoroquinolone derivative, orbifloxacin, in the presence of
chloride ion, Chem.Pharm.Bull., 1997, 45, 1828–1832.
SAMPLE
Matrix: tissue
Sample preparation: Condition a 3 mL 250 mg Supelclean ENVI C18 SPE cartridge
with 2 mL MeOH and 2 mL MeCN. Pack 250 mg 200–400 mesh AG MP-1 resin (BioRad) between frits in an empty 3 mL tube, condition with MeOH, water, and 5 mL 8 mM
NaOH. Add 5 mL MeCN to 1.5 g pureed fish, sonicate (Sonifier 450, 30% duty cycle, 40%
power) for 3 min, rotate for 10 min, centrifuge at 3000 rpm for 5 min, repeat extraction
with 5 mL MeCN. Combine the extracts and evaporate them to ca. 4 mL under a stream
of nitrogen at 45◦ , add to the C18 SPE cartridge, elute with another 1 mL MeCN. Dilute
all the eluates to 40 mL with 8 mM NaOH. Add this solution to the AG MP-1 cartridge,
wash with 2 mL water, wash with 2 mL MeOH, dry with a stream of nitrogen, elute
with 3 mL MeCN:2% formic acid 20:80, add 90 µL IS solution to the eluate, inject a
10 µL aliquot. (IS solution contained 500 ng/mL clenbuterol and 50 ng/mL penbutolol
in MeCN:water 20:80.)
HPLC VARIABLES
Column: 150 × 2.1 5 µm Zorbax Extend C18
Column temperature: 30
Mobile phase: Gradient. MeCN:2% formic acid:water 20:10:70 for 3 min, to 55:10:35
over 0.1 min, maintain at 55:10:35 for 6.9 min, return to initial conditions over 0.1 min,
re-equilibrate for 6.9 min.
Flow rate: 0.2
Injection volume: 10
Detector: MS, Micromass Quattro LC triple quadrupole, positive ion mode, nebulizer
gas nitrogen 100 L/h, drying gas nitrogen 700 L/h, collision gas argon at 1 µbar, source
block 110◦ , desolvation 350◦ , cone 32 V, collision energy 16 eV, m/z 396.36–352.11
CHROMATOGRAM
Retention time: 2.57
Internal standard: clenbuterol (m/z 277.00–203.00, cone 20 V, collision 15 eV) (3.13),
penbutolol (m/z 292.27–236.13, cone 28 V, collision 16 eV) (8.53)
Limit of detection: 5 ng/g
OTHER SUBSTANCES
Extracted: ciprofloxacin (m/z 332.15–288.07, cone 40 V, collision 16 eV; LOD 10 ng/g)
(2.17), danofloxacin (m/z 358.08–95.91, cone 38 V, collision 22 eV) (2.23), enrofloxacin
(m/z 360.24–316.14, cone 40 V, collision 16 eV) (2.41), flumequine (m/z 262.14–202.03,
cone 28 V, collision 34 eV) (9.31), nalidixic acid (m/z 233.09–187.05, cone 28 V, collision
28 eV) (9.14), oxolinic acid (m/z 262.12–160.02, cone 28 V, collision 40 eV) (8.11),
piromidic acid (m/z 289.17–243.06, cone 34 V, collision 28 eV) (10.05), sarafloxacin
(m/z 386.20–299.06, cone 40 V, collision 28 eV) (3.04)
474
Orbifloxacin
KEY WORDS
abalone; fish; SPE; trout
REFERENCE
Johnston, L.; Mackay, L.; Croft, M. Determination of quinolones and fluoroquinolones in fish tissue
and seafood by high-performance liquid chromatography with electrospray ionisation tandem mass
spectrometric detection, J.Chromatogr.A, 2002, 982, 97–109.
ANNOTATED BIBLIOGRAPHY
Morimura, T.; Ohno, T.; Matsukura, H.; Nobuhara, Y. Photodegradation kinetics of the new antibacterial
fluoroquinolone derivative, orbifloxacin, in aqueous solution, Chem.Pharm.Bull., 1995, 43, 1000–1004.
Morimura, T.; Ohno, T.; Matsukura, H.; Nobuhara, Y. Degradation kinetics of the new antibacterial
fluoroquinolone derivative, orbifloxacin, in aqueous solution, Chem.Pharm.Bull., 1995, 43, 1052–1054.
Morimura, T.; Nobuhara, Y.; Matsukura, H. Photodegradation products of a new antibacterial fluoroquinolone derivative, orbifloxacin, in aqueous solution, Biol.Pharm.Bull., 1997, 45, 373–377.
Schneider, M.J.; Donoghue, D.J. Multiresidue analysis of fluoroquinolone antibiotics in chicken tissue
using liquid chromatography-fluorescence-multiple mass spectrometry, J.Chromatogr.B, 2002, 780,
83–92. [ciprofloxacin; norfloxacin; danofloxacin; enrofloxacin; orbifloxacin; sarafloxacin; difloxacin;
LOQ 10 ng/g]
Schneider, M.J.; Donoghue, D.J. Multiresidue determination of fluoroquinolone antibiotics in eggs using
liquid chromatography-fluorescence-mass spectrometry, Anal.Chim.Acta, 2003, 483, 39–49. [norfloxacin; ciprofloxacin; danofloxacin; enrofloxacin; orbifloxacin; sarafloxacin; difloxacin; LOQ 10 ng/g]
Orlistat
Orlistat
Molecular formula: C29 H53 NO5
Molecular weight: 495.73
475
O
H
CH3
O
N
O
H3C
CAS Registry No: 96829-58-2
Merck Index: 13, 6935
O
O
CH3
(CH2)10CH3
SAMPLE
Matrix: blood
Sample preparation: Mix 1 mL plasma with 50 µL 100 ng/mL IS in MeOH, add 1 mL
MeCN, vortex, centrifuge at RCF 834 for 5 min. Remove the upper layer and add it to
5 mL hexane, rotate for 20 min, centrifuge at RCF 834 for 5 min. Evaporate the organic
layer to dryness under a stream of nitrogen at 25◦ , reconstitute the residue with 30 µL
MeCN:2 mM ammonium acetate 70:30, inject a 10 µL aliquot.
HPLC VARIABLES
Column: 50 × 2 5 µm Deltabond phenyl (Keystone)
Mobile phase: MeCN:2 mM ammonium acetate 90:10
Flow rate: 0.2
Injection volume: 10
Detector: MS, PE Sciex API-III, APCI, tandem triple quadrupole, positive ion mode,
declustering potential 38 V, sprayer 4600 V, multiplier −4200 V, nebulizer gas nitrogen
at 60 psi, curtain gas nitrogen at 1.2 L/min, collision gas argon, m/z 496–160
CHROMATOGRAM
Retention time: 1.2
Internal standard: d5 -orlistat (m/z 501–160)
Limit of detection: 0.1 ng/mL
Limit of quantitation: 0.2 ng/mL
KEY WORDS
plasma
REFERENCE
Bennett, P.K.; Li, Y.T.; Edom, R.; Henion, J. Quantitative determination of Orlistat (tetrahydrolipostatin, Ro 18–0647) in human plasma by high-performance liquid chromatography coupled with ion
spray tandem mass spectrometry, J.Mass Spectrom., 1997, 32, 739–749.
SAMPLE
Matrix: blood
Sample preparation: Mix 1 mL plasma, IS, and 1 mL MeCN, centrifuge. Mix the
supernatant with 5 mL hexane, shake, centrifuge. Remove the upper hexane layer and
evaporate it to dryness, reconstitute the residue with 50 µL MeCN:10 mM ammonium
acetate 70:30, inject a 20 µL aliquot.
HPLC VARIABLES
Guard column: 2 mm long
Column: 100 × 2 Spherisorb C6
Mobile phase: A MeCN:0.1% formic acid 95:5; or B MeOH:water 85:15 for 2.4 min, to
100:0 (step gradient), maintain at 100:0 for 3 min, re-equilibrate at 85:15 for 3 min.
Flow rate: A 0.15; B 0.15 for 2.4 min then 0.3
Injection volume: 20
476
Orlistat
Detector: MS Finnigan LCQ, quadrupolar ion trap, capillary electrospray, needle voltage
4 kV; nebulizer gas flow at 60% of maximum; capillary temperature 250◦ ; capillary
potential 20 V; lens potential 10 V, positive ion MS-MS mode, m/z 140–350
CHROMATOGRAM
Retention time: 1.1 (A), 2.4 (B)
Internal standard: d5 -orlistat
Limit of quantitation: 0.3 ng/mL
KEY WORDS
plasma
REFERENCE
Wieboldt, R.; Campbell, D.A.; Henion, J. Quantitative liquid chromatographic-tandem mass spectrometric determination of orlistat in plasma with a quadrupole ion trap, J.Chromatogr.B, 1998, 708,
121–129.
477
Oseltamivir
Oseltamivir
H3C
H3C
Molecular formula: C16 H28 N2 O4
Molecular weight: 312.40
CAS Registry No: 196618-13-0,
204255-11-8 (phosphate)
Merck Index: 13, 6958
H
O
O
N
H3C
H2N
CH3
O
O
SAMPLE
Matrix: blood
Sample preparation: Condition a 100 mg C18 SPE cartridge (Varian) with 1 mL
MeCN:water 75:25 and 1 mL 10 mM HCl in MeCN:water 5:95. Mix 100 µL plasma with
25 µL 1 M citric acid and 100 µL 1 µg/mL IS in 50 mM sodium dihydrogen phosphate,
add to the SPE cartridge, wash with 1 mL 10 mM HCl in MeCN:water 5:95, elute with
400 µL MeCN:water 75:25, add 50 µL 20 mM KCN in 200 mM pH 6.5 phosphate buffer
to the eluate, add 50 µL 20 mM naphthalene-2,3-dialdehyde in MeCN, vortex, heat at
40◦ for 45 min. Evaporate to dryness under reduced pressure at room temperature,
reconstitute the residue with 100 µL 50 mM sodium dihydrogen phosphate, centrifuge,
inject a 40 µL aliquot.
HPLC VARIABLES
Column: 150 × 4.6 5 µm Prodigy (ODS-2) (Phenomenex)
Column temperature: 40
Mobile phase: MeCN:water 27:73 containing 50 mM sodium acetate
Flow rate: 2
Injection volume: 40
Detector: F ex 420 em 472
CHROMATOGRAM
Retention time: 5.2 (for GS4071 the free acid active metabolite)
Internal standard: GS4057 ((3R, 4R, 5S)-4-acetamido- 5 -amino-3-(1-cyclopentanoxy)1-cyclohexene-1-carboxylic acid) (4.3)
Limit of detection: 20 ng/mL (S/N 3)
Limit of quantitation: 50 ng/mL
KEY WORDS
derivatization; plasma; rat; SPE
REFERENCE
Eisenberg, E.J.; Cundy, K.C. High-performance liquid chromatographic determination of GS4071, a
potent inhibitor of influenza neuraminidase, in plasma by precolumn fluorescence derivatization with
naphthalenedialdehyde, J.Chromatogr.B, 1998, 716, 267–273.
SAMPLE
Matrix: blood, tissue, urine
Sample preparation: Homogenize (Ultraturrax) 1 g lung or liver, centrifuge at 1300 g
at 4◦ for 10 min, filter (0.2 µm Gelman Z-spin) while centrifuging at 15 000 g for 15 min,
inject an aliquot of the filtrate. Filter (Millipore 10 000 MW cut-off) plasma or urine
while centrifuging at 15 000 g for 15 min, inject an aliquot of the ultrafiltrate.
HPLC VARIABLES
Column: 125 × 4 5 µm Inertsil C-18
Mobile phase: Gradient. A was 50 mM ammonium acetate. B was MeCN:50 mM ammonium acetate 60:40. A:B from 90:10 to 0:100 over 20 min.
478
Oseltamivir
Flow rate: 1
Detector: UV 220; Radioactivity (14 C)
CHROMATOGRAM
Retention time: 14.5
OTHER SUBSTANCES
Extracted: GS4071 (free acid active metabolite) (7), other metabolites
KEY WORDS
liver; lung; plasma; rat; ultrafiltrate
REFERENCE
Sweeny, D.J.; Lynch, G.; Bidgood, A.M.; Lew, W.; Wang, K.-Y.; Cundy, K.C. Metabolism of the influenza
neuraminidase inhibitor prodrug oseltamivir in the rat, Drug Metab.Dispos., 2000, 28, 737–741.
SAMPLE
Matrix: blood, urine
Sample preparation: Condition a 3 mL 7 mm Empore Mixed Phase Cation-MPC SPE
disc with 1 mL MeOH, three 3 mL portions of MeOH:50 mM ammonium acetate 90:10,
and 1 mL 5 mM pH 3.5 ammonium acetate. Mix 100 µL plasma or urine with 50 µL IS
solution and 1 mL 5 mM pH 3.5 ammonium acetate buffer, add to the SPE disc, wash
with 1 mL water, wash with 1 mL MeOH, wash with 1 mL MeOH:water 90:10, dry
under vacuum, elute with 1 mL MeOH:50 mM ammonium acetate 90:10. Evaporate the
eluate to dryness under a stream of nitrogen at ca. 50◦ , reconstitute the residue with
150 µL water, centrifuge at >500 rpm, inject a 100 µL aliquot. (IS solution contained
50 ng/ml of d3 -oseltamivir and 2 µg/ml of d3 -GS4071 in water).
HPLC VARIABLES
Column: 100 × 5 4 µm Nova-Pak CN HP radial compression
Mobile phase: MeOH:80 mM pH 3 formic acid 50:50
Flow rate: 0.5
Injection volume: 100
Detector: MS, Finnigan TSQ 7000 tandem quadrupole, electrospray, collision gas argon,
m/z 313–224
CHROMATOGRAM
Retention time: 5
Internal standard: d3 -oseltamivir (m/z 316–227); d3 -GS4071 (m/z 288–201)
Limit of quantitation: 1 ng/mL (plasma), 5 ng/mL (urine)
OTHER SUBSTANCES
Extracted: GS4071 (de-ethylated active metabolite) (LOQ 10 ng/mL (plasma), 30 ng/mL
(urine); m/z 285–198) (3.5)
KEY WORDS
ferret; human; marmoset; mouse; plasma; rabbit; rat; SPE
REFERENCE
Wiltshire, H.; Wiltshire, B.; Citron, A.; Clarke, T.; Serpe, C.; Gray, D.; Herron, W. Development of a
high-performance liquid chromatographic-mass spectrometric assay for the specific and sensitive
quantification of Ro 64–0802, an anti-influenza drug, and its pro-drug, oseltamivir, in human and
animal plasma and urine, J.Chromatogr.B, 2000, 745, 373–388.
479
Oxaliplatin
Oxaliplatin
Molecular formula: C8 H14 N2 O4 Pt
Molecular weight: 397.29
CAS Registry No: 61825-94-3
Merck Index: 13, 6981
H
H
N
O
O
O
O
Pt
N
H
H
SAMPLE
Matrix: blood
Sample preparation: Filter (Sartorius Centrisart I 10 000 MW cut-off) while centrifuging at 4000 g at 4◦ for 1 h, inject an aliquot of the ultrafiltrate.
HPLC VARIABLES
Column: 250 × 4 5 µm Hypercarb
Mobile phase: MeOH:250 mM pH 7.0 succinic acid buffer 90:10
Flow rate: 0.5
Injection volume: 25
Detector: UV 344 following post-column reaction. The column effluent mixed with
2.7 mM sodium N, N-diethyldithiocarbamate in MeOH pumped at 0.17 mL/min and the
mixture flowed through a 2.3 m long 0.51 mm ID length of PTFE tubing to the detector.
A 2 m length of the tubing was heated in a microwave field (ca. 130 W, Smithcreator,
Personal Chemistry, Sweden) at about 110◦ .
CHROMATOGRAM
Retention time: 12
Limit of quantitation: 40 ng/mL (S/N 10)
KEY WORDS
pharmacokinetics; post-column reaction; ultrafiltrate; whole blood
REFERENCE
Ehrsson, H.; Wallin, I. Liquid chromatographic determination of oxaliplatin in blood using post-column
derivatization in a microwave field followed by photometric detection, J.Chromatogr.B, 2003, 795,
291–294.
SAMPLE
Matrix: blood, urine
Sample preparation: Plasma. Filter (Amicon MPS-1 with a YMT membrane) while
centrifuging at 4◦ at 3000 g for 15 min, inject an aliquot of the ultrafiltrate. Urine.
Directly inject an aliquot of urine. (Protect from light.)
HPLC VARIABLES
Column: 250 × 4.6 Inertsil ODS-2
Column temperature: 40
Mobile phase: MeCN:10 mM pH 5.5 acetate buffer 5:95
Flow rate: 1
Injection volume: 100
Detector: UV 290 following post-column reaction. The column effluent mixed with the
reagent (maintained at 0◦ ) pumped at 0.3 mL/min and the mixture flowed through a
10 m ×0.5 mm ID PTFE coil at 60◦ to the detector. (The reagent was 10 mM pH 5.5
acetate buffer containing 40 mM sodium bisulfite.)
CHROMATOGRAM
Retention time: 11
480
Oxaliplatin
Limit of detection: 60 nM
OTHER SUBSTANCES
Extracted: carboplatin (6.5), tetraplatin (8)
KEY WORDS
plasma; post-column reaction; rabbit
REFERENCE
Kizu, R.; Yamamoto, T.; Yokoyama, T.; Tanaka, M.; Miyazaki, M. A sensitive postcolumn derivatization/UV detection system for HPLC determination of antitumor divalent and quadrivalent platinum
complexes, Chem.Pharm.Bull., 1995, 43, 108–114.
SAMPLE
Matrix: solutions
Sample preparation: Inject an aliquot of a 700 µg/mL solution of oxaliplatin in MeOH
containing 800 µg/mL IS.
HPLC VARIABLES
Column: 250 × 4.6 10 µm Vydac C18
Column temperature: 24
Mobile phase: MeCN:water 80:20
Flow rate: 0.8
Detector: UV 255
CHROMATOGRAM
Retention time: 3.4
Internal standard: flavone (4.6)
KEY WORDS
robust; validated
REFERENCE
Ficarra, R.; Calabrò, M.L.; Cutroneo, P.; Tommasini, S.; Melardi, S.; Semreen, M.; Furlanetto, S.; Ficarra,
P.; Altavilla, G. Validation of a LC method for the analysis of oxaliplatin in a pharmaceutical formulation using an experimental design, J.Pharm.Biomed.Anal., 2002, 29, 1097–1103.
ANNOTATED BIBLIOGRAPHY
Heudi, O.; Mercier-Jobard, S.; Cailleux, A.; Allain, P. Mechanisms of reaction of L-methionine with
carboplatin and oxaliplatin in different media: a comparison with cisplatin, Biopharm.Drug Dispos.,
1999, 20, 107–116.
Luo, F.R.; Yen, T.-Y.; Wyrick, S.D.; Chaney, S.G. High-performance liquid chromatographic separation
of the biotransformation products of oxaliplatin, J.Chromatogr.B, 1999, 724, 345–356.
481
Oxiconazole
Oxiconazole
N
Molecular formula: C18 H13 Cl4 N3 O
Molecular weight: 492.14
CAS Registry No: 64211-45-6, 64211-46-7 (nitrate)
Merck Index: 13, 7006
Cl
N
Cl
O
N
Cl
Cl
SAMPLE
Matrix: formulations
Sample preparation: Dissolve lotion containing 50 mg oxiconazole in 50 mL MeOH,
dilute a 5 mL aliquot to 50 mL with mobile phase, inject an aliquot. Shake cream
containing 50 mg oxiconazole with 50 mL chloroform:MeOH 50:50 for 30 min (Caution!
Chloroform is a carcinogen!), make up to 100 mL with MeOH, centrifuge at 4000 rpm
at 4◦ for 15 min, filter, dilute fivefold with MeOH.
HPLC VARIABLES
Guard column: LiChrocart 4-4
Column: 125 × 4 5 µm LiChrocart C8
Mobile phase: MeOH:20 mM ammonium acetate buffer 85:15
Flow rate: 1
Injection volume: 20
Detector: UV 254
CHROMATOGRAM
Retention time: 2.7
Limit of detection: 1.2 µg/mL
Limit of quantitation: 3.75 µg/mL
OTHER SUBSTANCES
Noninterfering: benzyl alcohol
KEY WORDS
cream; lotion; stability-indicating
REFERENCE
Milano, J.; Morsch, L.M.; Gonalves Cardoso, S. LC method for the analysis of Oxiconazole in pharmaceutical formulations, J.Pharm.Biomed.Anal., 2002, 30, 175–180.
SAMPLE
Matrix: solutions
Sample preparation: Inject an aliquot of a 100 µg/mL solution in MeOH.
HPLC VARIABLES
Column: 125 × 4 5 µm LiChrospher 100 RP8
Mobile phase: MeCN:20 mM ammonium acetate buffer 75:25
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Retention time: 4.93
482
Oxiconazole
OTHER SUBSTANCES
Simultaneous: bifonazole, photodegradation products
REFERENCE
Thoma, K.; Kübler, N. Untersuchung der Photostabilität von Antimykotika. 1. Mitt.: Photostabilität
von Azolantimykotika [Photodegradation of antimycotic drugs. Part 1: Photodegradation of azole
antimycotics], Pharmazie, 1996, 51, 885–892.
Panipenem
Molecular formula: C15 H21 N3 O4 S
Molecular weight: 339.42
CAS Registry No: 87726-17-8
Merck Index: 13, 7079
NH
OH H H
H3C
N
N
CH3
S
O
COOH
SAMPLE
Matrix: blood
Sample preparation: Vortex 10 µL plasma with 10 µL of 200 mM MOPS (3-(Nmorpholino)propanesulfonic acid) solution, add 100 µL MeOH, mix, centrifuge at
12 000 rpm for 5 min, inject a 25 µL aliquot of the supernatant.
HPLC VARIABLES
Column: 150 × 4.6 5 µm Inertsil ODS-2
Mobile phase: MeOH:buffer 35:65 (The buffer was 5 mM pH 5.8 sodium dihydrogen
phosphate containing 5 mM sodium n-dodecylsulfate.)
Flow rate: 0.8
Injection volume: 25
Detector: UV 300
CHROMATOGRAM
Retention time: 6
Limit of quantitation: 500 ng/mL
KEY WORDS
plasma
REFERENCE
Kokubun, H.; Kimura, T.; Murase, S.; Shimada, S.; Kubo, H.; Matsumoto, M.; Nowatari, M.; Matsuura, N.
Determination of panipenem in neonatal plasma by HPLC, Anal.Sci., 2000, 16, 1077–1078.
HPLC Methods for Recently Approved Pharmaceuticals. by George Lunn
Copyright 2005 John Wiley & Sons, Ltd. ISBN: 0-471-66941-5
483
484
Parecoxib
Parecoxib
H3C
N
H
Molecular formula: C19 H18 N2 O4 S
Molecular weight: 370.42
CAS Registry No: 198470-84-7,
197502-82-2 (Na salt)
O
N
H3C
O
O
S
O
SAMPLE
Matrix: formulations
Sample preparation: Inject a 10 µL aliquot.
HPLC VARIABLES
Column: 150 × 4.6 5 µm YMC ODS-AQ
Column temperature: 40
Mobile phase: MeCN:10 mM pH 3.0 phosphate buffer 40:60
Flow rate: 2
Injection volume: 10
Detector: UV 215
CHROMATOGRAM
Retention time: 9.5
OTHER SUBSTANCES
Simultaneous: valdecoxib (5)
KEY WORDS
injections; stability-indicating
REFERENCE
Crane, I.M.; Mulhern, M.G.; Nema, S. Stability of reconstituted parecoxib for injection with commonly
used diluents, J.Clin.Pharm.Ther., 2003, 28, 363–369.
485
Paricalcitol
Paricalcitol
CH3
H3C
CH3
CH3
OH
Molecular formula: C27 H44 O3
CH3
Molecular weight: 416.63
CAS Registry No: 131918-61-1
Merck Index: 13, 7111
H
HO
OH
SAMPLE
Matrix: blood
Sample preparation: Extract plasma with ether, inject an aliquot onto column A and
column B in series and elute with mobile phase. After 1.5 min, remove column A from the
circuit. Continue to elute column B with mobile phase and collect fractions. Regenerate
column A by backflushing with a stronger mobile phase
HPLC VARIABLES
Column: A 23 × 4 PVA-Sil; B 250 × 4.6 PVA-Sil
Mobile phase: MTBE:isopropanol 97.5:2.5
Detector: Radio receptor assay (after evaporation of fractions)
KEY WORDS
column-switching; normal phase; plasma
REFERENCE
Chang, M.; Qasawa, B.; Chu, S. Determination of 19-nor-1α,25 dihydroxy-vitamin D2 (ABT-358) in
plasma using a combination of HPLC and radio-receptor assay techniques (Abstract 2648), Pharm.Res.,
1997, 14, S427.
SAMPLE
Matrix: solutions
Sample preparation: Inject an aliquot of a solution in mobile phase.
HPLC VARIABLES
Column: 80 × 6.2 3 µm Zorbax SIL
Mobile phase: Hexane:isopropanol:MeOH 91:7:2
Flow rate: 1
Detector: UV 251
CHROMATOGRAM
Retention time: 11.9
OTHER SUBSTANCES
Simultaneous: metabolites
KEY WORDS
normal phase
486
Paricalcitol
REFERENCE
Shankar, V.N.; Propp, A.E.; Schroeder, N.; Surber, B.W.; Makin, H.L.J.; Jones, G. In vitro metabolism of
19-nor-1α,25-(OH)2 D2 in cultured cell lines: inducible synthesis of lipid- and water-soluble metabolites,
Arch.Biochem.Biophys., 2001, 387, 297–306.
SAMPLE
Matrix: solutions
Sample preparation: Inject an aliquot of a solution in mobile phase.
HPLC VARIABLES
Column: 250 × 4.6 6 µm Zorbax CN
Mobile phase: Hexane:isopropanol:MeOH 88:10:2
Flow rate: 1
Detector: UV 251
CHROMATOGRAM
Retention time: 8.57
OTHER SUBSTANCES
Simultaneous: metabolites
REFERENCE
Shankar, V.N.; Propp, A.E.; Schroeder, N.; Surber, B.W.; Makin, H.L.J.; Jones, G. In vitro metabolism of
19-nor-1α,25-(OH)2 D2 in cultured cell lines: inducible synthesis of lipid- and water-soluble metabolites,
Arch.Biochem.Biophys., 2001, 387, 297–306.
487
Pazufloxacin
Pazufloxacin
Molecular formula: C16 H15 FN2 O4
Molecular weight: 318.30
CAS Registry No: 127045-41-4
Merck Index: 13, 7128
CH3
O
N
H2N
F
COOH
O
SAMPLE
Matrix: blood
Sample preparation: Vortex 50 µL plasma with 350 µL 1 µg/mL IS in MeOH, cen-
trifuge. Evaporate the supernatant to dryness under a stream of nitrogen at 40◦ ,
reconstitute the residue with mobile phase, inject an aliquot.
HPLC VARIABLES
Column: 150 × 4.6 Cosmosil 5C18
Column temperature: 40
Mobile phase: MeCN:1 M ammonium acetate:50 mM citric acid 15:1:84
Flow rate: 1
Detector: F ex 335 em 450
CHROMATOGRAM
Internal standard: lomefloxacin
Limit of quantitation: 100 ng/mL
KEY WORDS
plasma; rat
REFERENCE
Hasegawa, T.; Nadai, M.; Haghgoo, S.; Yamaki, K.; Takagi, K.; Nabeshima, T. Influence of a newly developed quinolone, T-3761, on pharmacokinetics of theophylline in rats, Antimicrob.Agents Chemother.,
1995, 39, 2138–2140.
SAMPLE
Matrix: blood, urine
Sample preparation: Dilute plasma with an equal volume of MeOH, centrifuge, inject
an aliquot. Dilute urine with mobile phase, inject an aliquot.
HPLC VARIABLES
Column: 150 × 4 Develosil ODS-HG-5
Mobile phase: MeCN:200 mM pH 7.0 phosphate buffer:water 9:5:86 containing 0.68%
tetra-n-butylammonium hydrogen sulfate
Detector: UV 330
KEY WORDS
plasma; rat
REFERENCE
Hayakawa, H.; Takagi, K.; Takano, Y.F.; Kawamura, Y.; Tsuji, A. Determinant of the distribution volume
at steady state for novel quinolone pazufloxacin in rats, J.Pharm.Pharmacol., 2002, 54, 1229–1236.
488
Penciclovir
Penciclovir
Molecular formula: C10 H15 N5 O3
Molecular weight: 253.26
CAS Registry No: 39809-25-1
Merck Index: 13, 7154
O
H
N
N
H2N
N
N
OH
OH
SAMPLE
Matrix: blood
Sample preparation: Mix 200 µL blood with 600 µL 16% trichloroacetic acid, centrifuge, inject an aliquot of the supernatant.
HPLC VARIABLES
Guard column: C18 Guard-Pak (Waters)
Column: 100 × 8 Nova-Pak C18 (in a Z module)
Mobile phase: Gradient. A was 50 mM sodium hydrogen phosphate. B was MeOH:water
80:20 containing 5 mM sodium dihydrogen phosphate. A:B 99:1 for 1.5 min, to 5:95 over
18.5 min, re-equilibrate at initial conditions for 10 min.
Flow rate: 1.6
Detector: UV 254
CHROMATOGRAM
Retention time: 13.5
Limit of detection: 200 ng/mL
OTHER SUBSTANCES
Extracted: acyclovir (LOD 250 ng/mL) (11.6)
KEY WORDS
mouse; pharmacokinetics; whole blood
REFERENCE
Boyd, M.R.; Bacon, T.H.; Sutton, D. Antiherpesvirus activity of 9-(4-hydroxy-3-hydroxymethylbut-1-yl)
guanine (BRL 39123) in animals, Antimicrob.Agents Chemother., 1988, 32, 358–363.
SAMPLE
Matrix: blood
Sample preparation: Condition a 1 mL Bond Elut SCX SPE cartridge with 1 mL
MeOH, 1 mL water, and 1 mL 1 mM pH 7 sodium dihydrogen phosphate buffer. Mix
500 µL plasma with 50 µL 100 µg/mL IS in water, add 500 µL 16% trichloroacetic
acid, mix, centrifuge (12 cm rotor arm) at 3000 rpm for 5 min. Add the supernatant
to 500 µL 1 mM pH 7 sodium dihydrogen phosphate buffer, add to the SPE cartridge,
wash with 1 mL 1 mM pH 7 sodium dihydrogen phosphate buffer, elute with 500 µL
MeOH:250 mM pH 11 potassium dihydrogen phosphate 20:80, inject a 25–50 µL aliquot
of the eluate.
HPLC VARIABLES
Guard column: 15 × 3.2 7 µm Newguard RP-18
Column: 250 × 4.6 5 µm Spherisorb ODS2
Mobile phase: MeOH:10 mM pH 7 sodium dihydrogen phosphate buffer 10:90
Flow rate: 1
Injection volume: 25–50
Detector: UV 254
Penciclovir
489
CHROMATOGRAM
Retention time: 9
Internal standard: BRL 42377 (9-(4-hydroxy-2-hydroxymethylbut-1-yl)guanine) (10.5)
Limit of quantitation: 100 ng/mL
KEY WORDS
pharmacokinetics; plasma; SPE
REFERENCE
Fowles, S.E.; Pierce, D.M. High-performance liquid chromatographic method for the determination of
9-(4-hydroxy-3-hydroxymethylbut-1-yl)guanine (BRL-39123) in human plasma and urine, Analyst,
1989, 114, 1373–1375.
SAMPLE
Matrix: blood, urine
Sample preparation: Condition a Bond Elut SCX strong cation exchange SPE cartridge
with 1 mL MeOH, 1 mL water, and 1 mL 1 mM pH 7.0 disodium hydrogen phosphate
buffer. Mix 500 µL plasma or 100 µL urine with 100 µL 20–50 µg/mL IS in water and
100–500 µL 16% trichloroacetic acid, add the supernatant to the SPE cartridge, wash
with 1 mL MeOH:1 mM pH 7.0 disodium hydrogen phosphate buffer 20:80, elute with
1 mL MeOH:100 mM pH 11.0 dipotassium hydrogen phosphate buffer 25:75, inject an
aliquot.
HPLC VARIABLES
Column: 3 µm Apex 1 ODS
Mobile phase: Gradient. A was MeOH:10 mM pH 7.0 disodium hydrogen phosphate
buffer 7:93. B was MeOH:10 mM pH 7.0 disodium hydrogen phosphate buffer 35:65.
A:B from 100:0 to 0:100 over 4 min, maintain at 0:100 for 1.5 min, return to 0:100 over
1 min.
Flow rate: 2
Detector: UV 254
CHROMATOGRAM
Retention time: 1.5
Internal standard:
6-deoxy-9-(4-hydroxy-2-hydroxymethylbut-1-yl)guanine (BRL
44056) (UV 305) (2.8)
Limit of detection: 500 ng/mL (plasma); 50 µg/mL (urine)
OTHER SUBSTANCES
Extracted: famciclovir (UV 305) (6); metabolites
KEY WORDS
dog; human; plasma; rat; SPE
REFERENCE
Winton, C.F.; Fowles, S.E.; Pierce, D.M.; Hodge, A.V. Gradient high-performance liquid chromatographic
method for the analysis of the pro-drug famciclovir and its metabolites, including the active anti-viral
agent penciclovir, in plasma and urine, Anal.Proc., 1990, 27, 181–182.
490
Pentaerythritol tetranitrate
Pentaerythritol tetranitrate
Molecular formula: C5 H8 N4 O12
ONO2 ONO2
O2NO
ONO2
Molecular weight: 316.14
CAS Registry No: 78-11-5
Merck Index: 13, 7186
SAMPLE
Matrix: blood
Sample preparation: Vigorously shake 5 mL plasma with 8 mL ethyl acetate for 1 min,
sonicate for 5 min, centrifuge at 3000 rpm for 3 min, repeat extraction with 8 mL ethyl
acetate, repeat extraction with 6 mL ethyl acetate. Pass the extracts through a C18 SPE
cartridge, filter (0.5 µm) the eluate. Evaporate the eluate to 200 µL under a stream of
nitrogen at 35◦ , inject a 25 µL aliquot.
HPLC VARIABLES
Column: 300 × 3.9 10 µm µBondapak CN
Mobile phase: Iso-octane:dichloromethane:MeOH 75:20:5
Flow rate: 1.5
Injection volume: 25
Detector: TEA (Thermal Energy Analyzer; nitrosyl specific), Thermo Electron Corpo-
ration Model 502, pyrolyzer 500◦ , carrier gas nitrogen at 5 mL/min, reaction chamber
0.6 torr, cryogenic trap -78◦
CHROMATOGRAM
Retention time: 7
Limit of quantitation: 1 ng/mL
OTHER SUBSTANCES
Extracted: isosorbide dinitrate (4), nitroglycerin (5), metabolites
KEY WORDS
plasma
REFERENCE
Yu, W.C.; Goff, E.U. Measurement of plasma concentrations of vasodilators and metabolites by the TEA
Analyzer, Biopharm.Drug Dispos., 1983, 4, 311–319.
491
Pentosan polysulfate
Pentosan polysulfate
Molecular weight: 1500–5000
CAS Registry No: 37300-21-3, 37319-17-8 (sodium salt)
Merck Index: 13, 7212
O
HSO3O
O
OSO3H
n
n = 6−12
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: LiChrospher Si 100 diol
Column temperature: 25
Mobile phase: 200 mM NaCl
Flow rate: 1
Detector: Refractive Index
CHROMATOGRAM
Retention time: 2
REFERENCE
Muller, D.; Ndoume-Nze, M.; Jozefonvicz, J. High-pressure size-exclusion chromatography of anticoagulant materials, J.Chromatogr., 1984, 297, 351–358.
492
Perflubron
Perflubron
Molecular formula: C8 BrF17
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
Br
Molecular weight: 498.96
CAS Registry No: 423-55-2
Merck Index: 13, 7235
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: Dynamax Microsorb silica (Rainin)
Mobile phase: Hexane:isopropanol 50:1
Flow rate: 2
Injection volume: 20
Detector: UV 254
CHROMATOGRAM
Retention time: 1.6
OTHER SUBSTANCES
Simultaneous: phenol (3.8)
Interfering: 1-(4-perfluorobutylphenyl)-1-hexanone
KEY WORDS
normal phase
REFERENCE
Williams, T.D.; Jay, M.; Lehmler, H.-J.; Clark, M.E.; Stalker, D.J.; Bummer, P.M. Solubility enhancement of phenol and phenol derivatives in perfluorooctyl bromide, J.Pharm.Sci., 1998, 87, 1585–1589.
493
Perospirone
Perospirone
Molecular formula: C23 H30 N4 O2 S
Molecular weight: 426.58
CAS Registry No: 150915-41-6, 129273-38-7 (HCl)
Merck Index: 13, 7259
H
O
N
H
N
O
N
N
S
SAMPLE
Matrix: blood
Sample preparation: Vortex 2 mL plasma, 500 µL water, 500 µL 1 mM NaOH, and
100 µL 250 ng/mL IS in water for 10 s, add 5 mL n-hexane:chloroform 70:30 (Caution!
Chloroform is a carcinogen!), shake for 10 min, centrifuge at 1700 g at 4◦ for 10 min.
Evaporate the organic layer to dryness under reduced pressure at 45◦ , reconstitute the
residue with 750 µL mobile phase A, inject a 500 µL aliquot onto column A and elute to
waste with mobile phase A; after 13.5 min, elute the contents of column A onto column
B with mobile phase B; after another 3.5, min remove column A from the circuit, elute
column B with mobile phase B, monitor the effluent from column B. (Re-equilibrate
column A with mobile phase A for 18 min.)
HPLC VARIABLES
Column: A 35 × 4.6 10 µm TSK gel PW (methacrylate polymer) (Tosoh); B 150 × 4.6 5 µm
C18 STR ODS II (Shinwa, Kyoto)
Column temperature: 30 (column B only)
Mobile phase: A MeCN:50 mM pH 4.6 phosphate buffer:6 M perchloric acid 4.6:94.9:0.5;
B MeCN:50 mM pH 4.6 phosphate buffer:6 M perchloric acid 41.5:58.0:0.5
Flow rate: A 1.2; B 0.6
Injection volume: 500
Detector: F ex 315 em 405
CHROMATOGRAM
Retention time: 25
Internal standard: tiospirone (29.5)
Limit of detection: 60 pg/mL
Limit of quantitation: 100 pg/mL
OTHER SUBSTANCES
Extracted: metabolite (ID-15036) (21.5)
Noninterfering: alprazolam, bromperidol, chlorpromazine, diazepam, haloperidol, levomepromazine, nitrazepam, risperidone
KEY WORDS
column-switching; pharmacokinetics; plasma
REFERENCE
Yasui-Furukori, N.; Inoue, Y.; Tateishi, T. Determination of a new atypical antipsychotic agent perospirone and its metabolite in human plasma by automated column-switching high-performance liquid
chromatography, J.Chromatogr.B, 2003, 789, 239–245.
494
Phenazopyridine hydrochloride
Phenazopyridine hydrochloride
Molecular formula: C11 H12 ClN5
Molecular weight: 249.71
CAS Registry No: 136-40-3
Merck Index: 13, 7296
NH2
N
H2N
N
N
HCl
SAMPLE
Matrix: blood
Sample preparation: Vigorously mix 1 mL plasma, 50 µL 40 µg/mL IS, 200 µL
100 mM NaOH, and 4 mL MTBE, centrifuge at 3000 rpm for 5 min. Evaporate the
organic layer to dryness under a stream of nitrogen, reconstitute the residue with 1 mL
20 mM phosphoric acid, inject an 80 µL aliquot.
HPLC VARIABLES
Guard column: BDS C8
Column: 100 × 4.6 5 µm LiChrospher 60 RP-Select B
Mobile phase: MeCN:20 mM pH 2.7 potassium dihydrogen phosphate buffer 30:70
Flow rate: 1.5
Injection volume: 80
Detector: UV 405
CHROMATOGRAM
Retention time: 2.5
Internal standard: diltiazem (UV 238) (3.9)
Limit of detection: 10 ng/mL
Limit of quantitation: 50 ng/mL
KEY WORDS
plasma
REFERENCE
Farin, D.; Piva, G.; Kitzes-Cohen, R. Determination of phenazopyridine in human plasma by high
performance liquid chromatography, Chromatographia, 2000, 52, 179–180.
SAMPLE
Matrix: blood, urine
Sample preparation: Condition a 3 mL 130 mg Bond Elut Certify SPE cartridge with
3 mL MeOH and 3 mL 200 mM pH 6.0 phosphate buffer. Vortex 3 mL serum or urine
with 3 mL 400 mM pH 6.0 phosphate buffer for 1 min, add to the SPE cartridge at
1 mL/min, wash with 3 mL MeOH:200 mM pH 6.0 phosphate buffer 5:95, pass air
through the SPE cartridge for 10 s, elute with 750 µL MeOH:10% ammonia 100:20 at
0.5 mL/min. Add a few drops of 1 M HCl to the eluate and evaporate to dryness under a
stream of nitrogen, reconstitute the residue with 150 µL mobile phase, inject an aliquot.
HPLC VARIABLES
Guard column: 10 × 2.1 5 µm Hypersil C18
Column: 150 × 4.6 5 µm Hypersil C18
Column temperature: 45
Mobile phase: Gradient. A was MeCN:buffer 5:95. B was MeCN:buffer 50:50. A:B from
85:15 to 10:90 over 20 min, maintain at 10:90 for 5 min, return to initial conditions
over 3 min, re-equilibrate for 6 min. (The buffer was 50 mM pH 3.0 phosphate buffer
containing 375 mg/L sodium octyl sulfate and 3 mL/L triethylamine.)
Phenazopyridine hydrochloride
495
Flow rate: 1
Detector: UV 240
CHROMATOGRAM
Retention time: 17.1
OTHER SUBSTANCES
Extracted: acebutolol (UV 326,235) (12.8), acetaminophen (UV 247) (2.7), N-acetylprocainamide (UV 268) (4.9), amineptine (UV 268) (19.3), amitriptyline (UV 240,211) (22.3),
amobarbital (UV 211) (15.3), amoxapine (UV 296,211.5) (19.1), amphetamine (UV 254)
(11.2), aprobarbital (14.7), atenolol (UV 275,230) (4.3), atropine (UV 260) (11.3), barbital
(UV 212) (5.5), benzhexol (UV 260) (22.5), benztropine (UV 257,223) (22.9), benzoylecgonine (UV 278,235) (6.7), bromazepam (UV 235) (13.3), buprenorphine (UV 284,213)
(19.4), buspirone (UV 302,239,211) (16.2), butabarbital (UV 211) (10.1), caffeine (UV
270) (3.5), carbamazepine (UV 283,215) (15.1), chloramphenicol (UV 277) (10.6), chlordiazepoxide (UV 303,245) (15.1), chlormethiazole (UV 252,211) (12.4), chlorpromazine
(UV 310,255) (23.9), cimetidine (UV 219) (3.5), citalopram (UV 239) (19.0), clobazam (UV
292,231) (20.5), clomipramine (UV 250,211) (23.6), clonazepam (UV 310,240,211) (18.5),
clonidine (UV 265) (6.9), cloperastine (UV 238) (26.2), cloxazolam (UV 340,265,239.5)
(18.4), cocaine (UV 278,235) (14.7), codeine (UV 280,211) (5.0), desipramine (UV 250)
(20.5), diazepam (UV 313,229) (22.6), dihydrocodeine (UV 282,211) (4.8), diltiazem (UV
242,211.5) (19.2), diphenhydramine (UV 258) (19.4), disopyramide (UV 260) (14.9),
dothiepin (UV 303,263,231,211) (21.5), doxepin (UV 291) (19.1), ephedrine (UV 257)
(6.2), erythromycin (UV 211) (23.3), fentanyl (UV 262) (18.4), flecainide (UV 299) (19.5),
flumazenil (UV 250) (11.0), fluoxetine (UV 262,228) (22.6), fluphenazine (UV 310,257)
(23.7), flupenthixol (UV 268,231) (27.2), flurazepam (UV 315,231) (20.6), fluvoxamine
(UV 251) (20.8), furosemide (UV 344,275,235) (13.2), glutethimide (15.8), guaifenesin
(UV 275,224) (6.7), haloperidol (UV 245,220) (19.6), hydralazine (UV 304,260,211) (3.5),
hydroxyzine (UV 231) (21.2), ibuprofen (UV 263,219.5) (28.0), imipramine (UV 250,211)
(21.6), indomethacin (UV 299,240) (27.9), ketamine (UV 270) (12.7), ketoconazole (UV
295,240) (20.8), labetalol (UV 303) (15.7), lidocaine (UV 266) (13.7), lorazepam (UV
318,231.5) (18.4), lormetazepam (UV 315,231) (20.5), loxapine (UV 295,250) (19.8),
maprotiline (UV 272) (22.0), MDMA (UV 287,235) (13.0), methadone (23.0), methamphetamine (UV 254) (12.0), methaqualone (UV 308,267,227) (18.1), methylephedrine
(UV 254) (7.7), methylphenidate (UV 260) (15.0), metoclopramide (UV 311,275,215)
(13.0), metoprolol (UV 275,223) (13.8), mianserin (UV 280) (19.3), midazolam (UV 221)
(19.3), moclobemide (UV 238) (14.1), molindone (UV 301,256,211) (13.6), morphine
(UV 286,212) (2.7), nadolol (UV 272,219) (6.6), naloxone (UV 282,209) (5.0), naproxen
(UV 270,231.5) (20.2), nifedipine (UV 335,235) (20.8), nitrazepam (UV 310,260,219)
(16.3), nordiazepam (UV 310,231) (19.7), norfluoxetine (UV 262,228) (21.4), nortriptyline (UV 240) (21.0), ofloxacin (UV 327,295.5,230) (9.5), oxycodone (UV 280) (9.9),
oxymorphone (UV 280) (6.7), paroxetine (UV 295,235) (19.8), pentobarbital (UV 211)
(14.9), pericyazine (UV 269,232) (19.6), perphenazine (UV 320,255,210) (22.2), pethidine
(15.5), phenazopyridine (UV 240) (17.1), phencyclidine (UV 265) (17.8), pheniramine
(UV 260) (13.0), phenobarbital (9.9), phentermine (UV 254) (12.5), phenylephrine (UV
302,259,219) (9.2), phenylpropanolamine (UV 258) (4.9), phenyltoloxamine (UV 272)
(20.6), phenytoin (15.3), pholcodine (UV 284,211.5) (4.0), pimozide (UV 280) (25.3),
pinazepam (UV 310,227.5) (25.5), piroxicam (UV 248) (15.9), prazepam (UV 311,231.5)
(25.1), primidone (6.2), procainamide (UV 280,210) (3.6), promethazine (UV 297,250)
(21.7), propoxyphene (UV 259) (21.8), protriptyline (UV 290,211) (21.4), pseudoephedrine
(UV 257) (6.2), quinidine (UV 330,240,212) (13.8), ranitidine (UV 315,227) (4.6), salicylic acid (UV 300,234) (6.5), secobarbital (UV 211) (16.0), sertraline (UV 273) (23.2),
spironolactone (UV 240) (24.5), sulfamethoxazole (UV 269.5,211.5) (8.0), sulpiride (UV
293,213) (4.0), temazepam (UV 231.5,312) (19.5), terfenadine (UV 260,219) (27.4), theophylline (UV 270) (2.5), thiopentone (UV 288,238) (18.7), thioridazine (UV 314,265)
(24.5), thiothixene (UV 308,259,231) (22.0), tocainide (UV 276) (8.8), tolbutamide (UV
228) (18.9), trazodone (UV 250) (14.9), triazolam (UV 223) (19.3), triflupromazine (UV
496
Phenazopyridine hydrochloride
308,257) (25.8), trimeprazine (UV 250) (26.9), trimethoprim (UV 272) (10.9), trimipramine (UV 250,211) (23.9), verapamil (UV 280,230) (21.6), zopiclone (UV 305,218)
(12.3), zuclopenthixol (UV 325,267,227) (22.0)
Interfering: alprazolam (UV 225) (17.5), brompheniramine (UV 227, UV 263) (17.2),
chlorpheniramine (UV 263, UV 223) (17.3), clozapine (UV 211, UV 290) (17.0), fenfluramine (UV 263) (17.2), flunitrazepam (UV 220, UV 253, UV 313) (17.5), mesoridazine
(UV 220, UV 273) (17.2), oxazepam (UV 229, UV 310) (17.5), oxazolam (UV 234) (17.0),
pentazocine (UV 280) (16.7), phenolphthalein (UV 231, UV 278) (16.8), propranolol
(UV215, UV 230, UV 290) (17.2), propyphenazone (UV 249, UV 274) (17.0), risperidone
(UV 236, UV 276) (16.7)
KEY WORDS
serum; SPE
REFERENCE
Lai, C.-K.; Lee, T.; Au, K.-M.; Chan, A.Y.-W. Uniform solid-phase extraction procedure for toxicological
drug screening in serum and urine by HPLC with photodiode-array detection, Clin.Chem., 1997, 43,
312–325.
Phentermine
Phentermine
497
NH2
H3C CH3
Molecular formula: C10 H15 N
Molecular weight: 149.23
CAS Registry No: 122-09-8, 1197-21-3 (HCl)
Merck Index: 13, 7346
SAMPLE
Matrix: blood
Sample preparation: Condition a 200 mg Bond Elut C18 SPE cartridge with 1 mL
MeOH, 1 mL water, and 2 mL 10 mM pH 9.3 ammonium carbonate buffer. Centrifuge
serum, whole blood, or urine at 14 000 g for 5 min. Vortex 0.2–1 mL aliquots of the
supernatant with 2 mL 10 mM pH 9.3 ammonium carbonate buffer and IS, centrifuge
at 5000 g for 10 min, add 2 mL of the supernatant to the SPE cartridge, wash with 2 mL
10 mM pH 9.3 ammonium carbonate buffer, dry SPE cartridge under vacuum for 5 min,
elute with 500 µL MeOH:500 mM acetic acid 90:10. Add 10 µL 1 mM HCl to the eluate,
evaporate to dryness under a stream of nitrogen, reconstitute the residue with 100 µL
mobile phase, centrifuge at 14 000 g for 4 min, inject a 5–20 µL aliquot.
HPLC VARIABLES
Column: 125 × 3 Superspher RP 18
Mobile phase: MeCN:50 mM pH 3.0 ammonium formate buffer 25:75
Flow rate: 0.3
Injection volume: 5–20
Detector: MS, Finnigan MAT SSQ 7000, APCI, positive ion mode, sheath gas nitrogen
70 psi, auxiliary gas nitrogen 20 mL/min, capillary 190◦ , vaporizer 450◦ , corona current
5 µA
CHROMATOGRAM
Retention time: 4.3
Internal standard: d10 -methamphetamine
Limit of detection: 1 ng/mL
OTHER SUBSTANCES
Extracted: amphetamine (3.4), cathinone (2.6), ephedrine (3.1), fenfluramine (7.0), MDA
(3.5), MDEA (5.2), MDMA (4.2), methamphetamine (4.1), norfenfluramine (4.2), phenylpropanolamine (2.5)
KEY WORDS
serum; SPE; urine; whole blood
REFERENCE
Bogusz, M.J. Liquid chromatography-mass spectrometry as a routine method in forensic sciences: a proof
of maturity, J.Chromatogr.B, 2000, 748, 3–19.
SAMPLE
Matrix: blood
Sample preparation: Mix 100 µL plasma with 10 µL 2 µM IS in water, add 200 µL
100 mM pH 10.6 borate buffer, add 750 µL ethyl acetate, vortex for 1 min, centrifuge at
1700 g at 4◦ for 10 min. Add 10 µL acetic acid to the organic layer, evaporate to dryness
under reduced pressure at 45◦ over 15 min. Vortex the residue with 75 µL 4.7 mM
dansyl chloride in MeCN and 25 µL 20 mM pH 9.0 carbonate buffer, heat at 45◦ for
30 min, add 3 µL MeCN:methylamine 80:20, let stand for 10 min, add 4 µL 3 M HCl,
inject a 20 µL aliquot.
498
Phentermine
HPLC VARIABLES
Column: 250 × 4.6 5 µm Diasopak SP-120-5-ODS (Daiso, Osaka)
Mobile phase: Gradient. A was MeOH:100 mM acetic acid 60:40. B was MeCN. A:B
80:20 for 20 min, to 45:55 over 2 min, maintain at 45:55 for 17 min, return to initial
conditions.
Flow rate: 1
Injection volume: 20
Detector: F ex 325 em 530
CHROMATOGRAM
Retention time: 31
Internal standard: fluoxetine (45)
Limit of quantitation: 5 nM
OTHER SUBSTANCES
Extracted: 4-bromo-2,5-dimethoxyphenylethylamine (30), epinephrine (21), fenfluramine
(39), norepinephrine (16), 2-phenylethylamine (25)
KEY WORDS
derivatization; human; pharmacokinetics; plasma; rat
REFERENCE
Kaddoumi, A.; Nakashima, M.N.; Wada, M.; Kuroda, N.; Nakahara, Y.; Nakashima, K. HPLC of (±)fenfluramine and phentermine in plasma after derivatization with dansyl chloride, J.Liq.Chromatogr.
Rel.Technol., 2001, 24, 57–67.
SAMPLE
Matrix: blood, urine
Sample preparation: Condition a 3 mL 130 mg Bond Elut Certify SPE cartridge with
3 mL MeOH and 3 mL 200 mM pH 6.0 phosphate buffer. Vortex 3 mL serum or urine
with 3 mL 400 mM pH 6.0 phosphate buffer for 1 min, add to the SPE cartridge at
1 mL/min, wash with 3 mL MeOH:200 mM pH 6.0 phosphate buffer 5:95, pass air
through the SPE cartridge for 10 s, elute with 750 µL MeOH:10% ammonia 100:20 at
0.5 mL/min. Add a few drops of 1 M HCl to the eluate and evaporate to dryness under a
stream of nitrogen, reconstitute the residue with 150 µL mobile phase, inject an aliquot.
HPLC VARIABLES
Guard column: 10 × 2.1 5 µm Hypersil C18
Column: 150 × 4.6 5 µm Hypersil C18
Column temperature: 45
Mobile phase: Gradient. A was MeCN:buffer 5:95. B was MeCN:buffer 50:50. A:B from
85:15 to 10:90 over 20 min, maintain at 10:90 for 5 min, return to initial conditions
over 3 min, re-equilibrate for 6 min. (The buffer was 50 mM pH 3.0 phosphate buffer
containing 375 mg/L sodium octyl sulfate and 3 mL/L triethylamine.)
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Retention time: 12.5
OTHER SUBSTANCES
Extracted: acetaminophen (UV 247) (2.7), N-acetylprocainamide (UV 268) (4.9), alprazolam (UV 225) (17.5), amineptine (UV 268) (19.3), amitriptyline (UV 240,211) (22.3),
amobarbital (UV 211) (15.3), amoxapine (UV 296,211.5) (19.1), amphetamine (UV
254) (11.2), aprobarbital (14.7), atenolol (UV 275,230) (4.3), atropine (UV 260) (11.3),
Phentermine
499
barbital (UV 212) (5.5), benzhexol (UV 260) (22.5), benztropine (UV 257,223) (22.9),
benzoylecgonine (UV 278,235) (6.7), bromazepam (UV 235) (13.3), brompheniramine
(UV 263,227) (17.2), buprenorphine (UV 284,213) (19.4), buspirone (UV 302,239,211)
(16.2), butabarbital (UV 211) (10.1), caffeine (UV 270) (3.5), carbamazepine (UV 283,215)
(15.1), chloramphenicol (UV 277) (10.6), chlordiazepoxide (UV 303,245) (15.1), chlorpheniramine (UV 263,223) (17.3), chlorpromazine (UV 310,255) (23.9), cimetidine (UV
219) (3.5), citalopram (UV 239) (19.0), clobazam (UV 292,231) (20.5), clomipramine (UV
250,211) (23.6), clonazepam (UV 310,240,211) (18.5), clonidine (UV 265) (6.9), cloperastine (UV 238) (26.2), cloxazolam (UV 340,265,239.5) (18.4), clozapine (UV 290,211)
(17.0), cocaine (UV 278,235) (14.7), codeine (UV 280,211) (5.0), desipramine (UV 250)
(20.5), diazepam (UV 313,229) (22.6), dihydrocodeine (UV 282,211) (4.8), diltiazem (UV
242,211.5) (19.2), diphenhydramine (UV 258) (19.4), disopyramide (UV 260) (14.9), dothiepin (UV 303,263,231,211) (21.5), doxepin (UV 291) (19.1), ephedrine (UV 257) (6.2),
erythromycin (UV 211) (23.3), fenfluramine (UV 263) (17.2), fentanyl (UV 262) (18.4),
flecainide (UV 299) (19.5), flumazenil (UV 250) (11.0), flunitrazepam (UV 313,253,220)
(17.5), fluoxetine (UV 262,228) (22.6), fluphenazine (UV 310,257) (23.7), flupenthixol (UV
268,231) (27.2), flurazepam (UV 315,231) (20.6), fluvoxamine (UV 251) (20.8), furosemide
(UV 344,275,235) (13.2), glutethimide (15.8), guaifenesin (UV 275,224) (6.7), haloperidol
(UV 245,220) (19.6), hydralazine (UV 304,260,211) (3.5), hydroxyzine (UV 231) (21.2),
ibuprofen (UV 263,219.5) (28.0), imipramine (UV 250,211) (21.6), indomethacin (UV
299,240) (27.9), ketoconazole (UV 295,240) (20.8), labetalol (UV 303) (15.7), lidocaine
(UV 266) (13.7), lorazepam (UV 318,231.5) (18.4), lormetazepam (UV 315,231) (20.5),
loxapine (UV 295,250) (19.8), maprotiline (UV 272) (22.0), mesoridazine (UV 273,220)
(17.2), methadone (23.0), methaqualone (UV 308,267,227) (18.1), methylephedrine (UV
254) (7.7), methylphenidate (UV 260) (15.0), metoprolol (UV 275,223) (13.8), mianserin
(UV 280) (19.3), midazolam (UV 221) (19.3), moclobemide (UV 238) (14.1), molindone
(UV 301,256,211) (13.6), morphine (UV 286,212) (2.7), nadolol (UV 272,219) (6.6), naloxone (UV 282,209) (5.0), naproxen (UV 270,231.5) (20.2), nifedipine (UV 335,235) (20.8),
nitrazepam (UV 310,260,219) (16.3), nordiazepam (UV 310,231) (19.7), norfluoxetine
(UV 262,228) (21.4), nortriptyline (UV 240) (21.0), ofloxacin (UV 327,295.5,230) (9.5),
oxazepam (UV 310,229) (17.6), oxazolam (UV 234) (17.0), oxycodone (UV 280) (9.9), oxymorphone (UV 280) (6.7), paroxetine (UV 295,235) (19.8), pentazocine (UV 280) (16.7),
pentobarbital (UV 211) (14.9), pericyazine (UV 269,232) (19.6), perphenazine (UV
320,255,210) (22.2), pethidine (15.5), phenazopyridine (UV 240) (17.1), phencyclidine
(UV 265) (17.8), phenobarbital (9.9), phenolphthalein (UV 278,231) (16.8), phentermine
(UV 254) (12.5), phenylephrine (UV 302,259,219) (9.2), phenylpropanolamine (UV 258)
(4.9), phenyltoloxamine (UV 272) (20.6), phenytoin (15.3), pholcodine (UV 284,211.5)
(4.0), pimozide (UV 280) (25.3), pinazepam (UV 310,227.5) (25.5), piroxicam (UV 248)
(15.9), prazepam (UV 311,231.5) (25.1), primidone (6.2), procainamide (UV 280,210)
(3.6), promethazine (UV 297,250) (21.7), propoxyphene (UV 259) (21.8), propranolol (UV
290,230,215) (17.2), propyphenazone (UV 274,249) (17.0), protriptyline (UV 290,211)
(21.4), pseudoephedrine (UV 257) (6.2), quinidine (UV 330,240,212) (13.8), ranitidine
(UV 315,227) (4.6), risperidone (UV 276,236) (16.7), salicylic acid (UV 300,234) (6.5),
secobarbital (UV 211) (16.0), sertraline (UV 273) (23.2), spironolactone (UV 240) (24.5),
sulfamethoxazole (UV 269.5,211.5) (8.0), sulpiride (UV 293,213) (4.0), temazepam (UV
231.5,312) (19.5), terfenadine (UV 260,219) (27.4), theophylline (UV 270) (2.5), thiopentone (UV 288,238) (18.7), thioridazine (UV 314,265) (24.5), thiothixene (UV 308,259,231)
(22.0), tocainide (UV 276) (8.8), tolbutamide (UV 228) (18.9), trazodone (UV 250) (14.9),
triazolam (UV 223) (19.3), triflupromazine (UV 308,257) (25.8), trimeprazine (UV 250)
(26.9), trimethoprim (UV 272) (10.9), trimipramine (UV 250,211) (23.9), verapamil (UV
280,230) (21.6), zuclopenthixol (UV 325,267,227) (22.0)
Interfering: acebutolol (UV 235, UV 326) (12.8), chlormethiazole (UV 211, UV 252)
(12.4), ketamine (UV 270) (12.7), MDA (UV 235, UV 287) (12.4), MDMA (UV 235, UV
287) (13.0), methamphetamine (12.0), metoclopramide (UV 215, UV 275, UV 311) (13.0),
pheniramine (UV 260) (13.0), zopiclone (UV 218, UV 305) (12.3),
KEY WORDS
serum; SPE
500
Phentermine
REFERENCE
Lai, C.-K.; Lee, T.; Au, K.-M.; Chan, A.Y.-W. Uniform solid-phase extraction procedure for toxicological
drug screening in serum and urine by HPLC with photodiode-array detection, Clin.Chem., 1997, 43,
312–325.
ANNOTATED BIBLIOGRAPHY
Bogusz, M.J.; Krüger, K.-D.; Maier, R.-D. Analysis of underivatized amphetamines and related phenethylamines with high-performance liquid chromatography-atmospheric pressure chemical ionization mass
spectrometry, J.Anal.Toxicol., 2000, 24, 77–84. [SPE; LOD 1–5 ng/mL; amphetamine; fenfluramine;
methamphetamine; phentermine; cathinone; propylhexedrine; ephedrine; phenylpropanolamine; norfenfluramine; phenylethylamine]
Kaddoumi, A.; Kubota, A.; Nakashima, M.N.; Takahashi, M.; Nakashima, K. High performance liquid chromatography with UV detection for the simultaneous determination of sympathomimetic
amines using 4-(4,5-diphenyl-1H-imidazole-2-yl)benzoyl chloride as a label, Biomed.Chromatogr.,
2001, 15, 379–388. [human; rat; plasma; derivatization; LOD 50–100 nM; ephedrine; norephedrine;
2-phenylethylamine; fenfluramine; phentermine; cyclohexylamine; fluoxetine is internal standard;
pharmacokinetics]
Kaddoumi, A.; Nakashima, M.N.; Nakashima, K. Fluorometric determination of DL-fenfluramine, DLnorfenfluramine and phentermine in plasma by achiral and chiral high-performance liquid chromatography, J.Chromatogr.B, 2001, 763, 79–90. [fluorescence detection; derivatization; human; rat; LOD
10 nM; ephedrine; norephedrine]
Kaddoumi, A.; Nakashima, M.N.; Maki, T.; Matsumura, Y.; Nakamura, J.; Nakashima, K. Liquid chromatography studies on the pharmacokinetics of phentermine and fenfluramine in brain and blood
microdialysates after intraperitoneal administration to rats, J.Chromatogr.B, 2003, 791, 291–303.
[fluorescence detection; derivatization; LOQ 5 nM; fluoxetine is internal standard]
Unterhalt, B.; Wenning, C. Zur Trennung von Oxetacain und seinen Metaboliten [Separation of oxetacaine and its metabolites], Pharmazie, 2001, 56, 58–60. [oxetacaine; mephentermine; phentermine]
Phosphatidylcholine
Phosphatidylcholine
CAS Registry No: 8002-43-5
Merck Index: 13, 5447
501
O
O
R
O
R
O
O
O−
O
P
N+(CH3)3
O
R = fatty acid
SAMPLE
Matrix: blood
Sample preparation: Shake 2 mL blood, 4 mL chloroform (Caution! Chloroform is
a carcinogen!), and 2 mL MeOH vigorously for 2 min, cool in ice, centrifuge at 800 g.
Evaporate the lower chloroform layer to dryness under a stream of nitrogen, reconstitute
the residue with chloroform:MeOH 2:1, inject an aliquot.
HPLC VARIABLES
Guard column: 60 × 4.6 5 µm SI 60 (Merck)
Column: 250 × 4.6 5 µm Lichrosorb Diol
Column temperature: 55
Mobile phase: Gradient. A was MeCN. B was MeCN:water 80:20. A:B 88:12 for 5 min,
to 77:23 over 3 min, to 30:70 over 4 min, to 25:75 over 1 min, maintain at 25:75 for
5 min, return to initial conditions over 1 min.
Flow rate: 2.5
Detector: UV 201
CHROMATOGRAM
Retention time: 14
OTHER SUBSTANCES
Extracted: phosphatidylethanolamine (13), sphingomyelin (16)
KEY WORDS
whole blood
REFERENCE
Heinze, T.; Kynast, G.; Dudenhausen, J.W.; Saling, E. Effect of blood and meconium on the determination
of phospholipids in amniotic fluid using high pressure liquid chromatography, J.Perinat.Med., 1988,
16, 53–60.
SAMPLE
Matrix: blood
Sample preparation: Sonicate 300 µL whole blood, 600 µL water, and 5 mL MeOH
containing 0.01% BHT for 1 min, add 10 mL chloroform (Caution! Chloroform is a
carcinogen!), sonicate for 1 min, vortex for 30 s, let stand at room temperature for 1 h,
add 5 mL water, mix for 5 s, centrifuge at 2600 g for 10 min. Evaporate the lower
organic layer to dryness under a stream of nitrogen, reconstitute the residue with
300 µL chloroform:MeOH 95:5, inject a 10 µL aliquot.
HPLC VARIABLES
Guard column: 10 × 2 5 µm LiChrospher 100 diol
Column: 250 × 2 5 µm LiChrospher 100 diol
Column temperature: 30
Mobile phase: Gradient. A was chloroform. B was MeOH:formic acid 99.9:0.1 with
ammonia added to pH 5.3 (ca. 0.05% ammonia) and 0.05% triethylamine. A:B from 95:5
502
Phosphatidylcholine
to 70:30 over 11 min, to 20:80 over 3 min, maintain at 20:80 for 4 min, return to initial
conditions over 2 min, re-equilibrate for 10 min.
Flow rate: 0.3
Injection volume: 10
Detector: MS, Finnigan LCQ, electrospray, ionization needle 4.5 kV, capillary 230◦ ,
sheath gas flow 90 units
CHROMATOGRAM
Retention time: 7.07 (m/z 878.5; C18:0, C22:6), 7.28 (m/z 826.5; C16:0, C20:4), 7.84 (m/z
778.5; C16:0, C16:0)
Limit of detection: 0.1–5 ng
OTHER SUBSTANCES
Extracted: phosphatidylethanolamine (10), phosphatidylglycerol (6), phosphatidylinositol (13), phosphatidylserine (17)
KEY WORDS
whole blood
REFERENCE
Uran, S.; Larsen, A.; Jacobsen, P.B.; Skotland, T. Analysis of phospholipid species in human blood using
normal-phase liquid chromatography coupled with electrospray ionization ion-trap tandem mass
spectrometry, J.Chromatogr.B, 2001, 758, 265–275.
SAMPLE
Matrix: lung lavage fluid
Sample preparation: Centrifuge lung lavage fluid at 4◦ at 450 g for 10 min. Shake
10 mL supernatant and 40 mL chloroform:MeOH 2:1 at 4◦ for 3 min (Caution! Chloroform is a carcinogen!). Remove the lower organic phase and wash it with 2 mL 50 mM
NaCl, centrifuge, dry under a stream of nitrogen at 45◦ , reconstitute with 500 µL mobile
phase, vortex at 4◦ for 1 min, inject a 100 µL aliquot.
HPLC VARIABLES
Guard column: 20 × 2.1 5 µm Encapharm 100 spherical silica gel (Molnar, Berlin)
Column: 120 × 4.6 5 µm Encapharm 100 spherical silica gel (Molnar, Berlin)
Column temperature: 30
Mobile phase: Gradient. A was chloroform:MeOH:ammonium hydroxide 80:19.5:0.5.
B was chloroform:MeOH:water:ammonium hydroxide 60:34:5.5:0.5. A:B from 100:0 to
0:100 over 14 min, return to initial conditions over 7 min, re-equilibrate for 10 min.
Flow rate: 1
Injection volume: 100
Detector: Evaporative Light-Scattering Detector, SEDERE Sedex-45, evaporation temperature 50◦ , nebulization gas nitrogen, pressure 200 kPa, flow 6 L/min, response is
nonlinear but proportional to the power 1.7 of the mass and must be calibrated for each
compound
CHROMATOGRAM
Retention time: 13.11
Limit of detection: 100 ng
OTHER SUBSTANCES
Extracted: diarachidoylphosphatidylcholine
(12.75),
dilinoleylphosphatidylcholine
(12.69), diphosphatidylglycerol (6.30), lysophosphatidylcholine (18.46, 18.81), phosphatidic acid (13.78), phosphatidylethanolamine (9.00), phosphatidylglycerol (5.37),
phosphatidylinositol (10.75), phosphatidylserine (11.81), sphingomyelin (15.24, 15.72)
Phosphatidylcholine
503
REFERENCE
Bünger, H.; Pison, U. Quantitative analysis of pulmonary surfactant phospholipids by high-performance
liquid chromatography and light-scattering detection, J.Chromatogr.B, 1995, 672, 25–31.
ANNOTATED BIBLIOGRAPHY
Wada, M.; Nakashima, K.; Kuroda, N.; Akiyama, S.; Imai, K. Sensitive flow-injection method with
peroxylate chemiluminescence detection combined with preparative high-performance liquid chromatography for determination of choline-containing phospholipids in human serum, J.Chromatogr.B,
1996, 678, 129–136.
504
Phosphatidylglycerol
Phosphatidylglycerol
Merck Index: 13, 8030
SAMPLE
Matrix: blood
Sample preparation: Sonicate 300 µL whole blood, 600 µL water, and 5 mL MeOH
containing 0.01% BHT for 1 min, add 10 mL chloroform (Caution! Chloroform is a
carcinogen!), sonicate for 1 min, vortex for 30 s, let stand at room temperature for 1 h,
add 5 mL water, mix for 5 s, centrifuge at 2600 g for 10 min. Evaporate the lower
organic layer to dryness under a stream of nitrogen, reconstitute the residue with
300 µL chloroform:MeOH 95:5, inject a 10 µL aliquot.
HPLC VARIABLES
Guard column: 10 × 2 5 µm LiChrospher 100 diol
Column: 250 × 2 5 µm LiChrospher 100 diol
Column temperature: 30
Mobile phase: Gradient. A was chloroform (Caution! Chloroform is a carcinogen!). B
was MeOH:formic acid 99.9:0.1 with ammonia added to pH 5.3 (ca. 0.05% ammonia) and
0.05% triethylamine. A:B from 95:5 to 70:30 over 11 min, to 20:80 over 3 min, maintain
at 20:80 for 4 min, return to initial conditions over 2 min, re-equilibrate for 10 min.
Flow rate: 0.3
Injection volume: 10
Detector: MS, Finnigan LCQ, electrospray, ionization needle 4.5 kV, capillary 230◦ ,
sheath gas flow 90 units
CHROMATOGRAM
Retention time: 6 (m/z 721.5; C16:0, C16:0), 7.28 (m/z 749.5; C16:0, C18:0), 7.84 (m/z
777.5; C18:0, C18:0)
Limit of detection: 0.1–5 ng
OTHER SUBSTANCES
Extracted: phosphatidylcholine (7.5), phosphatidylethanolamine (10), phosphatidylinositol (13), phosphatidylserine (17)
KEY WORDS
whole blood
REFERENCE
Uran, S.; Larsen, A.; Jacobsen, P.B.; Skotland, T. Analysis of phospholipid species in human blood using
normal-phase liquid chromatography coupled with electrospray ionization ion-trap tandem mass
spectrometry, J.Chromatogr.B, 2001, 758, 265–275.
SAMPLE
Matrix: formulations
Sample preparation: Dilute liposome dispersions 10-fold with chloroform:MeOH 60:40,
centrifuge at 2700 g for 15 min, inject an aliquot of the supernatant. (Caution! Chloroform is a carcinogen!)
HPLC VARIABLES
Column: 250 × 4.6 5 µm Zorbax amino
Mobile phase: MeCN:MeOH:buffer 64:28:8 (The buffer was 10 mM phosphoric acid
adjusted to pH 4.8 with dilute ammonium hydroxide solution. To prepare mobile phase,
mix MeCN and MeOH and then add buffer.)
Flow rate: 1.5
Phosphatidylglycerol
505
Injection volume: 5–20
Detector: Refractive Index
CHROMATOGRAM
Retention time: 6
Limit of detection: 29 µg/mL
OTHER SUBSTANCES
Simultaneous: acyl lysophosphatidylcholine, acyl lysophosphatidylglycerol, phosphatidylcholine (5)
KEY WORDS
liposome dispersions
REFERENCE
Grit, M.; Crommelin, D.J.A.; Lang, J. Determination of phosphatidylcholine, phosphatidylglycerol and
their lyso forms from liposome dispersions by high-performance liquid chromatography using highsensitivity refractive index detection, J.Chromatogr., 1991, 585, 239–246.
SAMPLE
Matrix: lung lavage fluid
Sample preparation: Centrifuge lung lavage fluid at 4◦ at 450 g for 10 min. Shake
10 mL supernatant and 40 mL chloroform:MeOH 2:1 at 4◦ for 3 min (Caution! Chloroform is a carcinogen!). Remove the lower organic phase and wash it with 2 mL 50 mM
NaCl, centrifuge, dry under a stream of nitrogen at 45◦ , reconstitute with 500 µL mobile
phase, vortex at 4◦ for 1 min, inject a 100 µL aliquot.
HPLC VARIABLES
Guard column: 20 × 2.1 5 µm Encapharm 100 spherical silica gel (Molnar, Berlin)
Column: 120 × 4.6 5 µm Encapharm 100 spherical silica gel (Molnar, Berlin)
Column temperature: 30
Mobile phase: Gradient. A was chloroform:MeOH:ammonium hydroxide 80:19.5:0.5.
B was chloroform:MeOH:water:ammonium hydroxide 60:34:5.5:0.5. A:B from 100:0 to
0:100 over 14 min, return to initial conditions over 7 min, re-equilibrate for 10 min.
Flow rate: 1
Injection volume: 100
Detector: Evaporative Light-Scattering Detector, SEDERE Sedex-45, evaporation temperature 50◦ , nebulization gas nitrogen, pressure 200 kPa, flow 6 L/min, response is
nonlinear but proportional to the power 1.7 of the mass and must be calibrated for each
compound
CHROMATOGRAM
Retention time: 5.37
Limit of detection: 100 ng
OTHER SUBSTANCES
Extracted: diarachidoylphosphatidylcholine
(12.75),
dilinoleylphosphatidylcholine
(12.69), diphosphatidylglycerol (6.30), lysophosphatidylcholine (18.46, 18.81), phosphatidic acid (13.78), phosphatidylcholine (13.11), phosphatidylethanolamine (9.00),
phosphatidylinositol (10.75), phosphatidylserine (11.81), sphingomyelin (15.24, 15.72)
REFERENCE
Bünger, H.; Pison, U. Quantitative analysis of pulmonary surfactant phospholipids by high-performance
liquid chromatography and light-scattering detection, J.Chromatogr.B, 1995, 672, 25–31.
506
Phosphatidylglycerol
SAMPLE
Matrix: tissue
Sample preparation: Homogenize (Potter-Elvehjem) liver or lung with 20 volumes
chloroform:MeOH 2:1, filter (paper), wash with a volume of 50 mM NaCl equal to onefifth the volume of extract, centrifuge (J.Biol.Chem. 1957, 226, 497). Remove the lower
organic layer and evaporate it to dryness under a stream of nitrogen, reconstitute with
chloroform:MeOH 25:75, inject an aliquot. (Caution! Chloroform is a carcinogen!)
HPLC VARIABLES
Guard column: 10 × 4.6 5 µm Nucleosil 5 NH2
Column: 50 × 4.6 5 µm Nucleosil 5 NH2 + 175 × 4.6 5 µm Nucleosil 5 NH2
Column temperature: 50
Mobile phase: MeCN:MeOH:water:50% methylphosphonic acid in water 73:25:1.5:0.03,
adjusted to pH 3 with 25% ammonium hydroxide in water
Flow rate: 1
Detector: F ex 340 em 460 following post-column derivatization. The column effluent
mixed with the reagent pumped at 4.5 mL/min and the mixture flowed through a 2 m
×0.8 mm ID coil of PTFE tubing at 50◦ to the detector. (The reagent was 1 L water
to which was added 250 µL Brij 35 (30% w/v), 50 mg sodium azide, and 150 µL 3 mM
diphenylhexatriene in MeCN.)
CHROMATOGRAM
Retention time: 22
OTHER SUBSTANCES
Extracted: dipalmitolyphosphatidylcholine
KEY WORDS
gastric mucosa; liver; lung; pig; post-column reaction
REFERENCE
Bernhard, W.; Linck, M.; Creutzburg, H.; Postle, A.D.; Arning, A.; Martin-Carrera, I.; Sewing, K.-F.
High-performance liquid chromatographic analysis of phospholipids from different sources with combined fluorescence and ultraviolet detection, Anal.Biochem., 1994, 220, 172–180.
ANNOTATED BIBLIOGRAPHY
Alvarez, J.G.; Touchstone, J.C. Separation of acidic and neutral lipids by aminopropyl-bonded silica gel
column chromatography, J.Chromatogr., 1992, 577, 142–145.
Bonanno, L.M.; Denizot, B.A.; Tchoreloff, P.C.; Puisieux, F.; Cardot, P.J. Determination of phospholipids
from pulmonary surfactant using an on-line coupled silica/reversed-phase high-performance liquid
chromatography system, Anal.Chem., 1992, 64, 371–379.
Dethloff, L.A.; Gilmore, L.B.; Hook, G.E. Separation of pulmonary surfactant phospholipids by highperformance liquid chromatography, J.Chromatogr., 1986, 382, 79–87.
Ishizuka, T.; Ishikawa, K.; Maseki, M.; Tomoda, Y.; Tsuda, T. Determination of phosphatidylglycerol in
amniotic fluid for prediction of foetal lung maturity by microbore-column liquid chromatography,
J.Chromatogr., 1986, 380, 43–53.
Jaaskelainen, I.; Urtti, A. Liquid chromatography determination of liposome components using a lightscattering evaporative detector, J.Pharm.Biomed.Anal., 1994, 12, 977–982.
Jungalwala, F.B.; Evans, J.E.; McCluer, R.H. Compositional and molecular species analysis of phospholipids by high performance liquid chromatography coupled with chemical ionization mass spectrometry,
J.Lipid Res., 1984, 25, 738–749.
Kynast, G.; Schmitz, C. Simplified quantitative determination of phospholipids in amniotic fluid, alveolar
lavages and milk using high performance liquid chromatography (HPLC), J.Perinat.Med., 1989, 17,
203–212.
Nissen, H.P.; Kreysel, H.W. Analysis of phospholipids in human semen by high-performance liquid
chromatography, J.Chromatogr., 1983, 276, 29–35.
Phosphatidylglycerol
507
Pison, U.; Gono, E.; Joka, T.; Obertacke, U.; Obladen, M. High-performance liquid chromatography of
adult human bronchoalveolar lavage: assay for phospholipid lung profile, J.Chromatogr., 1986, 377,
79–89.
Sas, B.; Peys, E.; Helsen, M. Efficient method for (lyso)phospholipid class separation by high-performance
liquid chromatography using an evaporative light-scattering detector, J.Chromatogr.A, 1999, 864,
179–182.
Scarim, J.; Ghanbari, H.; Taylor, V.; Menon, G. Determination of phosphatidylcholine and disaturated
phosphatidylcholine content in lung surfactant by high performance liquid chromatography, J.Lipid
Res., 1989, 30, 607–611.
Smith, L.A.; Norman, H.A.; Cho, S.H.; Thompson, G.A. Jr. Isolation and quantitative analysis of phosphatidylglycerol and glycolipid molecular species using reversed-phase high-performance liquid
chromatography with flame ionization detection, J.Chromatogr., 1985, 346, 291–299.
508
Piketoprofen
Piketoprofen
O
Molecular formula: C22 H20 N2 O2
Molecular weight: 344.41
CAS Registry No: 60576-13-8
Merck Index: 13, 7503
CH3
H
N
N
O
CH3
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 × 4.6 (S,S)-Whelk-O (Regis)
Mobile phase: Hexane:isopropanol:acetic acid 70:30:0.5
Flow rate: 1
Injection volume: 5
Detector: UV 270
CHROMATOGRAM
Retention time: k′ 2.97 (α 1.17)
KEY WORDS
chiral
REFERENCE
Baeyens, W.R.G.; Van Der Weken, G.; Aboul-Enein, H.Y.; Reygaerts, S.; Smet, E. Comparison of the
enantiomeric separation of some 2-arylpropionic acids on a novel Pirkle-concept stationary packing by
narrow-bore and conventional liquid chromatography, Biomed.Chromatogr., 2000, 14, 58–60.
SAMPLE
Matrix: bulk
Sample preparation: Prepare a 1.0 mg/mL solution. Inject an aliquot.
HPLC VARIABLES
Column: 150 × 4.6 5 µm Chiralcel OJ-R
Mobile phase: MeCN:buffer 35:65 (A) or MeCN:MeOH:buffer 35:35:30 (B) (Prepare
the buffer by dissolving 70.25 g sodium perchlorate in water, adjust to pH 2.0 with
concentrated perchloric acid, make up to 1 L with water.)
Flow rate: 0.5
Detector: UV 230; UV 254
CHROMATOGRAM
Retention time: k′ 3.53 (mobile phase A, α = 1.29), k′ 0.61 (mobile phase B, α = 1.17)
KEY WORDS
chiral
REFERENCE
Van Overbeke, A.; Baeyens, W.; Oda, H.; Aboul-Enein, H.Y. Direct enantiomeric HPLC separation of
several 2-arylpropionic acids, barbituric acids and benzodiazepines on Chiralcel OJ-R chiral stationary
phase, Chromatographia, 1996, 43, 599–606.
509
Pilsicainide
Pilsicainide
Molecular formula: C17 H24 N2 O
Molecular weight: 272.38
CAS Registry No: 88069-67-4, 88069-49-2 (HCl hemihydrate)
Merck Index: 13, 7508
H
CH3
N
N
O
H3C
SAMPLE
Matrix: blood
Sample preparation: Mix 500 µL serum with 1 mL 100 mM NaOH, add 100 µL
10 µg/mL IS, add 5 mL chloroform (Caution! Chloroform is a carcinogen!), shake for
10 min, centrifuge at 2270 g for 5 min. Evaporate 4 mL of the organic layer to dryness
under a stream of nitrogen at 40◦ , reconstitute the residue with 50 µL MeCN, inject a
40 µL aliquot.
HPLC VARIABLES
Column: 100 × 5 10 µm Radial-pak 5CN
Mobile phase: MeCN:10 mM ammonium acetate 70:30
Flow rate: 2.5
Injection volume: 40
Detector: UV 210
CHROMATOGRAM
Internal standard: mexiletine
Limit of quantitation: 250 ng/mL
KEY WORDS
comparison with ELISA; rabbit; serum
REFERENCE
Saita, T.; Fujito, H.; Mori, M. ELISA for the quantification of pilsicainide, Biol.Pharm.Bull., 2001, 24,
1113–1116.
SAMPLE
Matrix: blood, urine
Sample preparation: Plasma. Shake 500 µL plasma, 200 µL 500 mM NaOH, and 2 mL
diethyl ether for 5 min, centrifuge at 3000 g for 5 min. Evaporate the organic layer to
dryness under a stream of nitrogen at 40◦ , reconstitute the residue with 200 µL mobile
phase, inject a 50 µL aliquot. Urine. Condition a Bond Elut CN SPE cartridge with
two 1 mL portions of water, 1 mL MeCN:200 mM sodium perchlorate 1:2, and three
1 mL portions of water. Heat 10 mL urine with 40 µL β-glucuronidase (8 units) at 37◦
for 16 h, add 1 mL to the SPE cartridge, centrifuge the SPE cartridge at 1000 g for
5 min, wash with 1 mL MeCN:water 1:2, centrifuge at 2000 g for 5 min, elute with 1 mL
MeCN:water 1:2, centrifuge at 2000 g for 5 min, inject a 100 µL aliquot of the eluate.
HPLC VARIABLES
Column: 150 × 4.6 STR ODS-II (Shimadzu)
Column temperature: 40
Mobile phase: MeCN:MeOH:25 mM pH 4.7 phosphate buffer 10:3:87
Flow rate: 1.5
Injection volume: 50–100
Detector: UV 210
510
Pilsicainide
CHROMATOGRAM
Limit of detection: 10 ng/mL
Limit of quantitation: 50 ng/mL (plasma), 1 µg/mL (urine)
OTHER SUBSTANCES
Noninterfering: cimetidine, probenecid
KEY WORDS
pharmacokinetics; plasma; SPE
REFERENCE
Shiga, T.; Hashiguchi, M.; Urae, A.; Kasanuki, H.; Rikihisa, T. Effect of cimetidine and probenecid on
pilsicainide renal clearance in humans, Clin.Pharmacol.Ther., 2000, 67, 222–228.
511
Pioglitazone
Pioglitazone
Molecular formula: C19 H20 N2 O3 S
N
O
O
S
N
H3C
Molecular weight: 356.45
CAS Registry No: 111025-46-8
Merck Index: 13, 7533
H
O
SAMPLE
Matrix: blood
Sample preparation: Mix 200 µL plasma with 100 µL 50 ng/mL IS in MeCN and 1 mL
100 mM pH 4 ammonium acetate buffer, add 4 mL MTBE:1-chlorobutane 50:50, shake
at high speed for 20 min, centrifuge at 4000 rpm for 20 min. Evaporate the organic
layer to dryness under a stream of nitrogen at 45◦ , reconstitute the residue with 100 µL
mobile phase, vortex for 1 min, centrifuge at 4000 rpm for 5 min, inject a 10 µL aliquot
of the supernatant.
HPLC VARIABLES
Column: 50 × 2 3 µm MetaChem Polaris C18-A
Mobile phase: MeCN:water 60:40 containing 10 mM ammonium acetate and 0.02%
trifluoroacetic acid
Flow rate: 0.2
Injection volume: 10
Detector: MS, Micromass Quattro LC, triple quadrupole, electrospray atmospheric pressure ionization, positive ion mode, capillary 3.5 kV, cone 35 V, extractor 3 V, desolvation
400◦ , ion source 80◦ , collision energy 35 eV, argon 1.9 mtorr, m/z 257–134
CHROMATOGRAM
Retention time: 1.37
Internal standard: (5-(4-(2-(5-(2-methyl-1,3-dioxolan-2-yl)-2-pyridyl)ethoxy)benzylidene)-2,4-thiazolidinedione (m/z 413–178) (1.26)
Limit of quantitation: 0.5 ng/mL (S/N 30)
OTHER SUBSTANCES
Extracted: metabolites
KEY WORDS
plasma
REFERENCE
Lin, Z.J.; Ji, W.; Desai-Krieger, D.; Shum, L. Simultaneous determination of pioglitazone and its two
active metabolites in human plasma by LC-MS/MS, J.Pharm.Biomed.Anal., 2003, 33, 101–108.
SAMPLE
Matrix: formulations
Sample preparation: Sonicate powdered tablet containing 500 mg metformin and
30 mg pioglitazone with 100 mL 100 mM HCl for 10 min with intermittent shaking, add
40 mL MeCN, sonicate for 25 min with intermittent shaking, cool to room temperature,
add 10 mL MeCN, make up to 200 mL with 100 mM HCl, mix well, centrifuge at 10
000 rpm for 10 min. Mix a 2 mL aliquot with 1 mL 250 µg/mL IS in MeCN:100 mM HCl
25:75, make up to 50 mL with diluent, inject an aliquot. (The diluent was MeCN:5 mM
disodium hydrogen phosphate 30:70, adjusted to pH 2.3 with HCl.)
HPLC VARIABLES
Column: 150 × 4.6 5 µm Zorbax XDB C18
512
Pioglitazone
Column temperature: 40
Mobile phase: MeCN:10 mM disodium hydrogen phosphate containing 5 mM sodium
dodecyl sulfate 34:66, adjusted to pH 7.1 with orthophosphoric acid
Flow rate: 1
Injection volume: 25
Detector: UV 226
CHROMATOGRAM
Retention time: 7.82
Internal standard: methylparaben (3.30)
Limit of detection: 3 ng/mL
Limit of quantitation: 10 ng/mL
OTHER SUBSTANCES
Extracted: metformin (LOD 22 ng/mL, LOQ 70 ng/mL) (5.06)
KEY WORDS
tablets; validation details
REFERENCE
Kolte, B.L.; Raut, B.B.; Deo, A.A.; Bagool, B.A.; Shinde, D.B. Simultaneous high-performance liquid
chromatographic determination of pioglitazone and metformin in pharmaceutical-dosage form, J.Chromatogr.Sci., 2004, 42, 27–31.
SAMPLE
Matrix: solutions
Sample preparation: Inject a 1 µL aliquot of a solution in MeOH:water 10:90.
HPLC VARIABLES
Column: 20 × 2 5 µm DASH BetaBasic C8 (ThermoHypersil Keystone)
Mobile phase: Gradient. A was MeCN:water:formic acid 5:95:0.1. B was MeCN:water:
formic acid 95:5:0.1. A:B 100:0 for 0.2 min, to 0:100 over 1.5 min.
Flow rate: 1.5
Injection volume: 1
Detector: MS, PE Sciex API-3000, TurboIonspray, electrospray 4500 V, ring 290 V,
orifice 60 V, drying gas 400◦ , 20% of column effluent entered the detector, m/z 357.2–134
CHROMATOGRAM
Retention time: 0.87
OTHER SUBSTANCES
Simultaneous: amitriptyline (m/z 278.3–233) (1.1), aprepitant (MK-869) (m/z
535.3–277) (1.4), diclofenac (m/z 296.1–215) (1.35), enoxacin (m/z 321.2–234) (0.7),
fenofibrate (m/z 360.9–233) (1.6), finasteride (m/z 373.2–317) (1.2), indinavir (m/z
614.4–421) (0.93), raloxifene (m/z 474.1–112) (0.97)
REFERENCE
Romanyshyn, L.A.; Tiller, P.R. Ultra-short columns and ballistic gradients: considerations for ultra-fast
chromatographic liquid chromatographic-tandem mass spectrometric analysis, J.Chromatogr.A, 2001,
928, 41–51.
ANNOTATED BIBLIOGRAPHY
Kiyota, Y.; Kondo, T.; Maeshiba, Y.; Hashimoto, A.; Yamashita, K.; Yoshimura, Y.; Motohashi, M.;
Tanayama, S. Studies on the metabolism of the new antidiabetic agent pioglitazone. Identification
of metabolites in rats and dogs, Arzneimittelforschung, 1997, 47, 22–28. [LC-MS]
Pioglitazone
513
Kolte, B.L.; Raut, B.B.; Deo, A.A.; Bagool, M.A.; Shinde, D.B. Liquid chromatographic method for the
determination of rosiglitazone in human plasma, J.Chromatogr.B, 2003, 788, 37–44. [pioglitazone is
internal standard]
Radhakrishna, T.; Rao, D.S.; Reddy, G.O. Determination of pioglitazone hydrochloride in bulk and pharmaceutical formulations by HPLC and MEKC methods, J.Pharm.Biomed.Anal., 2002, 29, 593–607.
[impurities]
Shen, Z.; Reed, J.R.; Creighton, M.; Liu, D.Q.; Tang, Y.S.; Hora, D.F.; Feeney, W.; Szewczyk, J.; Bakhtiar,
R.; Franklin, R.B.; Vincent, S.H. Identification of novel metabolites of pioglitazone in rat and dog,
Xenobiotica, 2003, 33, 499–509. [LC-MS; dog; rat; bile; plasma; urine]
Xue, Y.-J.; Turner, K.C.; Meeker, J.B.; Pursley, J.; Arnold, M.; Unger, S. Quantitative determination
of pioglitazone in human serum by direct-injection high-performance liquid chromatography mass
spectrometry and its application to a bioequivalence study, J.Chromatogr.B, 2003, 795, 215–226.
[LC-MS; column-switching; LOQ 9 ng/mL]
Yamashita, K.; Murakami, H.; Okuda, T.; Motohashi, M. High-performance liquid chromatographic
determination of pioglitazone and its metabolites in human serum and urine, J.Chromatogr.B, 1996,
677, 141–146. [SPE; LOD 10 ng/mL]
Zhong, W.Z.; Lakings, D.B. Determination of pioglitazone in dog serum using solid-phase extraction and
high-performance liquid chromatography with ultraviolet (229 nm) detection, J.Chromatogr., 1989,
490, 377–385.
Zhong, W.Z.; Williams, M.G. Simultaneous quantitation of pioglitazone and its metabolites in human
serum by liquid chromatography and solid phase extraction, J.Pharm.Biomed.Anal., 1996, 14,
465–473. [LOQ 20 ng/mL]
514
Pipercuronium bromide
Pipercuronium bromide
Molecular formula: C35 H62 Br2 N4 O4
Molecular weight: 762.71
CAS Registry No: 52212-02-9
Merck Index: 13, 7540
O
H3C
CH3 O
CH3
CH3
N
CH3
+
CH3
N
H
O
O
H
N
H
2Br−
N+
CH3
H
CH3
SAMPLE
Matrix: blood
Sample preparation: Condition a 1 mL Bond-Elut C18 SPE cartridge with 1 mL
MeOH:MeCN 2:1 and 1 mL water. Acidify 1 mL plasma with 150 µL 1 M sodium
dihydrogen phosphate. Add 20–200 ng IS to 1 mL acidified plasma, add to the SPE
cartridge, wash with 1 mL water, wash with 1 mL 100 mM pH 3 sodium dihydrogen
phosphate, elute with 400 µL mobile phase, discard first 100 µL eluate, inject a 200 µL
aliquot of the remaining eluate. (Although the original method was only validated
for vecuronium, it can also be used to determine pipecuronium; see: D’Honneur,G.;
Khalil,M.; Dominique,C.; Haberer,J.P.; Kleef,U.W.; Duvaldestin,P. Pharmacokinetics
and pharmacodynamics of pipecuronium in patients with cirrhosis. Anesth.Analg. 1993,
77, 1203–1206.)
HPLC VARIABLES
Column: 150 × 3.9 4 µm Nova-Pak C18
Mobile phase: Dioxane:water 20:80 containing 100 mM sodium dihydrogen phosphate
and 0.44 mM 9,10-dimethoxyanthracene-2-sulfonate, pH adjusted to 3 with phosphoric
acid. (Caution! Dioxane is a carcinogen!) (After each series of analyses, flush column
with 200 mL MeOH and then re-equilibrate with 120 mL mobile phase.)
Flow rate: 1
Injection volume: 200
Detector: F ex 380 em 452 following post-column extraction. The column effluent mixed
with dichloroethane pumped at 1.6 mL/min and the mixture flowed through a 1 m
×0.25 mm ID stainless steel coil to a phase separator (Anal.Chim.Acta 1987, 192, 267)
and then the organic phase flowed through the detector.
CHROMATOGRAM
Internal standard: 1-(3α,17β-diacetoxy-2β-piperidino-5α-androstan-16β,5α-yl)piperidine (16)
Limit of detection: 20 ng/mL
OTHER SUBSTANCES
Extracted: vecuronium (LOD 5 ng/mL) (13)
KEY WORDS
pharmacokinetics; SPE
REFERENCE
Paanakker, J.E.; Thio, J.M.S.L.; Van den Wildenberg, H.M.; Kaspersen, F.M. Assay of vecuronium in
plasma using solid-phase extraction, high-performance liquid chromatography and post-column ionpair extraction with fluorimetric detection, J.Chromatogr., 1987, 421, 327–335.
515
Pirlimycin
Pirlimycin
Molecular formula: C17 H31 ClN2 O5 S
Molecular weight: 410.96
CAS Registry No: 79548-73-5,
78822-40-9 (HCl)
Merck Index: 13, 7583
H
N
O
CH3
Cl
N
H OH O
OH
CH3
S
OH
CH3
SAMPLE
Matrix: milk
Sample preparation: Shake 3 mL extracting solution and 1 mL milk for 30 s, centrifuge at 1000 g for 4 min. Remove the supernatant and add 2 mL extracting solution
to the pellet. Shake for 30 s and centrifuge at 1000 g for 4 min. Combine the supernatants, add 6 mL 1-chlorobutane, add 500 µL water, shake for 30 s, centrifuge at
1000 g for 4 min. Remove the lower aqueous phase and add 2 mL water to the
organic layer. Shake for 30 s and centrifuge at 1000 g for 4 min. Combine the aqueous layers, evaporate to 2 mL under nitrogen at 55–60◦ , add 1 mL 15% ammonia,
mix. Within 30 s, add 15 mL dichloromethane and shake for 30 s. (Caution! Wear
gloves and eye protection, ammonia may escape under pressure!) Centrifuge at
1000 g for 4 min. Remove the water layer and evaporate the organic layer to dryness under nitrogen at 60◦ . Add 500 µL 250 µM NaOH, vortex for 10 s, add 500 µL
100 µg/mL 9-fluorenylmethyl chloroformate in MeCN, vortex for 10 s, let stand for
1 h, vortex for 10 s, let stand at room temperature for at least another 2 h. Inject
a 50 µL aliquot. (The extracting solution was 125 µL concentrated HCl in 100 mL
MeCN.)
HPLC VARIABLES
Column: 100 × 2.1 5 µm Hypersil ODS
Column temperature: 35
Mobile phase: MeOH:MeCN:1% acetic acid 30:30:40
Flow rate: 0.6
Injection volume: 50
Detector: UV 264
CHROMATOGRAM
Retention time: 8.8
Limit of quantitation: 100 ppb
KEY WORDS
cow; derivatization
REFERENCE
Heller, D.N. Determination of pirlimycin residue in milk by liquid chromatographic analysis of the
9-fluorenylmethyl chloroformate derivative, J.AOAC Int., 1997, 80, 975–981.
SAMPLE
Matrix: solutions
Sample preparation: Prepare a solution in mobile phase, inject a 25 µL aliquot.
HPLC VARIABLES
Column: 250 × 4.6 Zorbax C8
Mobile phase: MeCN:water 12:88 containing 0.25 g/L tetrabutylammonium perchlorate
and 2 mL/L 70% perchloric acid, apparent pH adjusted to 2.5 with 50% NaOH
Flow rate: 1.5
516
Pirlimycin
Injection volume: 25
Detector: UV 214
CHROMATOGRAM
Retention time: 15
OTHER SUBSTANCES
Simultaneous: benzyl alcohol, clindamycin (22.5), lincomycin (k′ 0.9)
REFERENCE
Theis, D.L. Ion-pairing liquid chromatographic method for the determination of pirlimycin hydrochloride,
J.Chromatogr., 1987, 402, 335–343.
SAMPLE
Matrix: tissue
Sample preparation: Mix 2 mL ground liver with 100 µL 10 µg/mL IS in water, add
10 mL MeCN:trifluoroacetic acid 99.75:0.25, homogenize (Polytron) at medium–high
speed for 30 s, filter, wash tube and filter cake twice with 5 mL portions of
MeCN:water:trifluoroacetic acid 84.75:15:0.25, filter the washes. Combine the filtrates
and shake with 25 mL n-butyl chloride for 10 s, centrifuge at 500 g for 1 min, remove
the aqueous layer, add 4 mL water to the organic layer, shake for 5–10 s, centrifuge
at 500 g for 1 min. Combine the aqueous layers, evaporate to 1.5–2.0 mL under a
stream of nitrogen at 80◦ , cool to room temperature, add 1 mL 15% ammonium
hydroxide, extract with 15 mL dichloromethane. Evaporate the organic layer to
dryness under a stream of nitrogen at 60◦ , reconstitute the residue with 2 mL
MeCN:100 mM pH 6.8 ammonium acetate buffer 20:80, inject a 100 µL aliquot. (Sample
preparation from Hornish,R.E.; Cazers,A.R.; Chester,S.T.,Jr.; Roof,R.D. Identification
and determination of pirlimycin residue in bovine milk and liver by high-performance
liquid chromatography-thermospray mass spectrometry. J.Chromatogr.B 1995, 674,
219–235.)
HPLC VARIABLES
Column: 150 × 2.1 Zorbax C18
Mobile phase: MeCN:100 mM ammonium formate 30:70
Flow rate: 0.18
Injection volume: 100
Detector: MS, Finnigan TSQ 7000, APCI, single quad mode, Q3 as analyzer, corona
4 kV, corona current 4.5 kV, capillary 180–200◦ , m/z 411–413
CHROMATOGRAM
Retention time: 14
Internal standard: iso-pirlimycin (10.5)
Limit of quantitation: 25 ng/g
OTHER SUBSTANCES
Extracted: pirlimycin sulfone (m/z 443), pirlimycin sulfoxide (m/z 427–429)
KEY WORDS
cow; liver
REFERENCE
Hornish, R.E.; Roof, R.D.; Wiest, J.R. Pirlimycin residue in bovine liver-a case of reverse metabolism,
Analyst, 1998, 123, 2463–2467.
Pirlimycin
517
ANNOTATED BIBLIOGRAPHY
Heller, D.N. Determination and confirmation of pirlimycin residue in bovine milk and liver by liquid chromatography/thermospray mass spectrometry: Interlaboratory study, J.AOAC Int., 1996, 79,
1054–1061.
Hornish, R.E.; Cazers, A.R.; Chester, S.T. Jr.; Roof, R.D. Identification and determination of pirlimycin
residue in bovine milk and liver by high-performance liquid chromatography-thermospray mass
spectrometry, J.Chromatogr.B, 1995, 674, 219–235. [SPE; LOQ 50 ng/mL; LOQ 25 ng/g]
Shah, J.A.; Weber, D.J. High-performance liquid chromatographic assay of pirlimycin in human serum
and urine using 9-fluorenylmethylchloroformate, J.Chromatogr., 1984, 309, 95–105. [derivatization]
518
Poloxalene
Poloxalene
Molecular weight: ca. 3000
CAS Registry No: 9003-11-6
Merck Index: 13, 7644
SAMPLE
Matrix: solutions
Sample preparation: Inject a 20 µL aliquot of a 3% solution in mobile phase
HPLC VARIABLES
Column: 300 × 7.5 10 µm HP Plgel 500A
Mobile phase: THF
Flow rate: 1
Injection volume: 20
Detector: Refractive Index
CHROMATOGRAM
Retention time: 9
REFERENCE
Wigman, L.S.; Abdel-Kader, H.; Menon, G.K. Size exclusion chromatography of poloxalene poloxamers:
polyethylene glycol-polypropylene glycol co-polymers used to control cattle bloat, J.Pharm.Biomed.Anal.,
1994, 12, 719–722.
SAMPLE
Matrix: solutions
Sample preparation: Inject a 20 µL aliquot of a 3% solution in mobile phase
HPLC VARIABLES
Column: 300 × 7.5 10 µm Spherogel TSK2000SW 250A
Mobile phase: MeOH:water 30:70
Flow rate: 1
Injection volume: 20
Detector: Refractive Index
CHROMATOGRAM
Retention time: 6
REFERENCE
Wigman, L.S.; Abdel-Kader, H.; Menon, G.K. Size exclusion chromatography of poloxalene poloxamers:
polyethylene glycol-polypropylene glycol co-polymers used to control cattle bloat, J.Pharm.Biomed.Anal.,
1994, 12, 719–722.
519
Pramipexole
Pramipexole
Molecular formula: C10 H17 N3 S
Molecular weight: 211.33
CAS Registry No: 104632-26-0
Merck Index: 13, 7790
H
H3C
N
S
NH2
N
SAMPLE
Matrix: blood
Sample preparation: Vortex 1 mL plasma, 50 µL 180 ng/mL IS in 1% acetic acid,
100 µL 1 M NaOH, and 6 mL MTBE for 5 min, centrifuge at 630 g for 5 min, freeze
in dry ice acetone. Evaporate the organic layer to dryness under a stream of nitrogen,
reconstitute the residue with 100 µL MeOH:water 95:5, inject a 70 µL aliquot.
HPLC VARIABLES
Column: 150 × 4.6 5 µm Zorbax SB CN
Mobile phase: MeOH:water:100 mM ammonium acetate 80:15:5
Flow rate: 1.2
Injection volume: 70
Detector: MS, PE Sciex API-III, triple quadrupole, orifice 45 V, nebulizer probe 470◦ ,
nebulizer gas at 550 kPa, auxiliary flow 1.5 L/min, corona discharge needle 3 µA, m/z
212–153
CHROMATOGRAM
Retention time: 3.4
Internal standard: BHT-920 (m/z 210–141) (2.2)
Limit of quantitation: 50 pg/mL
KEY WORDS
plasma; use polypropylene containers
REFERENCE
Lau, Y.Y.; Selenka, J.M.; Hanson, G.D.; Talaat, R.; Ichhpurani, N. Determination of pramipexole
(U-98,528) in human plasma by high-performance liquid chromatography with atmospheric pressure
chemical ionization tandem mass spectrometry, J.Chromatogr.B, 1996, 683, 209–216.
SAMPLE
Matrix: blood, urine
Sample preparation: Vortex 1 mL plasma or urine, 50 µL 30 ng/mL (plasma) or
15 µg/mL (urine) IS in 1% acetic acid, 100 µL 1 M NaOH, and 6 mL diethyl ether for
5 min, centrifuge at 2500 g for 5 min, freeze in dry ice/acetone. Extract the organic layer
with 100 (plasma) or 200 (urine) µL buffer. Freeze in dry ice/acetone, discard organic
layer, remove all traces of ether with a stream of nitrogen, thaw, inject a 50 µL aliquot.
(The buffer was 1.7 g potassium dihydrogen phosphate, 1.7 g sodium acetate, and 0.5 g
sodium heptanesulfonate in 500 mL water, adjusted to pH 3.5 with acetic acid.)
HPLC VARIABLES
Guard column: 15 × 3.2 7 µm Brownlee RP-8
Column: 250 × 4.6 5 µm Zorbax Rx C8
Mobile phase: MeCN:buffer 45:55 (The buffer was 10.2 g potassium dihydrogen phosphate, 10.2 g sodium acetate, and 4.5 g sodium heptanesulfonate in 3 L water, adjusted
to pH 3.5 with acetic acid.)
Flow rate: 1.2
Injection volume: 50
520
Pramipexole
Detector: E, ESA 5100A, 5011 analytical cell, 5020 guard cell, electrode 2 0.6 V
(detection), electrode 1 0.2 V (impurity screening), guard cell 0.65 V (mobile-phase
conditioning) (plasma); UV 286 (urine)
CHROMATOGRAM
Retention time: 14.4
Internal standard: BHT-920 (10.7)
Limit of quantitation: 50 pg/mL (plasma), 10 ng/mL (urine)
KEY WORDS
plasma
REFERENCE
Lau, Y.Y.; Hanson, G.D.; Ichhpurani, N. Determination of pramipexole (U-98,528) in human plasma
and urine by high-performance liquid chromatography with electrochemical and ultraviolet detection,
J.Chromatogr.B, 1996, 683, 217–223.
521
Pranlukast
Pranlukast
Molecular formula: C27 H23 N5 O4
Molecular weight: 481.50
CAS Registry No: 103177-37-3
Merck Index: 13, 7795
O
H
H
N
O
O
N N
N
N
O
SAMPLE
Matrix: blood
Sample preparation: Add IS to 200 µL plasma, wash with hexane:isopropanol 95:5,
add MeCN to the aqueous layer, centrifuge. Evaporate the supernatant to dryness,
reconstitute the residue with mobile phase, inject an aliquot.
HPLC VARIABLES
Column: 300 × 4.6 C18
Mobile phase: MeCN:70 mM ammonium formate buffer 45:55
Flow rate: 1
Detector: UV 262
CHROMATOGRAM
Internal standard: ONO-RS-425 (4-oxo-6-methyl-8-(4-phenylbutoxy)benzoylamino)-2(tetrazol-5-yl)-4H-1-benzopyran)
Limit of quantitation: 10 ng/mL
KEY WORDS
pharmacokinetics; plasma
REFERENCE
Brocks, D.R.; Upward, J.; Hust, R.; Köester, F.E.; Collie, H.; Qian, Y.; Dennis, M.J. The pharmacokinetics
of pranlukast in healthy young and elderly subjects, Int.J.Clin.Pharmacol.Ther., 1996, 34, 375–379.
SAMPLE
Matrix: blood
Sample preparation: Mix 100 µL plasma with 100 µL 250 ng/mL IS in 1 M citric acid
solution, inject a 30–50 µL aliquot onto column A and elute to waste with water at
1 mL/min; after 25 s, elute to waste with water at 7 mL/min; after another 15 s, elute to
waste with water at 1 mL/min; after 70 s, backflush column A with water for 70 s, elute
the contents of column A onto column B with the mobile phase.
HPLC VARIABLES
Column: A 10 × 2 40 µm Ph-EC-IST SPE cartridge (Jones Chromatography); B 20 × 2
unspecified guard column + 30 × 2 3 µm Hypersil BDS C-18
Mobile phase: Gradient. MeOH:20 mM ammonium acetate 25:75 for 0.3 min, to 70:30
over 0.1 min, maintain at 70:30 for 2 min, to 90:10 over 0.1 min, maintain at 90:10 over
0.8 min, return to initial conditions over 0.1 min.
Flow rate: 0.3
Injection volume: 30–50
Detector: MS, PE Sciex API-III Plus triple quadrupole, turbo ionspray, negative ion
mode, ionspray -3200 V, interface -650 V, orifice -68 V, collision energy 23 V, nebulizer
gas air at 42 psi and 0.6 L/min, curtain gas nitrogen at 1.0 L/min, auxiliary gas air
at 5.0 L/min, turbo-ionspray probe 350◦ , collision gas argon, ca. 60 µL/mL of column
effluent entered the detector, m/z 480.0–291.0
522
Pranlukast
CHROMATOGRAM
Internal standard: SK&F 108566 (m/z 423.0–244.0) (1.9)
Limit of quantitation: 10 ng/mL
KEY WORDS
column-switching; plasma
REFERENCE
Marchese, A.; McHugh, C.; Kehler, J.; Bi, H. Determination of pranlukast and its metabolites in human
plasma by LC/MS/MS with PROSPEKTTM on-line solid-phase extraction, J.Mass.Spectrom., 1998, 33,
1071–1079.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 × 4.6 10 µm Cosmosil 5C18-MS
Column temperature: 50
Mobile phase: Gradient. MeCN:10 mM pH 4.2 potassium phosphate buffer 0:100 for
2 min, to 75:25 over 7.5 min, maintain at 75:25 for 5.5 min. Isocratic. MeCN:buffer 50:50
Flow rate: 1.5
Detector: UV (wavelength not specified)
CHROMATOGRAM
Retention time: 13.4 (gradient) or 6.7 (isocratic)
OTHER SUBSTANCES
Simultaneous: acetazolamide (7.9), acyclovir (7.0), allopurinol (7.3), aniracetam (8.6),
benazepril (11.5), betamethasone (10.8), camostat mesylate (8.8), carbamazepine (10.8),
cilazapril (10.7), cimetidine (7.5), clofibrate (15.0), clonazepam (11.3), cyclophosphamide
(9.9), delapril (11.9), dexamethasone (10.9), diazepam (12.5), digoxin (9.0), dilazep (10.6),
diltiazem (10.2), docarpamine (11.4), eperisone (8.7), ethosuximide (9.0), fenbufen (11.8),
fluconazole (9.2), flutamide (12.7), fominoben (12.6), hydrocortisone acetate (12.1), imidapril (10.1), indomethacin (12.6), irinotecan (9.2), maprotiline (10.5), methotrexate
(8.0), nefiracetam (9.7), nifedipine (11.9), nitrazepam (11.2), pentobarbital (10.9), phenobarbital (10.0), phenytoin (10.8), pindolol (8.3), pranoprofen (10.4), prednisolone (10.3),
primidone (8.9), quinapril (10.5), spironolactone (12.4), sulpiride (7.6), sulthiame (9.3),
tolbutamide (11.6), tranilast (10.5), triamcinolone (11.2), warfarin (12.0), zonisamide
(9.5) (gradient retention times; isocratic conditions may differ)
REFERENCE
Sugiyama, T.; Matsuyama, R.; Usui, S.; Katagiri, Y.; Hirano, K. Selection of mobile phases in highperformance liquid chromatographic determination for medicines, Biol.Pharm.Bull., 2000, 23, 274–278.
523
Prednicarbate
Prednicarbate
O
H3C
Molecular formula: C27 H36 O8
O
CH3
HO
Molecular weight: 488.57
CAS Registry No: 73771-04-7
Merck Index: 13, 7805
O
O
CH3
O
H
O
H
H
O
SAMPLE
Matrix: cell incubations
Sample preparation: Sonicate cells, extract twice with 3 mL portions of ethyl acetate
by vortexing for 1 min, centrifuge at 1000 rpm for 5 min. Evaporate the combined
organic layers to dryness under a stream of nitrogen, reconstitute the residue with 1 mL
MeOH, vortex for 1 min, evaporate to dryness, reconstitute the residue with 100 µL
MeOH, centrifuge, inject a 20 µL aliquot.
HPLC VARIABLES
Column: 125 × 4 5 µm LiChrospher 100 RP-18
Mobile phase: Gradient. MeCN:water from 20:80 to 100:0 over 20 min.
Flow rate: 1
Injection volume: 20
Detector: UV 254
CHROMATOGRAM
Retention time: 15.1
Internal standard: betamethasone (9.4)
Limit of detection: 10 ng/mL
Limit of quantitation: 100 ng/mL
OTHER SUBSTANCES
Extracted: prednisolone (7.8), prednisolone 17-ethylcarbonate (11.1), prednisolone 21ethylcarbonate (12.5)
REFERENCE
Gysler, A.; Lange, K.; Korting, H.C.; Schäfer-Korting, M. Prednicarbate biotransformation in human
foreskin keratinocytes and fibroblasts, Pharm.Res., 1997, 14, 793–797.
524
Propionylpromazine
Propionylpromazine
Molecular formula: C20 H24 N2 OS
CH3
N
O
H3C
Molecular weight: 340.49
CAS Registry No: 3568-24-9
Merck Index: 13, 7921
N
CH3
S
SAMPLE
Matrix: formulations
Sample preparation: Vortex 100 mg formulation with 10 mL hexane, sonicate for
5 min, add 10 mL 50 mM HCl, vortex, centrifuge at 835 g for 2 min. Repeat the extraction
of the hexane layer with 10 mL 50 mM HCl. Combine the aqueous layers and make up
to 50 mL with 50 mM HCl. Dilute a 2 mL aliquot to 10 mL with MeCN:mobile-phase
buffer 70:30, filter (0.45 µm PTFE), inject a 1 µL aliquot. (Protect from light. Use of
hexane may not be necessary, depending on the nature of the formulation.)
HPLC VARIABLES
Column: 150 × 2 5 µm ODS/H C18 (Keystone)
Column temperature: 40
Mobile phase: MeCN:buffer 85:15 (Prepare the buffer by diluting 25 mL of a 200 mM
heptanesulfonic acid premixed solution (Alltech) to 1 L with water.)
Flow rate: 0.3
Injection volume: 1
Detector: UV 280
CHROMATOGRAM
Retention time: 3
Limit of detection: 0.09%
REFERENCE
Hurlbut, D.B.; Primus, T.M.; Goodall, M.J.; Volz, S.A.; Johnston, J.J. Determination of propionylpromazine hydrochloride in formulation matrices using reversed-phase ion-pair small bore liquid
chromatography, J.AOAC Int., 1999, 82, 1321–1328.
SAMPLE
Matrix: tissue
Sample preparation: Condition a Sep-Pak C18 SPE cartridge with 5 mL MeOH and
5 mL water. Homogenize kidney with a kitchen grinder. Weigh out a 5 g sample and
add 20 mL MeCN with continuous gentle mixing, mix vigorously on a vibromixer at
1500 rpm for 30 s, sonicate for 2 min, centrifuge at 4000 g for 5 min. Mix 7.5 mL sample
extract and 40 mL 10% NaCl and add to SPE cartridge, wash with 1 mL 10 mM sulfuric
acid, wash with 2 mL air, elute with 2 mL acidic MeCN. Place eluate in a washed
tube and evaporate to 300 µL at 70◦ under a stream of nitrogen, mix gently, add 1 mL
n-hexane, mix on a vibromixer for 30 s, centrifuge at 2000 g, inject a 50 µL aliquot of the
aqueous phase. (Acidic MeCN was 1 mL 50 mM sulfuric acid and 100 mL MeCN. The
washed tube was prepared by rinsing with concentrated ammonia, water, and acetone
and drying under a stream of nitrogen.)
HPLC VARIABLES
Guard column: 10 × 2.1 37–50 µm Bondapak C18
Column: 300 × 3.9 Bondapak C18
Mobile phase: MeCN:water 55:45 containing 2.46 g/L anhydrous sodium acetate, pH
adjusted to 6.5 with acetic acid
Flow rate: 1.2
Propionylpromazine
525
Injection volume: 50
Detector: UV 240
CHROMATOGRAM
Retention time: 18
Limit of detection: 4 ng/g
OTHER SUBSTANCES
Extracted: acepromazine (13), azaperol (Fex 246 em 351) (5), azaperone (8), carazolol
(5), haloperidol (8.5), chlorpromazine (25), xylazine (7)
KEY WORDS
kidney; pig; SPE
REFERENCE
Keukens, H.J.; Aerts, M.M.L. Determination of residues of carazolol and a number of tranquilizers in
swine kidney by high-performance liquid chromatography with ultraviolet and fluorescence detection,
J.Chromatogr., 1989, 464, 149–161.
SAMPLE
Matrix: tissue
Sample preparation: Condition a Bond-Elut C18 SPE cartridge with 5 mL MeOH and
5 mL water. Cut pig kidney or liver into small pieces and homogenize. 5 g Homogenate +
10 mL MeCN, shake, vortex for 30 s, sonicate for 3 min, vortex for 30 s, sonicate for
3 min, centrifuge at 10000 g for 20 min. Add 7.5 mL supernatant and 40 mL 10% NaCl
to the SPE cartridge at about 1 mL/min, do not allow cartridge to dry out, wash with
850 µL 10 mM sulfuric acid, dry with air, elute with 3.5 mL acidic MeCN. Evaporate
the eluate to dryness under a stream of nitrogen at 50◦ , reconstitute the residue in
300 µL 10 mM sulfuric acid, vortex briefly, add 1 mL hexane, vortex for 30 s, centrifuge
at 2000 g for 5 min, inject an aliquot of the aqueous layer. (Acidic MeCN was 1 mL
50 mM sulfuric acid in 100 mL MeCN.)
HPLC VARIABLES
Guard column: Hypersil 5 µm SAS C1
Column: 250 mm long 5 µm Hypersil SAS C1
Mobile phase: MeCN:water 50:50 containing 0.77 g/L ammonium acetate
Flow rate: 2
Detector: E, ESA Model 5100A Coulochem, first electrode +0.4 V, second electrode
(which was monitored) +0.7 V, Model 5020 guard cell after pump but before injector at
+0.75 V
CHROMATOGRAM
Retention time: 25
Limit of detection: 2 ng/g
OTHER SUBSTANCES
Extracted: acepromazine (18), azaperol (5), azaperone (6.5), carazolol (9), chlorpromazine
(32), haloperidol (12.5), xylazine (10)
KEY WORDS
kidney; liver; pig; SPE
REFERENCE
Rose, M.D.; Shearer, G. Determination of tranquilisers and carazolol residues in animal tissue using
high-performance liquid chromatography with electrochemical detection, J.Chromatogr., 1992, 624,
471–477.
526
Propionylpromazine
ANNOTATED BIBLIOGRAPHY
Delahaut, P.; Levaux, C.; Eloy, P.; Dubois, M. Validation of a method for detecting and quantifying
tranquillisers and a β-blocker in pig tissues by liquid chromatography-tandem mass spectrometry,
Anal.Chim.Acta, 2003, 483, 335–340. [SPE; LOD 1–10 ng/g; propionylpromazine; acepromazine;
chlorpromazine; xylazine; carazolol; azaperone; azaperol; haloperidol; isobutcar; levamisole is internal
standard]
Quintana, M.C.; Blanco, M.H.; Lacal, J.; Hernández, L. Analysis of pharmaceutical residues in bovine
liver by HPLC, J.Liq.Chromatogr.Rel.Technol., 2001, 24, 735–745. [LOD 21–298 ng/mL; dexamethasone; betamethasone; betamethasone acetate; xylazine; haloperidol; acepromazine; propionylpromazine; chlorpromazine]
Propoxycaine hydrochloride
527
Propoxycaine hydrochloride
Molecular formula: C16 H27 ClN2 O3
Molecular weight: 330.86
CAS Registry No: 550-83-4
Merck Index: 13, 7930
CH3
O
O
N
O
CH3
CH3
HCl
NH2
SAMPLE
Matrix: formulations
Sample preparation: Dilute with mobile phase, inject a 20 µL aliquot.
HPLC VARIABLES
Column: 300 × 3.9 µBondapak phenyl
Mobile phase: MeCN:buffer 30:70 (The buffer was 25 mM pH 3.0 potassium dihydrogen
phosphate containing 50 mM sodium heptanesulfonate.)
Flow rate: 1
Injection volume: 20
Detector: UV 254
CHROMATOGRAM
Retention time: 18.9
OTHER SUBSTANCES
Simultaneous: levonordefrin (3.6), norepinephrine (3.9), procaine (9.8)
KEY WORDS
injections; stability-indicating
REFERENCE
Storms, M.L.; Stewart, J.T. Stability-indicating HPLC assays for the determination of prilocaine and
procaine drug combinations, J.Pharm.Biomed.Anal., 2002, 30, 49–58.
528
Propylene glycol
Propylene glycol
HO
OH
Molecular formula: C3 H8 O2
Molecular weight: 76.09
CAS Registry No: 57-55-6
Merck Index: 13, 7947
SAMPLE
Matrix: blood, tissue
Sample preparation: Plasma. Mix 50 µL plasma with 10 µL 50 µg/mL IS and 50 µL
4 M NaOH, add 1 mL benzoyl chloride solution, vortex for 30 min, add 25 µL 1% glycine,
vortex for 15 min, let stand at room temperature for 45 min, centrifuge at 4000 rpm
for 5 min. Remove a 100 µL aliquot of the pentane layer and evaporate it to dryness
under a stream of nitrogen at 40◦ , reconstitute the residue with 1 mL MeOH, inject
a 10 µL aliquot. Tissue. Homogenize rat lung tissue with 4 vol of water. Mix 100 µL
homogenate with 10 µL 100 µg/mL IS, let stand at room temperature for 30 min, vortex
thoroughly, centrifuge at 4000 rpm for 10 min. Mix 50 µL of the supernatant with 50 µL
4 M NaOH, add 1 mL benzoyl chloride solution, vortex for 30 min, add 25 µL 1% glycine,
vortex for 15 min, let stand at room temperature for 45 min, centrifuge at 4000 rpm
for 5 min. Remove a 100 µL aliquot of the pentane layer and evaporate it to dryness
under a stream of nitrogen at 40◦ , reconstitute the residue with 200 µL MeOH, inject
a 10 µL aliquot. (Prepare benzoyl chloride solution by mixing 125 µL benzoyl chloride
with 10 mL pentane.)
HPLC VARIABLES
Column: 100 × 2.1 3.5 µm Symmetry Shield RP-18 (Waters)
Mobile phase: MeOH:water:formic acid 78:22:0.1
Flow rate: 0.25
Injection volume: 10
Detector: MS, Applied Biosystems API365, APCI, ionspray 4500 V, declustering potential 1 V, focusing potential 120 V, entrance potential -2.5 V, collision entrance potential
8.57 V, collision energy 13 V, collision exit potential 16 V, probe 450◦ , turbo gas flow
7 L/min, m/z 285.1–163.2
CHROMATOGRAM
Retention time: 3.5
Internal standard: 1,4-butanediol (m/z 299.0–177.2) (4.4)
Limit of detection: 269 ng/mL (plasma), 1.12 µg/g (lung)
Limit of quantitation: 448 ng/mL (plasma), 1.62 µg/g (lung)
KEY WORDS
derivatization; lung; plasma; rat
REFERENCE
Gao, S.; Wilson, D.M.; Edinboro, L.E.; McGuire, G.M.; Williams, S.G.P.; Karnes, H.T. Improvement of
sensitivity for the determination of propylene glycol in rat plasma and lung tissue using HPLC/tandem
MS and derivatization with benzoyl chloride, J.Liq.Chromatogr.Rel.Technol., 2003, 26, 3413–3431.
529
Propylhexedrine
Propylhexedrine
Molecular formula: C10 H21 N
H
N
CH3
CH3
Molecular weight: 155.28
CAS Registry No: 101-40-6
Merck Index: 13, 7952
SAMPLE
Matrix: blood
Sample preparation: Condition a 200 mg Bond-Elut C18 SPE cartridge with 1 mL
MeOH, 1 mL water, and 2 mL 10 mM pH 9.3 ammonium carbonate buffer. Centrifuge
whole blood or serum at 14 000 g for 5 min. Vortex 0.2–1 mL of the supernatant with IS
and 2 mL 10 mM pH 9.3 ammonium carbonate buffer, centrifuge at 5000 g for 10 min,
add 2 mL of the supernatant to the SPE cartridge and allow to pass through over 5 min,
wash with 2 mL 10 mM pH 9.3 ammonium carbonate buffer, dry with vacuum for 5 min,
elute with 500 µL MeOH:500 mM acetic acid 90:10. Add 10 µL 1 mM HCl to the eluate
and evaporate to dryness under a stream of nitrogen, reconstitute the residue with
100 µL mobile phase, centrifuge at 14 000 g for 4 min, inject a 5–20 µL aliquot of the
supernatant.
HPLC VARIABLES
Column: 125 × 3 4 µm Superspher 100 RP 18
Mobile phase: MeCN:50 mM pH 3.0 ammonium formate buffer 25:75
Flow rate: 0.8
Injection volume: 5–20
Detector: MS, Finnigan MAT SSQ 7000, APCI, positive ion mode, sheath gas nitrogen
70 psi, auxiliary gas nitrogen 20 mL/min, capillary 190◦ , vaporizer 450◦ , corona current
5 µA, m/z 204–156–157
CHROMATOGRAM
Retention time: 3.9
Internal standard: norfenfluramine (m/z 212–204–187–159 (4.4)
Limit of detection: 2 ng/mL
OTHER SUBSTANCES
Extracted: amphetamine (m/z 147–136–119–91) (3.7), cathinone (m/z 150–147–132)
(3), ephedrine (m/z 160-166–148) (3.1), fenfluramine (m/z 232–212–159) (7), MDA (m/z
180–163–147) (3.8), MDEA (m/z 215–208–163) (5.3), MDMA (m/z 199–194–163–133)
(4.3), methamphetamine (m/z 160–150–119–91) (4.2), norfenfluramine (m/z
212–204–187–159) (4.4), phentermine (m/z 160–150–133–91) (4.4), phenylethylamine
(m/z 147–122–105) (3.1), phenylpropanolamine (m/z 152–147–134) (2.8)
KEY WORDS
serum; SPE; whole blood
REFERENCE
Bogusz, M.J.; Krüger, K.-D.; Maier, R.-D. Analysis of underivatized amphetamines and related phenethylamines with high-performance liquid chromatography-atmospheric pressure chemical ionization mass
spectrometry, J.Anal.Toxicol., 2000, 24, 77–84.
530
Protirelin
Protirelin
H
N
O
H O
Molecular formula: C16 H22 N6 O4
N
Molecular weight: 362.38
CAS Registry No: 24305-27-9
Merck Index: 13, 9663
H
H
O
O
NH2
H
N
N
H
N
SAMPLE
Matrix: blood
Sample preparation: Mix 1 mL serum with 2 mL water and 7.5 mL cold EtOH/HCl,
let stand at 4◦ for 12 h, centrifuge at 2800 rpm at 4◦ for 30 min, adjust the pH of the
supernatant to 8.3 with concentrated ammonium hydroxide, let stand at 4◦ for 15 min,
centrifuge at 2800 rpm at 4◦ for 20 min, adjust the pH of the supernatant to 5.3 with
4 M HCl. For each 1 mL of extract, add 25 µL 2 M ammonium acetate, adjust pH to 5.3;
to each 10 mL of extract, slowly add 15 mL cold EtOH and 25 mL diethyl ether, let stand
at 4◦ for 12 h, centrifuge at 2800 rpm at 4◦ for 30 min. Collect the precipitate and dry it
under nitrogen, reconstitute with 100 mM pH 3.10 sodium dihydrogen phosphate, inject
an aliquot containing 1.1–3.3 nmol. (Prepare EtOH/HCl by adding 7.5 mL concentrated
HCl to 375 mL 95% EtOH. Sample preparation from Oyama,H.; Tenku,A.; Kakita,K.;
Matsumura,S.; Nishida,S.; Horino,M. Recovery of human C-peptide by acid-ethanol
extraction. Endocrinol.Japon. 1978, 25, 493–498.)
HPLC VARIABLES
Column: 250 × 4.5 Spherisorb S5 ODS2
Column temperature: 37
Mobile phase: Gradient. MeCN: 100 mM pH 3.10 sodium dihydrogen phosphate from
0:100 to 28:72 over 14 min, to 31.2:68.8 over 8 min, to 32.8:67.2 over 8 min, to 42.8 over
5 min, to 60:40 over 3.5 min, maintain at 60:40.
Flow rate: 1
Injection volume: 200
Detector: UV 220
CHROMATOGRAM
Retention time: 7.5
OTHER SUBSTANCES
Extracted: porcine C-peptide (21), porcine glucagon (24), porcine monocomponent insulin
(25), porcine pancreatic polypeptide (36), porcine proinsulin (30), somatostatin (24),
vasoactive intestinal polypeptide (19)
KEY WORDS
cord; plasma
REFERENCE
Knip, M. Analysis of pancreatic peptide hormones by reversed-phase high-performance liquid chromatography, Horm.Metab.Res., 1984, 16, 487–491.
SAMPLE
Matrix: blood
Sample preparation: Mix whole blood with ice-cold EtOH, centrifuge (?), evaporate to
dryness under reduced pressure or a stream of nitrogen at 35◦ , reconstitute with 0.1%
trifluoroacetic acid, centrifuge briefly, inject an aliquot of the supernatant.
Protirelin
531
HPLC VARIABLES
Column: 250 × 4.6 Ultrasphere ODS
Mobile phase: Gradient. MeCN:water:trifluoroacetic acid from 0:100:0.1 to 100:0:0.1
over 50 min
Flow rate: 1
Injection volume: 1000
Detector: RIA (of fractions)
CHROMATOGRAM
Retention time: 16
KEY WORDS
rat
REFERENCE
Sheward, W.J.; Harmar, A.J.; Fraser, H.M.; Fink, G. Thyrotropin-releasing hormone in rat pituitary
stalk blood and hypothalamus: studies with high performance liquid chromatography, Endocrinology,
1983, 113, 1865–1869.
532
Prulifloxacin
Prulifloxacin
Molecular formula: C21 H20 FN3 O6 S
Molecular weight: 461.46
CAS Registry No: 123447-62-1
O
O
O
H
N
CH3
N
N
F
CH3
S
COOH
O
SAMPLE
Matrix: blood, urine
Sample preparation: Inject urine directly. Condition a 200 mg Sep-Pak Vac PS-1 SPE
cartridge with 3 mL MeOH and 3 mL 1% acetic acid. Mix 500 µL plasma with 1 mL 1%
acetic acid, add to the SPE cartridge, wash with 1 mL 1% acetic acid, elute with 4 mL
MeCN:1% acetic acid 90:10. Evaporate the eluate to dryness under reduced pressure,
reconstitute the residue with MeOH, inject an aliquot.
HPLC VARIABLES
Column: 250 × 4.6 Capcell Pak C18 SG120 (Shiseido)
Column temperature: 40
Mobile phase: MeCN:MeOH:50 mM pH 2 sodium phosphate buffer 10:20:60
Flow rate: 1.0–1.7
Detector: UV 275; Radioactivity (14 C)
CHROMATOGRAM
Retention time: 15
OTHER SUBSTANCES
Extracted: metabolites
KEY WORDS
dog; monkey; plasma; rat
REFERENCE
Okuyama, Y.; Morino, A. Pharmacokinetics of prulifloxacin. 3rd communication: metabolism in rats,
dogs and monkeys, Arzneimittelforschung, 1997, 47, 293–298.
Pyrethrins
Pyrethrins
Molecular formula: C21 H28 O3 (pyrethrin I),
C22 H28 O5 (pyrethrin II)
Molecular weight: 328.44 (pyrethrin I),
372.45 (pyrethrin II)
CAS Registry No: 121-21-1 (pyrethrin I),
121-29-9 (pyrethrin II)
Merck Index: 13, 8054
533
O
H3C
R
CH3
CH3
O
CH2
O
CH3
R = CH3 (I) or COOCH3 (II)
SAMPLE
Matrix: formulations
Sample preparation: Dissolve 5 g formulation in 100 mL isopropanol that contains
5 mL IS, dilute with isopropanol, filter (0.45 µm PTFE), inject a 10 µL aliquot.
HPLC VARIABLES
Column: 200 × 4.6 3 µm Pinnacle C8 (Restek)
Column temperature: 50
Mobile phase: Gradient. MeCN:MeOH:water 40:15:45 for 10 min, to 45:15:40 over
5 min, maintain at 45:15:40 for 10 min, to 50:15:35 over 10 min, to 55:15:30 over
10 min. (For MS, all components contained 0.1% trifluoroacetic acid.)
Flow rate: 0.8
Injection volume: 10
Detector: UV 240; MS Finnigan TSQ 7000, electrospray, triple-stage quadrupole, positive ion mode, capillary 5.0 kV, electrospray vaporizer 400◦ , capillary 210◦ , collision gas
argon 1.5 mtorr
CHROMATOGRAM
Retention time: 21 (pyrethrin II), 37 (pyrethrin I)
Internal standard: 2,2-dimethylpropiophenone (9)
Limit of detection: 160 ng/mL (pyrethrin I, UV), 60 ng/mL (pyrethrin II, UV)
Limit of quantitation: 430 ng/mL (pyrethrin I, UV), 120 ng/mL (pyrethrin II, UV)
OTHER SUBSTANCES
Simultaneous: cinerin I (35), cinerin II (20), jasmolin I (42), jasmolin II (26), (S)methoprene (45), piperonyl butoxide (24)
KEY WORDS
validation details
REFERENCE
Wang, I.-H.; Subramanian, V.; Moorman, R.; Ko, J.; Johnson, D. A validated reversed-phase HPLC
method for analyzing pyrethrins, piperonyl butoxide, and (S)-methoprene in pesticide formulations,
LC.GC, 1999, 17, 260–275.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 × 4 5 µm Polygosil C18 (MetaChem)
Mobile phase: MeOH:isopropanol:EtOH 35:15:50
Flow rate: 0.5
Detector: UV 225
534
Pyrethrins
CHROMATOGRAM
Retention time: 15 (pyrethrin II), 31 (pyrethrin I)
MetaChem Catalog; 1995; page 46.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 150 × 8 Nucleosil 5 NO2
Mobile phase: n-Hexane:THF 96:4
Flow rate: 2
Detector: UV 220
CHROMATOGRAM
Retention time: 18 (pyrethrin I)
OTHER SUBSTANCES
Simultaneous: bioallethrin (15, 16), cinerin (14), jasmolin (12.5)
KEY WORDS
normal phase
REFERENCE
Ando, T.; Kurotsu, Y.; Uchiyama, M. High performance liquid chromatographic separation of the
stereoisomers of natural pyrethrins and related compounds, Agric.Biol.Chem., 1986, 50, 491–493.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: two 300 × 4 10 µm µPorasil columns in series
Mobile phase: Dichloromethane:water-saturated dichloromethane 50:50
Flow rate: 0.6
Injection volume: 10
Detector: UV 254; Refractive Index
CHROMATOGRAM
Retention time: 18–57 (numerous isomers)
KEY WORDS
normal phase
REFERENCE
Otieno, D.A.; Jondiko, I.J.; McDowell, P.G.; Kezdy, F.J. Quantitative analysis of the pyrethrins by HPLC,
J.Chromatogr.Sci., 1982, 20, 566–570.
SAMPLE
Matrix: urine
Sample preparation: Add 4 g solid NaCl, 3.5 mL MeCN, and 5 mL saturated NaCl
solution to 5 mL MeCN, shake for 1 min. Remove the MeCN layer and extract the
aqueous layer with 1 mL MeCN. Combine the MeCN layers and adjust to a known
volume (0.5–1 mL), mix, filter (0.45 µm), inject a 40 µL aliquot.
Pyrethrins
535
HPLC VARIABLES
Column: 150 × 3 3 µm Luna C18(2) (Phenomenex)
Column temperature: 30
Mobile phase: Gradient. MeCN:water 10:90 for 1 min, to 90:10 over 30 min, maintain
at 90:10 for 4 min, to 100:0 over 1 min, maintain at 100:0 for 10 min, return to initial
conditions over 1 min.
Flow rate: 0.5
Injection volume: 40
Detector: UV 235
CHROMATOGRAM
Retention time: 29.6 (pyrethrin I), 33.7 (pyrethrin II)
Limit of detection: 4 ng/mL (pyrethrin I), 40 ng/mL (pyrethrin II)
OTHER SUBSTANCES
Extracted: allethrin (31.8, LOD 5 ng/mL), bifenthrin (37, LOD 5 ng/mL), cyfluthrin
(34.3, LOD 5 ng/mL), fenvalerate (35.3, LOD 2 ng/mL), cis-permethrin (35.7, LO
5 ng/mL), trans-permethrin (36.3, LOD 5 ng/mL), phenothrin (36.4, LOD 5 ng/mL),
m-phenoxybenzyl alcohol (21, LOD 5 ng/mL), resmethrin (35.2, LOD 5 ng/mL), tetramethrin (31.4, LOD 5 ng/mL)
REFERENCE
Loper, B.L.; Anderson, K.A. Determination of pyrethrin and pyrethroid pesticides in urine and water
matrices by liquid chromatography with diode array detection, J.AOAC Int., 2003, 86, 1236–1240.
ANNOTATED BIBLIOGRAPHY
Essig, K.; Zhao, Z. Method development and validation of a high-performance liquid chromatographic
method for pyrethrum extract, J.Chromatogr.Sci., 2001, 39, 473–480. [pyrethrins; cinerin; jasmolin;
normal phase]
Wang, I.-H.; Moorman, R.; Burleson, J. Simultaneous determination of dipropyl pyridine-2,5-dicarboxylate, N-octyl bicycloheptene dicarboximide, piperonyl butoxide, and pyrethrins in pet shampoo by
reversed phase high-performance liquid chromatography, J.Liq.Chromatogr.Rel.Technol., 1996, 19,
3293–3304.
Quetiapine
N
O
OH
N
Molecular formula: C21 H25 N3 O2 S
Molecular weight: 383.52
N
CAS Registry No: 111974-69-7,
111974-72-2 (hemifumarate)
Merck Index: 13, 8127
S
SAMPLE
Matrix: blood
Sample preparation: Mix 400 µL plasma and 10 µL 2.5 µg/mL IS in MeCN:MeOH
50:50, add 25 µL 15% ammonium hydroxide, add 3 mL ethyl acetate, vortex for 30 s,
centrifuge for 5 min. Add the upper organic layer to 1 mL 200 mM HCl, vortex for 30 s,
centrifuge for 5 min, discard the organic layer. Basify the aqueous layer with 500 µL
15% ammonium hydroxide, add 3 mL ethyl acetate, vortex for 30 s, centrifuge for 5 min.
Evaporate the organic layer to dryness under a stream of nitrogen at 35◦ , reconstitute
the residue with 30 µL MeCN:MeOH:20 mM pH 7.4 phosphate buffer containing 30 mM
KCl 10:30:60, inject a 25 µL aliquot.
HPLC VARIABLES
Column: 150 × 2.1 5 µm Zorbax Stablebond phenyl SB-Ph
Mobile phase: MeCN:MeOH:20 mM pH 7.4 phosphate buffer containing 30 mM KCl
10:50:40
Flow rate: 0.25
Injection volume: 25
Detector: UV 225; E, Bioanalytical Systems LC-44, precell 0.25 V, analytical cell 0.55 V,
Ag/AgCl reference cell
CHROMATOGRAM
Retention time: 9 (UV 225)
Internal standard: M 214,652 (2-(2-[4-(8-chlorodibenzo[b,f][1,4]thiazepin-11-yl)piperazin-1-yl]ethoxy)ethanol) (UV 225) (16)
Limit of quantitation: 2.5 ng/mL
OTHER SUBSTANCES
Extracted: metabolite ICI 214,227 (7-hydroxylated) (E) (5), metabolite M 236,303 (7hydroxylated, N-dealkylated) (E) (14)
KEY WORDS
pharmacokinetics; plasma
REFERENCE
Davis, P.C.; Wong, J.; Gefvert, O. Analysis and pharmacokinetics of quetiapine and two metabolites in human plasma using reversed-phase HPLC with ultraviolet and electrochemical detection,
J.Pharm.Biomed.Anal., 1999, 20, 271–282.
SAMPLE
Matrix: blood
Sample preparation: Condition a 3 mL 3M-Empore mixed-phase SPE extraction disc
with 1 mL MeOH and 1 mL water. Mix 900 µL serum with 100 µL 3 µg/mL IS in serum
and 2 mL 100 mM pH 6.0 potassium dihydrogen phosphate buffer, add to the SPE disc,
wash with 1 mL water, wash with 1 mL 1 M acetic acid, wash with 1 mL n-hexane,
wash with 2 mL n-hexane:ethyl acetate 50:50, wash with 1 mL MeOH, elute with 1 mL
536
HPLC Methods for Recently Approved Pharmaceuticals. by George Lunn
Copyright 2005 John Wiley & Sons, Ltd. ISBN: 0-471-66941-5
Quetiapine
537
isopropanol:25% ammonia:dichloromethane 20:2:78. Evaporate the eluate to dryness,
reconstitute the residue with 250 µL MeCN:water 30:70, inject a 100 µL aliquot.
HPLC VARIABLES
Column: 250 × 4.6 5 µm Nucleosil 100-5-Protect 1 (endcapped)
Column temperature: 25
Mobile phase: MeCN:25 mM pH 7.0 potassium dihydrogen phosphate 40:60
Flow rate: 1
Injection volume: 100
Detector: UV 230
CHROMATOGRAM
Retention time: 11.7
Internal standard: melperone (8.8)
Limit of quantitation: 5 ng/mL
OTHER SUBSTANCES
Extracted: amisulpride (6.1), amitriptyline (23.4), m-chlorophenylpiperazine (8.0), citalopram (13.3), clomipramine (30.8), clozapine (30.9), desipramine (12.8), O-desmethylvenlafaxine (4.8), diazepam (11.0), doxepin (18.3), fluoxetine (17.8), 9-hydroxyrisperidone
(6.6), imipramine (20.6), maprotiline (15.3), melperone (8.8), mianserin (29.0), mirtazapine (16.6), moclobemide (5.6), nefazodone (32.5), norclomipramine (19.2), norclozapine
(14.4), nordoxepin (10.9), norfluoxetine (13.4), nortriptyline (14.5), paroxetine (15.3),
quetiapine (11.7), reboxetine (10.2), risperidone (11.1), sertraline (33.6), sulpiride (4.1),
trimipramine (21.5), venlafaxine (7.3), ziprasidone (26.4)
Simultaneous: benperidol (11.5), chlorprothixene (36.4), fluphenazine (31.0), haloperidol (15.3), normirtazapine (8.3), olanzapine (21.0), pimozide (44.1), promethazine (28.1),
thioridazine (43.2), trifluperidol (20.8), zolpidem (10.2)
Noninterfering: biperiden, buspirone, carbamazepine, lorazepam, perazine, valproic
acid, zopiclone, zotepine
Interfering: dibenzepin (11.5), fluvoxamine (11.6), normaprotiline (11.5), opipramol
(11.6)
KEY WORDS
serum; SPE
REFERENCE
Frahnert, C.; Rao, M.L.; Grasmäder, K. Analysis of eighteen antidepressants, four atypical antipsychotics and active metabolites in serum by liquid chromatography: a simple tool for therapeutic drug
monitoring, J.Chromatogr.B, 2003, 794, 35–47.
ANNOTATED BIBLIOGRAPHY
Hasselstrom, J.; Linnet, K. Fully automated on-line quantification of quetiapine in human serum by
solid phase extraction and liquid chromatography, J.Chromatogr.B, 2003, 798, 9–16. [LOD 10.3 nM;
trifluoperazine is internal standard]
Mandrioli, R.; Fanali, S.; Ferranti, A.; Raggi, M.A. HPLC analysis of the novel antipsychotic drug quetiapine in human plasma, J.Pharm.Biomed.Anal., 2002, 30, 969–977. [triprolidine is internal standard;
SPE; LOQ 4 ng/mL]
Thieme, D.; Sachs, H. Improved screening capabilities in forensic toxicology by application of liquid
chromatography-tandem mass spectrometry, Anal.Chim.Acta, 2003, 492, 171–186. [hair; LC-MS;
alprazolam; dothiepin; piritramide; cocaine; lorazepam; lormetazepam; clonazepam; flunitrazepam;
bromazepam; midazolam; flurazepam; nitrazepam; temazepam; medazepam; nordazepam; diazepam;
methylclonazepam; triazolam; oxazepam; haloperidol; benperidol; sulpiride; amisulpride; mirtazapine;
citalopram; olanzapine; paroxetine; fluoxetine; sertraline; zopiclone; zolpidem; risperidone; quetiapine;
fentanyl; pipamperone; meperidine; buprenorphine; propoxyphene; pentazocine; phenazocine; EDDP;
538
Quetiapine
tilidine; methadone; morphine; codeine; dihydrocodeine; acetylmorphine; amphetamine; ephedrine;
norephedrine; pseudoephedrine; methylephedrine; amphetaminil; benzphetamine; methylphenidate;
nikethamide; amfeparone; clobenzorex; atropine; scopolamine; ajmaline; aconitine; colchicine; strychnine; metoprolol; acebutolol; propranolol; sotalol; atenolol; bisoprolol; amiloride; triamterene; warfarin;
brodifacoum; coumatetralyl; phenprocoumon; methaqualone; clomethiazole; acetaminophen; methoxamine; vecuronium; neostigmine; LSD]
539
Quinfamide
Quinfamide
Molecular formula: C16 H13 Cl2 NO4
Molecular weight: 354.19
O
O
O
CAS Registry No: 62265-68-3
Merck Index: 13, 8147
N
Cl
O
Cl
SAMPLE
Matrix: blood, feces, urine
Sample preparation: Condition a Sep-Pak C18 SPE cartridge with 2 mL MeOH, 1 mL
5 µg/mL diazepam in MeOH (sic), and 2 mL water. Dissolve 10 mg lyophilized feces in
10 mL MeCN. Add 1 mL plasma, urine, or feces solution to the SPE cartridge, wash
with 12 mL water, elute with 1 mL MeCN, vortex the eluate, inject a 20 µL aliquot.
HPLC VARIABLES
Column: 250 × 4.6 5 µm Zorbax SB-CN
Mobile phase: MeCN:MeOH:water 10:50:40
Flow rate: 0.7
Injection volume: 20
Detector: UV 269
CHROMATOGRAM
Retention time: 9.7
Internal standard: diazepam (8.5)
Limit of quantitation: 80 ng/mL
OTHER SUBSTANCES
Extracted: 1-(dichloroacetyl)-1,2,3,4-tetrahydro-6-quinolol (metabolite) (6.5)
KEY WORDS
plasma; SPE
REFERENCE
Morales, J.M.; Jung, C.H.; Alarcón, A.; Barreda, A. Solid-phase extraction and liquid chromatographic
quantitation of quinfamide in biological samples, J.Chromatogr.B, 2000, 746, 133–139.
540
Quinupristin
Quinupristin
H3C
N
CH3
Molecular formula: C53 H67 N9 O10 S
Molecular weight: 1022.24
CAS Registry No: 120138-50-3
Merck Index: 13, 8176
CH3
N
N
N
O
CH3
N
H
S
N
O
O
O
NH
H
O
O
CH3
H
O
O
N
O
H
N
OH
SAMPLE
Matrix: blood
Sample preparation: Condition a CN SPE cartridge (Lida-Interchim) with 1 mL
MeOH, 1 mL water, and 1 mL buffer. Add 1 mL 3.8% sodium citrate and 2.5 mL
250 mM HCl to 10 mL whole blood. Shake gently by hand and centrifuge at 2000 g at
4◦ for 15 min. Add 1 mL buffer and 50 µL 100 µg/mL IS in MeOH to 1.35 mL acidified
plasma. Vortex for a few seconds, centrifuge at 4000 g at 4◦ for 5 min. Add either
supernatant to the SPE cartridge and dry the SPE cartridge with 3 mL air. Elute with
500 µL MeOH:water 70:30 containing 3.5 mM pentane sulfonic acid, inject an aliquot.
(The buffer was a mixture of 85 mM pH 3.0 citric acid monohydrate containing 81 mM
NaOH and 60 mM HCl.)
HPLC VARIABLES
Guard column: 10 µm µBondapak C18
Column: 125 × 4.6 5 µm Kromasil C18 (Higgins Analytical)
Mobile phase: Gradient. MeCN:buffer 30:70 for 11 min, then 32:68 for 4 min (step
gradient), to 40:60 over 0.6 min, maintain at 40:60 for 0.4 min, at 38:62 for 18 min (step
gradient), at 80:20 for 2 min (step gradient), re-equilibrate at 30:70 for 9 min. (Prepare
buffer by adding 800 µL 70% perchloric acid to 1 L water.)
Flow rate: 0.5 for 11 min, 1 for 25 min, 0.5 for 9 min
Injection volume: 500
Detector: UV 235
CHROMATOGRAM
Retention time: 22.1
Internal standard: dimethylamino-3-propyl thiomethylene-5 virginiamycin S (31.0)
Limit of quantitation: 25 pg/mL
OTHER SUBSTANCES
Extracted: metabolites, dalfopristin (12.7), pristinamycin II A (24.1)
KEY WORDS
plasma; SPE; whole blood
REFERENCE
Le Liboux, A.; Pasquier, O.; Montay, G. Simultaneous high-performance liquid chromatographic determination of quinupristin, dalfopristin and their main metabolites in human plasma, J.Chromatogr.B,
1998, 708, 161–168.
Quinupristin
541
SAMPLE
Matrix: formulations
Sample preparation: Inject a 5–10 µL aliquot of the infusion solution.
HPLC VARIABLES
Column: 125 × 4 5 µm LiChrospher-100 RP18
Column Temperature: 40 ± 1
Mobile phase: Gradient. A:B from 0:100 to 66:34 over 42.5 min, return to initial
conditions over 1.5 min, re-equilibrate for 5 min. A was MeCN:buffer 65:35. B was
MeCN:buffer 20:80. The buffer was 30 mM potassium dihydrogen phosphate adjusted
to pH 2.9 with phosphoric acid.
Flow rate: 1.1
Injection volume: 5–10
Detector: UV 254
CHROMATOGRAM
Retention time: 23.9, 23.1, 27.0
Limit of quantitation: 0.12%
OTHER SUBSTANCES
Simultaneous: impurities, dalfopristin (LOQ 0.05%) (8.5)
KEY WORDS
infusion; injection; stability-indicating
REFERENCE
Vasselle, B.; Gousset, G.; Bounine, J.-P. Development and validation of a high-performance liquid chromatographic stability-indicating method for the analysis of Synercid in quality control, stability and
compatibility studies, J.Pharm.Biomed.Anal., 1999, 19, 641–657.
ANNOTATED BIBLIOGRAPHY
Abdel-Hamid, M.E.; Phillips, O.A. LC-MS/MS determination of Synercid injections, J.Pharm.Biomed.
Anal., 2003, 32, 1167–1174. [pristinamycin; quinupristin; dalfopristin]
Rabeprazole
H
N
O
N
S
Molecular formula: C18 H21 N3 O3 S
O
N
Molecular weight: 359.45
CAS Registry No: 117976-89-3, 117976-90-6 (Na salt)
Merck Index: 13, 8181
OCH3
CH3
SAMPLE
Matrix: blood
Sample preparation: Mix 1 mL plasma with 100 µL 1% diethylamine in water, add
100 µL 0.1% diethylamine in MeOH, add 1 mL pH 10.38 Britton–Robinson buffer, add
4 mL ethyl acetate, shake for 10 min, centrifuge at 1500 g for 5 min, repeat extraction
twice. Combine the organic layers and evaporate to dryness under a stream of nitrogen
at 40◦ , reconstitute the residue with 100 µL 0.1% diethylamine in MeOH, inject a 30 µL
aliquot.
HPLC VARIABLES
Column: 150 × 4.6 5 µm Inertsil C8
Column temperature: 40
Mobile phase: MeCN:100 mM pH 7.00 phosphate buffer 28:72, adjusted to pH 7.00 with
phosphoric acid
Flow rate: 1.4
Injection volume: 30
Detector: UV 288
CHROMATOGRAM
Retention time: 6.5
Internal standard: IS735 (5-methyl-2-[{4-(3-methoxypropoxy)-3-methylpyridin-2-yl}
methylsulfinyl]-1H-benzimidazole sodium salt) (10.1)
Limit of quantitation: 5 ng/mL
OTHER SUBSTANCES
Extracted: metabolites
KEY WORDS
pharmacokinetics; plasma
REFERENCE
Nakai, H.; Shimamura, Y.; Kanazawa, T.; Yasuda, S.; Kayano, M. Determination of a new H+ -K+ ATPase
inhibitor (E3810) and its four metabolites in human plasma by high-performance liquid chromatography, J.Chromatogr.B, 1994, 660, 211–220.
SAMPLE
Matrix: formulations
Sample preparation: Weigh out powdered tablet containing 15 mg rabeprazole, dissolve in 100 mL water, filter, inject a 20 µL aliquot.
HPLC VARIABLES
Column: 250 × 4.6 4.6 µm VP-ODS Shim-pack
Mobile phase: MeOH:water 70:30
Flow rate: 2
Injection volume: 20
Detector: UV 284
542
HPLC Methods for Recently Approved Pharmaceuticals. by George Lunn
Copyright 2005 John Wiley & Sons, Ltd. ISBN: 0-471-66941-5
Rabeprazole
543
CHROMATOGRAM
Retention time: 5.2
Limit of detection: 25 ng/mL
Limit of quantitation: 76 ng/mL
OTHER SUBSTANCES
Simultaneous: degradants
KEY WORDS
comparison with HPTLC; validation data
REFERENCE
el-Gindy, A.; El-Yazby, F.; Maher, M.M. Spectrophotometric and chromatographic determination of
rabeprazole in presence of its degradation products, J.Pharm.Biomed.Anal., 2003, 31, 229–242.
ANNOTATED BIBLIOGRAPHY
Mano, N.; Oda, Y.; Takakuwa, S.; Chiku, S.; Nakata, H.; Asakawa, N. Plasma direct injection highperformance liquid chromatographic method for simultaneously determining E3810 enantiomers and
their metabolites by using flavoprotein-conjugated column, J.Pharm.Sci., 1996, 85, 903–907. [chiral;
dog; column-switching]
Takakuwa, S.; Chiku, S.; Nakata, H.; Yuzuriha, T.; Mano, N.; Asakawa, N. Enantioselective high-performance liquid chromatographic assay for determination of the enantiomers of a new anti-ulcer agent,
E3810, in Beagle dog plasma and rat plasma, J.Chromatogr.B, 1995, 673, 113–122. [chiral; LOQ
30 ng/mL]
544
Ractopamine
Ractopamine
OH
N
Molecular formula: C18 H23 NO3
Molecular weight: 301.38
CAS Registry No: 97825-25-7, 90274-24-1 (HCl)
Merck Index: 13, 8184
OH
H
HO
CH3
SAMPLE
Matrix: tissue, urine
Sample preparation: Condition a ChromP SPE cartridge (Supelco) with 6 mL ethyl
acetate, 6 mL MeOH, and 6 mL water. Condition a Screen DAU SPE cartridge (Supelco)
with 2 mL MeOH, 2 mL water, and 2 mL 100 mM pH 6 phosphate buffer. Freeze-dry
and grind 15 g tissue or 200–500 mg retina, add 12 mL MeOH, add 15 mL 2 M pH 5.2
acetate buffer, stir for 30 min, centrifuge at 2000 g for 15 min. Remove the supernatant
and evaporate the MeOH. Mix 10 mL urine with 2 mL 2 M pH 5.2 acetate buffer. Add
the tissue or urine preparations to 400 µL Helix pomatia preparation containing 25
U/µL (Sigma), heat at 60◦ for 15 h, centrifuge, add the supernatant to the ChromP
SPE cartridge, wash with 5 mL hexane, wash with 12 mL hexane:diethyl ether 70:30,
elute with 24 mL diethyl ether. Evaporate the eluate to dryness, reconstitute with 6 mL
100 mM pH 6 phosphate buffer, add to the DAU SPE cartridge, wash with 1 mL 1 M
acetic acid, wash with 6 mL MeOH, elute with 6 mL ethyl acetate:32% ammonium
hydroxide 97:3. Evaporate the eluate to dryness, reconstitute the residue with 50 µL
MeOH:water:acetic acid 5:95:0.5, inject a 10 µL aliquot.
HPLC VARIABLES
Guard column: 10 × 2 5 µm Nucleosil C18AB
Column: 50 × 2 5 µm Nucleosil C18AB
Mobile phase: Gradient. MeOH:0.5% acetic acid from 95:5 to 50:50 over 10 min, to 10:90
over 10 min, maintain at 10:90 for 10 min (?), re-equilibrate at initial conditions for
10 min. (sic).
Flow rate: 0.22
Injection volume: 10
Detector: MS, Micromass QuattroLC, triple quadrupole, nebulizing gas nitrogen 90 L/h,
desolvation gas nitrogen 600 L/h, source 120◦ , desolvation 350◦ , capillary 3.5 kV, sampling cone 25 V, collision gas argon 0.4 µbar, collision energy 15–25 V, m/z 302–284–150
CHROMATOGRAM
Retention time: 8.58
Internal standard: isoxsuprine (m/z 302–284–164–136–121–107–91) (10.35)
Limit of detection: 10 ng/kg
Limit of quantitation: 30 ng/kg
KEY WORDS
kidney; liver; lung; meat; pig; retina; SPE
REFERENCE
Antignac, J.-P.; Marchand, P.; Le Bizec, B.; Andre, F. Identification of ractopamine residues in tissue
and urine samples at ultra-trace level using liquid chromatography-positive electrospray tandem mass
spectrometry, J.Chromatogr.B, 2002, 774, 59–66.
SAMPLE
Matrix: tissue
Sample preparation: Condition a Sep-Pak Alumina A SPE cartridge with ethyl acetate.
Add IS to tissue, extract 10 g homogenized tissue three times with 20 mL portions of
MeOH, and combine the extracts. Evaporate an 8 mL aliquot to dryness, reconstitute
Ractopamine
545
with 1 mL 25 mM pH 5.0 sodium acetate buffer, add 20 µL β-glucuronidase (Helix
pomatia) (Sigma), heat at 65◦ for 2 h, mix with 2 mL 25 mM pH 10.3 sodium borate
buffer, extract three times with 7 mL portions of ethyl acetate, add the combined
extracts to the alumina SPE cartridge, wash with ethyl acetate, dry under vacuum,
elute with 10 mL MeOH. Evaporate the eluate to dryness, reconstitute the residue with
4 mL 1 M acetic acid, add to a 6 mL 500 mg Oasis MCX SPE cartridge, elute with 4 mL
2% ammonia in MeOH. Evaporate the eluate to dryness, reconstitute the residue with
500 µL 2% acetic acid, inject a 100 µL aliquot.
HPLC VARIABLES
Column: 250 × 4.6 5 µm Supelcosil LC-18-DB
Mobile phase: MeCN:water:glacial acetic acid 32:68:2 containing 0.87 g/L 1-pentanesulfonic acid
Flow rate: 1
Injection volume: 100
Detector: F ex 226 em 306
CHROMATOGRAM
Retention time: 5.4
Internal standard: ritodrine (4.7)
Limit of quantitation: 1 ng/g
KEY WORDS
cow; pig; muscle; SPE
REFERENCE
Shishani, E.; Chai, S.C.; Jamokha, S.; Aznar, G.; Hoffman, M.K. Determination of ractopamine in animal
tissues by liquid chromatography-fluorescence and liquid chromatography/tandem mass spectrometry,
Anal.Chim.Acta, 2003, 483, 137–145.
SAMPLE
Matrix: tissue
Sample preparation: Condition a Sep-Pak Alumina A SPE cartridge with ethyl acetate.
Add IS to tissue, extract 10 g homogenized tissue three times with 20 mL portions of
MeOH, and combine the extracts. Evaporate an 8 mL aliquot to dryness, reconstitute
with 1 mL 25 mM pH 5.0 sodium acetate buffer, add 20 µL β-glucuronidase (Helix
pomatia) (Sigma), heat at 65◦ for 2 h, mix with 2 mL 25 mM pH 10.3 sodium borate
buffer, extract three times with 7 mL portions of ethyl acetate, add the combined
extracts to the alumina SPE cartridge, wash with ethyl acetate, dry under vacuum,
elute with 10 mL MeOH. Evaporate the eluate to dryness, reconstitute the residue with
4 mL 1 M acetic acid, add to a 6 mL 500 mg Oasis MCX SPE cartridge, elute with 4 mL
2% ammonia in MeOH. Evaporate the eluate to dryness, reconstitute the residue with
200 µL MeOH for MS detection, inject an aliquot.
HPLC VARIABLES
Column: 50 × 2.1 5 µm Discovery RP-Amide C-16 (Supelco)
Mobile phase: Gradient. MeCN:pH 4.5 ammonium acetate buffer from 5:95 to 100:0
over 2 min.
Flow rate: 0.5
Detector: MS, PE Sciex API-III, positive mode, nebulizer 500◦ , interface 55◦ , nebulizer
gas 0.6 L/min, auxiliary flow 2.8 L/min, collision gas argon, m/z 302–164, 302–107,
302–121
CHROMATOGRAM
Internal standard: ritodrine (m/z 288–150)
Limit of quantitation: 1 ng/g
546
Ractopamine
KEY WORDS
cow; pig; muscle; SPE
REFERENCE
Shishani, E.; Chai, S.C.; Jamokha, S.; Aznar, G.; Hoffman, M.K. Determination of ractopamine in animal
tissues by liquid chromatography-fluorescence and liquid chromatography/tandem mass spectrometry,
Anal.Chim.Acta, 2003, 483, 137–145.
ANNOTATED BIBLIOGRAPHY
Turberg, M.P.; Macy, T.D.; Lewis, J.J.; Coleman, M.R. Determination of ractopamine hydrochloride in
swine, cattle, and turkey feeds by liquid chromatography with coulometric detection, J.AOAC Int.,
1994, 77, 840–847. [SPE; LOQ 1.25 ppm]
Turberg, M.P.; Macy, T.D.; Lewis, J.J.; Coleman, M.R. Determination of ractopamine hydrochloride in
swine and turkey tissues by liquid chromatography with coulometric detection, J.AOAC Int., 1995, 78,
1394–1402. [liver; kidney; muscle; fat; SPE; LOD 0.5 ppb]
Turberg, M.P.; Rodewald, J.M.; Coleman, M.R. Determination of ractopamine in monkey plasma
and swine serum by high-performance liquid chromatography with electrochemical detection,
J.Chromatogr.B, 1996, 675, 279–285. [SPE; LOQ 2 ng/mL]
547
Raloxifene
Raloxifene
HO
S
OH
Molecular formula: C28 H27 NO4 S
Molecular weight: 473.59
CAS Registry No: 84449-90-1, 82640-04-8 (HCl)
Merck Index: 13, 8190
O
O
N
SAMPLE
Matrix: blood
Sample preparation: Mix 100 ng IS with 300 µL plasma, add 600 µL acetone, mix, let
stand on ice for 10 min, centrifuge. Evaporate the supernatant to dryness under reduced
pressure, reconstitute the residue with 100 µL 500 mM pH 9.0 sodium carbonate and
100 µL dichloromethane, mix thoroughly, centrifuge, inject a 40 µL aliquot of the
organic layer.
HPLC VARIABLES
Column: 10 µm Zorbax C8 or LiChrosorb C8
Mobile phase: MeCN:100 mM pH 4.0 sodium acetate 50:50
Flow rate: 2
Injection volume: 40
Detector: E, BAS LC-3, glassy carbon electrode +0.65 V
CHROMATOGRAM
Retention time: 5.8
Internal standard: (4-(2-(dimethylamino)ethoxy)phenyl)(6-hydroxy-2-(4-hydroxyphenyl)benzo[b]thien-3-yl)methanone (3.9)
KEY WORDS
dog; monkey; plasma; rat
REFERENCE
Lindstrom, T.D.; Whitaker, N.G.; Whitaker, G.W. Disposition and metabolism of a new benzothiophene
antiestrogen in rats, dogs and monkeys, Xenobiotica, 1984, 14, 841–847.
SAMPLE
Matrix: solutions
Sample preparation: Inject a 1 µL aliquot of a solution in MeOH:water 10:90.
HPLC VARIABLES
Column: 20 × 2 5 µm DASH BetaBasic C8 (ThermoHypersil Keystone)
Mobile phase: Gradient. A was MeCN:water:formic acid 5:95:0.1. B was MeCN:water:
formic acid 95:5:0.1. A:B 100:0 for 0.2 min, to 0:100 over 1.5 min.
Flow rate: 1.5
Injection volume: 1
Detector: MS, PE Sciex API-3000, TurboIonspray, electrospray 4500 V, ring 290 V,
orifice 60 V, drying gas 400◦ , 20% of column effluent entered the detector, m/z 474.1–112
CHROMATOGRAM
Retention time: 0.97
OTHER SUBSTANCES
Simultaneous: amitriptyline (m/z 278.3–233) (1.1), aprepitant (MK-869) (m/z
535.3–277) (1.4), diclofenac (m/z 296.1–215) (1.35), enoxacin (m/z 321.2–234) (0.7),
548
Raloxifene
fenofibrate (m/z 360.9–233) (1.6), finasteride (m/z 373.2–317) (1.2), indinavir (m/z
614.4–421) (0.93), pioglitazone (357.2–134) (0.87)
REFERENCE
Romanyshyn, L.A.; Tiller, P.R. Ultra-short columns and ballistic gradients: considerations for ultra-fast
chromatographic liquid chromatographic-tandem mass spectrometric analysis, J.Chromatogr.A, 2001,
928, 41–51.
Ramosetron
Ramosetron
CH3
N
H
N
Molecular formula: C17 H17 N3 O
Molecular weight: 279.34
CAS Registry No: 132036-88-5
Merck Index: 13, 8195
549
O
N
SAMPLE
Matrix: blood, urine
Sample preparation: Shake 1 (animal) or 2 (human) mL plasma or 1 mL urine with
200 µL 100 µg/mL IS in water, 1 mL saturated sodium bicarbonate, and 5 mL ethyl
acetate for 15 min, centrifuge at 1200 g for 10 min. Remove the organic layer and add
it to 2.5 mL 400 mM HCl, shake for 15 min, centrifuge at 800 g for 5 min. Remove the
aqueous layer, add 2 mL saturated sodium bicarbonate solution to the aqueous layer,
stir, let stand at room temperature for 20 min, add 4.5 mL ethyl acetate, shake for
15 min, centrifuge at 800 g for 5 min. Evaporate the organic layer to dryness under
reduced pressure, reconstitute the residue with 100 µL mobile phase, inject a 60 µL
aliquot.
HPLC VARIABLES
Column: 250 × 4.6 TSK-gel ODS-80-Tm (Tosoh) (plasma) or 250 × 4.6 Cosmosil 5C18AR
(Nacalai Tesque) (urine)
Mobile phase: MeCN:100 mM potassium dihydrogen phosphate:100 mM phosphoric
acid 25:37.5:37.5 (plasma) or MeCN:100 mM potassium dihydrogen phosphate:100 mM
phosphoric acid 33:33:33 containing 400 µM sodium dodecyl sulfate (urine)
Flow rate: 1
Injection volume: 60
Detector: UV 311
CHROMATOGRAM
Retention time: 10
Internal standard: GSA 110 (9-methyl-3-[(5-methylimidazole-4-yl)methyl]-2,3,4,9tetrahydrothiopyrano[2,3-b]indole-4-one fumarate) (14)
Limit of detection: 200 pg/mL (S/N 3, human plasma), 500 pg/mL (rat, dog plasma),
1 ng/mL (human urine)
KEY WORDS
dog; human; plasma; rat
REFERENCE
Miura, H.; Takeshige, T.; Kobayashi, S.-i.; Higuchi, S. A simple method for the determination of YM060
in plasma and urine by high performance liquid chromatography, Biomed.Chromatogr., 1994, 8,
103–104.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 150 × 4 Crestpak C18S-10
Mobile phase: MeCN:buffer 15:85 (The buffer was 3% triethylamine adjusted to pH 2.0
with phosphoric acid.)
Flow rate: 2
Detector: Radioactivity (11 C)
550
Ramosetron
CHROMATOGRAM
Retention time: 7.3
REFERENCE
Ishiwata, K.; Ishii, K.; Ishii, S.-I.; Senda, M. Synthesis of 5-HT3 receptor antagonists, [11 C]Y-25130 and
[11 C]YM060, Appl.Radiat.Isot., 1995, 46, 907–910.
551
Rapacuronium bromide
Rapacuronium bromide
Br−
O
Molecular formula: C37 H61 BrN2 O4
Molecular weight: 677.81
CAS Registry No: 156137-99-4
Merck Index: 13, 8201
CH3 O
CH3
N
H
O
O
N+
H
H
H
CH3
SAMPLE
Matrix: bile, blood, stoma fluid, tissue, urine
Sample preparation: Caution! The method as described is applied to and validated
for rocuronium. However, Wierda et al. (Wierda,J.M.K.H.; Beaufort,A.M.; Kleef,U.W.;
Smeulers,N.J.; Agoston,S. Preliminary investigations of the clinical pharmacology of
three short-acting non-depolarizing neuromuscular blocking agents, Org 9453, Org
9489 and Org 9487. Can.J.Anaesth. 1994, 41, 213–220.) state that ‘‘the compounds
were determined by means of the HPLC method described for rocuronium bromide,
which was modified and validated for . . . Org 9487’’ [rapacuronium]. Homogenize
(Ultra-Turrax) 1 g tissue with 9 mL 1 M sodium dihydrogen phosphate for 10 min.
Acidify 1 mL plasma, urine, or bile with 200 µL 1 M sodium dihydrogen phosphate.
Homogenize 1 mL stoma fluid with 200 µL 1 M sodium dihydrogen phosphate. Make
up 50–1000 µL plasma, 200–1000 µL urine, 5–200 µL bile, 1000 µL stoma fluid, or
100–1000 µL tissue homogenate to 2 mL with water, add 1 mL buffer, add 150 ng IS,
add 7 mL dichloromethane, vortex for 15 s, centrifuge at 740 g for 5 min. Remove the
lower organic layer and evaporate it to dryness under a stream of air at 37◦ , reconstitute
the residue in 200 µL mobile phase, inject a 100 µL aliquot (or less). (The buffer was
prepared by mixing 6 mL of an aqueous solution containing 7.505 mg/mL glycine and
5.85 mg/mL NaCl, with 4 mL 100 mM NaOH and 6.2 g KI.)
HPLC VARIABLES
Guard column: 4 × 6 µBondapak C18
Column: 150 × 3.9 5 µm Lichrospher 100-RP18
Mobile phase: Dioxane:buffer 16:84 (Caution! Dioxane is a carcinogen!) (The buffer was
100 mM sodium dihydrogen phosphate containing 0.11 mM 9,10-dimethoxyanthracene2-sulfonate and 0.11 mM 1-heptanesulfonic acid, pH adjusted to 3.0 with orthophosphoric acid. After each series of analyses, flush column with 15 mL water and 75 mL
MeOH.)
Flow rate: 1
Injection volume: 100
Detector: F ex 385 em 452 following post-column extraction. The column effluent mixed
with dichloroethane pumped at 1 mL/min and the mixture flowed through a 1 m ×
0.25 mm ID stainless steel coil to a phase separator (Organon International) and
then the organic phase flowed through the detector (J.Chromatogr. 1987, 421, 327;
Anal.Chim.Acta 1987, 192, 267).
CHROMATOGRAM
Retention time: 9 (for rocuronium)
Internal standard: 1-(3α,17β-dihydroxy-2β-morpholino-5α-androstan-16β-yl)-1-methylpiperidinium bromide (Org 7402, Organon) (21)
Limit of detection: 3 ng (plasma), 4 ng (urine, bile), 5 ng (tissue)
Limit of quantitation: 10 ng/mL (plasma), 25 ng/mL (urine), 100 ng/mL (bile), 20 ng/mL
(stoma fluid)
552
Rapacuronium bromide
OTHER SUBSTANCES
Simultaneous: cefotaxime, ceftazidime, ceftriaxone, cefuroxime, cefamandole, cephradine, dixyrazine, meperidine, metocurine, metoprolol, sulfamethoxazole, trimethoprim
Interfering: alizapride, atracurium, ketamine, ketogan, lidocaine, metoclopramide,
nimodipine, prochlorperazine, tubocurarine
aprotinin, atropine, bupivacaine, chlorpromazine,
dalteparin, dexamethasone, diazepam, dopamine, droperidol, etomidate, fentanyl,
furosemide, gallamine, haloperidol, midazolam, morphine, neostigmine, nitroglycerin,
nitroprusside, oxytocin, pancuronium, pentobarbital, phenylephrine, phenytoin,
pipecuronium, piperacillin, promethazine, propofol, ranitidine, succinylcholine,
sufentanil, terbutaline, thiopental, vecuronium, verapamil
Noninterfering: alfentanil,
KEY WORDS
human; dog; plasma; liver; lung
REFERENCE
Kleef, U.W.; Proost, J.H.; Roggeveld, J.; Wierda, J.M.K.H. Determination of rocuronium and its putative
metabolites in body fluids and tissue homogenates, J.Chromatogr., 1993, 621, 65–76.
553
Remifentanil
Remifentanil
Molecular formula: C20 H28 N2 O5
Molecular weight: 376.45
CAS Registry No: 132875-61-7
Merck Index: 13, 8216
O
N
OCH3
N
H3C
O
OCH3
O
SAMPLE
Matrix: blood
Sample preparation: Mix 500 µL whole blood with 10 µL 50% citric acid and 25 µL
500 ng/mL IS in 1 mM HCl, add 500 µL 100 mM pH 7.4 phosphate buffer, vortex until
homogeneous, add 2 mL dichloromethane, shake mechanically for 10 min, centrifuge at
13 000 rpm for 10 min. Evaporate the lower dichloromethane layer to dryness under a
stream of nitrogen at room temperature, reconstitute the residue with 125 µL mobile
phase, inject a 20 µL aliquot.
HPLC VARIABLES
Column: 33 × 4.6 Pecosphere C18 (Perkin-Elmer)
Mobile phase: MeCN:chloroform 50:50 containing 2 mM ammonium acetate (Caution!
Chloroform is a carcinogen!)
Flow rate: 0.3
Injection volume: 20
Detector: MS, PE Sciex API-III Plus triple quadrupole, turbo ionspray, positive ion mode,
turbo 200◦ , orifice 60 V, auxiliary gas nitrogen at 4 L/min, curtain gas at 1.2 L/min, m/z
377–228
CHROMATOGRAM
Retention time: 1.3
Internal standard: d4 -remifentanil (381–232)
Limit of quantitation: 0.1 ng/mL
KEY WORDS
whole blood
REFERENCE
Bender, J.; van den Elshout, J.; Selinger, K.; Broeders, G.; Dankers, J.; van der Heiden, C. Determination
of remifentanil in human heparinised whole blood by tandem mass spectrometry with short-column
separation, J.Pharm.Biomed.Anal., 1999, 21, 559–567.
SAMPLE
Matrix: blood
Sample preparation: Condition a Bond Elut phenyl SPE cartridge with 1 mL MeOH
and 1 mL 50 mM pH 3 potassium phosphate buffer. Add 500 µL plasma and 750 µL
800 ng/mL IS in 50 mM pH 3 potassium phosphate buffer to the SPE cartridge, wash
with 1 mL 50 mM pH 3 potassium phosphate buffer, wash with 1 mL MeCN, elute with
1 mL MeOH. Evaporate the eluate to dryness under reduced pressure, reconstitute the
residue with 200 µL mobile phase, inject a 50–100 µL aliquot.
HPLC VARIABLES
Guard column: 4 × 3 C1
Column: 150 × 4.6 5 µm Spherisorb C1
Mobile phase: MeCN:MeOH:50 mM pH 3 potassium phosphate buffer:water 18:12:4.
8:65.2
Flow rate: 1.5
554
Remifentanil
Injection volume: 50–100
Detector: UV 210
CHROMATOGRAM
Retention time: 9
Internal standard: GI97559 (ethyl 3-{4-methoxycarbonyl-4-[(1-oxopropyl)-phenylamino]-1-piperidine}propanoate) (10.5)
Limit of detection: 0.5 ng/mL
Limit of quantitation: 8 ng/mL
OTHER SUBSTANCES
Extracted: demethoxyremifentanil (metabolite) (4)
KEY WORDS
dog; pharmacokinetics; plasma; SPE
REFERENCE
Kabbaj, M.; Varin, F. Simultaneous solid-phase extraction combined with liquid chromatography with
ultraviolet absorbance detection for the determination of remifentanil and its metabolite in dog plasma,
J.Chromatogr.B, 2003, 783, 103–111.
ANNOTATED BIBLIOGRAPHY
Haidar, S.H.; Liang, Z.; Selinger, K.; Hamlett, L.; Eddington, N.D. Determination of remifentanil, an
ultra-short-acting opioid anesthetic, in rat blood by high-performance liquid chromatography with
ultraviolet detection, J.Pharm.Biomed.Anal., 1996, 14, 1727–1732. [LOQ 2.5 ng/mL]
Selinger, K.; Lanzo, C.; Sekut, A. Determination of remifentanil in human and dog blood by HPLC with
UV detection, J.Pharm.Biomed.Anal., 1994, 12, 243–248. [LOQ 1 ng/mL]
Vishwanathan, K.; Stewart, J.T. HPLC determination of a propofol and remifentanil mixture,
J.Liq.Chromatogr.Rel.Technol., 1999, 22, 923–931. [LOD 200 ng/mL]
555
Repaglinide
Repaglinide
CH3
H3C
O
Molecular formula: C27 H36 N2 O4
Molecular weight: 452.58
CAS Registry No: 135062-02-1
Merck Index: 13, 8220
NH
COOH
O
CH3
N
SAMPLE
Matrix: blood
Sample preparation: Centrifuge plasma at 13 000 g for 2 min and dilute with 2 vol
of 200 mM HCl, inject a 160 µL aliquot onto column A and elute to waste with water;
after 3 min, backflush the contents of column A onto column B with mobile phase,
monitor the effluent from column B. At the end of the separation, wash column A with
MeCN:MeOH:dioxane 24:68:8. (Caution! Dioxane is a carcinogen!)
HPLC VARIABLES
Column: A 17 × 2.9 30–40 µm Perisorb RP-2 (Merck); B 17 × 4.6 5 µm ODS-Hypersil +
125 × 4 5 µm LiChrospher 100 RP-18
Mobile phase: MeCN:MeOH:dioxane:buffer 18.24:51.68:6.08:24 (Caution! Dioxane is a
carcinogen!) (The buffer was 3 g potassium dihydrogen phosphate and 0.5 g lithium
perchlorate in 1 L water, adjusted to pH 2.7 with orthophosphoric acid.)
Flow rate: 1
Injection volume: 160
Detector: E, Waters 460, 1.04 V (to prevent contamination, column effluent passes
through the detector only from 7.5–13.8 min)
CHROMATOGRAM
Retention time: 9
Limit of detection: 5 ng/mL
KEY WORDS
column-switching; plasma
REFERENCE
Greischel, A.; Beschke, K.; Rapp, H.; Roth, W. Quantitation of the new hypoglycaemic agent AG-EE 388
ZW in human plasma by automated high-performance liquid chromatography with electrochemical
detection, J.Chromatogr., 1991, 568, 246–252.
SAMPLE
Matrix: formulations
Sample preparation: Dissolve powdered tablet containing 25 mg repaglinide and
25 mg nimesulide in 25 mL MeOH, shake well, filter (0.45 µm), dilute filtrate with
mobile phase to a concentration of 1 µg/mL, inject a 20 µL aliquot.
HPLC VARIABLES
Guard column: 20 mm long Shim-pack ODS (Shimadzu)
Column: 150 × 4.6 5 µm Shim-pack RP-C18
Column temperature: 30
Mobile phase: MeOH:buffer 50:50 (The buffer was 0.1% triethylamine adjusted to pH 7
with 1% orthophosphoric acid.)
Flow rate: 1
Injection volume: 20
Detector: UV 235
556
Repaglinide
CHROMATOGRAM
Retention time: 3.4
Internal standard: nimesulide (2.04)
Limit of quantitation: 100 ng/mL
KEY WORDS
tablets
REFERENCE
Gandhimathi, M.; Ravi, T.K.; Renu, S.K. Determination of repaglinide in pharmaceutical formulations
by HPLC with UV detection, Anal.Sci., 2003, 19, 1675–1677.
ANNOTATED BIBLIOGRAPHY
Niemi, M.; Neuvonen, P.J.; Kivistö, K.T. The cytochrome P4503A4 inhibitor clarithromycin increases
the plasma concentrations and effects of repaglinide, Clin.Pharmacol.Ther., 2001, 70, 58–65. [LC-MS;
LOQ 50 pg/mL; indomethacin is internal standard]
Reddy, K.V.S.R.K.; Babu, J.M.; Mathad, V.T.; Eswaraiah, S.; Reddy, M.S.; Dubey, P.K.; Vyas, K. Impurity profile study of repaglinide, J.Pharm.Biomed.Anal., 2003, 32, 461–467.
Thomsen, M.S.; Chassard, D.; Evène, E.; Nielsen, K.K.; Jorgensen, M. Pharmacokinetics of repaglinide
in healthy Caucasian and Japanese subjects, J.Clin.Pharmacol., 2003, 43, 23–28. [LC-MS; LOQ
200 pg/mL; SPE]
Ricinoleic acid
Ricinoleic acid
Molecular formula: C18 H34 O3
557
CH3(CH2)5
(CH2)6COOH
HO
Molecular weight: 289.46
CAS Registry No: 141-22-0
Merck Index: 13, 8295
SAMPLE
Matrix: blood
Sample preparation: Vortex 20 µL plasma, 10 µL 2 mg/mL margaric acid in MeOH,
and 200 µL 500 mM KOH in EtOH for 30 s, centrifuge at 2500 g for 10 min. Remove
the supernatant and heat it at 100◦ for 30 min, cool, add 200 µL 1 M HCl, add 2 mL
chloroform (Caution! Chloroform is a carcinogen!), mix vigorously for 5 min, centrifuge
at 460 g for 5 min. Remove the organic layer and evaporate it to dryness under reduced
pressure at 43◦ , reconstitute the residue in 500 µL benzene (Caution! Benzene is
a carcinogen!), sonicate for 1 min, add 500 µL 2% oxalyl chloride in benzene, heat
at 70◦ for 30 min, evaporate to dryness under reduced pressure, reconstitute with
100 µL benzene, add 100 µL 40 mM 1-naphthylamine (Caution! 1-Naphthylamine is a
carcinogen!) in benzene, vortex for 30 s, heat at 37◦ for 30 min, evaporate to dryness,
reconstitute with MeOH:MeCN:water 72:13:15, vortex for 20 s, inject a 20 µL aliquot.
(This method was developed for Cremophor EL, which is saponified to ricinoleic acid and
then derivatized with 1-naphthylamine. However, the method should work for ricinoleic
acid in plasma.)
HPLC VARIABLES
Column: two 100 × 3 5 µm Spherisorb ODS-I glass columns in series
Mobile phase: MeCN:MeOH:10 mM pH 7.0 potassium phosphate buffer 13:72:15
Flow rate: 0.4
Injection volume: 20
Detector: UV 280
CHROMATOGRAM
Retention time: 10.0
Internal standard: margaric acid (27.8)
Limit of detection: 0.005%
Limit of quantitation: 0.01%
OTHER SUBSTANCES
Simultaneous: arachidonic acid, linoleic acid, linolenic acid, myristic acid, oleic acid,
palmitic acid, palmitoleic acid, stearic acid
KEY WORDS
derivatization; human; mouse; plasma
REFERENCE
Sparreboom, A.; van Tellingen, O.; Huizing, M.T.; Nooijen, W.J.; Beijnen, J.H. Determination of polyoxyethyleneglycerol triricinolate 35 (Cremophor EL) in plasma by pre-column derivatization and
reversed-phase high-performance liquid chromatography, J.Chromatogr.B, 1996, 681, 355–362.
558
Rifaximin
Rifaximin
Molecular formula: C43 H51 N3 O11
Molecular weight: 785.88
CAS Registry No: 80621-81-4
Merck Index: 13, 8304
CH3
O
CH3
HO
CH3
OH O
OH OH
CH3
O
H3C
CH3O
CH3
N
H
CH3
N
O
N
O
CH3
O
CH3
SAMPLE
Matrix: blood, urine
Sample preparation: Mix 1 mL plasma or 2 mL urine with 100 µL 1 µg/mL IS solution,
add 6 mL ethyl acetate, shake horizontally for 10 min, centrifuge. Remove the organic
layer and, for urine samples only, wash with 1 mL 200 mM NaOH. Evaporate a 5 mL
aliquot of the organic layer to dryness under reduced pressure, reconstitute the residue
with 200 µL MeCN:mobile phase 20:80, vortex, sonicate for 10 min, inject a 40 µL
aliquot.
HPLC VARIABLES
Column: 150 × 4.6 5 µm Kromasil C18
Mobile phase: MeCN:water:500 mM pH 7.2 phosphate buffer 47:48:5 containing 1 g/L
tetrabutylammonium phosphate
Injection volume: 40
Detector: E, ESA Coulochem Model 5100A, guard cell Model 5020 + 0.7 V, analytical
cell Model 5010, channel 1 + 0.28 V, channel 2 + 0.6 V
CHROMATOGRAM
Retention time: 7.5
Internal standard: 17α-estradiol (6.0)
Limit of quantitation: 2 ng/mL
KEY WORDS
plasma
REFERENCE
Descombe, J.J.; Dubourg, D.; Picard, M.; Palazzini, E. Pharmacokinetic study of rifaximin after oral
administration in healthy volunteers, Int.J.Clin.Pharmacol.Res., 1994, 14, 51–56.
559
Rilmazafone
Rilmazafone
CH3
O
N
CH3
Molecular formula: C21 H20 Cl2 N6 O3
N
Molecular weight: 475.34
CAS Registry No: 99593-25-6
Merck Index: 13, 8305
N
N
N
O
Cl
H
NH2
O
Cl
SAMPLE
Matrix: blood
Sample preparation: Mix 1 mL plasma with 100 µL 1 M pH 5.0 acetate buffer and
5 mL EtOH, centrifuge at 1000 rpm for 15 min. Evaporate 5 mL of the supernatant to
dryness, reconstitute the residue with 5 mL 100 mM pH 5.0 acetate buffer, add 4 mL
of this solution to a Sep-Pak C18 SPE cartridge, elute with 5 mL MeOH, evaporate
the eluate to dryness, reconstitute the residue with 200 µL 100 mM pH 11.6 sodium
carbonate buffer, inject a 5–10 µL aliquot.
HPLC VARIABLES
Guard column: 50 × 4 Nucleosil 10C18
Column: 150 × 4.6 Nucleosil 5C18
Mobile phase: MeCN:buffer 35:65 (Prepare the buffer by dissolving one vial of Waters
PIC reagent A in 650 mL water.)
Flow rate: 1.5
Injection volume: 5–10
Detector: UV 254
CHROMATOGRAM
Retention time: 7
OTHER SUBSTANCES
Extracted: metabolites
KEY WORDS
plasma; rat
REFERENCE
Matsubara, T.; Touchi, A.; Yamada, N.; Sugeno, K. Induction of rat liver microsomal drug-metabolizing
enzymes by a new sleep inducer 450191-S and plasma levels of 450191-S-metabolites, J.Pharmacobiodyn., 1986, 9, 249–256.
560
Risedronate sodium
Risedronate sodium
O
N
HO
Molecular formula: C7 H10 NNaO7 P2
Molecular weight: 305.09
CAS Registry No: 115436-72-1, 105462-24-6 (free acid)
Merck Index: 13, 8315
OH
P
P
O
OH
OH
ONa
SAMPLE
Matrix: urine
Sample preparation: Condition a 1 mL 30 mg HLB SPE cartridge (Waters) with
2 mL MeOH, 1 mL water, and 1 mL 10 mM mM 1-octyltriethylammonium phosphate.
Immediately upon collection, add 25 µL 6 M HCl to each 1 mL urine, freeze at – 20◦ until
ready to analyze. Mix 5 mL urine with 25 µL 125 µg/mL IS in water, add 50 µL 1.25 M
calcium chloride solution, add 65 µL 30% NaOH, vortex; if no precipitate is observed, add
10 µL aliquots of 30% NaOH until a visible precipitate forms, centrifuge at 5020 g for
10 min, discard the supernatant, dissolve the precipitate in 50 µL 1 M HCl; if necessary,
add 25 µL aliquots of 1 M HCl until the precipitate is completely dissolved, dilute with
5 mL water, add 50 µL NaOH to produce a second precipitate, centrifuge, discard the
aqueous layer. Repeat the process. Dissolve the third precipitate in 500 µL 50 mM
ethylene glycol-bis(β-aminoethyl ether)-N, N, N ′ ,N ′ -tetraacetic acid (EGTA), sonicate
for 5 min, vortex, add 4.5 mL water, add 100 µL 500 mM 1-octyltriethylammonium
phosphate, vortex for 10 s, add to the SPE cartridge at 0.3–0.5 mL/min, wash with
1 mL water, wash with 1 mL MeOH:water 5:95, elute with 1 mL MeOH by centrifuging
at 70 g for 5 min. Evaporate the eluate to dryness under a stream of nitrogen at 50◦ ,
reconstitute the residue with 500 µL 10 mM pH 6.25 sodium phosphate containing
1 mM etidronate, inject a 100 µL aliquot onto column A and elute to waste with mobile
phase A; after 3.5 min, divert the effluent from column A onto column B and continue to
elute with mobile phase A; after another 3 min, remove column A from the circuit, elute
column B with mobile phase B, monitor the effluent from column B.
HPLC VARIABLES
Column: A 50 × 4.6 3.5 µm X-Terra RP 18 (Waters); B 150 × 4.6 4 µm Synergi Polar RP
(Phenomenex)
Column temperature: 30 (A and B)
Mobile phase: A MeCN:10 mM sodium phosphate containing 5 mM 1-octyltriethylammonium phosphate and 1 mM etidronate 8:92, apparent pH 6.25; B MeCN:11 mM
sodium phosphate containing 5 mM 1-octyltriethylammonium phosphate and 1.1 mM
etidronate 13:87, apparent pH 6.25
Flow rate: 1
Injection volume: 100
Detector: UV 262
CHROMATOGRAM
Retention time: 18.5
Internal standard: 2-(3-pyridinyl)oxy-1-hydroxymethane diphosphonic acid tetraammonium salt (23)
Limit of quantitation: 7.5 ng/mL
KEY WORDS
column-switching; SPE
REFERENCE
Vallano, P.T.; Shugarts, S.B.; Kline, W.F.; Woolf, E.J.; Matuszewski, B.K. Determination of risedronate
in human urine by column-switching ion-pair high-performance liquid chromatography with ultraviolet
detection, J.Chromatogr.B, 2003, 794, 23–33.
561
Rizatriptan
Rizatriptan
Molecular formula: C15 H19 N5
Molecular weight: 269.34
CAS Registry No: 144034-80-0, 145202-66-0 (benzoate)
Merck Index: 13, 8324
H
N
N
N
N
N
CH3
CH3
SAMPLE
Matrix: blood
Sample preparation: Condition a 1 mL 100 mg Bond Elut C2 SPE cartridge with two
1 mL portions of MeOH and two 1 mL portions of water. Mix 1 mL plasma with 100 µL
100 ng/mL IS in water, add to the SPE cartridge, wash with 1 mL water, wash with two
1 mL portions of MeOH:water 30:70, elute with 1 mL MeOH:10 mM pH 5.0 ammonium
acetate 60:40. Evaporate the eluate to dryness at 50◦ , reconstitute the residue with
200 µL mobile phase, sonicate for 10 min, vortex, inject a 25 µL aliquot.
HPLC VARIABLES
Column: 250 × 4.6 5 µm Spherisorb CN
Mobile phase: MeCN:MeOH:water:trifluoroacetic acid 54:4:42:0.1
Flow rate: 1
Injection volume: 25
Detector: MS, PE Sciex API-III triple quadrupole, nebulizer probe 500◦ , positive ionization, nebulizer gas at 80 psi, auxiliary gas at 2 L/min, corona discharge +3 µA,
0.1143 mm orifice, orifice 45 V, collision gas argon, m/z 270–201
CHROMATOGRAM
Retention time: 5
Internal standard: L-743,214 (N, N-diethyl analogue) (m/z 298–229) (5.5)
Limit of quantitation: 0.5 ng/mL
KEY WORDS
pharmacokinetics; plasma; SPE
REFERENCE
McLoughlin, D.A.; Olah, T.V.; Ellis, J.D.; Gilbert, J.D.; Halpin, R.A. Quantitation of the 5HT1D agonists MK-462 and sumatriptan in plasma by liquid chromatography-atmospheric pressure chemical
ionization mass spectrometry, J.Chromatogr.A, 1996, 726, 115–124.
562
Rofecoxib
Rofecoxib
O
O
Molecular formula: C17 H14 O4 S
Molecular weight: 314.36
CAS Registry No: 162011-90-7
Merck Index: 13, 8330
H3C
S
O
O
SAMPLE
Matrix: blood
Sample preparation: Add 50 µL 100 mM NaOH to 100 µL serum dropwise while
gently vortexing, mix thoroughly, add 1 mL 600 ng/mL IS in ethyl acetate, vortex for
30 s, centrifuge at 1200 g for 10 min. Evaporate the organic layer to dryness under a
stream of nitrogen at 37◦ , reconstitute the residue with 100 µL mobile phase, inject a
40 µL aliquot.
HPLC VARIABLES
Column: 150 × 3.9 5 µm Novapak C18
Mobile phase: MeCN:50 mM sodium acetate 40:60, pH 6.4
Flow rate: 1
Injection volume: 40
Detector: UV 273
CHROMATOGRAM
Retention time: 3.23
Internal standard: 5-ethyl-5-tolyl barbituric acid (2.17)
Limit of quantitation: 20 ng/mL
OTHER SUBSTANCES
Noninterfering: EDTA, heparin, ibuprofen, indomethacin, naproxen
KEY WORDS
plasma
REFERENCE
Aravind, M.K.; Prescilla, R.; Ofenstein, J.P. A rapid and sensitive high-performance liquid chromatography assay for rofecoxib in human serum, J.Chromatogr.Sci., 2002, 40, 26–28.
SAMPLE
Matrix: blood
Sample preparation: Mix 1 mL plasma with 100 µL 10 µg/mL IS in MeCN and 1 mL
pH 9.8 carbonate buffer, add 8 mL MTBE, rotate for 15 min. Evaporate the organic
layer to dryness, reconstitute the residue with 500 µL MeCN, vortex for 1 min, add
50 µL water, vortex and sonicate for 15 min, inject a 25 µL aliquot.
HPLC VARIABLES
Guard column: 20 × 3 YMC ODS AQ
Column: 100 × 3 3 µm YMC ODS AQ
Mobile phase: MeCN:water 50:50
Flow rate: 0.4
Injection volume: 25
Detector: MS, PE Sciex API-III Plus triple quadrupole, ionspray, negative ionization,
orifice – 40 V, corona – 40 µA, nebulizer gas air at 80 psi, collision gas argon, m/z
313–257
Rofecoxib
563
CHROMATOGRAM
Retention time: 5
Internal standard: 4-(4-methanesulfonylphenyl)-3-(4-methylphenyl)-5H-furan-2-one
(m/z 327–271) (6.5)
Limit of quantitation: 100 pg/mL
KEY WORDS
pharmacokinetics; plasma
REFERENCE
Chavez-Eng, C.M.; Constanzer, M.L.; Matuszewski, B.K. Determination of Rofecoxib (MK-0966), a
cyclooxygenase-2 inhibitor, in human plasma by high-performance liquid chromatography with tandem
mass spectrometric detection, J.Chromatogr.B, 2000, 748, 31–39.
SAMPLE
Matrix: blood
Sample preparation: Vortex 1 mL plasma with 50 µL MeCN and 25 µL 400 ng/mL IS
in MeCN, add 1 mL 100 mM pH 5 acetate buffer, vortex, add 8 mL hexane:dichloromethane 50:50, mix on a flat-bed shaker for 5 min, centrifuge at 1500 g for 5 min,
freeze in dry ice/acetone. Evaporate the organic layer to dryness under a stream of
nitrogen at 50◦ , reconstitute the residue with 1 mL mobile phase, inject a 150 µL
aliquot.
HPLC VARIABLES
Guard column: 20 × 4 5 µm BDS-Hypersil C18
Column: 100 × 4.6 5 µm BDS-Hypersil C18
Mobile phase: MeCN:water 35:65
Flow rate: 1.2
Injection volume: 150
Detector: F ex 250 em 375 following post-column reaction. The column effluent flowed
through a 10 m ×0.3 mm ID reaction coil irradiated at 254 nm (Astec Beam Boost) to
the detector.
CHROMATOGRAM
Retention time: 6
Internal standard: 4-(4-methanesulfonylphenyl)-3-(4-methylphenyl)-5H-furan-2one (9)
Limit of quantitation: 0.5 ng/mL
KEY WORDS
plasma; post-column photochemical derivatization
REFERENCE
Woolf, E.; Fu, I.; Matuszewski, B. Determination of rofecoxib, a cyclooxygenase-2 specific inhibitor,
in human plasma using high-performance liquid chromatography with post-column photochemical
derivatization and fluorescence detection, J.Chromatogr.B, 1999, 730, 221–227.
ANNOTATED BIBLIOGRAPHY
Abdel-Hamid, M.E. LC-MS analysis of selected sulfur-containing non-steroid antiinflammatory agents:
applications to pharmaceutical products, J.Liq.Chromatogr.Rel.Technol., 2000, 23, 3095–3107. [LCMS; LOQ 100 ng/mL; rofecoxib; sulindac; celecoxib; piroxicam; tenoxicam]
Chavez-Eng, C.M.; Constanzer, M.L.; Matuszewski, B.K. High-performance liquid chromatographictandem mass spectrometric evaluation and determination of stable isotope labeled analogs of rofecoxib
in human plasma samples from oral bioavailability studies, J.Chromatogr.B, 2002, 767, 117–129.
[LOQ 100 pg/mL]
564
Rofecoxib
Ehrich, E.W.; Dallob, A.; De Lepeleire, I.; Van Hecken, A.; Riendeau, D.; Yuan, W.; Porras, A.;
Wittreich, J.; Seibold, J.R.; De Schepper, P.; Mehlisch, D.R.; Gertz, B.J. Characterization of rofecoxib
as a cyclooxygenase-2 isoform inhibitor and demonstration of analgesia in the dental pain model,
Clin.Pharmacol.Ther., 1999, 65, 336–347. [fluorescence detection]
Halpin, R.A.; Geer, L.A.; Zhang, K.E.; Marks, T.M.; Dean, D.C.; Jones, A.N.; Melillo, D.; Doss, G.;
Vyas, K.P. The absorption, distribution, metabolism and excretion of rofecoxib, a potent and selective
cyclooxygenase-2 inhibitor, in rats and dogs, Drug Metab.Dispos., 2000, 28, 1244–1254. [LOQ 1 ng/mL;
plasma; post-column photochemical derivatization; fluorescence detection]
Halpin, R.A.; Porras, A.G.; Geer, L.A.; Davis, M.R.; Cui, D.; Doss, G.A.; Woolf, E.; Musson, D.;
Matthews, C.; Mazenko, R.; Schwartz, J.I.; Lasseter, K.C.; Vyas, K.P.; Baillie, T.A. The disposition
and metabolism of rofecoxib, a potent and selective cyclooxygenase-2 inhibitor, in human subjects,
Drug Metab.Dispos., 2002, 30, 684–693. [plasma; urine; bile; feces; LOQ 0.5 ng/mL; post-column
photochemical derivatization; fluorescence detection]
Hsieh, J.Y.-K.; Lin, L.; Matuszewski, B.K. High-throughput liquid chromatographic determination
of rofecoxib in human plasma using a fully automated on-line solid-phase extraction
system, J.Liq.Chromatogr.Rel.Technol., 2001, 24, 799–812. [LOQ 0.5 ng/mL; plasma; post-column
photochemical derivatization; fluorescence detection; SPE]
Krishna Reddy, K.V.S.R.; Babu, J.M.; Dubey, P.K.; Chandra Sekhar, B.; Om Reddy, G.; Vyas, K. Isolation
and characterisation of process-related impurities in rofecoxib, J.Pharm.Biomed.Anal., 2002, 29,
355–360.
Mao, B.; Abrahim, A.; Ge, Z.; Ellison, D.K.; Hartman, R.; Prabhu, S.V.; Reamer, R.A.; Wyvratt, J.
Examination of rofecoxib solution decomposition under alkaline and photolytic stress conditions,
J.Pharm.Biomed.Anal., 2002, 28, 1101–1113. [stability-indicating]
Matthews, C.Z.; Woolf, E.J.; Matuszewski, B.K. Improved procedure for the determination of rofecoxib in
human plasma involving 96-well solid-phase extraction and fluorescence detection, J.Chromatogr.A,
2002, 949, 83–89. [LOQ 0.5 ng/mL; plasma; post-column photochemical derivatization; fluorescence
detection; SPE]
Niederberger, E.; Tegeder, I.; Schäfer, C.; Seegel, M.; Grösch, S.; Geisslinger, G. Opposite effects of
rofecoxib on nuclear factor-kappaB and activating protein-1 activation, J.Pharmacol.Exp.Ther., 2003,
304, 1153–1160. [plasma; post-column photochemical derivatization; fluorescence detection]
Radhakrishna, T.; Rao, D.S.; Reddy, G.O. LC determination of rofecoxib in bulk and pharmaceutical
formulations, J.Pharm.Biomed.Anal., 2001, 26, 617–628.
Slaughter, D.; Takenaga, N.; Lu, P.; Assang, C.; Walsh, D.J.; Arison, B.H.; Cui, D.; Halpin, R.A.;
Geer, L.A.; Vyas, K.P.; Baillie, T.A. Metabolism of rofecoxib in vitro using human liver subcellular
fractions, Drug Metab.Dispos., 2003, 31, 1398–1408. [LC-MS]
Vallano, P.T.; Mazenko, R.S.; Woolf, E.J.; Matuszewski, B.K. Monolithic silica liquid chromatography
columns for the determination of cyclooxygenase II inhibitors in human plasma, J.Chromatogr.B,
2002, 779, 249–257. [LOQ 0.5 ng/mL; plasma; post-column photochemical derivatization; fluorescence
detection; SPE]
Werner, U.; Werner, D.; Mundkowski, R.; Gillich, M.; Brune, K. Selective and rapid liquid
chromatography-mass spectrometry method for the quantification of rofecoxib in pharmacokinetic
studies with humans, J.Chromatogr.B, 2001, 760, 83–90. [LC-MS; plasma; LOQ 1 ng/mL; celecoxib is
internal standard]
Werner, U.; Werner, D.; Pahl, A.; Mundkowski, R.; Gillich, M.; Brune, K. Investigation of the
pharmacokinetics of celecoxib by liquid chromatography-mass spectrometry, Biomed.Chromatogr.,
2002, 16, 56–60. [LC-MS; rofecoxib is internal standard]
565
Ropinirole
Ropinirole
Molecular formula: C16 H24 N2 O
Molecular weight: 260.37
CAS Registry No: 91374-21-9, 91374-20-8 (HCl)
Merck Index: 13, 8338
H
N
O
H3C
H3C
N
SAMPLE
Matrix: blood
Sample preparation: Condition a 3 mL low displacement C18 SPE cartridge (Baker)
with 3 column vol of MeOH and 3 column vol of water. Mix 1 mL plasma with 50 µL
2 µg/mL IS in water, add to the SPE cartridge, wash with 10 mL water, wash with 10 mL
MeCN, elute with 3.5 mL MeCN:water:ammonium hydroxide 100:2:0.5. Evaporate the
eluate to dryness under a stream of nitrogen at 35◦ , immediately reconstitute the
residue with 300 µL mobile phase, mix vigorously, inject a 10–100 µL aliquot.
HPLC VARIABLES
Column: 250 × 4.6 5 µm Ultrasphere ODS
Mobile phase: MeCN:70 mM pH 3.8 ammonium formate buffer 25:75 containing 0.3%
EDTA and 0.005% sodium octyl sulfate
Flow rate: 1
Injection volume: 10–100
Detector: UV 250
CHROMATOGRAM
Retention time: 9.4
Internal standard: 4-(2-di-N,N-propylaminoethyl)-7-methoxy-2-(3H)-indoline
HCl
(11.5)
Limit of detection: 5 ng/mL
Limit of quantitation: 10 ng/mL
KEY WORDS
dog; human; pharmacokinetics; plasma; rat; SPE
REFERENCE
Swagzdis, J.E.; Mico, B.A. Liquid chromatographic determination of 4-(2-di-N,N-propylaminoethyl)-2(3H)-indolone in rat, dog, and human plasma with ultraviolet detection, J.Pharm.Sci., 1986, 75,
90–91.
566
Rosiglitazone
Rosiglitazone
Molecular formula: C18 H19 N3 O3 S
S
CH3
N
N
O
O
O
N
H
Molecular weight: 357.43
CAS Registry No: 122320-73-4, 155141-29-0 (maleate)
Merck Index: 13, 8346
SAMPLE
Matrix: blood
Sample preparation: Vortex 1 mL plasma with 50 µL 10 µg/mL IS in pH 2.3 buffer for
1 min, add 200 µL 20 mM pH 9.3 disodium tetraborate solution, vortex for 1 min, add
5 mL dichloromethane, shake horizontally for 10 min, centrifuge at 735 g for 10 min.
Evaporate the organic layer to dryness under a stream of nitrogen at room temperature,
reconstitute the residue with 5 mL n-hexane:dichloromethane 80:20, vortex for 30 s,
add 350 µL pH 2.3 buffer, vortex for 2 min, centrifuge at 735 g for 10 min, inject a
100 µL aliquot. (Prepare pH 2.3 buffer by dissolving 0.136 g of potassium dihydrogen
phosphate in 800 ml of water, adjusting to pH 2.3 with 10% HCl, and diluting to 1 L
with water.)
HPLC VARIABLES
Column: 150 × 4.6 5 µm Zorbax SB C18
Column temperature: 30
Mobile phase: MeOH:buffer 30:70 (Prepare the buffer by dissolving 1.41 g of disodium
hydrogen phosphate and 1.56 g of sodium dihydrogen phosphate in 800 ml water,
adjusting to pH 2.6 with orthophosphoric acid and diluting to 1 L with water.)
Flow rate: 1.2
Injection volume: 100
Detector: UV 245
CHROMATOGRAM
Retention time: 8.3
Internal standard: pioglitazone (18.0)
Limit of quantitation: 5 ng/mL
KEY WORDS
plasma
REFERENCE
Kolte, B.L.; Raut, B.B.; Deo, A.A.; Bagool, M.A.; Shinde, D.B. Liquid chromatographic method for the
determination of rosiglitazone in human plasma, J.Chromatogr.B, 2003, 788, 37–44.
SAMPLE
Matrix: blood
Sample preparation: Mix 250 µL plasma with 10 µg IS for 15 s, add 3 mL ethyl
acetate, vortex for 1 min, centrifuge at 2000 rpm for 10 min. Evaporate the organic
layer to dryness under a stream of nitrogen at 40◦ , reconstitute the residue with 750 µL
mobile phase, inject a 50 µL aliquot.
HPLC VARIABLES
Column: 250 × 4.6 5 µm Hichrom KR100-5C18-250A
Mobile phase: MeCN:MeOH:10 mM potassium dihydrogen phosphate 50:10:40,
adjusted to pH 6.5 with triethylamine
Rosiglitazone
567
Flow rate: 1
Injection volume: 50
Detector: F ex 247 em 367
CHROMATOGRAM
Retention time: 8
Internal standard: celecoxib (16)
Limit of quantitation: 5 ng/mL
KEY WORDS
plasma
REFERENCE
Mamidi, R.N.V.S.; Benjamin, B.; Ramesh, M.; Srinivas, N.R. Simple method for the determination of
rosiglitazone in human plasma using a commercially available internal standard, Biomed.Chromatogr.,
2003, 17, 417–420.
ANNOTATED BIBLIOGRAPHY
Cox, P.J.; Ryan, D.A.; Hollis, F.J.; Harris, A.-M.; Miller, A.K.; Vousden, M.; Cowley, H. Absorption, disposition, and metabolism of rosiglitazone, a potent thiazolidinedione insulin sensitizer, in humans,
Drug Metab.Dispos., 2000, 28, 772–780. [plasma; urine; feces; UV detection; LC-MS; SPE]
Di Cicco, R.A.; Allen, A.; Carr, A.; Fowles, S.; Jorkasky, D.K.; Freed, M.I. Rosiglitazone does not alter
the pharmacokinetics of metformin, J.Clin.Pharmacol., 2000, 40, 1280–1285. [dialysis; fluorescence
detection; LOQ 2.5 ng/mL]
Mamidi, R.N.V.S.; Chaluvadi, M.R.; Benjamin, B.; Ramesh, M.; Katneni, K.; Babu, A.P.; Bhanduri, J.;
Rao, N.M.U.; Rajagopalan, R. HPLC method for the determination of rosiglitazone in human plasma
and its application in a clinical pharmacokinetic study, Arzneimittelforschung, 2002, 52, 560–564.
[fluorescence detection; LOQ 5 ng/mL]
Muxlow, A.-M.; Fowles, S.; Russell, P. Automated high-performance liquid chromatography method for
the determination of rosiglitazone in human plasma, J.Chromatogr.B, 2001, 752, 77–84. [dialysis;
fluorescence detection; LOQ 3 ng/mL]
Radhakrishna, T.; Satyanarayana, J.; Satyanarayana, A. LC determination of rosiglitazone in bulk and
pharmaceutical formulation, J.Pharm.Biomed.Anal., 2002, 29, 873–880.
Rao, M.N.V.S.; Mullangi, R.; Katneni, K.; Ravikanth, B.; Babu, A.P.; Rani, U.P.; Naidu, M.U.R.; Srinivas, N.R.; Rajagopalan, R. Lack of effect of sucralfate on the absorption and pharmacokinetics of
rosiglitazone, J.Clin.Pharmacol., 2002, 42, 670–675. [fluorescence detection; LOQ 5 ng/mL]
568
Rosuvastatin calcium
Rosuvastatin calcium
F
Molecular formula: 2C22 H27 FN3 O6 S.Ca
Molecular weight: 1001.14
CAS Registry No: 147098-20-2
H OH
O
O
OH
N
H
S
H3C
N
CH3
CO2− Ca++
N
CH3
CH3
2
SAMPLE
Matrix: blood
Sample preparation: Condition a 30 mg Oasis HLB SPE cartridge (in 96 well plate)
with 1 mL MeOH and 1 mL 0.5% acetic acid. Mix 500 µL plasma with 500 µL 100 mM
pH 4 acetate buffer, 50 µL 150 ng/mL IS in MeOH:1 M acetic acid 50:50, and 750 µL 1 M
acetic acid, vortex for 2 s, centrifuge at 738 g for 7 min, add 1.7 mL of the supernatant to
the SPE cartridge, wash with 1 mL MeOH:0.5% acetic acid 30:70, elute with 1 mL 0.5%
acetic acid in MeOH. Evaporate the eluate to dryness under a stream of nitrogen at 40◦ ,
reconstitute the residue with 130 µL 0.5% acetic acid, centrifuge at 1700 or 9500 g for
10 min, inject a 100 µL aliquot.
HPLC VARIABLES
Column: 150 × 4.6 5 µm Luna C18(2)
Mobile phase: MeOH:0.2% formic acid 70:30 (divert first 2 min of run to waste)
Flow rate: 1
Injection volume: 100
Detector: MS, PE Sciex API 365, TurboIonspray 450◦ , 0.2 mL/min entered detector,
positive ion mode, ionspray 3000 V, ring 160 V, orifice 44 V, nebulizer gas nitrogen at 14
units, TurboIonspray gas nitrogen at 7 L/min, collision gas nitrogen 4 units, curtain gas
nitrogen 8 units, deflector −250 V, electron multiplier 2900 V, collision energy −47.5 V,
m/z 482.2–258.2
CHROMATOGRAM
Retention time: 3.6
Internal standard: d6 -rosuvastatin (m/z 488.2–264.2)
Limit of quantitation: 100 pg/mL
KEY WORDS
plasma; SPE
REFERENCE
Hull, C.K.; Penman, A.D.; Smith, C.K.; Martin, P.D. Quantification of rosuvastatin in human plasma by
automated solid-phase extraction using tandem mass spectrometric detection, J.Chromatogr.B, 2002,
772, 219–228.
Sarafloxacin
Molecular formula: C20 H17 F2 N3 O3
Molecular weight: 385.36
CAS Registry No: 98105-99-8, 91296-87-6 (HCl)
Merck Index: 13, 8447
F
H
N
N
N
F
COOH
O
SAMPLE
Matrix: blood
Sample preparation: Mix 200 µL plasma with 800 µL 750 ng/mL IS in 100 mM pH 7.4
phosphate buffer, add 6 mL chloroform (Caution! Chloroform is a carcinogen!), shake at
200 oscillations/min for 30 min, centrifuge at 13 000 g for 6 min. Evaporate the organic
layer to dryness under a stream of nitrogen at 40◦ , reconstitute the residue with 200 µL
phosphate-buffered saline, inject a 10–80 µL aliquot.
HPLC VARIABLES
Guard column: Guard-Pak Novapak C18
Column: 150 × 3.9 5 µm Novapak C18
Mobile phase: MeCN:buffer 18:82 (The buffer was 20 mM potassium dihydrogen phosphate containing 6 mM phosphoric acid and 12 mM tetraethylammonium bromide
adjusted to pH 3.0 with 2 M NaOH.)
Flow rate: 1
Injection volume: 10–80
Detector: F ex 338 em 425
CHROMATOGRAM
Retention time: 5.6
Internal standard: norfloxacin (2.2)
Limit of detection: 6 ng/mL
Limit of quantitation: 12 ng/mL
OTHER SUBSTANCES
Extracted: difloxacin (6.2)
KEY WORDS
plasma; rabbit
REFERENCE
Garcia, M.A.; Solans, C.; Aramayona, J.J.; Rueda, S.; Bregante, M.A. Simultaneous determination of
difloxacin and its primary metabolite sarafloxacin in rabbit plasma, Chromatographia, 2000, 51,
487–490.
SAMPLE
Matrix: blood
Sample preparation: Condition a 1 mL Bond Elut C2 SPE cartridge with two 1 mL
portions of MeOH and three 1 mL portions of 2 mM phosphoric acid. Add 300 µL
500 mM phosphoric acid to the SPE cartridge, add 250 µL serum, add 50 µL 500 ng/mL
IS in mobile phase, add 250 µL water, wash with 100 µL water, wash with 100 µL
100 mM phosphoric acid, elute with five 100 µL aliquots of MeOH:500 mM phosphoric
acid 70:30, dilute the eluate with 500 µL water, inject a 20 µL aliquot.
569
570
Sarafloxacin
HPLC VARIABLES
Guard column: 10 × 3 5 µm PLRP-S (Polymer Laboratories)
Column: 150 × 4.6 5 µm PLRP-S (Polymer Laboratories)
Mobile phase: MeCN:MeOH:2 mM phosphoric acid 20:8:72
Flow rate: 0.9
Injection volume: 20
Detector: F ex 278 em 440
CHROMATOGRAM
Retention time: 5
Internal standard: enrofloxacin (3.5)
Limit of detection: 5 ng/g
KEY WORDS
fish; serum; SPE
REFERENCE
Steffenak, I.; Hormazabal, V.; Yndestad, M. A rapid assay for the determination of sarafloxacin (A-55620)
in fish serum by high performance liquid chromatography, J.Liq.Chromatogr., 1991, 14, 1983–1988.
ANNOTATED BIBLIOGRAPHY
Holtzapple, C.K.; Pishko, E.J.; Stanker, L.H. Separation and quantification of two fluoroquinolones in
serum by on-line high-performance immunoaffinity chromatography, Anal.Chem., 2000, 72, 4148–4153.
[LOD 1.7 ng/mL; fluorescence detection]
Holtzapple, C.K.; Buckley, S.A.; Stanker, L.H. Determination of fluoroquinolones in serum using an
on-line clean-up column coupled to high-performance immunoaffinity-reversed-phase liquid chromatography, J.Chromatogr.B, 2001, 754, 1–9. [LOD 1.7 ng/mL; fluorescence detection]
Selamectin
Selamectin
Molecular formula: C43 H63 NO11
Molecular weight: 769.96
CAS Registry No: 220119-17-5
Merck Index: 13, 8500
571
OCH3
HO
H3C
CH3
O
H
O
CH3
O
H
O
H3C
O
O
OH
H
O
H
CH3
N
HO
SAMPLE
Matrix: blood
Sample preparation: Condition a 1 mL 100 mg Isolute C18 SPE cartridge (Jones
Chromatography) with 1 mL MeOH and 1 mL water. Vortex 0.2 (cat) or 1 (dog) mL
plasma with 10 µL 1 µg/mL IS in MeOH and 1 mL MeCN:water 30:70, add to the SPE
cartridge, wash with 1 mL water, dry under vacuum, elute with two 500 µL portions of
MeOH. Evaporate the eluate to dryness under a stream of nitrogen at 37◦ , make sure
sample is completely dry, reconstitute the residue with 100 µL triethylamine:MeCN
50:50, mix thoroughly, add 150 µL trifluoroacetic acid:MeCN 33:67, mix thoroughly.
Evaporate the sample to 75 µL under a stream of nitrogen at 40◦ (ca. 20 min), add
250 µL 2.0 M ammonia in MeOH, mix gently. Evaporate the sample to 75 µL under
a stream of nitrogen at 40◦ (ca. 10 min), add 200 µL MeCN, mix thoroughly, inject a
100 µL aliquot.
HPLC VARIABLES
Column: 250 × 3.2 Spherisorb RPB
Mobile phase: MeCN:THF:water 68:15:17
Flow rate: 0.5
Injection volume: 100
Detector: F ex 360 em 450
CHROMATOGRAM
Retention time: 14
Internal standard: UK-127,053 (methoxyselamectin) (11.5)
Limit of quantitation: 0.2 ng/mL (dog), 1 ng/mL (cat)
KEY WORDS
cat; derivatization; dog; plasma; SPE
REFERENCE
Walker, D.K.; Fenner, K.S. A sensitive method for the measurement of the novel pet endectocide,
selamectin (UK-124,114), in dog and cat plasma by chemical derivatisation and high-performance
liquid chromatography with fluorescence detection, J.Pharm.Biomed.Anal., 2000, 24, 105–111.
572
Sermorelin
Sermorelin
Molecular formula: C149 H246 N44 O42 S
Molecular weight: 3357.94
CAS Registry No: 86168-78-7
Merck Index: 13, 8535
SAMPLE
Matrix: blood
Sample preparation: Condition a Sep-Pak C18 SPE cartridge with MeOH, 0.01%
trifluoroacetic acid in MeCN, and 0.01% trifluoroacetic acid in water. Mix 200 µL serum
with 800 µL cold 50 mM pH 0.8 (sic) phosphate buffer, add to the SPE cartridge, elute
with 2 mL MeCN:water:trifluoroacetic acid 40:60:0.01, lyophilize the eluate, reconstitute
with initial mobile phase, inject an aliquot.
HPLC VARIABLES
Column: 150 × 3.9 10 µm µBondapak C18
Mobile phase: Gradient. MeCN:water:trifluoroacetic acid from 20:80:0.01 to 36:64:0.01
over 40 min, wash at 40:60:0.01 for 10 min.
Flow rate: 1.5
Detector: UV 214
CHROMATOGRAM
Retention time: 33
OTHER SUBSTANCES
Extracted: metabolites
KEY WORDS
rat; serum; SPE
REFERENCE
Boulanger, L.; Roughly, P.; Gaudreau, P. Catabolism of rat growth hormone-releasing factor(1–29) amide
in rat serum and liver, Peptides, 1992, 13, 681–689.
SAMPLE
Matrix: blood
Sample preparation: Condition a Sep-Pak C18 SPE cartridge with 2 mL 80% acetic
acid and 4 mL 10 mM trifluoroacetic acid. Mix 1 mL plasma with 200 µL 1 M trifluoroacetic acid, place on ice, add to the SPE cartridge, wash with 3 mL 100 mM
trifluoroacetic acid, pass 20 mL air through the cartridge, elute with 3 mL 80% acetic
acid, inject an aliquot.
HPLC VARIABLES
Column: 150 × 3.9 5 µm Delta-Pak C18 (Waters)
Mobile phase: Gradient. A was MeCN:water:trifluoroacetic acid 95:5:0.1. B was 0.1%
trifluoroacetic acid. A:B from 34:66 to 50:50 over 60 min.
Flow rate: 1
Detector: UV 215
CHROMATOGRAM
Retention time: 20
Sermorelin
573
OTHER SUBSTANCES
Extracted: metabolites
KEY WORDS
pig; plasma; SPE
REFERENCE
Su, C.M.; Jensen, L.R.; Heimer, E.P.; Felix, A.M.; Pan, Y.C.; Mowles, T.F. In vitro stability of growth
hormone releasing factor (GRF) analogs in porcine plasma, Horm.Metab.Res., 1991, 23, 15–21.
ANNOTATED BIBLIOGRAPHY
Zarandi, M.; Serfozo, P.; Zsigo, J.; Deutch, A.H.; Janaky, T.; Olsen, D.B.; Bajusz, S.; Schally, A.V. Potent
agonists of growth hormone-releasing hormone. II, Pept.Res., 1992, 5, 190–193.
574
Sibutramine
Sibutramine
Molecular formula: C17 H26 ClN
Molecular weight: 279.86
CH3
Cl
CH3
N
H3C
CH3
CAS Registry No: 106650-56-0, 125494-59-9 (HCl monohydrate)
Merck Index: 13, 8559
SAMPLE
Matrix: blood
Sample preparation: Mix 1 mL plasma, 50 µL 80 µg/mL IS in MeOH, 1 mL saturated
sodium bicarbonate, and 5 mL cyclohexane, centrifuge. Evaporate the organic layer to
dryness under a stream of nitrogen, reconstitute the residue with 100 µL mobile phase,
inject a 50 µL aliquot.
HPLC VARIABLES
Column: 250 × 4.6 5 µm Hypersil ODS-2 C18
Column temperature: 25
Mobile phase: MeOH:10 mM pH 3.5 ammonium acetate buffer 75:25 (divert first 3 min
to waste)
Flow rate: 1
Injection volume: 50
Detector: MS, quadrupole, electrospray, drying gas nitrogen 10.5 L/min, nebulizer
45 psi, drying gas 350◦ , capillary 4 kV, positive ion mode, fragmenter 70 V, m/z 280
CHROMATOGRAM
Retention time: 5
Internal standard: phenoprolamine HCl (N-(2-(3,4-dimethoxyphenyl)ethyl)-N-(2-(2,6dimethylphenoxy)-1-methylethyl)amine) (m/z 344) (3.5)
Limit of quantitation: 50 ng/mL
OTHER SUBSTANCES
Extracted: metabolites
KEY WORDS
plasma
REFERENCE
Ding, L.; Hao, X.; Huang, X.; Zhang, S. Simultaneous determination of sibutramine and its N-desmethyl
metabolites in human plasma by liquid chromatography-electrospray ionization-mass spectrometry:
method and clinical applications, Anal.Chim.Acta, 2003, 492, 241–248.
SAMPLE
Matrix: solutions
Sample preparation: Inject a 10 µL aliquot of a 1 mg/mL solution.
HPLC VARIABLES
Guard column: 50 mm long Chiralcel OD
Column: 250 × 4.6 10 µm Chiralcel OD
Column temperature: 30
Mobile phase: Hexane:EtOH:trifluoroacetic acid 93:7:0.05
Flow rate: 1
Injection volume: 10
Detector: UV 225
Sibutramine
575
CHROMATOGRAM
Retention time: 10 (–), 14 (+)
KEY WORDS
chiral
REFERENCE
Radhakrishna, T.; Narayana, C.L.; Rao, D.S.; Vyas, K.; Reddy, G.O. LC method for the determination
of assay and purity of sibutramine hydrochloride and its enantiomers by chiral chromatography,
J.Pharm.Biomed.Anal., 2000, 22, 627–639.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 × 4.6 5 µm Microsorb-MV (Varian)
Mobile phase: MeOH:water:triethylamine 80:20:0.3, pH adjusted to 4.5 with 85% phosphoric acid
Flow rate: 1.1
Injection volume: 20
Detector: UV 225
CHROMATOGRAM
Retention time: 4
KEY WORDS
stability-indicating
REFERENCE
Segall, A.I.; Collado, E.A.; Ricci, R.A.; Pizzorno, M.T. Reversed-phase HPLC determination of sibutramine hydrochloride in the presence of its oxidatively-induced degradation products, J.Liq.Chromatogr.Rel.Technol., 2003, 26, 977–986.
576
Sildenafil
Sildenafil
O
H
CAS Registry No: 139755-83-2,
171599-83-0 (citrate)
Merck Index: 13, 8563
O
O
Molecular formula: C22 H30 N6 O4 S
Molecular weight: 474.59
N
N
S
N
H3C
CH3
N
N
N
O
CH3
CH3
SAMPLE
Matrix: blood
Sample preparation: Condition a 25 mg Certify mixed-mode SPE cartridge in a 96
well plate (Varian) with 500 µL MeOH and 500 µL 5% acetic acid. Mix 350 µL plasma
with 20 µL 500 ng/mL IS in MeOH:water 50:50 and 350 µL 5% acetic acid, add to the
SPE cartridge, wash with 500 µL 5% acetic acid, wash with 500 µL MeOH, dry for
3 min, elute with two 350 µL portions of MeCN:ammonium hydroxide 98:2. Evaporate
the eluate to dryness, reconstitute the residue with 200 µL 0.05% trifluoroacetic acid in
MeCN, inject a 10 µL aliquot.
HPLC VARIABLES
Column: 50 × 3 5 µm Betasil silica (Keystone)
Mobile phase: Unspecified. However, in similar analyses, the authors have reported
MeCN:water:trifluoroacetic acid 90:10:0.1 (J.Pharm.Biomed.Anal. 2003, 32, 609), MeCN:
water:formic acid 90:10:0.1 (J.Chromatogr.B 1999, 735, 255), and MeCN:water:trifluoroacetic acid 95:5:0.05 (J.Chromatogr.B 2001, 754, 387)
Flow rate: 0.4
Injection volume: 10
Detector: MS, PE Sciex API 3000, electrospray, positive ion mode, ionspray needle
5000 V, turbo gas 400◦ , auxiliary gas 8 L/min, nebulizer gas 12 units, curtain gas 8
units, collision gas 4 units, declustering 46 V, focusing 200 V, collision energy 77 V, m/z
475–283
CHROMATOGRAM
Retention time: 1.7
Internal standard: 1-[[3-(7-methoxy-1-methyl-3-propyl-1H-pyrazolo[4,3-d]pyrimidin5-yl)-4-ethoxyphenyl]sulfonyl]-4-methylpiperazine (m/z 489–297) (1.6)
Limit of quantitation: 1 ng/mL
OTHER SUBSTANCES
Extracted: desmethylsildenafil (metabolite) (m/z 461–283) (1.6)
KEY WORDS
plasma; SPE
REFERENCE
Eerkes, A.; Addison, T.; Naidong, W. Simultaneous assay of sildenafil and desmethylsildenafil in human
plasma using liquid chromatography-tandem mass spectrometry on silica column with aqueous-organic
mobile phase, J.Chromatogr.B, 2002, 768, 277–284.
SAMPLE
Matrix: blood
Sample preparation: Vortex 1 mL plasma with 100 µL 20 µg/mL IS in MeOH and
100 µL 1 M NaOH for 5 s, add 3 mL ethyl acetate, vortex for 5 min, centrifuge at 2950 g
Sildenafil
577
for 10 min. Evaporate the organic layer to dryness under a stream of nitrogen at 40◦ ,
reconstitute the residue with 200 µL mobile phase, inject a 100 µL aliquot.
HPLC VARIABLES
Column: 150 × 4.6 5 µm Inertsil 5 ODS-2
Mobile phase: MeCN:buffer 55:45 (The buffer was 30 mM potassium dihydrogen phosphate adjusted to pH 6.0 with 1 M NaOH.)
Flow rate: 0.5
Injection volume: 100
Detector: UV 230
CHROMATOGRAM
Retention time: 7.5
Internal standard: butylparaben (12)
Limit of quantitation: 10 ng/mL
KEY WORDS
pharmacokinetics; plasma
REFERENCE
Sheu, M.-T.; Wu, A.-B.; Yeh, G.-C.; Hsia, A.; Ho, H.-O. Development of a liquid chromatographic method
for bioanalytical applications with sildenafil, J.Chromatogr.B, 2003, 791, 255–262.
SAMPLE
Matrix: formulations
Sample preparation: Weigh out powdered tablets containing 100 mg of active, dissolve
in 100 mL MeCN:water 50:50, add IS, dilute with mobile phase, inject a 20 µL aliquot.
HPLC VARIABLES
Column: 250 × 4.6 5 µm LiChrospher C18
Mobile phase: MeCN:water 52:48
Flow rate: 1
Injection volume: 20
Detector: UV 245
CHROMATOGRAM
Retention time: 7.2
Internal standard: piroxicam (2.2)
Limit of detection: 15 ng/mL
Limit of quantitation: 50 ng/mL
KEY WORDS
stability-indicating; tablets
REFERENCE
Dinesh, N.D.; Vishukumar, B.K.; Nagaraja, P.; Gowda, N.M.M.; Rangappa, K.S. Stability indicating
RP-LC determination of sildenafil citrate (Viagra) in pure form and in pharmaceutical samples,
J.Pharm.Biomed.Anal., 2002, 29, 743–748.
ANNOTATED BIBLIOGRAPHY
Aboul-Enein, H.Y.; Hefnawy, M.M. Rapid determination of sildenafil citrate in pharmaceutical
preparations using monolithic silica HPLC column, J.Liq.Chromatogr.Rel.Technol., 2003, 26,
2897–2908. [LOD 25 ng/mL]
Cooper, J.D.H.; Muirhead, D.C.; Taylor, J.E.; Baker, P.R. Development of an assay for the simultaneous
determination of sildenafil (Viagra) and its metabolite (UK-103,320) using automated sequential trace
578
Sildenafil
enrichment of dialysates and high-performance liquid chromatography, J.Chromatogr.B, 1997, 701,
87–95.
Daraghmeh, N.; Al-Omari, M.; Badwan, A.A.; Jaber, A.M.Y. Determination of sildenafil citrate
and related substances in the commercial products and tablet dosage form using HPLC,
J.Pharm.Biomed.Anal., 2001, 25, 483–492.
Jeong, C.K.; Lee, H.-Y.; Jang, M.-S.; Kim, W.B.; Lee, H.S. Narrowbore high-performance liquid
chromatography for the simultaneous determination of sildenafil and its metabolite UK-103,320
in human plasma using column switching, J.Chromatogr.B, 2001, 752, 141–147. [LOQ 10 ng/mL]
Kim, J.; Ji, H.Y.; Kim, S.J.; Lee, H.W.; Lee, S.-S.; Kim, D.S.; Yoo, M.; Kim, W.B.; Lee, H.S. Simultaneous
determination of sildenafil and its active metabolite UK-103,320 in human plasma using liquid
chromatography-tandem mass spectrometry, J.Pharm.Biomed.Anal., 2003, 32, 317–322. [LOQ
2 ng/mL]
Lee, M.; Min, D.I. Determination of sildenafil citrate in plasma by high-performance liquid
chromatography and a case for the potential interaction of grapefruit juice with sildenafil citrate,
Ther.Drug Monit., 2001, 23, 21–26. [LOD 10 ng/mL]
Liaw, J.; Chang, T.-W. Determination of transdermal sildenafil in nude mouse skin by reversed-phase
high-performance liquid chromatography, J.Chromatogr.B, 2001, 765, 161–166. [LOQ 5 ng/mL]
Nagaraju, V.; Sreenath, D.; Rao, J.T.; Rao, R.N. Separation and determination of synthetic impurities
of sildenafil (Viagra) by reversed-phase high-performance liquid chromatography, Anal.Sci., 2003, 19,
1007–1011.
Segall, A.I.; Vitale, M.F.; Perez, V.L.; Palacios, M.L.; Pizzorno, M.T. Reversed-phase HPLC
determination of sildenafil citrate in the presence of its oxidative-induced degradation products,
J.Liq.Chromatogr.Rel.Technol., 2000, 23, 1377–1386. [stability-indicating]
Tracqui, A.; Ludes, B. HPLC-MS for the determination of sildenafil citrate (Viagra) in biological fluids.
Application to the salivary excretion of sildenafil after oral intake, J.Anal.Toxicol., 2003, 27, 88–94.
[saliva; plasma; LOD 0.2 ng/mL; LOQ 0.5 ng/mL]
Walker, D.K.; Ackland, M.J.; James, G.C.; Muirhead, G.J.; Rance, D.J.; Wastall, P.; Wright, P.A.
Pharmacokinetics and metabolism of sildenafil in mouse, rat, rabbit, dog and man, Xenobiotica,
1999, 29, 297–310. [SPE; LOQ 1 ng/mL]
Warrington, J.S.; Shader, R.I.; von Moltke, L.L.; Greenblatt, D.J. In vitro biotransformation of sildenafil
(Viagra): identification of human cytochromes and potential drug interactions, Drug Metab.Dispos.,
2000, 28, 392–397. [buspirone is internal standard]
Warrington, J.S.; von Moltke, L.L.; Harmatz, J.S.; Shader, R.I.; Greenblatt, D.J. The effect of age on
sildenafil biotransformation in rat and mouse liver microsomes, Drug Metab.Dispos., 2003, 31,
1306–1309.
Simethicone
579
Simethicone
CAS Registry No: 8050-81-5
Merck Index: 13, 3241
SAMPLE
Matrix: formulations
Sample preparation: Add formulation (oral liquid or crushed tablet) to 25 mL dichloromethane and 25 mL diluted HCl (2:1), shake vigorously for 5 min, let stand for 1 h. Dry
5 mL of the lower organic layer over 500 mg anhydrous sodium sulfate, filter (0.45 µm
nylon), inject an aliquot of the filtrate.
HPLC VARIABLES
Column: 250 × 4.6 5 µm Alltima C8
Mobile phase: Gradient. MeCN:chloroform from 45:55 to 15:85 over 5 min, return
to initial conditions over 5 min, re-equilibrate for 5 min. (Caution! Chloroform is a
carcinogen!)
Flow rate: 1
Detector: Evaporative Light Scattering Detector, Alltech model 500, drift tube 95◦ ,
nebulizer 2 L/min
CHROMATOGRAM
Retention time: 9.2
KEY WORDS
crushed tablet; oral liquid
REFERENCE
Moore, D.E.; Liu, T.X.; Miao, W.G.; Edwards, A.; Elliss, R. A RP-LC method with evaporative light scattering detection for the assay of simethicone in pharmaceutical formulations, J.Pharm.Biomed.Anal.,
2002, 30, 273–278.
580
Sivelestat
Sivelestat
Molecular formula: C20 H22 N2 O7 S
Molecular weight: 434.47
CAS Registry No: 127373-66-4,
201677-61-4 (Na salt tetrahydrate)
Merck Index: 13, 8629
O
O
S
O
H3C
H3C
O
CH3
N
H
O
N
COOH
H
SAMPLE
Matrix: blood, urine
Sample preparation: Mix plasma or urine with IS, filter (0.22 µm), inject a 40 (plasma)
or 10 (urine) µL aliquot onto column A and elute to waste with mobile phase A; after
7 min, backflush the contents of column A onto column B with mobile phase B; after
3 min, remove column A from the circuit and backflush the contents of column B onto
column C with mobile phase C; after 3 min, remove column B from the circuit, continue
to elute column C with mobile phase C, monitor the effluent from column C.
HPLC VARIABLES
Column: A 10 × 4.6 5 µm SPS-C18 + 150 × 4.6 5 µm SPS-C18 Semipermeable-Surface
(Regis); B 150 × 4.6 5 µm YMC-A602; C 150 × 4.6 5 µm Capcell Pak C18 (Shiseido)
Mobile phase: A MeCN:50 mM pH 7 phosphate buffer 10:90; B MeCN:water 10:90; C
Gradient. MeCN:20 mM pH 3.8 potassium dihydrogen phosphate buffer 20:80 for
10 min, to 40:50 over 20 min, maintain at 40:50 for 15 min.
Flow rate: 1
Injection volume: 40 (plasma), 10 (urine)
Detector: UV 240
CHROMATOGRAM
Retention time: 32
Internal standard: ONO-EI-547 (N-[2′ -[4-(2,2-dimethylpropionyloxy)-3-methylphenylsulfonylamino]-5′ -methylbenzoyl]aminoacetic acid) (38), ONO-EI-537 (N-[2′ -[4-hydroxy-3-methylphenylsulfonylamino]benzoyl]aminoacetic acid) (20)
Limit of quantitation: 156 ng/mL (plasma), 1.56 µg/mL (urine)
KEY WORDS
column-switching; plasma
REFERENCE
Shintani, T.; Takamoto, M.; Sawada, M.; Aishita, H.; Nakagawa, T. Simultaneous determination of
human neutrophil elastase inhibitor (ONO-5046) and its metabolite in plasma and urine by direct
injection column-switching HPLC, J.Pharm.Biomed.Anal., 1994, 12, 397–405.
SAMPLE
Matrix: blood, urine
Sample preparation: Mix plasma or urine with IS, dilute 500 µL plasma or 100 µL
urine with water and 1 M HCl, add to a Sep-Pak C18 SPE cartridge, elute with MeOH.
Add the eluate to a Bond Elut SAX SPE cartridge, elute with MeOH:100 mM HCl
60:40, extract the eluate with ethyl acetate. Evaporate the organic layer to dryness,
reconstitute the residue with 150 µL mobile phase, inject a 50 µL aliquot.
HPLC VARIABLES
Column: 150 × 4.6 3 µm Chemcosorb 3Dph
Mobile phase: Gradient. MeCN:20 mM pH 3.8 potassium dihydrogen phosphate buffer
from 20:80 to 40:50 over 40 min, maintain at 40:50 for 15 min.
Sivelestat
581
Flow rate: 1
Injection volume: 50
Detector: UV 240
CHROMATOGRAM
Internal standard: ONO-EI-547 (N-[2′ -[4-(2,2-dimethylpropionyloxy)-3-methylphenylsulfonylamino]-5′ -methylbenzoyl]aminoacetic acid), ONO-EI-537 (N-[2′ -[4-hydroxy-3methylphenylsulfonylamino]benzoyl]aminoacetic acid)
Limit of quantitation: 125 ng/mL (plasma), 1.25 µg/mL (urine)
KEY WORDS
comparison with column-switching (above); plasma; SPE
REFERENCE
Shintani, T.; Takamoto, M.; Sawada, M.; Aishita, H.; Nakagawa, T. Simultaneous determination of
human neutrophil elastase inhibitor (ONO-5046) and its metabolite in plasma and urine by direct
injection column-switching HPLC, J.Pharm.Biomed.Anal., 1994, 12, 397–405.
582
Sodium oxybate
Sodium oxybate
Molecular formula: C4 H7 NaO3
Molecular weight: 126.09
HO
COONa
CAS Registry No: 502-85-2, 591-81-1 (free acid)
Merck Index: 13, 4840
SAMPLE
Matrix: solutions
Sample preparation: Mix 200 µL of a solution in MeOH:water 10:90 with 150 µL
20 mM 0.5 mM tetrakis(decyl)ammonium bromide in 5 mM pH 7.0 phosphate buffer
and 100 µL 4.2 mg/mL 2-bromoacetyl-6-methoxynaphthalene in acetone, stir at 70◦ for
65 min, add 150 µL 80 µg/mL IS in MeCN, sonicate for 1 min, inject a 50 µL aliquot.
(Preparation of 2-bromoacetyl-6-methoxynaphthalene is given in Organic Syntheses,
Collective Volume 6, page 175; available free of cost at www.orgsyn.org.)
HPLC VARIABLES
Column: 250 × 4.5 Hypersil 5 ODS
Column temperature: 35
Mobile phase: MeCN:MeOH:25 mM pH 4.5 phosphate buffer 18.7:15.3:66
Flow rate: 1.7
Injection volume: 50
Detector: F ex 300 em 460
CHROMATOGRAM
Retention time: 35
Internal standard: 4-(6-methoxy-2-naphthyl)-4-oxobutanoic acid (Synthesis of 4-(6methoxy-2-naphthyl)-4-oxobutanoic acid is as follows. Dissolve 5 g 2-acetyl-6-methoxynaphthalene (6′ -methoxy-2′ -acetonaphthone) in the minimum amount of warm glacial
acetic acid, add 2.5 g glyoxylic acid, reflux for 24 h, evaporate to dryness under reduced
pressure, dissolve the residue in chloroform, extract three times with 5% sodium
carbonate solution (Caution! Chloroform is a carcinogen!). Combine the extracts and
acidify them with concentrated HCl, filter to recover the product, recrystallize from
MeOH/water to obtain 4-(6-methoxy-2-naphthyl)-4-oxo-2-butenoic acid (mp 165–168◦ ).
Dissolve 4 g 4-(6-methoxy-2-naphthyl)-4-oxo-2-butenoic acid in the minimum amount
of THF, add palladium on charcoal, hydrogenate until 450 mL hydrogen are absorbed,
filter, evaporate to dryness under reduced pressure, recrystallize from acetic acid to
obtain 4-(6-methoxy-2-naphthyl)-4-oxobutanoic acid as a white solid (mp 148◦ ) (Farmaco
Ed.Sci. 1982, 37, 171).) (25)
KEY WORDS
derivatization
REFERENCE
Gatti, R.; Bousquet, E.; Bonazzi, D.; Cavrini, V. Determination of carboxylic acid salts in pharmaceuticals
by high-performance liquid chromatography after pre-column fluorogenic labelling, Biomed.Chromatogr., 1996, 10, 19–24.
Somatropin
583
Somatropin
Molecular formula: C990 H1529 N263 O299 S7
Molecular weight: 22124.12
CAS Registry No: 9002-72-6,
12629-01-5 (human)
Merck Index: 13, 8789
SAMPLE
Matrix: blood
Sample preparation: Prepare an immunoaffinity chromatography column by circulating 5 mL 500 µg/mL polyclonal antibodies raised against porcine somatotropin in
200 mM pH 8.3 sodium bicarbonate containing 500 mM NaCl through a 1 mL Hitrap
NHS-activated immunoaffinity chromatography column (Amersham Pharmacia) for
12 h. Wash and deactivate the column by passing in sequence three times 6 mL 500 mM
pH 8.3 ethanolamine containing 500 mM NaCl and 6 mL 100 mM pH 4 sodium acetate
containing 500 mM NaCl. Slowly pass 10 mL serum (0.5 drop/s) through the column,
wash with 5 mL 200 mM pH 8.3 sodium bicarbonate containing 500 mM NaCl, elute
with 5 mL EtOH:water 70:30, concentrate with a diafiltration device (MW cut-off 10 000
Da, Vivasciences) while centrifuging at 6000 g for 30 min, inject a 25 µL aliquot of the
residue. Alternatively, dissolve 1 g urea in 10 mL plasma, filter with a diafiltration
device (MW cut-off 30 000 Da, Vivasciences) while centrifuging at 6000 g for 30 min,
pass 5 mL through a Hiload 26/60 Superdex 75 pg GPC column (Amersham Pharmacia),
concentrate the appropriate fraction with a diafiltration device (MW cut-off 10 000 Da,
Vivasciences) while centrifuging at 6000 g for 30 min, inject a 25 µL aliquot of the
residue.
HPLC VARIABLES
Column: 50 × 1 3.5 µm Zorbax Extend-C18
Mobile phase: Gradient. MeCN:1% acetic acid from 10:90 to 90:10 over 5 min
Flow rate: 0.06
Injection volume: 25
Detector: MS, Agilent 1100 MSD, electrospray, positive ion, nebulizer 20 psi, cap-
illary 5000 V, drying gas 7 L/min at 350◦ , voltage applied to capillary 190 V, m/z
1279.2–1359.1 (natural porcine), m/z 1270.5–1349.5 (recombinant porcine)
CHROMATOGRAM
Limit of detection: 10 ng/mL
KEY WORDS
immunoaffinity; pig; plasma; serum; ultrafiltrate
REFERENCE
Blokland, M.H.; Sterk, S.S.; van Ginkel, L.A.; Stephany, R.W.; Heck, A.J.R. Analysis for endogenous and
recombinant porcine somatotropine in serum, Anal.Chim.Acta, 2003, 483, 201–206.
584
Squalane
Squalane
Molecular formula: C30 H62
Molecular weight: 422.81
CAS Registry No: 111-01-3
Merck Index: 13, 8846
SAMPLE
Matrix: cell cultures
Sample preparation: Centrifuge cell culture at 2772 g at 4◦ for 20 min, extract the
supernatant with diethyl ether. Evaporate the extracts to dryness, reconstitute the
residue with n-propanol, inject an aliquot.
HPLC VARIABLES
Column: Nucleosil-100 C18
Mobile phase: n-Propanol
Flow rate: 0.5
Detector: Refractive Index
REFERENCE
Berekaa, M.M.; Steinbüchel, A. Microbial degradation of the multiply branched alkane 2,6,10,15,19,23hexamethyltetracosane (squalane) by Mycobacterium fortuitum and Mycobacterium ratisbonense,
Appl.Environ.Microbiol., 2000, 66, 4462–4467.
Squalene
585
Squalene
Molecular formula: C30 H50
Molecular weight: 410.72
CAS Registry No: 111-02-4
Merck Index: 13, 8847
SAMPLE
Matrix: cell cultures
Sample preparation: Centrifuge cell culture at 2772 g at 4◦ for 20 min, extract the
supernatant with diethyl ether. Evaporate the extracts to dryness, reconstitute the
residue with n-propanol, inject an aliquot.
HPLC VARIABLES
Column: Nucleosil-100 C18
Mobile phase: n-Propanol
Flow rate: 0.5
Detector: UV (wavelength not specified)
REFERENCE
Berekaa, M.M.; Steinbüchel, A. Microbial degradation of the multiply branched alkane 2,6,10,15,19,23hexamethyltetracosane (squalane) by Mycobacterium fortuitum and Mycobacterium ratisbonense,
Appl.Environ.Microbiol., 2000, 66, 4462–4467.
SAMPLE
Matrix: food
Sample preparation: Mix 500 mg solid food or milk powder, 5 g liquid milk, or
100–200 mg oil or fat with 10 mL EtOH, add 2 mL 50% KOH solution, heat at 70◦
for 8 min with periodic agitation, cool, add 20 mL hexane:diisopropyl ether 75:25, shake
mechanically for 5 min, add 30 mL water, invert 10 times, centrifuge at 180 g for 10 min.
Evaporate 10 mL of the organic layer to dryness at <45◦ , reconstitute the residue with
1 mL EtOH or hexane, inject a 20–50 µL aliquot.
HPLC VARIABLES
Guard column: Guard-Pak C18
Column: 5 µm Rad-Pak C18
Mobile phase: MeOH
Flow rate: 1
Injection volume: 20–50
Detector: UV 212
CHROMATOGRAM
Retention time: 32
OTHER SUBSTANCES
Simultaneous: campesterol (28), cholesterol (24), β-sitosterol (30), α-tocopherol (15),
δ-tocopherol (12), γ -tocopherol (14)
REFERENCE
Indyk, H.E. Simultaneous liquid chromatographic determination of cholesterol, phytosterols and tocopherols in foods, Analyst, 1990, 115, 1525–1530.
586
Squalene
ANNOTATED BIBLIOGRAPHY
Domnas, A.; Biswas, S.S.; Gallagher, P.A. Squalene metabolism in two species of Lagenidium, Can.J.
Microbiol., 1994, 40, 523–531.
Piretti, M.V.; Pagliuca, G.; Tarozzi, G. Simultaneous reversed-phase high-performance liquid chromatographic separation of non-polar isoprenoid lipids and their determination, J.Chromatogr.B, 1995, 674,
177–185.
Sulpice, J.C.; Ferezou, J. Squalene isolation by HPLC and quantitative comparison by HPLC and GLC,
Lipids, 1984, 19, 631–635.
587
Stanozolol
Stanozolol
CH3 OH
CH3
CH3
Molecular formula: C21 H32 N2 O
Molecular weight: 328.49
N
CAS Registry No: 10418-03-8
Merck Index: 13, 8873
H
H
H
H
N
H
SAMPLE
Matrix: blood
Sample preparation: Condition a 1 mL 100 mg Bond Elut C1 SPE cartridge with 2
column vol of MeCN and 2 column vol of water. Mix 500 µL serum with 25 µL 5 µg/mL
IS in MeCN, add 500 µL water, vortex for 5 s, add to the SPE cartridge, wash with 2
column vol of water, wash with 1 column vol of MeCN:water 10:90, elute with 1 mL
MeCN:water 45:55, concentrate the eluate to 250 µL under a stream of nitrogen at 45◦ ,
inject a 50 µL aliquot.
HPLC VARIABLES
Guard column: 15 × 4.6 7 µm silica (Brownlee)
Column: 220 × 4.6 5 µm silica (Brownlee)
Column temperature: 60
Mobile phase: MeCN:100 mM pH 2.5 sodium phosphate buffer 15:85
Flow rate: 1
Injection volume: 50
Detector: UV 230
CHROMATOGRAM
Retention time: 12.7
Internal standard: spironolactone (7.8)
Limit of detection: 7 ng/mL
OTHER SUBSTANCES
Extracted: fluoxymesterone (UV 247) (5.4), methandrostenolone (UV 247) (9.3), methyltestosterone (UV 247) (9.6), nandrolone (UV 247) (8.2), testosterone (UV 247) (8.6),
zeranol (UV 263) (3.6)
KEY WORDS
serum: SPE
REFERENCE
Lampert, B.L.; Stewart, J.T. Determination of anabolic steroids and zeranol in human serum by isocratic
reverse phase HPLC on silica, J.Liq.Chromatogr., 1989, 12, 3231–3249.
588
Succimer
Succimer
Molecular formula: C4 H6 O4 S2
Molecular weight: 182.22
HOOC
SH
HOOC
SH
CAS Registry No: 304-55-2
Merck Index: 13, 8949
SAMPLE
Matrix: blood
Sample preparation: Mix 100–250 µL whole blood or plasma with 50 µL 50–125 mM
dithiothreitol and 100 mM pH 8.3 ammonium bicarbonate buffer containing IS to a final
volume of 2 mL, purge headspace with nitrogen, mix vigorously for 1 min, let stand in
the dark at room temperature for 30 min, filter (2 mL Centricon 30) while centrifuging
at 6000 rpm for 1 h. Add 200 µL 80 mM monobromobimane in 100 mM ammonium
bicarbonate buffer to the ultrafiltrate, purge with nitrogen, mix vigorously for 1 min, let
stand in the dark at room temperature for 10 min, wash with 2 vol of dichloromethane,
shake for 15 s, centrifuge at 2500 rpm for 2 min, repeat the wash. Add 17 µL 6 M HCl
to the aqueous layer, inject an aliquot.
HPLC VARIABLES
Column: 250 × 4.6 5 µm Spherisorb ODS-2
Mobile phase: Gradient. A was 10 mM tetrabutylammonium bromide in MeOH containing 10 mM sodium acetate. B was 10 mM tetrabutylammonium bromide in 10 mM
pH 4.1 acetate buffer. A:B 52.5:47.5 for 10 min, to 90:10 over 2 min, maintain at 90:10
for 5 min, return to initial conditions over 2 min, re-equilibrate at initial conditions for
11 min.
Flow rate: 1
Injection volume: 10
Detector: F ex 356 em 450 (see also Maiorino,R.M.; Barry,T.J.; Aposhian,H.V. Determination and metabolism of dithiol chelating agents: Electrolytic and chemical reduction
of oxidized dithiols in urine. Anal.Biochem. 1987, 160, 217–226.)
CHROMATOGRAM
Retention time: 7.1
Internal standard: 2,3-dimercaptopropane-1-sulfonic acid (10.2)
Limit of quantitation: 5 µM
OTHER SUBSTANCES
Extracted: dithiothreitol (5.5)
KEY WORDS
derivatization; plasma; whole blood
REFERENCE
Maiorino, R.M.; Akins, J.M.; Blaha, K.; Carter, D.E.; Aposhian, H.V. Determination and metabolism of
dithiol chelating agents: X. In humans, meso-2,3-dimercaptosuccinic acid is bound to plasma proteins
via mixed disulfide formation, J.Pharmacol.Exp.Ther., 1990, 254, 570–577.
Succinylcholine chloride
589
Succinylcholine chloride
Molecular formula: C14 H30 Cl2 N2 O4
Molecular weight: 361.31
CAS Registry No: 71-27-2, 55-94-7 (bromide),
541-19-5 (iodide)
Merck Index: 13, 8959 (chloride), 8958
(bromide), 8960 (iodide)
O
(H3C)3N+
O
O
N+(CH3)3
2Cl−
O
SAMPLE
Matrix: blood
Sample preparation: Condition a Bond Elut C2 SPE cartridge with 3 mL TMAH
buffer and 3 mL water. Mix 1 mL plasma with 1 mL water and 100 µL 100 ng/mL IS
in 100 mM pH 5.0 phosphate buffer, add to the SPE cartridge, wash with 3 mL water,
wash with 3 mL MeCN, wash with 3 mL MeOH, elute with two 250 µL portions of
TMAH buffer, inject a 150 µL aliquot of the eluate. (Prepare TMAH buffer by dissolving
275.6 mg tetramethylammonium chloride in 1 mL water and adding 240 mL MeOH,
adjust to apparent pH 3.0 with 100 mM HCl, make up to 250 mL with MeOH.)
HPLC VARIABLES
Column: 125 × 4.6 5 µm Spherisorb CN
Column temperature: 35
Mobile phase: MeCN:MeOH:30 mM phosphoric acid 35:25:45, adjusted to apparent pH
5.00 with concentrated ammonium hydroxide
Flow rate: 2
Injection volume: 150
Detector: E, ESA Coulochem II, 5010 analytical cell, detector 1 450 mV (screen), detector
2 750 mV (monitored)
CHROMATOGRAM
Retention time: 9.2
Internal standard: pipecuronium (19.8)
Limit of quantitation: 250 ng/mL
OTHER SUBSTANCES
Simultaneous: mivacurium (21), rocuronium (18), vecuronium (21)
KEY WORDS
plasma; SPE
REFERENCE
Gao, H.; Roy, S.; Donati, F.; Varin, F. Determination of succinylcholine in human plasma by highperformance liquid chromatography with electrochemical detection, J.Chromatogr.B, 1998, 718,
129–134.
SAMPLE
Matrix: blood
Sample preparation: Vortex thoroughly 400 µL plasma with 40 µL 500 mg/mL trichloroacetic acid, centrifuge at 12 500 g for 5 min, inject a 100 µL aliquot.
HPLC VARIABLES
Column: 250 × 4.6 8 µm Cp-tm-Spher C8 (Chrompack)
Mobile phase: MeCN:MeOH:50 mM pH 5.0 potassium phosphate buffer 35:5:60
Flow rate: 1.2
590
Succinylcholine chloride
Injection volume: 100
Detector: F ex 257 em 282
CHROMATOGRAM
Retention time: 6.3
Limit of quantitation: 100 ng/mL
KEY WORDS
pharmacokinetics; plasma
REFERENCE
Lagerwerf, A.J.; Vanlinthout, L.E.; Vree, T.B. Rapid determination of succinylcholine in human plasma
by high-performance liquid chromatography with fluorescence detection, J.Chromatogr., 1991, 570,
390–395.
591
Sulfabromomethazine
Sulfabromomethazine
CH3
Molecular formula: C12 H13 BrN4 O2 S
Molecular weight: 357.23
CAS Registry No: 116-45-0
Merck Index: 13, 8983
S
H2N
Br
O N
O
N
N
CH3
H
SAMPLE
Matrix: tissue
Sample preparation: Cut 20 g tissue into small pieces and add to 25 mL dichloromethane, let stand for 20–30 min with frequent stirring with a glass rod, filter through
glass wool, repeat extraction twice more. Combine the dichloromethane layers and
shake with 10 mL 1.5 M sulfuric acid for 15 min. Remove the aqueous phase and add
it to 10 mL dichloromethane, shake for 10 min, discard the organic phase, repeat the
wash, add 1 mL 10% dipotassium hydrogen phosphate to the aqueous phase, add 3 mL
40% NaOH, mix well, allow to cool, adjust pH to 5–6 with 1.5 M sulfuric acid or 40%
NaOH, add 5 mL dichloromethane, extract for 15 min, repeat the extraction. Pass the
organic layers through anhydrous sodium sulfate, evaporate to 1 g on a hot plate at
60–70◦ (in a hood!), inject a 10 µL aliquot. For confirmation of sulfabromomethazine,
add 1 mL MeOH to the extract, add 2 drops acetic anhydride, heat at 80–90◦ on a hot
plate until acetic acid fumes are no longer seen, reconstitute with 1 g dichloromethane,
inject a 10 µL aliquot. (Note: Extraction from the sample matrix is only demonstrated
for sulfamethazine, but it should work for sulfabromomethazine.)
HPLC VARIABLES
Column: 250 mm long MicroPak CN-10
Mobile phase: Isooctane:chloroform:MeOH:acetic acid 30.5:65:4:0.5 (Caution! Chloroform is a carcinogen!)
Flow rate: 0.33
Injection volume: 10
Detector: UV 254
CHROMATOGRAM
Retention time: 3.56 (4.16 for acetyl sulfabromomethazine)
Limit of detection: 20 ppb (for sulfamethazine)
OTHER SUBSTANCES
Extracted: acetylsulfachlorpyridazine (6.68), acetylsulfadiazine (6.78), acetylsulfadimethoxine (4.70), acetylsulfaethoxypyridazine (5.23), acetylsulfamethazine (5.34), sulfachlorpyridazine (5.60), sulfadiazine (5.55), sulfadimethoxine (3.99), sulfaethoxypyridazine (4.60), sulfamethazine (4.34)
Noninterfering: sulfamerazine, sulfathiazole, sulfanilamide, sulfapyridine, sulfaquinoline
KEY WORDS
cow; liver; fat; kidney; muscle
REFERENCE
Seymour, D.; Rupe, B.D. High-pressure liquid chromatographic determination of sulfamethazine residues
in beef tissues, J.Pharm.Sci., 1980, 69, 701–703.
592
Sulfachlorpyridazine
Sulfachlorpyridazine
Molecular formula: C10 H9 ClN4 O2 S
Molecular weight: 284.73
O N
O
S
H2N
N
Cl
N
H
CAS Registry No: 80-32-0, 23282-55-5 (Na salt)
Merck Index: 13, 8985
SAMPLE
Matrix: honey
Sample preparation: Condition a 200 mg Oasis HLB SPE cartridge with 3 mL MeCN
and two 2 mL portions of water. Dissolve 7.5 g honey in 15 mL 2 M HCl, let stand at
room temperature for 30 min, add 30 mL 300 mM citric acid, mix, filter. Adjust the pH
of a 20 mL aliquot of the filtrate to 3.5–4.5 with 25% ammonia, immediately add to the
SPE cartridge and pass through within 10–15 min, wash with three 3 mL portions of
water, let dry for 4 min, elute with 3 mL MeCN. Evaporate the eluate to a small volume
under reduced pressure at 40◦ , add 500 µL mobile phase, vortex, inject a 10 µL aliquot.
HPLC VARIABLES
Guard column: 2 mm long 5 µm Nucleosil 100 C18 HD
Column: 50 × 2 5 µm Nucleosil 100-5 C18 HD
Mobile phase: Gradient. A was MeCN:water:formic acid 5:94.7:0.3. B was MeCN:formic
acid 99.7:0.3. A:B from 100:0 to 70:30 over 10 min, maintain at 70:30 for 2 min, return
to initial conditions over 0.1 min, re-equilibrate for 6.9 min.
Flow rate: 0.2
Injection volume: 10
Detector: MS, Micromass Quattro LCZ, triple quadrupole, electrospray, capillary 3.25 kV,
cone 25 V, extractor 3 V, RF lens 0.3 V, source 90◦ , desolvation 250◦ , cone gas flow 50 L/h,
desolvation gas nitrogen 560 L/h, nebulizer gas flow is factory preset, LM 1 resolution 12,
HM 1 resolution 12, ion energy 1 V, entrance voltage −5 V, exit voltage 0 V, collision cell
pressure 2 µbar argon, LM 2 resolution 12, HM 2 resolution 12, ion energy 2 V, multiplier
voltage 650 V, m/z 285–156
CHROMATOGRAM
Retention time: 12.9
Limit of detection: 1 ppb
OTHER SUBSTANCES
Extracted: chlortetracycline (m/z 479–444) (LOD 1 ppb) (13.1), flumequine (m/z 262–244)
(LOD 1 ppb) (17.9), oxytetracycline (m/z 461–426) (LOD 4 ppb) (11.1), sulfacetamide (m/z
215–156) (LOD 4 ppb) (5.6), sulfachloropyrazine (m/z 285–156) (LOD 1 ppb) (15.8), sulfadiazine (m/z 251–156) (LOD 2 ppb) (6.5), sulfadimethoxine (m/z 311–156) (LOD 1 ppb)
(16.0), sulfadoxine (m/z 311–156) (LOD 0.4 ppb) (13.5), sulfaguanidine (m/z 215–156)
(LOD 2 ppb) (2.0), sulfamerazine (m/z 265–156) (LOD 1 ppb) (8.9), sulfamethazine (m/z
279–186) (LOD 1 ppb) (10.4), sulfamethizole (m/z 271–156) (LOD 0.5 ppb) (11.5), sulfamethoxazole (m/z 254–156) (LOD 1 ppb) (14.0), sulfamethoxypyridazine (m/z 281–156)
(LOD 0.4 ppb) (11.5), sulfanilamide (m/z 173–156) (LOD 4 ppb) (2.4), sulfapyridine (m/z
250–156) (LOD 11 ppb) (8.3), sulfathiazole (m/z 256>) (LOD 156 ppb) (1 8.6), sulfisoxazole
(m/z 268–156) (LOD 1 ppb) (14.9), tetracycline (m/z 445–410) (LOD 4 ppb) (10.5)
KEY WORDS
SPE
REFERENCE
Kaufmann, A.; Roth, S.; Ryser, B.; Widmer, M.; Guggisberg, D. Quantitative LC/MS-MS determination
of sulfonamides and some other antibiotics in honey, J.AOAC Int., 2002, 85, 853–860.
Sulfachlorpyridazine
593
SAMPLE
Matrix: solutions
Sample preparation: Inject an aliquot of a solution in MeOH.
HPLC VARIABLES
Column: 300 × 3.9 µBondapak C18
Mobile phase: MeCN:water:acetic acid 12.5:86.5:1
Flow rate: 1.6
Injection volume: 10
Detector: UV 254
CHROMATOGRAM
Retention time: 13
OTHER SUBSTANCES
Simultaneous: sulfabenzamide (25), sulfacetamide (4), sulfadiazine (5), sulfadimethoxine (40), sulfamerazine (7), sulfamethazine (8), sulfamethizole (9), sulfamethoxazole
(16), sulfanilamide (2.5), sulfapyridine (6), sulfisoxazole (20)
REFERENCE
Roos, R.W. High pressure liquid chromatographic determination of sulfisoxazole in pharmaceuticals and
separation patterns of other sulfonamides, J.Assoc.Off.Anal.Chem., 1981, 64, 851–854.
SAMPLE
Matrix: tissue
Sample preparation: Condition a 500 mg Chromabond SA cation-exchange SPE cartridge (Macherey-Nagel) with 6 mL hexane, dry under vacuum for 10 min, condition
with 6 mL dichloromethane:acetone:acetic acid 50:50:2, do not allow to go dry. Homogenize (Polytron) 10 g sample with 60 mL dichloromethane:acetone 50:50 for 30 s, rinse
the apparatus with 2–3 mL dichloromethane:acetone 50:50, centrifuge the mixture at
2500 rpm for 10 min. Filter (cotton wool) the supernatant and wash it through with a
little dichloromethane:acetone 50:50, add 5 mL acetic acid to the filtrate, mix, remove
one-tenth of this mixture and add it to the SPE cartridge at 2 mL/min, do not allow the
SPE cartridge to run dry, wash with 5 mL water, wash with 5 mL MeOH, dry under
vacuum for 10 min, pass gaseous ammonia through the SPE cartridge until the acid is
neutralized (when air is passed through the cartridge, moist pH paper should turn blue),
elute with 3 mL MeOH at 1–2 mL/min, carefully evaporate to dryness under reduced
pressure (100 mbar) at 40◦ , reconstitute with 500 µL initial mobile phase, centrifuge,
inject a 50 µL aliquot of the supernatant.
HPLC VARIABLES
Column: 125 × 4 5 µm LiChrospher 100 RP-18
Mobile phase: Gradient. A was MeCN:20 mM pH 5 sodium acetate buffer 5.5:94.5. B
was MeCN:EtOH:20 mM pH 5 sodium acetate buffer 50:10:40. A:B from 100:0 to 0:100
over 32 min (concave gradient), return to initial conditions over 4 min, re-equilibrate at
initial conditions for 10 min.
Flow rate: 0.8
Injection volume: 50
Detector: UV 270; F ex 395 em 495 following post-column reaction. The column effluent
mixed with ice-cold reagent pumped at 0.3 mL/min and this mixture flowed through a
2.3 m × 0.5 mm ID coil in a cooled ultrasonic bath to the detector. (Prepare reagent by
dissolving 25 mg fluorescamine in 25 mL MeCN and adding 75 mL buffer and 200 µL
mercaptoethanol. The buffer was 20 mM sodium dihydrogen phosphate adjusted to pH
3 with 1 M phosphoric acid.)
594
Sulfachlorpyridazine
CHROMATOGRAM
Retention time: 22
Limit of detection: 0.5–5 ppb
OTHER SUBSTANCES
Extracted: sulfadiazine (10), sulfadimethoxine (28), sulfadoxine (24), sulfaguanidine (3),
sulfamerazine (16.5), sulfamethazine (20), sulfamethizole (18), sulfamethoxypyridazine
(21), sulfanilamide (3.7), sulfapyridine (15.7), sulfathiazole (15)
KEY WORDS
kidney; muscle; post-column reaction; SPE
REFERENCE
Pacciarelli, B.; Reber, S.; Douglas, C.; Dietrich, S.; Etter, R. Bestimmung von 12 Sulfonamiden in Fleisch
und Nieren mittels HPLC und Nachsäulenderivatisierung [Determination of 12 sulfonamides in meat
and kidney by HPLC and post column derivatization], Mitt.geb.Lebensmittelunters.Hyg., 1991, 82,
45–55.
SAMPLE
Matrix: tissue
Sample preparation: Cut 20 g tissue into small pieces and add to 25 mL dichloromethane, let stand for 20–30 min with frequent stirring with a glass rod, filter through
glass wool, repeat extraction twice more. Combine the dichloromethane layers and
shake with 10 mL 1.5 M sulfuric acid for 15 min. Remove the aqueous phase and add
it to 10 mL dichloromethane, shake for 10 min, discard the organic phase, repeat the
wash, add 1 mL 10% dipotassium hydrogen phosphate to the aqueous phase, add 3 mL
40% NaOH, mix well, allow to cool, adjust pH to 5–6 with 1.5 M sulfuric acid or 40%
NaOH, add 5 mL dichloromethane, extract for 15 min, repeat the extraction. Pass the
organic layers through anhydrous sodium sulfate, evaporate to 1 g on a hot plate at
60–70◦ (in a hood!), inject a 10 µL aliquot. For confirmation of sulfachlorpyridazine,
add 1 mL MeOH to the extract, add 2 drops acetic anhydride, heat at 80–90◦ on a hot
plate until acetic acid fumes are no longer seen, reconstitute with 1 g dichloromethane,
inject a 10 µL aliquot. (Note: Extraction from the sample matrix is only demonstrated
for sulfamethazine, but it should work for sulfachlorpyridazine.)
HPLC VARIABLES
Column: 250 mm long MicroPak CN-10
Mobile phase: Isooctane:chloroform:MeOH:acetic acid 30.5:65:4:0.5 (Caution! Chloroform is a carcinogen!)
Flow rate: 0.33
Injection volume: 10
Detector: UV 254
CHROMATOGRAM
Retention time: 5.60 (6.68 for acetylsulfachlorpyridazine)
Limit of detection: 20 ppb (for sulfamethazine)
OTHER SUBSTANCES
Extracted: acetylsulfabromomethazine (4.16), acetylsulfadiazine (6.78), acetylsulfadimethoxine (4.70), acetylsulfaethoxypyridazine (5.23), acetylsulfamethazine (5.34), sulfabromomethazine (3.56), sulfadiazine (5.55), sulfadimethoxine (3.99), sulfaethoxypyridazine (4.60), sulfamethazine (4.34)
Noninterfering: sulfamerazine, sulfathiazole, sulfanilamide, sulfapyridine, sulfaquinoline
Sulfachlorpyridazine
595
KEY WORDS
cow; liver; fat; kidney; muscle
REFERENCE
Seymour, D.; Rupe, B.D. High-pressure liquid chromatographic determination of sulfamethazine residues
in beef tissues, J.Pharm.Sci., 1980, 69, 701–703.
ANNOTATED BIBLIOGRAPHY
Combs, M.T.; Ashraf-Khorassani, M.; Taylor, L.T. Method development for the separation of sulfonamides by supercritical fluid chromatography, J.Chromatogr.Sci., 1997, 35, 176–180. [sulfamethazine;
sulfamerazine; sulfapyridine; sulfadimethoxine; sulfadiazine; sulfaquinoxaline; sulfachlorpyridazine;
sulfathiazole]
Combs, M.T.; Ashraf-Khorassani, M.; Taylor, L.T. HPLC/atmospheric pressure chemical ionization-mass
spectroscopy of eight regulated sulfonamides, J.Pharm.Biomed.Anal., 1999, 19, 301–308. [LOD
0.05–6 ng; sulfathiazole; sulfamethazine; sulfamerazine; sulfapyridine; sulfadimethoxine; sulfadiazine; sulfaquinoxaline; sulfachlorpyridazine]
McGrane, M.; O’Keeffe, M.; Smyth, M.R. The analysis of sulphonamide drug residues in pork muscle
using automated dialysis, Anal.Lett., 1999, 32, 481–495. [LOD 40 ng/g; sulfadiazine; sulfathiazole;
sulfapyridine; sulfamerazine; sulfamethizole; sulfamethazine; sulfamethoxypyridazine; sulfachlorpyridazine; sulfisoxazole]
Thomas, G.K.; Millar, R.G.; Anstis, P.W. Stability of sulfonamide antibiotics in spiked pig liver tissue during frozen storage, J.AOAC Int., 1997, 80, 988–995. [sulfamethazine; sulfadimethoxine;
sulfachlorpyridazine; sulfathiazole; sulfaquinoxaline]
596
Sulfaethoxypyridazine
Sulfaethoxypyridazine
Molecular formula: C12 H14 N4 O3 S
Molecular weight: 294.32
O N
O
S
H2N
N
O
CH3
N
H
CAS Registry No: 963-14-4
SAMPLE
Matrix: tissue
Sample preparation: Condition a 3 mL 500 mg Sep-Pak C18 SPE cartridge with
20 mL MeOH and 20 mL water. Shake 5 g homogenized tissue and 25 mL chloroform
mechanically for 2 min (Caution! Chloroform is a carcinogen!), centrifuge at 3000 g for
5 min, repeat the extraction. Combine the lower organic layers, add 10 mL 10% NaCl
in 100 mM NaOH, shake vigorously for 1 min. Remove the upper aqueous layer and
centrifuge it at 1500 g for 10 min. Remove 8 mL of the upper aqueous layer and add
it to 10 mL 1 M pH 6 sodium dihydrogen phosphate, vortex for 20 s, add to the SPE
cartridge, wash with 20 mL water, elute with 1 mL MeOH. Add 2 mL 50 mM sodium
dihydrogen phosphate to the eluate, filter (0.45 µm), inject a 20–50 µL aliquot. (SPE
cartridge must have a carbon loading of 14%.)
HPLC VARIABLES
Column: 250 × 4.6 5 µm Spherisorb C18 ODS
Mobile phase: MeOH:50 mM sodium dihydrogen phosphate 30:70
Flow rate: 0.8
Injection volume: 20–50
Detector: UV 265
CHROMATOGRAM
Retention time: 24.5
Limit of detection: 2 ng/g (for sulfamethazine)
OTHER SUBSTANCES
Extracted: sulfamethazine (10.5)
Simultaneous: sulfachlorpyridazine (12.1), sulfadiazine (3.8), sulfadimethoxine (45),
sulfadoxine (17.2), sulfamerazine (6.4), sulfamethoxazole (14), sulfamethoxypyridazine
(11.7), sulfanilamide (8.0), sulfathiazole (4.2)
KEY WORDS
kidney; liver; muscle; pig; SPE; the method is validated for sulfamethazine and sulfaethoxypyridazine is the internal standard
REFERENCE
Boison, J.O.K.; Keng, L.J.-Y. Determination of sulfamethazine in bovine and porcine tissues by reversedphase liquid chromatography, J.AOAC Int., 1994, 77, 558–564.
SAMPLE
Matrix: tissue
Sample preparation: Cut 20 g tissue into small pieces and add to 25 mL dichloromethane, let stand for 20–30 min with frequent stirring with a glass rod, filter through
glass wool, repeat extraction twice more. Combine the dichloromethane layers and
shake with 10 mL 1.5 M sulfuric acid for 15 min. Remove the aqueous phase and add
it to 10 mL dichloromethane, shake for 10 min, discard the organic phase, repeat the
wash, add 1 mL 10% dipotassium hydrogen phosphate to the aqueous phase, add 3 mL
40% NaOH, mix well, allow to cool, adjust pH to 5–6 with 1.5 M sulfuric acid or 40%
NaOH, add 5 mL dichloromethane, extract for 15 min, repeat the extraction. Pass the
Sulfaethoxypyridazine
597
organic layers through anhydrous sodium sulfate, evaporate to 1 g on a hot plate at
60–70◦ (in a hood!), inject a 10 µL aliquot. For confirmation of sulfaethoxypyridazine,
add 1 mL MeOH to the extract, add 2 drops acetic anhydride, heat at 80–90◦ on a hot
plate until acetic acid fumes are no longer seen, reconstitute with 1 g dichloromethane,
inject a 10 µL aliquot. (Note: Extraction from the sample matrix is only demonstrated
for sulfamethazine, but it should work for sulfaethoxypyridazine.)
HPLC VARIABLES
Column: 250 mm long MicroPak CN-10
Mobile phase: Isooctane:chloroform:MeOH:acetic acid 30.5:65:4:0.5 (Caution! Chloroform is a carcinogen!)
Flow rate: 0.33
Injection volume: 10
Detector: UV 254
CHROMATOGRAM
Retention time: 4.60 (5.23 for acetylsulfaethoxypyridazine)
Limit of detection: 20 ppb (for sulfamethazine)
OTHER SUBSTANCES
Extracted: acetylsulfabromomethazine (4.16), acetylsulfachlorpyridazine (6.68), acetylsulfadiazine (6.78), acetylsulfadimethoxine (4.70), acetylsulfamethazine (5.34), sulfabromomethazine (3.56), sulfachlorpyridazine (5.60), sulfadiazine (5.55), sulfadimethoxine
(3.99), sulfamethazine (4.34)
Noninterfering: sulfamerazine, sulfathiazole, sulfanilamide, sulfapyridine, sulfaquinoline
KEY WORDS
cow; liver; fat; kidney; muscle
REFERENCE
Seymour, D.; Rupe, B.D. High-pressure liquid chromatographic determination of sulfamethazine residues
in beef tissues, J.Pharm.Sci., 1980, 69, 701–703.
598
Sulfamerazine
Sulfamerazine
Molecular formula: C11 H12 N4 O2 S
Molecular weight: 264.31
O N
O
S
H2N
N
N
CH3
H
CAS Registry No: 127-79-7, 127-58-2 (Na salt)
Merck Index: 13, 8998
SAMPLE
Matrix: blood
Sample preparation: Vortex 1 mL plasma with 5–20 µg IS, 1 mL 10 mM pH 6.8
dipotassium hydrogen phosphate buffer, and 10 mL ethyl acetate:isopropanol 98:2 for
10 s, centrifuge at 1000 g for 10 min. Evaporate the organic layer to dryness under a
stream of nitrogen at 60◦ , reconstitute the residue with 100 µL MeOH, inject a 5–50 µL
aliquot.
HPLC VARIABLES
Column: 250 × 4.6 5 µm Spherisorb ODS1
Mobile phase: MeCN:MeOH:20 mM sodium dihydrogen phosphate 5:4:91
Flow rate: 1.5
Injection volume: 5–50
Detector: E, EDT Research LCA 15, +1.0 V
CHROMATOGRAM
Retention time: 21.5
Internal standard: sulfamethazine (36)
Limit of quantitation: 1 µg/mL
OTHER SUBSTANCES
Extracted: sulfadiazine (13.0), sulfapyridine (18.5)
KEY WORDS
pharmacokinetics; plasma; sheep
REFERENCE
Mallett, D.N.; Gulaid, A.A.; Dennis, M.J. High-performance liquid chromatographic method with electrochemical detection for the concomitant assay of sulphadiazine, sulphamerazine and sulphapyridine
in plasma, J.Chromatogr., 1988, 428, 190–195.
SAMPLE
Matrix: blood
Sample preparation: Mix 1 mL plasma with 1 mL MeOH, centrifuge at 2000 g for
10 min, inject a 20 µL aliquot.
HPLC VARIABLES
Guard column: Corasil/C18
Column: µBondapak C18
Mobile phase: MeOH:water 40:60
Flow rate: 0.8
Injection volume: 20
Detector: UV 254
CHROMATOGRAM
Retention time: 5.40
Limit of quantitation: 10 ng/mL
Sulfamerazine
599
OTHER SUBSTANCES
Extracted: acetylsulfisoxazole (4.40), sulfaguanidine (3.90), sulfisoxazole (4.20), sulfamethazine (6.10), sulfanilamide (3.05), sulfapyridine (5.00), sulfathiazole (3.30)
KEY WORDS
plasma
REFERENCE
Suber, R.L.; Edds, G.T. High performance liquid chromatographic determinations of sulfonamides by
ionic suppression, J.Liq.Chromatogr., 1980, 3, 257–268.
ANNOTATED BIBLIOGRAPHY
Goehl, T.J.; Mathur, L.K.; Strum, J.D.; Jaffe, J.M.; Pitlick, W.H.; Shah, V.P.; Poust, R.I.; Colaizzi, J.L.
Simple high-pressure liquid chromatographic determination of trisulfapyrimidines in human serum,
J.Pharm.Sci., 1978, 67, 404–406. [sulfamerazine; sulfadiazine; sulfamethazine]
600
Sulfanitran
Sulfanitran
Molecular formula: C14 H13 N3 O5 S
Molecular weight: 335.34
CAS Registry No: 122-16-7
Merck Index: 13, 9019
NO2
O
O
S
O
H3C
N
N
H
H
SAMPLE
Matrix: feed, premix
Sample preparation: Shake ground feed containing 1.5 mg sulfanitran with 80 mL
MeOH at 60◦ for 20 min, cool, let stand for 2 h, make up to 100 mL with MeOH,
mix well, filter (Whatman No. 2 paper), filter (0.50 µm), inject a 20 µL aliquot. Shake
premix containing 150 mg sulfanitran with 100 mL DMF on a rotary shaker for 1 h,
filter (Whatman No. 2 paper), filter (0.50 µm), dilute 1 mL filtrate to 50 mL with DMF,
inject a 20 µL aliquot.
HPLC VARIABLES
Guard column: 100 × 3 (OD) Bondapak C18/Corasil
Column: 300 × 4 µBondapak C18
Mobile phase: MeCN:water 45:55
Flow rate: 1
Injection volume: 20
Detector: UV 254
CHROMATOGRAM
Retention time: 6
OTHER SUBSTANCES
Extracted: dinsed (9)
REFERENCE
Eaves, K.L.; Colvin, B.M.; Hanks, A.R.; Bushway, R.J. High pressure liquid chromatographic determination of sulfanitran and dinsed in medicated feeds and premixes, J.Assoc.Off.Anal.Chem., 1977, 60,
1064–1066.
SAMPLE
Matrix: tissue
Sample preparation: Prepare a 30 × 6 column of 80–200 mesh neutral alumina
(Brockman Activity I) and wash with two 2 mL portions of chloroform:ethyl acetate
50:50 (Caution! Chloroform is a carcinogen!). Blend (Polytron) 2.5 g partially frozen
tissue with 20 mL chloroform:ethyl acetate:DMSO 50:50:0.8 at medium speed for 45 s,
centrifuge at 3500 rpm for 5 min (10 min for liver), pass the organic layer through a
plug of glass wool. Pass 15 mL filtrate through the alumina column, wash with three
1 mL portions of chloroform, wash with 3 mL chloroform, force remaining chloroform
out with air pressure until dry, pass air through the column for an additional 5 min,
elute with MeOH:50 mM pH 6.0 potassium phosphate buffer 50:50, collect first 2 mL
eluate, inject a 50 µL aliquot. (Protect from light.)
HPLC VARIABLES
Column: 250 × 4.6 5 µm Supelcosil LC-18
Mobile phase: MeOH:buffer 42.5:57.5 (The buffer was 50 mM potassium dihydrogen
phosphate containing 1 mM EDTA, adjusted to pH 6.0 with 1 M NaOH.)
Flow rate: 1
Injection volume: 50
Sulfanitran
601
Detector: E, Bioanalytical Systems BAS Model LC-4B, glassy carbon electrode −0.8V,
Ag/AgCl reference electrode
CHROMATOGRAM
Retention time: 13.2
Limit of detection: <6 ng/g
OTHER SUBSTANCES
Extracted: aklomide (5.3), furazolidone (4.0), nitrofurazone (4.1), nitromide (6.7), zoalene
(6.2)
KEY WORDS
chicken; liver; muscle; SPE
REFERENCE
Parks, O.W. Liquid chromatographic-electrochemical detection screening procedure for six nitro-containing drugs in chicken tissues at low ppb level, J.Assoc.Off.Anal.Chem., 1989, 72, 567–569.
602
Sultamicillin
Sultamicillin
Molecular formula: C25 H30 N4 O9 S2
Molecular weight: 594.66
O
S
N
NH2 H
O
H H
N
CH3 H3C
O
CH3H3C
N
O
CAS Registry No: 76497-13-7, 83105-70-8 (tosylate)
Merck Index: 13, 9083
H
S
O
O
O
O
O
SAMPLE
Matrix: bulk
Sample preparation: Inject a 20 µL aliquot of a solution in MeCN:25 mM pH 7.0
phosphate buffer 70:30.
HPLC VARIABLES
Column: 150 × 4.6 5 µm Kromasil C18
Column temperature: 20
Mobile phase: MeCN:25 mM pH 7.0 phosphate buffer 48:52
Flow rate: 1
Injection volume: 20
Detector: UV 215
CHROMATOGRAM
Retention time: 4.92
Limit of detection: 200 ng/mL
OTHER SUBSTANCES
Extracted: degradants, impurities
REFERENCE
Laviana, L.; Fernández-Marı́, F.; Bayod, M.; Blanco, D. HPLC for in-process control in the production of
sultamicillin, J.Pharm.Biomed.Anal., 2003, 26, 321–328.
SAMPLE
Matrix: formulations
Sample preparation: Sonicate a finely powdered tablet with 50 mL MeCN for 10 min,
make up to 100 mL with MeCN, filter (Whatman No. 42 paper), dilute a 1 mL aliquot to
100 mL with MeCN, inject a 20 µL aliquot.
HPLC VARIABLES
Column: 250 × 3.9 5 µm Shodex C18
Mobile phase: MeCN:20 mM sodium dihydrogen phosphate 20:60, pH adjusted to 3.0
with dilute phosphoric acid
Injection volume: 20
Detector: UV 215
CHROMATOGRAM
Retention time: 7.8
Limit of detection: 10 µg/mL
Limit of quantitation: 40 µg/mL
OTHER SUBSTANCES
Extracted: p-toluenesulfonic acid (3.7)
Sultamicillin
603
KEY WORDS
tablets
REFERENCE
Argekar, A.P.; Kunjir, S.S. Quantitative estimation of sultamicillin p-toluenesulfonate in pharmaceutical
preparations by reverse-phase high performance liquid chromatography, J.Pharm.Biomed.Anal., 1996,
15, 423–427.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 × 4.6 10 µm Cosmosil 5C18-MS
Column temperature: 50
Mobile phase: Gradient. MeCN:10 mM pH 2.5 potassium phosphate buffer 0:100 for
2 min, to 75:25 over 7.5 min, maintain at 75:25 for 5.5 min. Isocratic. MeCN:buffer
15:85.
Flow rate: 1.5
Detector: UV (wavelength not specified)
CHROMATOGRAM
Retention time: 8.3 (gradient) or 4.3 (isocratic)
OTHER SUBSTANCES
Simultaneous: acetaminophen (7.9), alacepril (10.9), ampicillin (7.9), aspirin (10.0),
caffeine (8.5), carbenicillin (9.5), cefotiam (7.2), chlorpromazine (10.8), cromolyn (8.9),
enalapril (9.9), loperamide (11.6), ofloxacin (8.3), procainamide (7.4), procaine (7.9),
propranolol (9.6), tegafur (8.4), temocapril (12.3), theophylline (8.0), tulobuterol (8.9)
(gradient retention times; isocratic conditions may differ)
REFERENCE
Sugiyama, T.; Matsuyama, R.; Usui, S.; Katagiri, Y.; Hirano, K. Selection of mobile phases in highperformance liquid chromatographic determination for medicines, Biol.Pharm.Bull., 2000, 23, 274–278.
Tacalcitol
OH
H 3C
CH3
Molecular formula: C27 H44 O3
Molecular weight: 416.63
CAS Registry No: 57333-96-7
Merck Index: 13, 9114
CH3
H
CH3
H
CH2
HO
OH
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 25 × 6.2 Zorbax SIL
Mobile phase: n-Hexane:isopropanol 90:10
Flow rate: 1.5
Detector: Radioreceptor assay
CHROMATOGRAM
Retention time: 20
REFERENCE
Shigeno, C.; Yamamoto, I.; Dokoh, S.; Hino, M.; Aoki, J.; Yamada, K.; Morita, R.; Kameyama, M.; Torizuka, K. Identification of 1,24(R)-dihydroxyvitamin D3 -like bone-resorbing lipid in a patient with
cancer-associated hypercalcemia, J.Clin.Endocrinol.Metab., 1985, 61, 761–768.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 25 × 4.6 Zorbax ODS
Mobile phase: MeOH:water 80:20
Flow rate: 1
Detector: Radioreceptor assay
CHROMATOGRAM
Retention time: 19
REFERENCE
Shigeno, C.; Yamamoto, I.; Dokoh, S.; Hino, M.; Aoki, J.; Yamada, K.; Morita, R.; Kameyama, M.; Torizuka, K. Identification of 1,24(R)-dihydroxyvitamin D3 -like bone-resorbing lipid in a patient with
cancer-associated hypercalcemia, J.Clin.Endocrinol.Metab., 1985, 61, 761–768.
604
HPLC Methods for Recently Approved Pharmaceuticals. by George Lunn
Copyright 2005 John Wiley & Sons, Ltd. ISBN: 0-471-66941-5
605
Talipexole
Talipexole
Molecular formula: C10 H15 N3 S
S
H2C
N
NH2
N
Molecular weight: 209.32
CAS Registry No: 101626-70-4,
36085-73-1 (di HCl)
Merck Index: 13, 9129
SAMPLE
Matrix: blood, urine
Sample preparation: Vortex 1 mL urine or plasma with 50 µL 1% acetic acid, 100 µL
1 M NaOH, and 6 mL diethyl ether for 5 min, centrifuge at 2500 g for 5 min, freeze
in dry ice/acetone. Add the organic layer to 100 (plasma) or 200 (urine) µL buffer,
mix, freeze in dry ice/acetone, discard the organic layer, remove traces of organic
solvent with a stream of nitrogen, thaw, inject a 50 µL aliquot. (Prepare the buffer
by dissolving 1.7 g potassium dihydrogen phosphate, 1.7 g sodium acetate, and 0.5 g
sodium heptanesulfonate in 500 mL water, adjust pH to 3.5 with acetic acid. Use only
polypropylene containers.)
HPLC VARIABLES
Guard column: 15 × 3.2 7 µm Brownlee RP-8
Column: 250 × 4.6 5 µm Zorbax Rx C8
Mobile phase: MeCN:buffer 13:87 (Prepare mobile phase by dissolving 10.2 g potassium
dihydrogen phosphate, 10.2 g sodium acetate, and 4.5 g sodium heptanesulfonate in 3 L
water, adjust pH to 3.5 with acetic acid, add 450 mL MeCN.)
Flow rate: 1.2
Injection volume: 50
Detector: E, ESA 5100A detector, 5020 guard cell 0.65 V, 5011 analytical cell, electrode
1 0.2 V (impurity screening), electrode 2 0.6 V (monitored); UV 286
CHROMATOGRAM
Retention time: 10
Limit of quantitation: 50 pg/mL (plasma, E), 10 ng/mL (urine, UV) (for pramipexole)
OTHER SUBSTANCES
Extracted: pramipexole (15)
KEY WORDS
plasma; method is validated for pramipexole and talipexole is IS; recovery of talipexole
from plasma is 98.2% and from urine is 95.1%
REFERENCE
Lau, Y.Y.; Hanson, G.D.; Ichhpurani, N. Determination of pramipexole (U-98,528) in human plasma
and urine by high-performance liquid chromatography with electrochemical and ultraviolet detection.
J.Chromatogr.B, 1996, 683, 217–223.
SAMPLE
Matrix: blood
Sample preparation: Vortex 1 mL plasma with 50 µL 1% acetic acid, 100 µL 1 M
NaOH, and 6 mL MTBE for 5 min, centrifuge at 630 g for 5 min, freeze in dry ice/acetone.
Evaporate the organic layer to dryness under a stream of nitrogen, reconstitute the
residue with 100 µL MeOH:water 95:5, inject a 70 µL aliquot. (Use only polypropylene
containers.)
606
Talipexole
HPLC VARIABLES
Column: 150 × 4.6 5 µm Zorbax SB CN
Mobile phase: MeOH:water:100 mM ammonium acetate 80:15:5
Flow rate: 1.2
Injection volume: 70
Detector: MS, PE Sciex API-III triple quadrupole, nebulizer 470◦ , gas-phase chemical
ionization, positive ion mode, Corona discharge 3 µA, nebulizer gas at 550 kPa, auxiliary
gas 1.5 L/min, orifice 45 V, m/z 210–141
CHROMATOGRAM
Retention time: 2.2
Limit of quantitation: 50 pg/mL (for pramipexole)
OTHER SUBSTANCES
Extracted: pramipexole (m/z 212–153) (3.4)
KEY WORDS
plasma; method is validated for pramipexole, and talipexole is IS; recovery of talipexole is
85.4%
REFERENCE
Lau, Y.Y.; Hanson, G.D.; Ichhpurani, N. Determination of pramipexole (U-98,528) in human plasma
and urine by high-performance liquid chromatography with electrochemical and ultraviolet detection,
J.Chromatogr.B, 1996, 683, 217–223.
607
Taltirelin
Taltirelin
Molecular formula: C17 H23 N7 O5
Molecular weight: 405.41
CAS Registry No: 103300-74-9,
201677-75-0 (tetrahydrate)
Merck Index: 13, 9135
N
H
O
N
N
NH
O
H
H3C
N
N
O
O
NH2
O
SAMPLE
Matrix: blood
Sample preparation: Condition a 3 mL 60 mg Oasis HLB divinylbenzene-N-vinylpyrrolidone SPE cartridge with 1 mL MeOH, 1 mL water, and 1 mL 200 mM sodium
carbonate. Mix 500 µL plasma with 50 µL mobile phase A and 1 mL 200 mM sodium
carbonate, add to the SPE cartridge, wash with 2 mL water, elute with 1 mL MeOH.
Evaporate the eluate to dryness under a stream of nitrogen at 40◦ , reconstitute the
residue with 250 µL 50 ng/mL IS in mobile phase A, inject a 25 µL aliquot.
HPLC VARIABLES
Column: 150 × 2 5 µm Daisopak SP-120-30DS-BP (Daiso, Osaka)
Column temperature: 40
Mobile phase: Gradient. A was MeOH:0.1% formic acid 7.5:92.5. B was MeOH:0.1%
formic acid 90:10. A:B 100:0 for 3 min, to 0:100 over 1 min, maintain at 0:100 for 3 min,
return to initial conditions over 0.1 min, re-equilibrate for 12.9 min.
Flow rate: 0.2
Injection volume: 25
Detector: MS, PE Sciex API 3000, TurboIonSpray, positive ionization, source 475◦ ,
source flow rate 7 L/min, ionspray 5000 V, nebulizer gas air at 14 units, curtain gas
nitrogen at 12 units, collision gas nitrogen at 4 units, collision energy – 80 eV, m/z
406–264
CHROMATOGRAM
Retention time: 3.1
Internal standard: N-[(S)-hexahydro-1-methyl-2,6-dioxo-4-pyrimidinylcarbonyl]-L-histidine (m/z 310–264) (2.7)
Limit of quantitation: 17 pg/mL
OTHER SUBSTANCES
Extracted: taltirelin acid-type (4.9)
KEY WORDS
plasma; SPE
REFERENCE
Horimoto, S.; Mayumi, T.; Tagawa, K.; Yamakita, H.; Yoshikawa, M. Determination of taltirelin, a new
stable thyrotropin-releasing hormone analogue, in human plasma by high-performance liquid chromatography turbo-ionspray ionization tandem mass spectrometry, J.Pharm.Biomed.Anal., 2002, 30,
1361–1369.
608
Technetium Tc 99m bicisate
Technetium Tc 99m bicisate
Molecular formula: C12 H21 N2 O5 S99m
2 Tc
Molecular weight: 436.44
CAS Registry No: 121281-41-2
Merck Index: 13, 9181
O
H3C
O
H
O
N O N
99m
Tc
S
O
CH3
S
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 × 4.6 Dynamax C8 (Rainin)
Mobile phase: Gradient. MeCN:50 mM ammonium acetate (unspecified)
Flow rate: 1
Detector: UV 254; Radioactivity (99 Tc)
CHROMATOGRAM
Retention time: 7.1
REFERENCE
Walovitch, R.C.; Franceschi, M.; Picard, M.; Cheesman, E.H.; Hall, K.M.; Makuch, J.; Watson, M.W.;
Zimmerman, R.E.; Watson, A.D.; Ganey, M.V.; Williams, S.J.; Holman, B.L. Metabolism of 99m Tc-L,Lethyl cysteinate dimer in healthy volunteers, Neuropharmacology, 1991, 30, 283–292.
Tegaserod
Tegaserod
NH
H
Molecular formula: C16 H23 N5 O
Molecular weight: 301.39
CAS Registry No: 145158-71-0,
189188-57-6 (maleate)
Merck Index: 13, 9194
609
N
N
N
H
CH3
H3CO
N
H
SAMPLE
Matrix: microsomal incubations
Sample preparation: Add an equal volume of cold MeOH, 70% perchloric acid, or 10%
trichloroacetic acid, cool on dry ice, centrifuge at 100 000 g for 10 min, inject an aliquot
of the supernatant.
HPLC VARIABLES
Guard column: 20 × 4.6 5 µm Supelco LC-18
Column: 150 × 4.6 5 µm Supelco LC-18
Column temperature: 40
Mobile phase: Gradient. MeCN:10 mM pH 5.4 ammonium acetate 0:100 for 10 min, to
10:90 over 20 min, to 90:10 over 41 min, to 100:0 over 19 min.
Flow rate: 1
Detector: Radioactivity (14 C) (scintillator flow 3 mL/min); MS, Finnigan MAT TSQ 700
triple-stage quadrupole, electrospray, 0.25 mL/min of column effluent mixed with
0.1 mL/min MeCN and entered the detector, sheath liquid methanol at 0.1 mL/min,
sheath gas nitrogen 45 psi, auxiliary gas nitrogen 5 units, spray 4.5 kV, transfer
capillary 240◦ , collision offset 30 V
CHROMATOGRAM
Retention time: 67
OTHER SUBSTANCES
Extracted: metabolites
KEY WORDS
human; liver
REFERENCE
Vickers, A.E.M.; Zollinger, M.; Dannecker, R.; Tynes, R.; Heitz, F.; Fischer, V. In vitro metabolism of
tegaserod in human liver and intestine: assessment of drug interactions, Drug Metab.Dispos., 2001,
29, 1269–1276.
610
Telithromycin
Telithromycin
N
Molecular formula: C43 H65 N5 O10
N
N
Molecular weight: 812.00
CAS Registry No: 191114-48-4
Merck Index: 13, 9199
O
H3C
H3C
O
CH3
N
O
OCH3
CH3
H3C
O
CH3
O
O
H 3C
N
CH3
HO
O
CH3
O
CH3
SAMPLE
Matrix: blood
Sample preparation: Mix plasma with MeCN, centrifuge. Evaporate the supernatant
to dryness under a stream of nitrogen, reconstitute the residue with MeCN:MeOH:50 mM
ammonium acetate 18.1:21.9:60 containing IS, inject an aliquot.
HPLC VARIABLES
Column: 125 × 4 5 µm Purospher RP-18e
Mobile phase: MeCN:MeOH:50 mM ammonium acetate 24:29:52
Flow rate: 1
Detector: F ex 263 em 460
CHROMATOGRAM
Internal standard: RU 66260
Limit of quantitation: 5 ng/mL
KEY WORDS
guinea pig; pharmacokinetics; plasma
REFERENCE
Edelstein, P.H.; Edelstein, M.A. In vitro activity of the ketolide HMR 3647 (RU 6647) for Legionella
spp., its pharmacokinetics in guinea pigs, and use of the drug to treat guinea pigs with Legionella
pneumophila pneumonia, Antimicrob.Agents Chemother., 1999, 43, 90–95.
611
Telmesteine
Telmesteine
O
O
N
Molecular formula: C7 H11 NO4 S
Molecular weight: 205.23
CAS Registry No: 122946-43-4
S
CH3
COOH
H
SAMPLE
Matrix: bile, blood, urine
Sample preparation: Plasma. Mix 300 µL plasma with 600 µL MeCN, centrifuge at
1000 g for 10 min. Evaporate the supernatant to dryness under a stream of nitrogen,
reconstitute the residue with 50 µL initial mobile phase, inject a 30 µL aliquot. Urine,
bile. Add 1 mL urine or bile to an activated Sep-Pak C18 SPE cartridge, wash with two
1 mL portions of water, elute with 1 mL MeOH. Evaporate the eluate to dryness under
a stream of nitrogen, reconstitute the residue with 100 µL initial mobile phase, inject a
30 µL aliquot.
HPLC VARIABLES
Column: 150 × 4.6 5 µm YMC-Pak C8
Mobile phase: Gradient. MeCN:0.1% acetic acid from 20:80 to 40:60 over 25 min,
maintain at 40:60 for 5 min
Flow rate: 1
Injection volume: 30
Detector: Radioactivity (14 C); MS, Agilent LC-MSD Trap, 10% of column effluent entered
MS detector
CHROMATOGRAM
Retention time: 21.1
OTHER SUBSTANCES
Extracted: glucuronide conjugate (15.8)
KEY WORDS
pharmacokinetics; plasma; rat; SPE
REFERENCE
Lee, J.; Son, J.; Rhee, S.W.; Kim, D.H. Absorption, distribution, metabolism and excretion of telmesteine,
a mucolitic agent, in rat, Xenobiotica, 2003, 33, 755–765.
612
Telmisartan
Telmisartan
Molecular formula: C33 H30 N4 O2
CH3
N
N
Molecular weight: 514.62
CAS Registry No: 144701-48-4
Merck Index: 13, 9209
CH3
N
N
CH3
COOH
SAMPLE
Matrix: blood
Sample preparation: Inject a 100 µL aliquot of plasma onto column A and elute to
waste with mobile phase A; after 3 min, backflush the contents of column A onto column
B using mobile phase B; after 3 min, elute column B with mobile phase B, monitor the
effluent from column B.
HPLC VARIABLES
Column: A 17 × 4.6 unspecified enrichment column; B 125 × 4.6 Nucleosil 100-5 C18
Mobile phase: A 50 mM pH 4.5 ammonium acetate; B MeCN:50 mM pH 4.5 ammonium
acetate 10:90; C Gradient. MeCN:50 mM pH 4.5 ammonium acetate 30:70 for 3 min, to
60:40 over 12 min
Flow rate: 1
Injection volume: 100
Detector: F ex 305 em 365
CHROMATOGRAM
Retention time: 18
Limit of quantitation: 2.5 ng/mL
OTHER SUBSTANCES
Extracted: metabolites
KEY WORDS
column-switching; plasma; rat
REFERENCE
Ebner, T.; Heinzel, G.; Prox, A.; Beschke, K.; Wachsmuth, H. Disposition and chemical stability of telmisartan 1-O-acylglucuronide, Drug Metab.Dispos., 1999, 27, 1143–1149.
SAMPLE
Matrix: urine
Sample preparation: Condition a 1 mL 100 mg Bond Elut C8 SPE cartridge with
2 mL MeOH and 1 mL 100 mM pH 6 phosphate buffer. Shake 1 mL urine with 500 µL
100 mM pH 6 phosphate buffer, centrifuge at 3500 rpm (16.1 g) at 4◦ for 5 min, add a
1 mL aliquot to the SPE cartridge, wash with 1 mL MeOH:100 mM pH 6 phosphate
buffer 30:70, dry under full vacuum for 20 min, elute with 1 mL MeOH. Evaporate
the eluate to dryness under a stream of nitrogen at 40◦ , reconstitute the residue with
500 µL mobile phase, inject a 20 µL aliquot.
HPLC VARIABLES
Guard column: 20 × 3.9 4 µm Novapak C18
Column: 150 × 3.9 4 µm Novapak C18
Mobile phase: MeCN:5 mM pH 6.0 phosphate buffer 45:55
Flow rate: 0.5
Telmisartan
613
Injection volume: 20
Detector: F ex 305 em 365
CHROMATOGRAM
Retention time: 2.5
Limit of quantitation: 1 ng/mL
KEY WORDS
SPE
REFERENCE
Torrealday, N.; González, L.; Alonso, R.M.; Jiménez, R.M.; Ortiz Lastra, E. Experimental design approach for the optimisation of a HPLC-fluorimetric method for the quantitation of the angiotensin II
receptor antagonist telmisartan in urine, J.Pharm.Biomed.Anal., 2003, 32, 847–857.
614
Temocapril
Temocapril
Molecular formula: C23 H28 N2 O5 S2
Molecular weight: 476.62
CAS Registry No: 111902-57-9
Merck Index: 13, 9215
H3C
O
O
S
N
H
S
N
O
COOH
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 × 4.6 Mightysil RP-18
Mobile phase: MeOH:water:phosphoric acid 50:50:0.05
Flow rate: 1
Detector: UV 220
OTHER SUBSTANCES
Simultaneous: enalapril
REFERENCE
Kitagawa, S.; Takeda, J.; Sato, S. Uptake of enalapril by rabbit small intestinal brush-border membrane
vesicles, Biol.Pharm.Bull., 1999, 22, 762–764.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 × 4.6 10 µm Cosmosil 5C18-MS
Column temperature: 50
Mobile phase: Gradient. MeCN:10 mM pH 2.5 potassium phosphate buffer 0:100 for
2 min, to 75:25 over 7.5 min, maintain at 75:25 for 5.5 min. Isocratic. MeCN:buffer
50:50.
Flow rate: 1.5
Detector: UV (wavelength not specified)
CHROMATOGRAM
Retention time: 12.3 (gradient) or 5.7 (isocratic)
OTHER SUBSTANCES
Simultaneous: acetaminophen (7.9), alacepril (10.9), ampicillin (7.9), aspirin (10.0),
caffeine (8.5), carbenicillin (9.5), cefotiam (7.2), chlorpromazine (10.8), cromolyn (8.9),
enalapril (9.9), loperamide (11.6), ofloxacin (8.3), procainamide (7.4), procaine (7.9), propranolol (9.6), sultamicillin tosylate (8.3), tegafur (8.4), theophylline (8.0), tulobuterol
(8.9) (gradient retention times; isocratic conditions may differ)
REFERENCE
Sugiyama, T.; Matsuyama, R.; Usui, S.; Katagiri, Y.; Hirano, K. Selection of mobile phases in highperformance liquid chromatographic determination for medicines, Biol.Pharm.Bull., 2000, 23, 274–278.
615
Temozolomide
Temozolomide
Molecular formula: C6 H6 N6 O2
Molecular weight: 194.15
CAS Registry No: 85622-93-1
Merck Index: 13, 9218
NH2
O
N
N
N
N
N
CH3
O
SAMPLE
Matrix: blood
Sample preparation: Vortex 500 µL plasma with 500 µL 1 µg/mL IS in water, 50 µL
1 M HCl, and 5 mL ethyl acetate for 10 min, centrifuge at 4500 g for 5 min. Evaporate
the organic layer to dryness under a stream of nitrogen at 45◦ , reconstitute the residue
with 300 µL mobile phase, inject a 20 µL aliquot.
HPLC VARIABLES
Column: 150 × 4.6 5 µm Ultrasphere ODS
Mobile phase: MeCN:0.1% acetic acid 10:90
Flow rate: 1
Injection volume: 20
Detector: UV 316
CHROMATOGRAM
Retention time: 2.7
Internal standard: ethazolastone (5.0)
Limit of quantitation: 100 ng/mL
KEY WORDS
pharmacokinetics; plasma
REFERENCE
Kim, H.; Likhari, P.; Parker, D.; Statkevich, P.; Marco, A.; Lin, C.-C.; Nomeir, A.A. High-performance
liquid chromatographic analysis and stability of anti-tumor agent temozolomide in human plasma,
J.Pharm.Biomed.Anal., 2001, 24, 461–468.
SAMPLE
Matrix: blood, urine
Sample preparation: Condition a 100 mg Chromabond C18 SPE cartridge (MachereyNagel) with two 1 mL portions of MeOH and two 1 mL portions of 0.5% acetic acid. Mix
1 mL plasma with 100 µL 1 M HCl. Mix 10 mL urine with 1 mL 1 M HCl. Mix 235 µL
acidified plasma or urine with 115 µL 20 (plasma) or 160 (urine) µg/mL IS in 100 mM
HCl. Add a 160 µL aliquot of this solution to the SPE cartridge, pull through under
light vacuum, let stand for 1 min (important!), wash with 750 µL 0.5% acetic acid, dry
under vacuum for 5 min, elute with 1.25 mL MeOH. Evaporate the eluate to dryness
under a stream of nitrogen at room temperature, reconstitute the residue with 200 µL
0.5% acetic acid, centrifuge, inject a 30 µL aliquot.
HPLC VARIABLES
Guard column: 20 × 4 5 µm HP ODS-Hypersil
Column: 100 × 4.6 5 µm HP ODS-Hypersil
Mobile phase: MeOH:0.5% acetic acid 10:90
Flow rate: 1
Injection volume: 30
Detector: UV 330
616
Temozolomide
CHROMATOGRAM
Retention time: 3.2
Internal standard: ethazolastone (7.4)
Limit of quantitation: 200 ng/mL (plasma), 2 µg/mL (urine)
KEY WORDS
pharmacokinetics; plasma; SPE
REFERENCE
Shen, F.; Decosterd, L.A.; Gander, M.; Leyvraz, S.; Biollax, J.; Lejeune, F. Determination of temozolomide in human plasma and urine by high-performance liquid chromatography after solid-phase
extraction, J.Chromatogr.B, 1995, 667, 291–300.
ANNOTATED BIBLIOGRAPHY
Chowdhury, S.K.; Laudicina, D.; Blumenkrantz, N.; Wirth, M.; Alton, K.B. An LC/MS/MS method for the
quantitation of MTIC (5-(3-N-methyltriazen-1-yl)-imidazole-4-carboxamide), a bioconversion product
of temozolomide, in rat and dog plasma, J.Pharm.Biomed.Anal., 1999, 19, 659–668.
Kim, H.K.; Lin, C.-C.; Parker, D.; Veals, J.; Lim, J.; Likhari, P.; Statkevich, P.; Marco, A.; Nomeir, A.A.
High-performance liquid chromatographic determination and stability of 5-(3-methyltriazen-1-yl)imidazo-4-carboximide, the biologically active product of the antitumor agent temozolomide, in human
plasma, J.Chromatogr.B, 1997, 703, 225–233.
Ma, J.; Li, S.; Reed, K.; Guo, P.; Gallo, J.M. Pharmacodynamic-mediated effects of the angiogenesis
inhibitor SU5416 on the tumor disposition of temozolomide in subcutaneous and intracerebral glioma
xenograft models, J.Pharmacol.Exp.Ther., 2003, 305, 833–839. [plasma; rat]
617
Tenofovir disoproxil fumarate
Tenofovir disoproxil fumarate
Molecular formula: C19 H30 N5 O10 P.C4 H4 O4
Molecular weight: 635.51
CAS Registry No: 202138-50-9,
147127-20-6 (tenofovir only)
Merck Index: 13, 9223
NH2
N
N
O
N
CH3
O
N
O
P
O
O
O
CH3
O
O
O
CH3
CH3
HOOC
COOH
O
CH3
SAMPLE
Matrix: blood
Sample preparation: Mix 200 µL plasma with 20 µL 2.5 µg/mL IS in water, add
600 µL MeOH, vortex for 30 s, centrifuge at 2200 g for 10 min. Evaporate the supernatant to dryness under a stream of nitrogen at 60◦ , reconstitute the residue with
200 µL reagent, heat at 80◦ for 40 min, cool at – 20◦ for 10 min, inject a 40–80 µL
aliquot. (The reagent was 0.34% chloroacetaldehyde in 100 mM pH 4.5 acetate buffer,
prepared just before use.)
HPLC VARIABLES
Guard column: 15 × 3 (Cluzeau)
Column: 250 × 3 5 µm C8 plus satisfaction (Cluzeau, France)
Column temperature: 35
Mobile phase: MeCN:5 mM pH 6 phosphate buffer containing 5 mM tetrabutylammonium chloride 15:85
Flow rate: 0.5
Injection volume: 40–80
Detector: F ex 236 em 420
CHROMATOGRAM
Retention time: 11.7 (of tenofovir)
Internal standard: adefovir (9.5)
Limit of quantitation: 5 ng/mL
KEY WORDS
derivatization; plasma
REFERENCE
Jullien, V.; Tréluyer, J.-M.; Pons, G.; Rey, E. Determination of tenofovir in human plasma by highperformance liquid chromatography with spectrofluorimetric detection, J.Chromatogr.B, 2003, 785,
377–381.
SAMPLE
Matrix: blood
Sample preparation: Mix 200 µL plasma with 1 µg/mL IS and 400 µL 0.1% trifluoroacetic acid in MeCN, centrifuge. Evaporate the supernatant to dryness under reduced
pressure at room temperature, reconstitute the residue with 200 µL 0.34% chloroacetaldehyde in 100 mM pH 4.5 sodium acetate buffer, vortex, centrifuge. Heat the
supernatant at 95◦ for 40 min, evaporate to dryness, reconstitute with 200 µL mobile
phase A, inject a 20 µL aliquot.
618
Tenofovir disoproxil fumarate
HPLC VARIABLES
Guard column: 15 × 3.2 Brownlee RP-18 Newguard
Column: 150 × 4.6 Zorbax RX-C18
Column temperature: 35
Mobile phase: Gradient. A was MeCN:buffer 5:95. B was MeCN:buffer 65:35. A:B
100:0 for 8 min, to 0:100 for 10 min, return to initial conditions (step gradient), reequilibrate for 8 min. (The buffer was 20 mM pH 6.0 phosphate buffer containing 5 mM
tetrabutylammonium hydrogen phosphate.)
Flow rate: 1.5
Injection volume: 20
Detector: F ex 236 em 420
CHROMATOGRAM
Internal standard: 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA)
KEY WORDS
derivatization; dog; plasma; method validated for tenofovir only
REFERENCE
Shaw, J.-P.; Sueoka, C.M.; Oliyai, R.; Lee, W.A.; Arimilli, M.N.; Kim, C.U.; Cundy, K.C. Metabolism and
pharmacokinetics of novel oral prodrugs of 9-[(R)-2-(phosphonomethoxy)propyl]adenine (PMPA) in
dogs, Pharm.Res., 1997, 14, 1824–1829.
SAMPLE
Matrix: blood, microsomal incubations
Sample preparation: Mix 50 µL plasma or microsomal incubation with 100 µL 0.1%
trifluoroacetic acid in MeCN, centrifuge for 5 min at 14 000 rpm, inject a 50 µL aliquot
of the supernatant.
HPLC VARIABLES
Guard column: 15 × 3.2 Brownlee RP-18 Newguard
Column: 250 × 4.6 5 µm Zorbax RX-C18
Column temperature: 35
Mobile phase: Gradient. MeCN:20 mM pH 6.0 phosphate buffer from 0:100 to 65:35
over 15 min, return to initial conditions (step gradient), re-equilibrate for 7 min.
Flow rate: 1
Injection volume: 50
Detector: UV 262
KEY WORDS
dog; intestine; liver; plasma; method validated for tenofovir and tenofovir disoproxil
REFERENCE
Shaw, J.-P.; Sueoka, C.M.; Oliyai, R.; Lee, W.A.; Arimilli, M.N.; Kim, C.U.; Cundy, K.C. Metabolism and
pharmacokinetics of novel oral prodrugs of 9-[(R)-2-(phosphonomethoxy)propyl]adenine (PMPA) in
dogs, Pharm.Res., 1997, 14, 1824–1829.
ANNOTATED BIBLIOGRAPHY
Cundy, K.C.; Sueoka, C.; Lynch, G.R.; Griffin, L.; Lee, W.A.; Shaw, J.-P. Pharmacokinetics and bioavailability of the anti-human immunodeficiency virus nucleotide analog 9-[(R)-2-(phosphonomethoxy)propyl]adenine (PMPA) in dogs, Antimicrob.Agents Chemother., 1998, 42, 687–690. [derivatization;
adefovir is internal standard; fluorescence detection; LOQ 25 ng/mL; tenofovir only]
Sentenac, S.; Fernandez, C.; Thuillier, A.; Lechat, P.; Aymard, G. Sensitive determination of tenofovir
in human plasma samples using reversed-phase liquid chromatography, J.Chromatogr.B, 2003, 793,
317–324. [SPE; tenofovir only; LOQ 10 ng/mL]
Tenofovir disoproxil fumarate
619
Sparidans, R.W.; Crommentuyn, K.M.L.; Schellens, J.H.M.; Beijnen, J.H. Liquid chromatographic assay
for the antiviral nucleotide analogue tenofovir in plasma using derivatization with chloroacetaldehyde,
J.Chromatogr.B, 2003, 791, 227–233. [tenofovir only; derivatization; adefovir is internal standard;
LOQ 20 ng/mL; fluorescence detection]
Van Gelder, J.; Witvrouw, M.; Pannecouque, C.; Henson, G.; Bridger, G.; Naesens, L.; De Clercq, E.;
Annaert, P.; Shafiee, M.; Van den Mooter, G.; Kinget, R.; Augustijns, P. Evaluation of the potential of
ion pair formation to improve the oral absorption of two potent antiviral compounds, AMD3100 and
PMPA, Int.J.Pharm., 1999, 186, 127–136. [tenofovir only]
620
Teprenone
Teprenone
Molecular formula: C23 H38 O
Molecular weight: 330.55
CAS Registry No: 6809-52-5
Merck Index: 13, 9228
O
CH3
SAMPLE
Matrix: blood
Sample preparation: Condition a 200 mg Bond Elut C18 SPE cartridge with 1 column
vol of MeOH and 2 column vol of water. Vortex 1 mL plasma, 100 µL 10 µg/mL IS in
MeOH, and 1 mL water, add 1 mL EtOH, add 100 µL 50% KOH solution, heat in a
boiling water bath for 10 min, cool, add 3 mL hexane, shake for 10 min, centrifuge at
1670 g for 5 min, repeat extraction twice more. Combine the organic layers, evaporate
to dryness under a stream of nitrogen at 45◦ , reconstitute the residue with 200 µL 0.5%
HCl in isopropanol, add 200 µL 0.2% dansylhydrazine in isopropanol, heat at 35◦ for
60 min, add 1 mL water, add to the SPE cartridge, wash twice with 3 mL portions of
water, wash with 1 mL MeCN, elute with three 500 µL portions of MeOH:chloroform
50:50 (Caution! Chloroform is a carcinogen!). Evaporate the eluate to dryness under
a stream of nitrogen at 45◦ , reconstitute the residue with 200 µL EtOH, sonicate,
centrifuge at 1670 g for 5 min, inject a 50 µL aliquot.
HPLC VARIABLES
Column: 150 × 6 5 µm YMC-A312 C18
Column temperature: 30
Mobile phase: MeCN:water:triethylamine 92.5:7.5:0.015
Flow rate: 1.8
Injection volume: 50
Detector: F ex 365 em 515
CHROMATOGRAM
Retention time: 9 (cis-5, syn), 10 (trans-5, syn), 13 (cis-5, anti), 14 (trans-5, anti)
Internal standard: 7,11,15,19-tetramethyl-6,10,14,18-eicosatetraen-3-one (15, 16 (syn/
anti))
Limit of quantitation: 8 ng/mL (cis), 12 ng/mL (trans)
KEY WORDS
derivatization; pharmacokinetics; plasma; SPE; teprenone is a cis:trans (39.8:60.2) mixture
at the 5-double bond and produces syn and anti derivatives at the ketone
REFERENCE
Seki, T.; Hashida, N.; Kanazawa, T. Determination of tetraprenylacetone in human plasma by highperformance liquid chromatography with fluorescence derivatization using dansylhydrazine, J. Chromatogr., 1988, 424, 410–415.
Teriparatide
621
Teriparatide
Molecular formula: C181 H291 N55 O51 S2
Molecular weight: 4117.72
CAS Registry No: 52232-67-4, 99294-94-7 (acetate)
Merck Index: 13, 9241
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 × 10 Vydac C-18
Mobile phase: Gradient. MeCN:0.1% trifluoroacetic acid from 20:80 to 60:40 over 40 min.
Detector: UV (wavelength not specified)
REFERENCE
Oldenburg, K.R.; D’Orfani, A.L.; Selick, H.E. A method for the high-level expression of a parathyroid
hormone analog in Escherichia coli, Protein Expr.Purif., 1994, 5, 278–284.
622
Tetrachlorvinphos
Tetrachlorvinphos
Molecular formula: C10 H9 Cl4 O4 P
Molecular weight: 365.96
CAS Registry No: 22248-79-9
Merck Index: 13, 9267
O
H3CO
H3CO
P
Cl
Cl
O
Cl
Cl
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 × 2.2 6–8 µm silica
Mobile phase: n-Hexane:EtOH 98.5:1.5
Flow rate: 0.55
Injection volume: 5
Detector: Transport flame ionization
CHROMATOGRAM
Retention time: 3 (β isomer), 4 (α isomer)
KEY WORDS
normal phase
REFERENCE
Szalontai, G. High-performance liquid chromatography of organophosphorus insecticides, J.Chromatogr.,
1976, 124, 9–16.
Tetrahydrozoline
Tetrahydrozoline
Molecular formula: C13 H16 N2
623
H
N
N
Molecular weight: 200.28
CAS Registry No: 84-22-0, 522-48-5 (HCl)
Merck Index: 13, 9292
SAMPLE
Matrix: blood
Sample preparation: Mix 1 mL whole blood with 20 µL 1 µg/mL dibenzepin in
MeOH:water 50:50, add 300 µL pH 11 Tris buffer, mix, add 500 µL butyl acetate,
vortex for 2 min, centrifuge. Preserve the aqueous layer (A). Remove the organic layer
and add it to 75 µL 10 mM ammonium acetate buffer containing 0.1% formic acid (pH
3.2), evaporate (?). Add 75 µL MeCN, sonicate for 5 min, centrifuge at 5000 rpm for
5 min, keep the extract as B. Add 20 µL 1 µg/mL enalapril in MeOH:water 50:50 and
120 mg NaCl to the aqueous layer (A), mix, add 500 µL pH 3 phosphate buffer, add
600 µL 8.5% phosphoric acid, add 5 mL dichloromethane:isopropanol 95:5, shake at
250 cycles/min in a bench-top shaker for 30 min, centrifuge at 5000 rpm for 5 min.
Remove the lower organic layer and evaporate it to dryness under a stream of air at
45◦ . Reconstitute the residue with 150 µL initial mobile phase, sonicate, centrifuge,
combine with extract B, inject a 30 µL aliquot. (Sample preparation from Gergov,M.;
Robson,J.N.; Ojanperä,I.; Heinonen,O.P.; Vuori,E. Simultaneous screening and quantitation of 18 antihistamine drugs in blood by liquid chromatography ionspray tandem
mass spectrometry. Forensic Sci.Inter. 2001, 121, 108–115.)
HPLC VARIABLES
Guard column: 40 mm long 4 µm Purospher RP-18 LiChro Cart 4-4
Column: 100 × 2.1 4 µm Genesis C18 (Jones Chromatography)
Column temperature: 35
Mobile phase: Gradient. MeCN:buffer from 20:80 to 100:0 over 10 min, maintain at
0:100 for 3 min, re-equilibrate at initial conditions for 5 min. (The buffer was 10 mM
ammonium acetate containing 0.1% formic acid (pH 3.2).)
Flow rate: 0.2
Injection volume: 30
Detector: MS, PE Sciex API 365 triple-stage quadrupole LC-MS-MS, PE Sciex TurboIonSpray interface, positive ion mode, needle voltage 5.2 kV, nebulizer gas air at 60 psi,
curtain gas nitrogen at 40 psi, collision cell gas nitrogen at 40 psi, turbo ionspray heater
375◦ , heater gas flow 7 L/min
CHROMATOGRAM
Retention time: 3.6
Internal standard: dibenzepin, enalapril
Limit of detection: 100 ng/mL
OTHER SUBSTANCES
Extracted: acebutolol (3.8, LOD 0.1 µg/mL), acetaminophen (2.5, LOD <5 µg/mL), acrivastine (5.7, LOD <0.02 µg/mL), alprazolam (6.1, LOD <0.02 µg/mL), alprenolol (5.4,
LOD 0.01 µg/mL), amantadine (3.4, LOD 0.1 µg/mL), amiloride (2.0, LOD 0.1 µg/mL),
aminophenazone (2.8, LOD <5 µg/mL), amiodarone (10.2, LOD 0.05 µg/mL), amitriptyline (6.6, LOD <0.02 µg/mL), astemizole (5.8, LOD <0.02 µg/mL), atenolol (1.7, LOD
0.30 µg/mL), azacyclonol (5.1, LOD 0.02 µg/mL), benzhexol (6.6, LOD <0.02 µg/mL),
benzoylecgonine (3.3, LOD 0.01 µg/mL), betaxolol (5.5, LOD 0.01 µg/mL), biperidine
(6.2, LOD <0.02 µg/mL), bisoprolol (5.0, LOD <0.02 µg/mL), brompheniramine (5.3,
LOD 0.002 µg/mL), bupivacaine (5.1, LOD <0.02 µg/mL), buprenorphine (5.9, LOD 0.01
µg/mL), buspirone (5.1, LOD 0.002 µg/mL), caffeine (2.8, LOD 1 µg/mL), carbamazepine
624
Tetrahydrozoline
(6.1, LOD <0.02 µg/mL), carbinoxamine (5.1, LOD 0.002 µg/mL), carisoprodol (6.7,
LOD <5 µg/mL), carvedilol (6.2, LOD <0.02 µg/mL), celiprolol (4.3, LOD 0.05 µg/mL),
cetirizine (6.3, LOD 0.05 µg/mL), chlorcyclizine (6.6, LOD <0.02 µg/mL), chlordiazepoxide (5.7, LOD <0.02 µg/mL), chlormezanone (5.8, LOD <5 µg/mL), chloroquine (2.7,
LOD 0.02 µg/mL), chlorpheniramine (5.1, LOD 0.002 µg/mL), chlorpromazine (7.0, LOD
0.02 µg/mL), chlorpropamide (6.7, LOD <5 µg/mL), chlorprothixene (7.0, LOD <0.02
µg/mL), cinnarizine (7.9, LOD <0.02 µg/mL), citalopram (5.7, LOD <0.02 µg/mL),
clemastine (7.7, LOD 0.02 µg/mL), clobazam (7.3, LOD <0.02 µg/mL), clobutinol (5.3,
LOD 0.02 µg/mL), clomethiazole (6.2, LOD 0.5 µg/mL), clomipramine (7.1, LOD <0.02
µg/mL), clonazepam (6.6, LOD <0.02 µg/mL), clonidine (2.8, LOD 0.1 µg/mL), clozapine (5.6, LOD <0.02 µg/mL), cocaine (4.6, LOD <0.02 µg/mL), codeine (2.5, LOD
0.1 µg/mL), coumatetralyl (8.4, LOD 0.05 µg/mL), cyclizine (5.8, LOD <0.02 µg/mL),
dextropropoxyphene (6.6, LOD 0.05 µg/mL), demoxepam (5.8, LOD 0.02 µg/mL), dextromethorphan (5.5, LOD <0.02 µg/mL), diazepam (8.1, LOD 0.02 µg/mL), diltiazem
(5.8, LOD <0.02 µg/mL), diphenhydramine (5.7, LOD <0.02 µg/mL), dipyridamole (5.4,
LOD 0.005 µg/mL), disopyramine (4.4, LOD <0.02 µg/mL), dixyrazine (6.8, LOD 0.005
µg/mL), doxapram (4.8, LOD <0.02 µg/mL), doxepin (5.9, LOD <0.02 µg/mL), dronabinol (12.3, LOD 0.05 µg/mL), ebastine (9.6, LOD 0.005 µg/mL), embutramide (6.7, LOD
0.005 µg/mL), ergotamine (5.5, LOD 0.005 µg/mL), ethenzamide (5.0, LOD 0.05 µg/mL),
ethylmorphine (3.2, LOD 0.05 µg/mL), ethylparathion (9.7, LOD <5 µg/mL), etodroxizine (6.4, LOD <0.02 µg/mL), felodipine (9.6, LOD 0.02 µg/mL), fenazepam (7.5, LOD
<0.02 µg/mL), fenfluramine (5.3, LOD <0.02 µg/mL), fenkamfamine (5.1, LOD <0.02
µg/mL), fentanyl (5.5, LOD <0.02 µg/mL), fexofenadine (6.3, LOD <0.02 µg/mL), flecainide (5.9, LOD <0.02 µg/mL), fluconazole (4.0, LOD 0.1 µg/mL), flumazenil (5.2, LOD
<0.02 µg/mL), flunitrazepam (7.1, LOD 0.002 µg/mL), fluoxetine (6.8, LOD 0.1 µg/mL),
flupentixol (7.5, LOD 0.18 µg/mL), fluvoxamine (6.3, LOD 0.02 µg/mL), glibenclamide
(8.5, LOD <0.02 µg/mL), glipizide (6.8, LOD <0.05 µg/mL), haloperidol (6.1, LOD <0.02
µg/mL), histapyrrodine (6.3, LOD 0.02 µg/mL), hydrocodone (3.0, LOD 0.05 µg/mL),
hydroxychloroquine (2.4, LOD <0.3 µg/mL), hydroxyzine (6.3, LOD <0.02 µg/mL),
imipramine (6.4, LOD 0.05 µg/mL), indomethacin (8.6, LOD 0.05 µg/mL), isoniazid (2.2,
LOD 3 µg/mL), isradipine (8.6, LOD 0.05 µg/mL), ketamine (3.6, LOD <0.05 µg/mL),
ketobemidone (3.3, LOD <0.05 µg/mL), ketoprofen (7.3, LOD 0.1 µg/mL), ketorolac (6.2,
LOD 0.05 µg/mL), labetalol (4.9, LOD 0.05 µg/mL), lamotrigine (4.0, LOD 0.1 µg/mL),
levocabastine (5.8, LOD 0.01 µg/mL), levomepromazine (6.5, LOD 0.02 µg/mL), lidocaine (3.7, LOD <0.05 µg/mL), loratadine (9.3, LOD 0.002 µg/mL), lorazepam (6.6, LOD
0.02 µg/mL), lormetazepam (7.4, LOD <0.02 µg/mL), LSD (4.7, LOD <0.02 µg/mL),
malathion (8.9, LOD 10 µg/mL), maprotiline (6.4, LOD <0.02 µg/mL), MDMA (3.3, LOD
0.02 µg/mL), meclozine (8.5, LOD <0.02 µg/mL), medazepam (6.3, LOD <0.02 µg/mL),
meloxicam (7.1, LOD 0.01 µg/mL), melperone (5.0, LOD <0.02 µg/mL), meperidine (4.7,
LOD <0.02 µg/mL), mepivacaine (3.7, LOD <0.02 µg/mL), meprobamate (4.9, LOD
0.1 µg/mL), mesoridazine (5.4, LOD <0.02 µg/mL), methamphetamine (3.3, LOD 0.05
µg/mL), methadone (6.7, LOD <0.02 µg/mL), methylparathion (8.6, LOD 10 µg/mL),
methylphenidate (4.2, LOD <0.02 µg/mL), metoclopramide (3.8, LOD <0.02 µg/mL),
metoprolol (4.1, LOD 0.02 µg/mL), metronidazole (2.6, LOD 1 µg/mL), mexiletine (4.4,
LOD 0.05 µg/mL), mianserin (5.7, LOD <0.02 µg/mL), midazolam (5.9, LOD <0.02
µg/mL), mirtazapine (4.4, LOD <0.02 µg/mL), mizolastine (5.5, LOD 0.01 µg/mL),
moclobemide (3.7, LOD 0.05 µg/mL), molindone (4.0, LOD <0.02 µg/mL), monoacetylmorphine (2.7, LOD 0.1 µg/mL), morphine (2.0, LOD 0.1 µg/mL), nicotine (2.2, LOD
0.05 µg/mL), nifedipine (7.5, LOD 0.02 µg/mL), nikethamide (3.6, LOD <0.02 µg/mL),
nitrazepam (6.5, LOD <0.02 µg/mL), nizatidine (1.7, LOD 1 µg/mL), nomifensine (4.6,
LOD <0.02 µg/mL), nortriptyline (6.4, LOD <0.02 µg/mL), norverapamil (6.2, LOD
1 µg/mL), noscapine (5.0, LOD <0.02 µg/mL), olanzapine (3.0, LOD 0.05 µg/mL),
ondansetron (4.6, LOD <0.02 µg/mL), orphenadrine (6.1, LOD <0.02 µg/mL), oxazepam
(6.3, LOD <0.02 µg/mL), oxcarbazepine (5.3, LOD 0.02 µg/mL), oxprenolol (4.7, LOD
0.02 µg/mL), oxycodone (2.8, LOD 0.05 µg/mL), papaverine (4.8, LOD <0.02 µg/mL),
paroxetine (6.2, LOD 0.02 µg/mL), pemoline (3.3, LOD 0.05 µg/mL), pentazocine (5.0,
LOD <0.02 µg/mL), pentifylline (7.3, LOD <5 µg/mL), pentoxyverine (6.6, LOD <0.02
µg/mL), perphenazine (6.9, LOD 0.002 µg/mL), phenazone (3.9, LOD 0.05 µg/mL), phencyclidine (5.3, LOD 0.05 µg/mL), pheniramine (4.1, LOD 0.02 µg/mL), phenylbutazone
(9.0, LOD <5 µg/mL), phenylpropanolamine (2.5, LOD 0.3 µg/mL), phenytoin (6.1,
Tetrahydrozoline
625
LOD 0.05 µg/mL), pindolol (3.3, LOD 0.05 µg/mL), piroxicam (6.6, LOD 0.02 µg/mL),
pitofenone (5.4, LOD <0.02 µg/mL), pizotifen (6.5, LOD <0.02 µg/mL), practolol (1.8,
LOD 0.1 µg/mL), prazosin (4.1, LOD 0.05 µg/mL), prilocaine (3.8, LOD <0.02 µg/mL),
primidone (4.0, LOD <5 µg/mL), procainamide (2.2, LOD 0.05 µg/mL), prochlorperazine (7.5, LOD 0.02 µg/mL), promazine (6.2, LOD <0.02 µg/mL), promethazine (6.0,
LOD 0.05 µg/mL), propafenone (6.3, LOD <0.02 µg/mL), propranolol (5.4, LOD 0.02
µg/mL), propyphenazone (6.6, LOD 0.50 µg/mL), pseudoephedrine (2.6, LOD 1 µg/mL),
quinine (4.2, LOD 0.02 µg/mL), ranitidine (1.8, LOD 0.1 µg/mL), risperidone (4.9, LOD
<0.02 µg/mL), rocurone (3.8, LOD 0.1 µg/mL), ropivacaine (4.6, LOD <0.02 µg/mL),
salicylamide (4.2, LOD <5 µg/mL), selegiline (4.1, LOD 0.05 µg/mL), sertindole (7.2,
LOD <0.02 µg/mL), sertraline (6.8, LOD 0.02 µg/mL), sulindac (6.5, LOD 0.02 µg/mL),
simazine (6.0, LOD 0.1 µg/mL), sincocaine (6.5, LOD <0.02 µg/mL), sisapride (5.9,
LOD <0.02 µg/mL), sotalol (2.1, LOD 0.1 µg/mL), strychnine (5.3, LOD 0.05 µg/mL),
sulpiride (1.9, LOD 0.1 µg/mL), sulthiame (4.1, LOD 0.05 µg/mL), temazepam (7.2, LOD
<0.02 µg/mL), terbutaline (2.3, LOD 0.1 µg/mL), terfenadine (8.1, LOD 0.002 µg/mL),
terodiline (6.7, LOD <0.02 µg/mL), tetracaine (5.7, LOD <0.02 µg/mL), theobromine
(2.3, LOD <5 µg/mL), theophylline (2.4, LOD <5 µg/mL), thioridazine (7.5, LOD 0.02
µg/mL), timolol (3.8, LOD 0.05 µg/mL), thiothixene (6.7, LOD 0.02 µg/mL), tolbutamide
(7.1, LOD <5 µg/mL), toremifene (8.7, LOD 0.02 µg/mL), tramadol (4.2, LOD 0.02
µg/mL), trazodone (5.2, LOD <0.02 µg/mL), triamterene (3.2, LOD 0.1 µg/mL), triazolam (6.7, LOD 0.002 µg/mL), trimeprazine (6.4, LOD <0.02 µg/mL), trimethoprim
(3.1, LOD 0.05 µg/mL), trimipramine (6.7, LOD <0.02 µg/mL), venlafaxine (4.9, LOD
0.02 µg/mL), verapamil (6.5, LOD <0.02 µg/mL), warfarin (7.9, LOD <0.02 µg/mL),
yohimbine (4.5, LOD <0.02 µg/mL), zolpidem (4.7, LOD <0.02 µg/mL), zopiclone (4.0,
LOD 0.1 µg/mL)
KEY WORDS
whole blood
REFERENCE
Gergov, M.; Ojanperä, I.; Vuori, E. Simultaneous screening for 238 drugs in blood by liquid chromatography-ionspray tandem mass spectrometry with multiple-reaction monitoring, J.Chromatogr.B, 2003,
795, 41–53.
626
Thalidomide
Thalidomide
O
N
O
Molecular formula: C13 H10 N2 O4
Molecular weight: 258.23
CAS Registry No: 50-35-1
Merck Index: 13, 9326
N
O
O
H
SAMPLE
Matrix: blood
Sample preparation: Condition a 1 mL 100 mg C18 SPE cartridge (Varian) with 1
column vol of MeOH and one column volume of water. Vortex 1 mL 25 mM pH 2.5
phosphate buffer with 1 mL plasma, add 100 µL 100 µg/mL labetalol in MeOH, vortex,
add to the SPE cartridge, wash with 2 column vol of water, dry under vacuum, elute
with 1 mL anhydrous diethyl ether, evaporate the eluate to dryness under a stream of
nitrogen, reconstitute the residue with 130 µL mobile phase, inject a 50 µL aliquot.
HPLC VARIABLES
Guard column: Bioptic AV-1 (Meta Chem Technologies, Torrance CA)
Column: 150 × 4.5 Bioptic AV-1 (Meta Chem Technologies, Torrance CA)
Mobile phase: Isopropanol:100 mM pH 4 phosphate buffer 2:98
Flow rate: 0.6
Injection volume: 50
Detector: UV 300
CHROMATOGRAM
Retention time: 8.4 (S-(−)), 10.5 (R-(+))
Internal standard: labetalol (6.7)
Limit of detection: 50 ng/mL
Limit of quantitation: 100 ng/mL
OTHER SUBSTANCES
Extracted: metabolites
KEY WORDS
chiral; plasma; SPE
REFERENCE
Haque, A.; Stewart, J.T. Determination of racemic thalidomide in human plasma by use of an avidin
column and solid phase extraction, J.Liq.Chromatogr.Rel.Technol., 1998, 21, 2151–2163.
SAMPLE
Matrix: blood
Sample preparation: Mix 500 µL serum with 20 µL 250 µg/mL IS in MeOH, add
500 µL 10% trichloroacetic acid solution with constant vortexing for 30 s, centrifuge at
2700 g for 5 min, inject a 20 µL aliquot.
HPLC VARIABLES
Guard column: 20 × 4.6 Discovery C18 (Supelco)
Column: 125 × 4.6 5 µm Discovery C18 (Supelco)
Mobile phase: MeOH:buffer 75:25 (The buffer was 10 mM potassium dihydrogen phosphate adjusted to pH 3.0 with 43% phosphoric acid.)
Flow rate: 1.5
Injection volume: 20
Detector: UV 220
Thalidomide
627
CHROMATOGRAM
Retention time: 7.9
Internal standard: phenacetin (15.0)
Limit of quantitation: 222 ng/mL
OTHER SUBSTANCES
Simultaneous: clofazimine (8.3), dapsone (3.5), indinavir (4.0), isoniazid (2.1), lamivudine (2.2), nevirapine (15.0), rifampin (2.5), saquinavir (2.5), stavudine (2.1), trimethoprim (4.8), ritonavir (2.2), zidovudine (4.0)
Noninterfering: cyclosporine, rifabutin, sulfamethoxazole, prednisolone
KEY WORDS
pharmacokinetics; serum
REFERENCE
Toraño, J.S.; Verbon, A.; Guchelaar, H.-J. Quantitative determination of thalidomide in human serum
with high-performance liquid chromatography using protein precipitation with trichloroacetic acid and
ultraviolet detection, J.Chromatogr.B, 1999, 734, 203–210.
ANNOTATED BIBLIOGRAPHY
Boughton, B.J.; Sheehan, T.M.; Wood, J.; O’Brien, D.; Butler, M.; Simpson, A.; Hale, K.A. High-performance liquid chromatographic assay of plasma thalidomide: stabilization of specimens and determination of a tentative therapeutic range for chronic graft-versus-host disease, Ann.Clin.Biochem., 1995,
32, 79–83.
Delon, A.; Favreliere, S.; Couet, W.; Courtois, P.; Bouquet, S. Rapid and sensitive determination of
thalidomide in human plasma by high- performance liquid chromatography, J.Liq.Chromatogr., 1995,
18, 297–309. [SPE; pharmacokinetics; ciprofloxacin is internal standard; LOQ 62.5 ng/mL; simultaneous acyclovir, flucytosine, metronidazole, azathioprine, ceftazidime, cefotaxime; non-interfering
cyclosporine, cyclophosphamide, prednisolone, hydroxyzine, nifedipine, diltiazem, amphotericin, clonazepam, clobazam, diazepam]
Eriksson, T.; Bjorkman, S.; Fyge, A.; Ekberg, H. Determination of thalidomide in plasma and blood by
high-performance liquid chromatography: avoiding hydrolytic degradation, J.Chromatogr., 1992, 582,
211–216.
Eriksson, T.; Bjorkman, S.; Roth, B.; Fyge, A.; Hoglund, P. Enantiomers of thalidomide: blood distribution and the influence of serum albumin on chiral inversion and hydrolysis, Chirality, 1998, 10,
223–228. [chiral]
Haque, A.; Stewart, J.T. Determination of racemic thalidomide in human plasma by use of an avidin
column and solid phase extraction (Abstract 4163), Pharm.Res., 1997, 14, S684–S685.
Huupponen, R.; Pykkö, K. Stability of thalidomide in human plasma, Clin.Chem., 1995, 41, 1199.
Teo, S.K.; Chandula, R.S.; Harden, J.L.; Stirling, D.I.; Thomas, S.D. Sensitive and rapid method for the
determination of thalidomide in human plasma and semen using solid-phase extraction and liquid
chromatography-tandem mass spectrometry, J.Chromatogr.B, 2002, 767, 145–151. [LOQ 2 ng/mL]
628
Thialbarbital
Thialbarbital
H
O
N
Molecular formula: C13 H16 N2 O2 S
Molecular weight: 264.35
CAS Registry No: 467-36-7
Merck Index: 13, 9363
S
N
H
O
H2C
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 150 × 2 Chiralcel OJ-R
Mobile phase: MeCN:water 30:70
Flow rate: 0.2
Injection volume: 8
Detector: UV 235
CHROMATOGRAM
Retention time: 21, 27 (enantiomers)
KEY WORDS
chiral
REFERENCE
Aboul-Enein, H.Y.; Schmid, M.G.; Tuscher, C.; Gübitz, G.; Laffranchini, M.; Bojarski, J. Chiral separation of several thiobarbiturates on a cellulose tris(4-methylbenzoate) phase under reversed-phase
conditions, J.Liq.Chromatogr.Rel.Technol., 2001, 24, 69–77.
Thyrotropin
629
Thyrotropin
Molecular formula: C1039 H1602 N274 O307 S27
Molecular weight: 23709.28
CAS Registry No: 9002-71-5
Merck Index: 13, 9870
SAMPLE
Matrix: solutions
HPLC VARIABLES
Guard column: Vydac 214 FSK 54
Column: 250 × 4.6 5 µm C4 Vydac 214 TP 54
Column temperature: 25
Mobile phase: Gradient. A was 50 mM pH 7.0 phosphate buffer. B was MeCN:50 mM
pH 7.0 phosphate buffer 50:50. A:B from 75:25 to 0:100 over 40 min. (Place a silica
column packed with 7.9–12.4 µm LiChrosorb Si-60 silica between pump and injector.)
Flow rate: 0.5
Injection volume: 5–200
Detector: UV 280
CHROMATOGRAM
Retention time: 33–36
Limit of detection: 200 ng
REFERENCE
Ezequiel de Oliveira, J.; de Mendonça, F.; Peroni, C.N.; Bartolini, P.; Ribela, M.T.C.P. Determination of
Chinese hamster ovary cell-derived recombinant thyrotropin by reversed-phase liquid chromatography,
J.Chromatogr.B, 2003, 787, 345–355.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Guard column: 75 × 7.5 SW
Column: 600 × 7.5 10 µm G2000 SW
Mobile phase: 20 mM pH 7.0 phosphate buffer containing 150 mM NaCl
Flow rate: 1
Injection volume: 5–50
Detector: UV 280 (?)
CHROMATOGRAM
Retention time: 17
REFERENCE
Ezequiel de Oliveira, J.; de Mendonça, F.; Peroni, C.N.; Bartolini, P.; Ribela, M.T.C.P. Determination of
Chinese hamster ovary cell-derived recombinant thyrotropin by reversed-phase liquid chromatography,
J.Chromatogr.B, 2003, 787, 345–355.
630
Tiagabine
Tiagabine
Molecular formula: C20 H25 NO2 S2
Molecular weight: 375.56
CAS Registry No: 115103-54-3,
145821-59-6 (HCl)
Merck Index: 13, 9493
H3C
S
N
S
COOH
CH3
SAMPLE
Matrix: blood
Sample preparation: Condition a 3 mL Bond Elut C8 SPE cartridge with 5 mL MeOH
and 2 mL water. Mix 1 mL plasma with 200 µL water and 200 µL 1 µg/mL IS in
MeOH:water 50:50, centrifuge at 1000 g for 15 min, add the supernatant to the SPE
cartridge, wash with 2 mL water, wash with 2 mL MeOH:water 5:95, elute with 1 mL
MeOH. Evaporate the eluate to dryness under a stream of air at 40◦ , reconstitute the
residue with 200 µL mobile phase, inject a 50–60 µL aliquot.
HPLC VARIABLES
Column: 250 × 4.6 5 µm Ultrasphere ODS C18
Mobile phase: MeCN:MeOH:water 37:10:53 containing 10 mM sodium dihydrogen phosphate and 5 mM sodium octanesulfonate
Flow rate: 1.2
Injection volume: 50–60
Detector: E, Environmental Sciences Coulochem Model 5100A, porous graphite electrode – 0.50 V (screen), porous graphite electrode +0.76 V (monitored), guard cell
+0.95 V (before injector)
CHROMATOGRAM
Retention time: 16
Internal standard: (R)-N-(4-(3-methyl-2-thienyl)-4-(2-thienyl)but-3-enyl)nipecotic acid
(12)
Limit of quantitation: 2 ng/mL
KEY WORDS
plasma; SPE
REFERENCE
Gustavson, L.E.; Chu, S.Y. High-performance liquid chromatographic procedure for the determination of
tiagabine concentrations in human plasma using electrochemical detection, J.Chromatogr., 1992, 574,
313–318.
631
Tiletamine hydrochloride
Tiletamine hydrochloride
Molecular formula: C12 H17 NOS.HCl
Molecular weight: 259.80
CAS Registry No: 14176-50-2
O
HCl
S
N
H
CH3
SAMPLE
Matrix: blood, tissue
Sample preparation: Add IS to serum or tissue, make alkaline with pH 9.5 borate
buffer, extract with ethyl acetate. Extract the organic layer with 1 mL 100 mM HCl.
Basify the aqueous layer with 100 mg sodium borate, extract with ethyl acetate.
Evaporate the organic layer to dryness under a stream of nitrogen, reconstitute the
residue with MeCN:water 25:75 (serum) or mobile phase (tissue), inject a 70 µL aliquot.
HPLC VARIABLES
Column: 250 × 4 5 µm LiChrospher 60 RP-select B C8
Mobile phase: MeCN:50 mM pH 6.8 phosphate buffer 26:74
Flow rate: 1
Injection volume: 70
Detector: UV 233
CHROMATOGRAM
Retention time: 18.9
Internal standard: pindolol (9.7)
Limit of quantitation: 10 ng/mL
OTHER SUBSTANCES
Extracted: zolazepam (LOQ 2 ng/mL) (12.5)
KEY WORDS
bear; muscle; serum
REFERENCE
Semple, H.A.; Gorecki, D.K.J.; Farley, S.D.; Ramsay, M.A. Pharmacokinetics and tissue residues of
Telazol in free-ranging polar bears, J.Wildlife Dis., 2000, 36, 653–662.
SAMPLE
Matrix: blood, urine
Sample preparation: Mix 1 mL pH 7 buffer with 5 mL whole blood or urine, add 1 µg
IS1, add 6 µg IS2, add 7 mL hexane:toluene:isoamyl alcohol 90:5:5, vortex, centrifuge,
remove the organic layer. Add 1 mL pH 9.5 buffer to the aqueous layer, extract with 6 mL
hexane:isoamyl alcohol 99:1. Combine the organic layers, evaporate to dryness under
a stream of nitrogen, reconstitute the residue with 250 µL MeCN:water:trifluoroacetic
acid 45:55:0.05, evaporate to half volume to remove MeCN, inject a 50 µL aliquot.
HPLC VARIABLES
Guard column: 5 µm Altima C18
Column: 20 × 2.1 5 µm Altima C18
Column temperature: 40
Mobile phase: Gradient. A was MeCN:water:trifluoroacetic acid 10:90:0.05. B was
MeCN:water:trifluoroacetic acid 80:20:0.05. A:B 100:0 for 5 min, to 0:100 over 50 min,
re-equilibrate at initial conditions for 15 min.
632
Tiletamine hydrochloride
Flow rate: 0.25
Injection volume: 50
Detector: MS, HP 5989B, electron impact; UV 205; UV 290
CHROMATOGRAM
Internal standard: SKF-525 A (IS1), 5-ethyl-5-p-tolylbarbituric acid (IS2)
OTHER SUBSTANCES
Extracted: zolazepam
KEY WORDS
whole blood
REFERENCE
Cording, C.J.; Deluca, R.; Camporese, T.; Spratt, E. A fatality related to the veterinary anesthetic Telazol,
J.Anal.Toxicol., 1999, 23, 552–555.
SAMPLE
Matrix: tissue
Sample preparation: Homogenize 1.5 g tissue with 300 µL 1 µg/mL IS in water and
6 mL 500 mM pH 9.5 sodium borate buffer, add 10 mL ethyl acetate, vortex for 20 min,
repeat the extraction. Combine the organic layers and extract twice with 2 mL portions
of 100 mM HCl. Combine the aqueous layers and make basic with 200 mg sodium
borate, extract with 12 mL ethyl acetate. Evaporate the organic layer to dryness under
a stream of nitrogen, reconstitute the residue with 200 µL MeCN:water 20:80, inject a
70 µL aliquot.
HPLC VARIABLES
Column: 250 × 4 5 µm LiChrospher 60 RP-select B C8
Mobile phase: MeCN:50 mM pH 5.5 sodium phosphate buffer 16:84
Flow rate: 1
Injection volume: 70
Detector: UV 233
CHROMATOGRAM
Retention time: 45
Internal standard: ripazapam (51.5)
OTHER SUBSTANCES
Extracted: tiletamine metabolite CI-398 (23.5), zolazepam (58), zolazepam metabolites
KEY WORDS
bear; fat; kidney; muscle
REFERENCE
Semple, H.A.; Gorecki, D.K.J.; Farley, S.D.; Ramsay, M.A. Pharmacokinetics and tissue residues of
Telazol in free-ranging polar bears, J.Wildlife Dis., 2000, 36, 653–662.
633
Tilmicosin
Tilmicosin
O
Molecular formula: C46 H80 N2 O13
Molecular weight: 869.13
CAS Registry No: 108050-54-0
Merck Index: 13, 9516
CH3
CH3
H3C
H3C
HO
CH3
O
O
OCH3
H3C
H3C
O
OCH3
O
OH
HO
O
O
N
CH3
OH
CH3
CH3
SAMPLE
Matrix: blood
Sample preparation: Condition a 500 mg Bond Elut C18 SPE cartridge with MeOH
and water. Add 2 mL serum to the SPE cartridge, wash with water, wash with 5%
ammonium hydroxide, wash with water, elute with 2 mL MeOH:acetic acid 5:95.
Evaporate the eluate to dryness under a stream of nitrogen, reconstitute the residue with
1 mL diluent, inject an aliquot. (The diluent was MeOH:water:1 M dibutylammonium
phosphate 50:47.5:2.5.)
HPLC VARIABLES
Column: 250 × 4.6 5 µm Hi-Chrom Reversible phenyl (Regis)
Mobile phase: Gradient. A was MeCN:water 50:50 adjusted to pH 2.5 with orthophosphoric acid. B was water adjusted to pH 2.5 with orthophosphoric acid. C was 168 mL
dibutylamine in 700 mL water, adjust to pH 2.5 with orthophosphoric acid, make up to
1 L with water. A:B:C 100:0:0 for 3 min, to 55:30:15 over 1 min, maintain at 55:30:15
for 7 min, return to initial conditions over 1 min, re-equilibrate for 8 min.
Flow rate: 1.5
Injection volume: 50
Detector: UV 280
CHROMATOGRAM
Retention time: 11
Limit of detection: 2.6 ng/mL
Limit of quantitation: 50 mg/mL
KEY WORDS
cow; pharmacokinetics; serum; sheep
REFERENCE
Modric, S.; Webb, A.I.; Derendorf, H. Pharmacokinetics and pharmacodynamics of tilmicosin in sheep
and cattle, J.vet.Pharmacol.Therap., 1998, 21, 444–452.
634
Tiludronic acid
Tiludronic acid
Molecular formula: C7 H9 ClO6 P2 S
O
OH
P
Cl
S
Molecular weight: 318.61
CAS Registry No: 89987-06-4, 149845-07-8 (Na salt),
155453-10-4 (Na salt hemihydrate)
Merck Index: 13, 9518
P
O
OH
OH
OH
SAMPLE
Matrix: blood, urine
Sample preparation: Mix 200 µL plasma or urine with 400 µL 5 µg/mL IS in water,
add 100 µL 1 M NaOH, add 100 µL 180 mM calcium chloride, add 1 mL water, vortex,
centrifuge at 11 600 g. Discard the liquid phase and dissolve the residue in 100 µL 1 M
HCl, add 200 µL 1 M NaOH, add 100 µL 180 mM calcium chloride (only for plasma
samples), add 1 mL water, vortex, centrifuge at 11 600 g. Discard the liquid phase and
vortex the residue with 200 µL mobile phase containing 100 mM disodium EDTA until
dissolution is complete, inject a 20–100 µL aliquot.
HPLC VARIABLES
Column: 150 × 4.1 5 µm PRP-1 (Hamilton)
Column temperature: 22
Mobile phase: MeCN:buffer 13:87 (The buffer was 50 mM pH 11.8 disodium hydrogen
phosphate containing 5 mM tetrabutylammonium phosphate.)
Flow rate: 1
Injection volume: 20–100
Detector: UV 280
CHROMATOGRAM
Retention time: 3
Internal standard: (3-trifluoromethyl)-thiomethylene bisphosphonic acid (4)
Limit of quantitation: 50 ng/mL
KEY WORDS
baboon; human; monkey; mouse; plasma
REFERENCE
Fels, J.-P.; Guyonnet, J.; Berger, Y.; Cautreels, W. Determination of (4-chlorophenyl)thiomethylene bisphosphonic acid, a new bisphosphonate, in biological fluids by high-performance liquid chromatography,
J.Chromatogr., 1988, 430, 73–79.
635
Tirilazad
Tirilazad
O
CH3
N
H3C
Molecular formula: C38 H52 N6 O2
Molecular weight: 624.86
CAS Registry No: 110101-66-1
Merck Index: 13, 9540
N
H
N
N
CH3
N
H
O
N
SAMPLE
Matrix: blood
Sample preparation: Condition a 1 mL 100 mg Bond Elut C18 SPE cartridge with
2 mL MeOH and 2 mL water. Vortex 500 µL plasma with 500 µL 5 µg/mL IS in MeCN
for 1 min, centrifuge at 1500 g at 4◦ , add 800 µL of the supernatant to the SPE cartridge,
wash with two 1 mL portions of MeCN:water 50:50, elute with two 1 mL portions of
MeOH. Evaporate the eluate to dryness under a stream of nitrogen at 40◦ , reconstitute
the residue with 350 µL MeCN, vortex for 1 min, dilute with 150 µL water, inject an
aliquot.
HPLC VARIABLES
Guard column: 15 × 3.2 7 µm NewGuard RP-8 (Brownlee)
Column: 250 × 4.6 5 µm Suplecosil LC-8
Mobile phase: MeCN:buffer 75:25 (The buffer was 22 mM triethylamine adjusted to pH
5.0 with glacial acetic acid.)
Flow rate: 1
Injection volume: 100
Detector: UV 254
CHROMATOGRAM
Retention time: 10.5
Internal standard: 21-[4-(2,6-di-1-pyrrolidinyl-4-pyrimidinyl)-1-piperazinyl]-3α-hydroxy-16α-methyl-5α-pregna-20-one (16)
Limit of quantitation: 12 ng/mL (S/N 10)
KEY WORDS
plasma; rat
REFERENCE
Cox, J.W.; Pullen, R.H. High-performance liquid chromatographic determination of a 21-aminosteroid
antioxidant in plasma, J.Chromatogr., 1988, 424, 293–302.
SAMPLE
Matrix: blood
Sample preparation: Add 150 µL water and 300 µL IS in MeCN to 100 µL plasma,
centrifuge, add 450 µL of the supernatant to a conditioned (unspecified) Advanced
Automated Sample Processor C8 SPE cartridge containing 0.5 mL water (?), wash with
500 µL MeCN:water 25:75, elute the contents directly onto column A in series with
column B using mobile phase, remove column A from the circuit, continue to elute
column B with mobile phase, monitor the effluent from column B.
HPLC VARIABLES
Column: A 15 × 3.2 7 µm Brownlee New Guard C18; B 250 × 4.6 Supelco LC8
Mobile phase: MeCN:water:triethylamine:acetic acid 80:20:0.1:0.2
Flow rate: 1.5
Detector: UV 254
636
Tirilazad
CHROMATOGRAM
Retention time: 8
Internal standard: (16α)-21-[4-(2,6-di-1-pyrrolidinyl-4-pyrimidinyl)-1-piperazinyl]-16methylpregna-3-ol-20-one (PNU-76824) (15)
Limit of quantitation: 5.4 ng/mL
KEY WORDS
column-switching; pharmacokinetics; plasma; rat
REFERENCE
Wang, Y.; Mesfin, G.-M.; Rodriguez, C.A.; Slater, J.G.; Schuette, M.R.; Cory, A.L.; Higgins, M.J. Venous
irritation, pharmacokinetics, and tissue distribution of tirilazad in rats following intravenous administration of a novel supersaturated submicron lipid emulsion, Pharm.Res., 1999, 16, 930–938.
SAMPLE
Matrix: microsomal incubations
Sample preparation: Vortex 500 µL microsomal incubation with 500 µL MeCN for
20 s, centrifuge at 2100 g at 4◦ for 25 min, inject an aliquot of the supernatant.
HPLC VARIABLES
Column: 250 × 4.6 5 µm Kromasil C18
Mobile phase: Gradient. MeCN:100 mM ammonium acetate from 10:90 to 100:0 in
60 min, maintain at 100:0 for 40 min
Flow rate: 0.5
Detector: MS, Finnigan 4021 quadrupole, thermal pneumatic nebulizer with momentum
separator, positive ion CI mode with ammonia as moderating gas, tip 180◦ , expansion
region 80◦ , helium 150 psi with a 360 × 75 µm fused silica capillary; Radioactivity (14 C)
CHROMATOGRAM
Retention time: 80
OTHER SUBSTANCES
Extracted: metabolites
KEY WORDS
human; liver
REFERENCE
Wienkers, L.C.; Steenwyk, R.C.; Sanders, P.E.; Pearson, P.G. Biotransformation of tirilazad in human:
1. Cytochrome P450 3A-mediated hydroxylation of tirilazad mesylate in human liver microsomes,
J.Pharmacol.Exp.Ther., 1996, 277, 982–990.
ANNOTATED BIBLIOGRAPHY
Cox, J.W.; Pullen, R.H. Irregular retention properties of 21-aminosteroid antioxidants in octylsilane
bonded-phase high-performance liquid chromatography, J.Chromatogr., 1988, 424, 285–292.
Fleishaker, J.C.; Straw, R.N.; Cross, C.J. Pharmacokinetics of tirilazad and U-89678, an active, reduced
metabolite, following acute head trauma in adults, J.Pharm.Sci., 1997, 86, 434–437. [LOQ 5 ng/mL]
Hoerle, S.L.; Snider, B.G. Determination of degradation products occurring in acidic solutions of a
21-aminosteroid (tirilazad) using a gradient HPLC method, J.Liq.Chromatogr., 1995, 18, 3269–3282.
Wienkers, L.C.; Steenwyk, R.C.; Mizsak, S.A.; Pearson, P.G. In vitro metabolism of tirilazad mesylate in
male and female rats. Contribution of cytochrome P4502C11 and 4 -5α-reductase, Drug Metab.Dispos.,
1995, 23, 383–392.
Tirofiban
Tirofiban
COOH
H
Molecular formula: C22 H36 N2 O5 S
637
N
O
N
H
Molecular weight: 440.60
CAS Registry No: 144494-65-5,
150915-40-5 (HCl monohydrate)
Merck Index: 13, 9541
S
O
CH3
O
SAMPLE
Matrix: blood
Sample preparation: Vortex 500 µL plasma with 50 µL 100 ng/mL IS and 500 µL
100 mM perchloric acid briefly, add 5 mL 1-chlorobutane, shake for 10 min, centrifuge,
extract the aqueous layer twice more with 5 mL portions of ethyl acetate. Combine
the organic layers and evaporate them to dryness under a stream of nitrogen at 40◦ ,
reconstitute the residue with 250 µL EtOH:toluene 50:50, evaporate to dryness under
a stream of nitrogen at 40◦ , add 400 µL 10 mM dimethylaminopyridine in MeCN, add
40 µL trifluoroacetic anhydride, heat at 40◦ for 30 min, remove excess reagents with a
stream of nitrogen, reconstitute the residue with 150 µL mobile phase, inject a 25 µL
aliquot.
HPLC VARIABLES
Column: 250 × 4.6 5 µm Zorbax RX-C18
Mobile phase: MeCN:1 mM ammonium acetate 41.5:58.5, pH 6.0
Injection volume: 25
Detector: MS, Sciex Model API-III triple-quadrupole, API, negative ion mode, orifice
potential – 60 V, electron multiplier +4.4 kV, interface 50◦ , nebulizer 500◦ , nebulizer
air 1 L/min, curtain gas 700 mL/min, m/z 535
CHROMATOGRAM
Retention time: 5.2
Internal standard: N-phenylsulfonyl-O-[4-(butane-4-piperidinyl]-L-tyrosine (L-702,
128) (5.1)
Limit of quantitation: 400 pg/mL
KEY WORDS
derivatization; pharmacokinetics; plasma
REFERENCE
Ellis, J.D.; Hand, E.L.; Gilbert, J.D. Use of LC-MS/MS to cross-validate a radioimmunoassay for the
fibrinogen receptor antagonist, Aggrastat (tirofiban hydrochloride) in human plasma, J. Pharm.
Biomed. Anal., 1997, 15, 561–569.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 × 4.6 5 µm Symmetry C18 (Waters)
Column temperature: 40
Mobile phase: MeCN:10 mM pH 2.3 potassium phosphate buffer 22:78
Flow rate: 1.5
Injection volume: 50
Detector: UV 227
638
Tirofiban
KEY WORDS
stability-indicating
REFERENCE
Bergquist, P.A.; Manas, D.; Hunke, W.A.; Reed, R.A. Stability and compatibility of tirofiban hydrochloride during simulated Y-site administration with other drugs, Amer.J.Health-Syst.Pharm., 2001, 58,
1218–1223.
ANNOTATED BIBLIOGRAPHY
Bergquist, P.A.; Zimmerman, J.; Kenney, R.R.; Han, R.Y.-H.; Hunke, W.A. Stability of tirofiban hydrochloride in three commonly used i.v. solutions and polyvinyl chloride administration sets, Am.J.HealthSyst.Pharm., 1999, 56, 1627–1629. [stability-indicating]
Garabito, M.J.; Jimenez, L.; Bautista, F.J.; Santos-Rubio, M.D.; Perez-Rodrigo, I. Stability of tirofiban
hydrochloride in 0.9% sodium chloride injection for 30 days, Am.J.Health-Syst.Pharm., 2001, 58,
1850–1851. [stability-indicating]
Vickers, S.; Theoharides, A.D.; Arison, B.; Balani, S.K.; Cui, D.; Duncan, C.A.; Ellis, J.D.; Gorham, L.M.;
Polsky, S.L.; Prueksaritanont, T.; Ramjit, H.G.; Slaughter, D.E.; Vyas, K.P. In vitro and in vivo studies
on the metabolism of tirofiban, Drug Metab.Dispos., 1999, 27, 1360–1366. [rat; dog; bile; urine; feces;
radiolabeled; metabolites]
639
Tiropramide
Tiropramide
Molecular formula: C28 H41 N3 O3
Molecular weight: 467.64
CAS Registry No: 55837-29-1, 53567-47-8 (HCl)
Merck Index: 13, 9543
O
H3C
H3C
N
H
N
O
O
N
CH3
CH3
SAMPLE
Matrix: blood
Sample preparation: Filter (0.22 µm PVDF) plasma, inject a 100 µL aliquot onto
column A and elute to waste with mobile phase A at 0.5 mL/min; after 4.5 min, direct
the effluent from column A onto column B and then to waste (continue to use mobile
phase A); after another 3 min, backflush the contents of column B onto column C with
mobile phase B at 0.1 mL/min, monitor the effluent from column C for 10.5 min.
HPLC VARIABLES
Column: A 20 × 4 5 µm Capcell Pak MF Ph-1 polymer-coated mixed function (change
after 35 samples); B 35 × 2 5 µm Capcell Pak C18 UG 120; C 250 × 1.5 Capcell Pak
C18 UG 120
Column temperature: 30 (columns A and C only)
Mobile phase: A MeCN:50 mM pH 7.0 potassium phosphate buffer 12:88; B MeCN:10
mM pH 7.0 potassium phosphate buffer 50:50
Flow rate: A 0.5; B 0.1
Injection volume: 100
Detector: UV 230
CHROMATOGRAM
Retention time: 14
Limit of quantitation: 5 ng/mL
KEY WORDS
column-switching; pharmacokinetics; plasma
REFERENCE
Baek, S.K.; Lee, S.S.; Park, E.J.; Sohn, D.H.; Lee, H.S. Semi-micro high-performance liquid chromatographic analysis of tiropramide in human plasma using column-switching, J.Pharm.Biomed.Anal.,
2003, 31, 185–189.
SAMPLE
Matrix: blood
Sample preparation: Mix 100 µL plasma with 10 µL 50 ng/mL IS in MeOH and
100 µL 50 µM NaOH, add 800 µL MTBE, vortex at high speed for 5 min, centrifuge at
5000 g for 5 min. Evaporate the organic layer to dryness under a stream of nitrogen at
35◦ , reconstitute the residue with 40 µL MeCN:water 50:50, vortex for 2 min, inject a
10 µL aliquot.
HPLC VARIABLES
Column: 100 × 2 3 µm Luna C8
Column temperature: 30
Mobile phase: MeCN:10 mM pH 4.5 ammonium formate buffer 50:50
Flow rate: 0.2
Injection volume: 10
640
Tiropramide
Detector: MS, Micromass Quattro LC, tandem quadrupole, positive ionization electrospray, ion source 120◦ , desolvation 250◦ , cone 42 V, collision energy 24 eV, collision gas
argon, m/z 469–367
CHROMATOGRAM
Retention time: 2.0
Internal standard: cisapride (cone 45 V, m/z 467–184) (1.9)
Limit of quantitation: 2 ng/mL
KEY WORDS
pharmacokinetics; plasma
REFERENCE
Lee, H.W.; Ji, H.Y.; Kim, H.H.; Cho, H.-Y.; Lee, Y.-B.; Lee, H.S. Determination of tiropramide in human
plasma by liquid chromatography-tandem mass spectrometry, J.Chromatogr.B, 2003, 796, 395–400.
641
Tizanidine
Tizanidine
N
S
N
Cl
Molecular formula: C9 H8 ClN5 S
N
Molecular weight: 253.72
CAS Registry No: 51322-75-9, 64461-82-1 (HCl)
Merck Index: 13, 9561
N
H
N
H
SAMPLE
Matrix: formulations
Sample preparation: Weigh out ground tablets containing 2 mg tizanidine, add 20 mL
mobile phase, sonicate for 5 min, make up to 50 mL with mobile phase, filter, discard
first 10 mL filtrate. Dilute 2 mL of the subsequent filtrate to 10 mL with mobile phase,
inject a 20 µL aliquot.
HPLC VARIABLES
Column: 150 × 5 5 µm Hypersil CN
Mobile phase: MeCN:MeOH:buffer 18:57:50 (Prepare the buffer by dissolving 2.5 mL
50 mM sodium 1-heptanesulfonate in water and 800 µL triethylamine in 800 mL water,
adjust pH to 3.3 with glacial acetic acid, make up to 1 L with water.)
Flow rate: 1
Injection volume: 20
Detector: UV 227
CHROMATOGRAM
Retention time: 7
Limit of quantitation: 51 ng/mL
OTHER SUBSTANCES
Simultaneous: degradants, impurities
KEY WORDS
stability-indicating; tablets
REFERENCE
Qi, M.-L.; Wang, P.; Wang, L. Validated liquid chromatography method for assay of tizanidine in drug
substance and formulated products, Anal.Chim.Acta, 2003, 478, 171–177.
SAMPLE
Matrix: solutions
Sample preparation: Inject an aliquot of a 100 µg/mL solution in MeOH.
HPLC VARIABLES
Column: 150 × 4.6 5 µm JASCO-RP-C18
Column temperature: 50
Mobile phase: Carbon dioxide:MeOH:acetic acid 90.9:8.918:0.182
Flow rate: 1.5
Injection volume: 20
Detector: UV 235
CHROMATOGRAM
Retention time: 8.81
Internal standard: guaifenesin (5.40)
Limit of detection: 600 ng/mL
642
Tizanidine
OTHER SUBSTANCES
Simultaneous: baclofen (13.64), chlorzoxazone (3.03), methocarbamol (3.92)
KEY WORDS
outlet pressure 12.75 Mpa; SFC
REFERENCE
Bhoir, I.C.; Raman, B.; Sundaresan, M.; Bhagwat, A.M. Development of an isocratic SFC method for four
centrally active muscle relaxant drugs, Anal.Lett., 1998, 31, 1533–1542.
Tolcapone
Tolcapone
Molecular formula: C14 H11 NO5
Molecular weight: 273.24
CAS Registry No: 134308-13-7
Merck Index: 13, 9585
643
O
HO
HO
CH3
NO2
SAMPLE
Matrix: blood
Sample preparation: Mix 200 µL plasma with 25 µL IS in MeCN, add 300 µL MeCN,
let stand at 4◦ for 15 min, centrifuge for 5 min. Mix 100 µL of the supernatant with
100 µL 50 mM pH 2 sodium dihydrogen phosphate buffer, inject an 80 µL aliquot.
HPLC VARIABLES
Guard column: 10 × 4 5 µm Hypersil ODS
Column: 125 × 4 5 µm Hypersil ODS
Mobile phase: MeOH:THF:buffer 55:45:5 (Prepare the buffer by mixing 550 mL MeOH
containing 11.5 g/L N-hexylmethylamine with 450 mL 50 mM sodium dihydrogen phosphate, adjust apparent pH to 2.1 with phosphoric acid, add 50 mL THF.)
Flow rate: 1
Injection volume: 80
Detector: UV 270
CHROMATOGRAM
Retention time: 7
Internal standard: 3-hydroxy-4-methoxy-4′ -methyl-5-nitrobenzophenone (10.5)
Limit of quantitation: 50 ng/mL
OTHER SUBSTANCES
Extracted: 3-O-methyl metabolite (9)
KEY WORDS
dog; human; monkey; mouse; plasma; rat
REFERENCE
Heizmann, P.; Schmitt, M.; Leube, J.; Martin, H.; Saner, A. Determination of the catechol-O-methyltransferase inhibitor tolcapone and three of its metabolites in animal and human plasma and urine by
reversed-phase high-performance liquid chromatography with ultraviolet detection, J.Chromatogr.B,
1999, 730, 153–160.
SAMPLE
Matrix: urine
Sample preparation: Mix 25 µL urine with 25 µL IS in MeCN and 200 µL MeCN:10%
phosphoric acid 12.5:87.5, vortex, centrifuge, inject a 50 µL aliquot.
HPLC VARIABLES
Guard column: 10 × 4 3 µm Spherisorb ODS-1
Column: 125 × 4 3 µm Spherisorb ODS-1
Column temperature: 25
Mobile phase: MeCN:THF:50 mM sodium dihydrogen phosphate 17:13:70, adjusted to
apparent pH 2.1 with phosphoric acid
Flow rate: 1
Injection volume: 50
Detector: UV 270
644
Tolcapone
CHROMATOGRAM
Retention time: 18
Internal standard: 3-hydroxy-4-methoxy-4′ -methyl-5-nitrobenzophenone (27)
Limit of quantitation: 200 ng/mL
OTHER SUBSTANCES
Extracted: 4′ -carboxylic acid metabolite (8), glucuronide metabolite (5), 3-O-methyl
metabolite (22)
REFERENCE
Heizmann, P.; Schmitt, M.; Leube, J.; Martin, H.; Saner, A. Determination of the catechol-O-methyl
transferase inhibitor tolcapone and three of its metabolites in animal and human plasma and urine by
reversed-phase high-performance liquid chromatography with ultraviolet detection, J.Chromatogr.B,
1999, 730, 153–160.
ANNOTATED BIBLIOGRAPHY
Dingemanse, J.; Jorga, K.M.; Schmitt, M.; Gieschke, R.; Fotteler, B.; Zürcher, G.; Da Prada, M.; van
Brummelen, P. Integrated pharmacokinetics and pharmacodynamics of the novel-O-methyltransferase
inhibitor tolcapone during first administration to humans, Clin.Pharmacol.Ther., 1995, 57, 508–517.
[LOQ 50 ng/mL]
Lave, T.; Dupin, S.; Schmitt, M.; Kapps, M.; Meyer, J.; Morgenroth, B.; Chou, R.C.; Jaeck, D.; Coassolo, P. Interspecies scaling of tolcapone, a new inhibitor of catechol-O-methyltransferase (COMT). Use
of in vitro data from hepatocytes to predict metabolic clearance in animals and humans, Xenobiotica,
1996, 26, 839–851.
645
Topiramate
Topiramate
Molecular formula: C12 H21 NO8 S
Molecular weight: 339.36
CAS Registry No: 97240-79-4
Merck Index: 13, 9625
O
O
O
S
O
H 3C
H3C
O
O
NH2
CH3
O
O
CH3
SAMPLE
Matrix: blood, CSF
Sample preparation: Vortex 200 µL plasma or CSF with 25 µL 20 µg/mL IS in
MeCN:water 50:50 and 400 µL MeCN, centrifuge at 10 900 rpm for 5 min. Mix a
250 µL aliquot of the supernatant with 100 µL 0.1% formic acid, inject a 10 µL aliquot.
HPLC VARIABLES
Column: 50 × 2 3 µm Luna C18(2)
Column temperature: 40
Mobile phase: MeOH:200 µM ammonium acetate:formic acid 40:60:0.1
Flow rate: 0.25
Injection volume: 10
Detector: MS, PE Sciex API 3000, nebulizer 375◦ , nebulizer gas nitrogen at 75 psi,
corona needle – 3 mA, auxiliary gas nitrogen at 10 units, curtain gas nitrogen at 8
units, negative ion mode, collision gas nitrogen at 5 units, collision energy 60 eV, m/z
338.2–78.1
CHROMATOGRAM
Retention time: 2.95
Internal standard: 1,2:3,4-bis-O-(1-methylethylidene-α-D-galactopyranose sulfamate)
(collision energy 30 eV, m/z 338.2–96.1) (3.50)
Limit of quantitation: 40 ng/mL
KEY WORDS
comparison with FPIA; plasma
REFERENCE
Christensen, J.; Hojskov, C.S.; Poulsen, J.H. Liquid chromatography tandem mass spectrometry assay
for topiramate analysis in plasma and cerebrospinal fluid: Validation and comparison with fluorescencepolarization immunoassay, Ther.Drug Monit., 2002, 24, 658–664.
SAMPLE
Matrix: blood, urine
Sample preparation: Plasma. Mix 500 µL plasma with 30 µL 100 µg/mL IS in MeOH,
extract with 10 mL diethyl ether. Evaporate the organic layer to dryness, reconstitute
the residue with 75 µL MeOH:10 mM ammonium acetate 50:50, inject a 10 µL aliquot.
Urine. Mix 100 µL urine with 900 µL water and 30 µL 100 µg/mL IS in MeOH, extract
with 10 mL diethyl ether. Evaporate the organic layer to dryness, reconstitute the
residue with 150 µL MeOH:10 mM ammonium acetate 50:50, inject a 10 µL aliquot.
HPLC VARIABLES
Column: 100 × 2.1 3.5 µm Symmetry C18 (Waters)
Mobile phase: Gradient. MeOH:10 mM ammonium acetate 5:95 for 2 min, to 50:50 over
4 min.
Flow rate: 0.25
Injection volume: 10
646
Topiramate
Detector: MS, Bruker Esquire-LC, electrospray, positive ion mode, nebulizer 20 psi,
capillary 4300 V, drying gas 300◦ and 8 L/min, m/z 357
CHROMATOGRAM
Retention time: 14
Internal standard: d12 -topiramate (m/z 369)
Limit of quantitation: 625 ng/mL
OTHER SUBSTANCES
Extracted: metabolites
KEY WORDS
pharmacokinetics; plasma
REFERENCE
Britzi, M.; Soback, S.; Isoherranen, N.; Levy, R.H.; Perucca, E.; Doose, D.R.; Maryanoff, B.E.; Bialer, M.
Analysis of topiramate and its metabolites in plasma and urine of healthy subjects and patients with
epilepsy by use of a novel liquid chromatography-mass spectrometry assay, Ther.Drug Monit., 2003,
25, 314–322.
ANNOTATED BIBLIOGRAPHY
Christensen, J.; Hojskov, C.S.; Dam, M.; Poulsen, J.H. Plasma concentration of topiramate correlates
with cerebrospinal fluid concentration, Ther.Drug Monit., 2001, 23, 529–535.
Contin, M.; Riva, R.; Albani, F.; Baruzzi, A. Simple and rapid liquid chromatographic-turbo ion spray
mass spectrometric determination of topiramate in human plasma, J.Chromatogr.B, 2001, 761,
133–137. [LOQ 250 ng/mL]
Duong, H.T.; Guh, H.Y.; Ko, C.Y.; Micheel, A.P.; Thakur, M.S. A HPLC assay for coating integrity of
topiramate sprinkle formulation, J.Pharm.Biomed.Anal., 2002, 29, 69–74. [sprinkles; LOD 0.07%;
refractive index detector]
Langman, L.J.; Kaliciak, H.A.; Boone, S.A. Fatal acute topiramate toxicity, J.Anal.Toxicol., 2003, 27,
323–324. [topiramate; LC-MS; gabapentin; vigabatrin; droperidol is internal standard]
647
Topotecan
Topotecan
Molecular formula: C23 H23 N3 O5
CH3
N
CH3
HO
Molecular weight: 421.44
CAS Registry No: 123948-87-8, 119413-54-6 (HCl)
Merck Index: 13, 9626
O
N
N
O
H3C
OH O
SAMPLE
Matrix: blood
Sample preparation: Vortex 200 µL plasma with 800 µL cold (dry ice) MeOH for 10 s,
centrifuge at 7000 g for 2 min. Dilute 2 vol of supernatant with 1 vol of water (to measure
lactone form) or 1.5% phosphoric acid (to measure total amount), inject a 20 µL aliquot.
HPLC VARIABLES
Guard column: 10 × 3 ChromGuard RP SS (Varian)
Column: 150 × 3 3.5 µm SB-C18 (Agilent)
Column temperature: 50
Mobile phase: MeOH:buffer 27:73 (The buffer was 75 mM potassium dihydrogen phosphate containing 0.2% triethylamine, adjusted to pH 6.5 with KOH.)
Flow rate: 0.8
Injection volume: 20
Detector: F ex 376 em 530
CHROMATOGRAM
Retention time: 13.7
Limit of quantitation: 250 pg/mL
OTHER SUBSTANCES
Extracted: N-desmethyltopotecan (LOQ 50 pg/mL) (7.6)
KEY WORDS
plasma
REFERENCE
Bai, F.; Kirstein, M.N.; Hanna, S.K.; Iacono, L.C.; Johnston, B.; Stewart, C.F. Determination of plasma
topotecan and its metabolite N-desmethyl topotecan as both lactone and total form by reversed-phase
liquid chromatography with fluorescence detection, J.Chromatogr.B, 2003, 784, 225–232.
SAMPLE
Matrix: blood
Sample preparation: Vortex 900 µL plasma with 100 µL buffer for 20 s. Add a 200 µL
aliquot to 800 µL cold (–40◦ ) MeOH, vortex for 20 s, centrifuge for 2 min. Mix a 500 µL
aliquot of the supernatant with 500 µL buffer, inject a 200 µL aliquot. (The buffer was
8 mM disodium hydrogen phosphate containing 1 mM potassium dihydrogen phosphate,
137 mM NaCl, and 3 mM KCl and was adjusted to pH 2.0 for lactone or pH 11.0 for
carboxylate forms.)
HPLC VARIABLES
Guard column: Guard-Pak C18 NovaPak
Column: 150 × 3.9 4 µm NovaPak C18
648
Topotecan
Mobile phase: MeCN:buffer 12:88 (The buffer was 3% triethylamine adjusted to pH 5.5
with glacial acetic acid.)
Flow rate: 1
Injection volume: 200
Detector: F (tunable) ex 390 em 520, F (filter) ex 350–470 em 510–650
CHROMATOGRAM
Retention time: 2.5 (carboxylate), 5.2 (lactone)
Limit of detection: 300 pg/mL (lactone, filter F), 260 pg/mL (lactone, tunable F),
150 pg/mL (carboxylate, filter F), 100 pg/mL (carboxylate, tunable F)
Limit of quantitation: 750 pg/mL (lactone, filter F), 500 pg/mL (lactone, tunable F),
500 pg/mL (carboxylate, filter F), 250 pg/mL (carboxylate, tunable F)
KEY WORDS
plasma
REFERENCE
Warner, D.L.; Burke, T.G. Comparison of filter and tunable fluorescence detection for the HPLC simultaneous quantitation of lactone and carboxylate forms of topotecan in plasma, J.Liq.Chromatogr.Rel.
Technol., 1997, 20, 1523–1537.
ANNOTATED BIBLIOGRAPHY
Beijnen, J.H.; Smith, B.R.; Keijer, W.J.; van Gijn, R.; ten Bokkel Huinink, W.W.; Vlasveld, L.T.; Rodenhuis, S.; Underberg, W.J. High-performance liquid chromatographic analysis of the new antitumour
drug SK&F 104864-A (NSC 609699) in plasma, J.Pharm.Biomed.Anal., 1990, 8, 789–794.
Boucaud, M.; Pinguet, F.; Poujol, S.; Astre, C.; Bressolle, F. Sensitive high performance liquid chromatographic fluorescence determination of topotecan in human plasma and parotid saliva, J.Liq.Chromatogr.Rel.Technol., 2000, 23, 2373–2390. [LOQ 50 pg/mL]
Loos, W.J.; Stoter, G.; Verweij, J.; Schellens, J.H.M. Sensitive high-performance liquid chromatographic
fluorescence assay for the quantitation of topotecan (SKF 104864-A) and its lactone ring-opened product
(hydroxy acid) in human plasma and urine, J.Chromatogr.B, 1996, 678, 309–315. [LOQ 0.1 ng/mL;
fluorescence detection]
Loos, W.J.; van Zomeren, D.M.; Gelderblom, H.; Verweij, J.; Nooter, K.; Stoter, G.; Sparreboom, A. Determination of topotecan in human whole blood and unwashed erythrocytes by high-performance liquid
chromatography, J.Chromatogr.B, 2002, 766, 99–105. [LOQ 200 pg/mL; fluorescence detection]
Rosing, H.; Doyle, E.; Davies, B.E.; Beijnen, J.H. High-performance liquid chromatographic determination of the novel antitumour drug topotecan as the total of the lactone plus carboxylate forms in human
plasma, J.Chromatogr.B, 1995, 668, 107–115. [LOQ 0.05 ng/mL; fluorescence detection]
Rosing, H.; Doyle, E.; Beijnen, J.H. The impact of column temperature in the high performance liquid
chromatographic analysis of topotecan in rat and dog plasma, J.Pharm.Biomed.Anal., 1996, 15,
279–286. [LOQ 0.1 ng/mL; fluorescence detection]
Rosing, H.; van Zomeren, D.M.; Doyle, E.; ten Bokkel Huinink, W.W.; Schellens, J.H.M.; Bult, A.; Beijnen, J.H. Quantification of topotecan and its metabolite N-desmethyltopotecan in human plasma,
urine and faeces by high-performance liquid chromatographic methods, J.Chromatogr.B, 1999, 727,
191–203. [LOQ 100 pg/mL; fluorescence detection]
649
Tosufloxacin
Tosufloxacin
Molecular formula: C19 H15 F3 N4 O3
Molecular weight: 404.34
CAS Registry No: 100490-36-6. 104051-69-6
(HCl), 115964-29-9 (tosylate)
Merck Index: 13, 9632
F
H2N
F
N
N
N
F
COOH
O
SAMPLE
Matrix: bile, blood, urine
Sample preparation: Dilute urine 1:20. Dilute bile 1:10. Vortex 500 µL serum, diluted
urine, or diluted bile with 3.2 mL dichloromethane, rotate at 20 rpm for 10 min, centrifuge at 1000 g for 10 min. Remove 3 mL of the lower organic phase and add it to
200 µL pH 2.5 acetic acid, rotate at 20 rpm for 10 min, centrifuge at 1000 g for 10 min,
inject a 20 µL aliquot of the aqueous layer.
HPLC VARIABLES
Column: 150 × 4.6 5 µm Ultrasphere C18
Mobile phase: MeCN:buffer 18:82, pH adjusted to 2 with 14.6 M phosphoric acid (The
buffer was 10 mM sodium dihydrogen phosphate containing 5 mM tetrabutylammonium
bromide.)
Flow rate: 2
Injection volume: 20
Detector: F ex 265 em 433
CHROMATOGRAM
Retention time: 2
Limit of detection: 20 ng/mL (serum), 500 ng/mL (urine), 250 ng/mL (bile)
OTHER SUBSTANCES
Noninterfering: amikacin, aztreonam, carbamazepine, cephalosporins, ciprofloxacin,
clavulanic acid, difloxacin, digitoxin, digoxin, fleroxacin, fosfomycin, furosemide, gentamicin, imipenem, lidocaine, netilmicin, norfloxacin, ofloxacin, pefloxacin, penicillins,
phenobarbital, phenytoin, primidone, procainamide, quinidine, rifampin, salicylic acid,
teicoplanin, theophylline, tobramycin, valproic acid, vancomycin
KEY WORDS
human; pig; serum
REFERENCE
Koechlin, C.; Jehl, F.; Linger, L.; Monteil, H. High-performance liquid chromatography for the determination of three new fluoroquinolones, fleroxacin, temafloxacin and A-64730, in biological fluids,
J.Chromatogr., 1989, 491, 379–387.
SAMPLE
Matrix: wastewater
Sample preparation: Condition a mixed-phase (C8 and benzenesulfonate) cationexchange SPE disc (Varian) with 2 mL MeOH and 2 mL water at pH 3. Adjust pH of
wastewater to 3, pass 50–150 mL through the SPE disc at 1 mL/min, dry under vacuum
for 5 min, elute with 2.5 mL 5% ammonia in MeOH:water 15:85. Neutralize the eluate
with 500 µL 85% phosphoric acid, inject a 200 µL aliquot.
650
Tosufloxacin
HPLC VARIABLES
Guard column: 20 × 3 5 µm Discovery RP-AmideC16 (Supelco)
Column: 250 × 3 5 µm Discovery RP-AmideC16 (Supelco)
Column temperature: 50
Mobile phase: Gradient. MeCN:25 mM pH 2.4 orthophosphoric acid from 5:95 to 7:93
over 17 min, maintain at 7:93 for 5 min, to 17:83 over 13 min, to 85:15 (step gradient),
maintain at 85:15 for 5 min, return to initial conditions over 2 min, re-equilibrate for
10 min.
Flow rate: 0.7
Injection volume: 200
Detector: F ex 278 em 445
CHROMATOGRAM
Retention time: 35
OTHER SUBSTANCES
Extracted: ciprofloxacin (19.5), danofloxacin (23), difloxacin (28), enrofloxacin (22.5),
fleroxacin (14), lomefloxacin (21), norfloxacin (18), ofloxacin (15.5), pipemidic acid (6),
KEY WORDS
SPE
REFERENCE
Golet, E.M.; Alder, A.C.; Hartmann, A.; Ternes, T.A.; Giger, W. Trace determination of fluoroquinolone
antibacterial agents in urban wastewater by solid-phase extraction and liquid chromatography with
fluorescence detection, Anal.Chem., 2001, 73, 3632–3638.
ANNOTATED BIBLIOGRAPHY
Lyon, D.J.; Cheung, S.W.; Chan, C.Y.; Cheng, A.F. Rapid HPLC assay of clinafloxacin, fleroxacin, levofloxacin, sparfloxacin and tosufloxacin, J.Antimicrob.Chemother., 1994, 34, 446–448. [in conjunction
with Chan,C.Y.; Lam,A.W.; French,G.L. Rapid HPLC assay of fluoroquinolones in clinical specimens.
J.Antimicrob.Chemother. 1989, 23, 597–604.]
651
Travoprost
Travoprost
CH3
Molecular formula: C26 H35 F3 O6
Molecular weight: 500.55
CAS Registry No: 157283-68-6
CH3
O
H
HO
H
OH
O
H
H
O
H
OH
F
F
F
SAMPLE
Matrix: blood
Sample preparation: Condition a SPEC 3 mL 15 mg MP1 nonpolar reversed-phase/
strong cation-exchange SPE Cartridge (Ansys) with 500 µL MeOH and 500 µL 40 mM
formic acid. Mix 1 mL plasma with 50 µL 400 IU/mL rabbit liver esterase (Sigma) in
buffered saline, heat at 37◦ for 45 min. (This hydrolyzes travoprost to the free acid.) Mix
1 mL plasma with 15 µL 20 ng/mL IS and 1 mL 100 mM formic acid, add to the SPE cartridge, rinse tube with 0.5–1 mL water, add rinse to the SPE cartridge, wash with 500 µL
water, dry under vacuum, wash with two 500 µL portions of toluene:dichloromethane
60:40, dry under vacuum, elute with 600 µL toluene:methylformate 20:80. Evaporate
the eluate to dryness under a stream of nitrogen at 30◦ , reconstitute the residue with
125 µL MeOH:water 50:50, inject a 35 µL aliquot.
HPLC VARIABLES
Column: 150 × 2 5 µm Phenomenex Columbus C18
Mobile phase: MeOH:5 mM pH 6.3 ammonium formate 70:30
Flow rate: 0.2
Injection volume: 35
Detector: MS, Micromass Quattro LC, electrospray, capillary 3.0 kV, sample cone 40 V,
extraction cone 2 V, RF lens 0.3 V, source temperature 125◦ , drying gas 250◦ , MS1
parameters LM resolution 14, HM resolution 14, ion energy 1.2 V, entrance and exit set
to 0 and 1, collision energy 30 eV, MS2 parameters LM resolution 15.0, HM resolution
15.0, ion energy 1.2 V, multiplier 650 V, nebulizing gas 75 L/h, drying gas 570 L/h,
collision gas argon, m/z 457–161
CHROMATOGRAM
Retention time: 5.3 (as travoprost free acid)
Internal standard: AL-5848X (tetradeutero travoprost free acid) (m/z 461–161)
Limit of quantitation: 10 pg/mL
KEY WORDS
plasma; SPE
REFERENCE
McCue, B.A.; Cason, M.M.; Curtis, M.A.; Faulkner, R.D.; Dahlin, D.C. Determination of travoprost and
travoprost free acid in human plasma by electrospray HPLC/MS/MS, J.Pharm.Biomed.Anal., 2002,
28, 199–208.
652
Trenbolone
Trenbolone
CH3 OH
H
Molecular formula: C18 H22 O2
Molecular weight: 270.37
CAS Registry No: 10161-33-8, 10161-34-9 (acetate),
23454-33-3 (cyclohexylmethylcarbonate)
Merck Index: 13, 9659
H
O
SAMPLE
Matrix: blood
Sample preparation: Serum. Condition a 3 mL Oasis HLB SPE cartridge with 3 mL
MeOH and 3 mL water. Add 1 mL serum containing 100 ng IS to the SPE cartridge,
wash with 3 mL water, elute with 2 mL MeOH. Evaporate the eluate to dryness under
a stream of nitrogen at 45◦ , reconstitute the residue with 100 µL MeOH:water 50:50,
inject a 5 µL aliquot. Urine. Condition a 360 mg Sep-Pak C18 SPE cartridge with 5 mL
MeOH and 10 mL water. Adjust the pH of 5 mL urine containing 100 ng IS to 5.5
with acetate buffer, add 50 µL β-glucuronidase-aryl sulfatase (Type H-2 Helix Pomatia,
Boehringer Mannheim), heat at 37◦ overnight, cool, add to the SPE cartridge, wash with
5 mL water, elute with 3 mL MeOH. Evaporate the eluate to dryness under a stream of
nitrogen at 45◦ , reconstitute the residue with 100 µL MeOH:water 50:50, inject a 5 µL
aliquot.
HPLC VARIABLES
Guard column: 4 × 2 Phenomenex C18
Column: 250 × 2.1 5 µm Hypersil C18
Mobile phase: MeOH:2.88 mM ammonium formate 65:35
Flow rate: 0.15
Injection volume: 5
Detector: MS, PE Sciex API 365, turbo ionspray, positive mode, source 4000 V, orifice 30 V, ring 250 V, nebulizer gas nitrogen at 8 units, curtain gas nitrogen at 8
units, collision gas nitrogen at 3 units, collision energy – 42 eV, vaporizer 450◦ , m/z
271.2–253.3–227.2–199.2
CHROMATOGRAM
Retention time: 13.8
Internal standard: methyltestosterone (m/z 303.1–109.1–97.0) (22.9)
Limit of detection: 350 pg/mL
Limit of quantitation: 1 ng/mL
KEY WORDS
cow; serum; SPE
REFERENCE
Buiarelli, F.; Cartoni, G.P.; Coccioli, F.; De Rossi, A.; Neri, B. Determination of trenbolone and its
metabolite in bovine fluids by liquid chromatography-tandem mass spectrometry, J.Chromatogr.B,
2003, 784, 1–15.
Treprostinil
Treprostinil
Molecular formula: C23 H34 O5
Molecular weight: 390.51
CAS Registry No: 81846-19-7
H
O
653
COOH
HO
H
H
HO
H
H
CH3
SAMPLE
Matrix: blood
Sample preparation: Extract 1 mL plasma with hexane:ethyl acetate 70:30. Evaporate
the organic layer to dryness under a stream of nitrogen, reconstitute the residue with
MeOH:water:100 mM ammonium formate:formic acid 50:47.5:2.5:0.05, inject a 25 µL
aliquot.
HPLC VARIABLES
Column: 100 × 2 Betasil C18 (Keystone)
Mobile phase: MeCN:water:100 mM ammonium formate:formic acid 33.25:61.75:5:0.1
Flow rate: 0.3
Injection volume: 25
Detector: MS, PE Sciex API-III or API 365, ionspray, atmospheric pressure ionization,
negative ionization
CHROMATOGRAM
Retention time: 2.5
Internal standard: LRXA-97 JO2 (a dimethylene homolog) (3.5)
Limit of quantitation: 25 pg/mL
KEY WORDS
pharmacokinetics; plasma
REFERENCE
Wade, M.; Baker, F.J.; Roscigno, R.; DellaMaestra, W.; Hunt, T.L.; Lai, A.A. Absolute bioavailability and pharmacokinetics of treprostinil sodium administered by acute subcutaneous infusion,
J.Clin.Pharmacol., 2004, 44, 83–88.
SAMPLE
Matrix: formulations
Sample preparation: Inject a 20 µL aliquot of a 4–130 µg/mL solution in water, 0.9%
NaCl, or 5% dextrose.
HPLC VARIABLES
Column: 250 × 4.6 5 µm Hypersil ODS
Mobile phase: Gradient. MeCN:water:trifluoroacetic acid 40:60:0.1 for 17 min, 78:22:0.1
for 18 min (step gradient (?)), initial conditions for 7 min (step gradient (?)).
Flow rate: 2
Injection volume: 20
Detector: UV 217
CHROMATOGRAM
Retention time: 15.0
Limit of detection: 0.025%
Limit of quantitation: 0.05%
654
Treprostinil
OTHER SUBSTANCES
Simultaneous: m-cresol (preservative) (3.4), degradants
KEY WORDS
injections; stability-indicating
REFERENCE
Phares, K.R.; Weiser, W.E.; Miller, S.P.; Myers, M.A.; Wade, M. Stability and preservative effectiveness
of treprostinil sodium after dilution in common intravenous diluents, Am.J.Health-Syst.Pharm., 2003,
60, 916–922.
655
Trichlorfon
Trichlorfon
Molecular formula: C4 H8 Cl3 O4 P
O
H3CO P
H3CO
CCl3
OH
Molecular weight: 257.44
CAS Registry No: 52-68-6
Merck Index: 13, 9696
SAMPLE
Matrix: blood
Sample preparation: Vortex 1 mL plasma and 5 mL chloroform for 15 s, centrifuge at
800 g at 0–5◦ for 15 min (Caution! Chloroform is a carcinogen!). Remove a 4.5 mL aliquot
of the organic layer and add it to 3.5 mL 5 M HCl, vortex for 15 s, centrifuge at 800 g
at 0–5◦ for 15 min. Remove a 4 mL aliquot of the chloroform layer and add it to 200 mg
anhydrous calcium sulfate, vortex, centrifuge at 800 g at 0–5◦ for 5 min. Evaporate
the chloroform layer to dryness in an acid-washed vial under a stream of nitrogen at
room temperature, reconstitute the residue with 120 µL water, vortex, centrifuge at
11 000 g at 0–5◦ for 5 min, inject a 100 µL aliquot. (However, Aden Abdi et al. claim that
substantial base-catalyzed conversion of trichlorfon to dichlorvos can occur under these
conditions and plasma should be acidified with phosphoric acid before extraction. (Aden
Abdi,Y.; Villén,T.; Gustafsson,L.L.; Ericsson,O.; Sjöqvist,F. Methodological commentary
on the analysis of metrifonate and dichlorvos in biological samples. J.Chromatogr.
1993, 612, 336–337.) These workers recommend mixing blood with an equal volume of
740 mM phosphoric acid immediately upon collection. Trichlorfon can still be extracted
from the acidified sample. (Aden Abdi,Y.; Villén,T. Pharmacokinetics of metrifonate
and its rearrangement product dichlorvos in whole blood. Pharmacol.Toxicol. 1991, 68,
137–139.))
HPLC VARIABLES
Guard column: 37–50 µm C18/Corasil
Column: 300 × 3.9 10 µm C18
Mobile phase: MeOH:THF:1 mM pH 3.0 sodium octanesulfonate 10:0.1:89.9
Flow rate: 2
Injection volume: 200
Detector: UV 210
CHROMATOGRAM
Retention time: 10.8
Limit of detection: 1 µg/mL
KEY WORDS
plasma
REFERENCE
Unni, L.K.; Hannant, M.E.; Becker, R.E. High-performance liquid chromatographic method using ultraviolet detection for measuring metrifonate and dichlorvos levels in human plasma, J.Chromatogr.,
1992, 573, 99–103.
656
Triethanolamine
Triethanolamine
OH
HO
N
OH
Molecular formula: C6 H15 NO3
Molecular weight: 149.19
CAS Registry No: 102-71-6
Merck Index: 13, 9739
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 100 × 4 Alltech Universal Cation
Mobile phase: 3 mM methanesulfonic acid
Flow rate: 1
Injection volume: 100
Detector: Conductivity (nonsuppressed mode, indirect conductivity detection)
CHROMATOGRAM
Retention time: 6
OTHER SUBSTANCES
Simultaneous: ammonium (4), calcium (12), ethanolamine (4.2), lithium (3), magnesium (10), potassium (5.5), sodium (3.5),
REFERENCE
Saari-Nordhaus, R.; Anderson, J.M. Jr. Alternative approach to enhancing cation selectivity in ion
chromatography, J.Chromatogr.A, 2004, 1039, 123–127.
657
Trifluridine
Trifluridine
O
H
Molecular formula: C10 H11 F3 N2 O5
Molecular weight: 296.20
CAS Registry No: 70-00-8
Merck Index: 13, 9758
CF3
N
HO
O
N
O
OH
SAMPLE
Matrix: blood, tissue
Sample preparation: Mix 1 mL blood with 2 mL ice-cold MeCN, centrifuge, filter,
inject a 5–20 µL aliquot. Homogenize brain tissue with 0.5 mL water, add 2 mL MeCN,
centrifuge, filter, inject a 5–20 µL aliquot.
HPLC VARIABLES
Guard column: C18 Corasil (Whatman)
Column: 100 × 3 Chrompack C18
Mobile phase: MeCN:50 mM sodium dihydrogen phosphate 15:85
Flow rate: 0.3–0.8
Injection volume: 5–20
Detector: UV 254, UV 280
KEY WORDS
brain; rat; whole blood
REFERENCE
Rand, K.H.; Bodor, N.; el Koussi, A.A.; Raad, I.; Miyake, A.; Houck, H.; Gildersleeve, N. Potential treatment of herpes simplex virus encephalitis by brain-specific delivery of trifluorothymidine using a
dihydropyridine in equilibrium pyridinium salt type redox delivery system, J.Med.Virol., 1986, 20,
1–8.
658
Triptorelin
Triptorelin
Molecular formula: C64 H82 N18 O13
Molecular weight: 1311.45
CAS Registry No: 57773-63-4,
140194-24-7 (acetate)
Merck Index: 13, 9818
SAMPLE
Matrix: blood
Sample preparation: Mix 500 µL plasma with 2 mL EtOH for 30 min, centrifuge at
3000 g for 15 min. Evaporate the supernatant to ca. 500 µL under a stream of nitrogen
at 40◦ for 1 h, lyophilize, reconstitute with mobile phase, inject a 600 µL aliquot.
HPLC VARIABLES
Column: Partisil PXS 10/25 SCX
Mobile phase: EtOH:200 mM pH 4.6 ammonium acetate 10:90
Flow rate: 1.6
Injection volume: 600
Detector: Radioimmunoassay of fractions
CHROMATOGRAM
Retention time: 14
KEY WORDS
plasma
REFERENCE
Barron, J.L.; Millar, R.P.; Searle, D.I. Metabolic clearance and plasma half-disappearance time of DTRP6 and exogenous luteinizing hormone-releasing hormone, J.Clin.Endocrinol.Metab., 1982, 54,
1169–1173.
SAMPLE
Matrix: blood
Sample preparation: Condition a Sep-Pak C18 SPE cartridge with 3 mL MeOH
and 5 mL water. Add 500 µL plasma to the SPE cartridge, wash with 5 mL 0.1%
trifluoroacetic acid, elute with 5 mL MeOH:water:trifluoroacetic acid 80:19:1. Evaporate
the eluate, freeze the residual aqueous solution, freeze, lyophilize, reconstitute with
300 µL 0.1% trifluoroacetic acid, inject a 100 µL aliquot.
HPLC VARIABLES
Column: µBondapak C18
Mobile phase: Gradient. A was 11 mM trifluoroacetic acid in MeCN:water 70:30. B was
11 mM trifluoroacetic acid in 3 mM acetic acid. A:B from 5:95 to 95:5 over 20 min.
Flow rate: 1
Injection volume: 100
Detector: Radioimmunoassay of fractions; UV 210
CHROMATOGRAM
Retention time: 18
KEY WORDS
dog; pharmacokinetics; plasma; rat; SPE
Triptorelin
659
REFERENCE
Ezan, E.; Drieu, K.; Chapelat, M.; Rougeot, C.; Dray, F. Radioimmunoassay of [D-Trp6 ]-luteinizing
hormone-releasing hormone: its application to animal pharmacokinetic studies after single injection
and long-acting formulation administration, Regul.Pept., 1986, 14, 155–167.
660
Troleandomycin
Troleandomycin
O
O
H3C
Molecular formula: C41 H67 NO15
H3C
Molecular weight: 813.97
CAS Registry No: 2751-09-9
Merck Index: 13, 9839
H3C
H3C
CH3
OAc
N
CH3
O AcO
H3C
O
O
CH3
O
O
CH3
O
OCH3
OAc
CH3
SAMPLE
Matrix: solutions
Sample preparation: Inject a 10 µL aliquot of a 40 mg/mL solution in MeCN.
HPLC VARIABLES
Column: 250 × 4.6 5 µm Xterra RP18
Column temperature: 30
Mobile phase: MeCN:200 mM pH 6.0 ammonium acetate:water 45:5:50
Flow rate: 1
Injection volume: 10
Detector: UV 205
CHROMATOGRAM
Retention time: 22
Limit of detection: 0.02% (S/N 3)
Limit of quantitation: 0.05%
REFERENCE
Chepkwony, H.K.; Roets, E.; Hoogmartens, J. Liquid chromatography of troleandomycin, J.Chromatogr.A, 2001, 914, 53–58.
661
Troxipide
Troxipide
H
N
O
Molecular formula: C15 H22 N2 O4
Molecular weight: 294.35
CAS Registry No: 99777-81-8
Merck Index: 13, 9860
OCH3
N
H
OCH3
OCH3
SAMPLE
Matrix: tissue
Sample preparation: Homogenize 5–30 mg gastric mucosal tissue with 1.2 mL 20 mM
pH 7.4 phosphate buffer, centrifuge at 3000 rpm for 10 min. Mix 1 mL of the supernatant
with 100 µL IS solution and 1.5 mL MeCN, let stand at 5–6◦ for 1 h, centrifuge at
3000 rpm at 4◦ for 10 min. Dry 2 mL of the supernatant, reconstitute with 1 mL EtOH,
dry, reconstitute with 120 µL injection solvent (unspecified), inject a 60 µL aliquot.
HPLC VARIABLES
Guard column: 10 × 4 5 µm Capcell Pak C18 UG 120
Column: 50 × 4.6 3 µm Capcell Pak C18 UG 120
Mobile phase: MeCN:50 mM pH 5.0 acetate buffer containing 0.08% sodium 1-hexanesulfonate (ratio not given)
Flow rate: 0.6
Injection volume: 60
Detector: UV 260
CHROMATOGRAM
Internal standard: 3-amino-1-[3,4,5-trimethoxybenzoyl]piperidine HCl
KEY WORDS
gastric mucosa
REFERENCE
Kusugami, K.; Ina, K.; Hosokawa, T.; Kobayashi, F.; Kusajima, H.; Momo, K.; Nishio, Y. Troxipide, a
novel antiulcer compound, has inhibitory effects on human neutrophil migration and activation
induced by various stimulants, Dig.Liver Dis., 2000, 32, 305–311.
Ubenimex
NH2
O
COOH CH3
N
Molecular formula: C16 H24 N2 O4
OH
Molecular weight: 308.37
CAS Registry No: 58970-76-6
Merck Index: 13, 9910
CH3
H
SAMPLE
Matrix: blood
Sample preparation: Mix 20 µL serum with 20 µL water and 200 µL 4.2 mM acetic
acid, heat in a boiling water bath for 5 min, centrifuge at 1000 g for 5 min. Mix 100 µL
of the supernatant with 50 µL 1.5 M ammonium hydroxide and 25 µL 3 mM sodium
periodate, let stand at room temperature for 20 min, add 25 µL 12 mM sodium sulfite to
decompose excess periodate, add 200 µL DDB solution, heat at 37◦ for 50 min, add 50 µL
1 M NaOH, inject a 100 µL aliquot. (Prepare DDB solution by dissolving 26.5 mg 4,5dimethoxy-1,2-diaminobenzene monohydrochloride in 100 mL 300 mM HCl, use within
3 h. Dihydrochloride available from Molecular Probes or Dojindo Laboratories)
HPLC VARIABLES
Column: 150 × 4 5 µm LiChrosorb RP-18
Mobile phase: MeCN:100 mM pH 8.7 tris-HCl buffer 25:75
Flow rate: 0.8
Injection volume: 100
Detector: F ex 320 em 390
CHROMATOGRAM
Retention time: 4.5
Limit of detection: 200 ng/mL
OTHER SUBSTANCES
Extracted: p-hydroxybestatin (10.5)
KEY WORDS
derivatization; serum
REFERENCE
Ishida, J.; Yamaguchi, M.; Kai, M.; Ohkura, Y.; Nakamura, M. Determination of bestatin in serum
by high-performance liquid chromatography with fluorescence detection, J.Chromatogr., 1984, 305,
381–389.
662
HPLC Methods for Recently Approved Pharmaceuticals. by George Lunn
Copyright 2005 John Wiley & Sons, Ltd. ISBN: 0-471-66941-5
663
Unoprostone isopropyl ester
Unoprostone isopropyl ester
Molecular formula: C25 H44 O5
Molecular weight: 424.61
CAS Registry No: 120373-24-2, 120373-36-6 (free acid)
Merck Index: 13, 9917
H
OH
H
O
O
HO
H
H
O
CH3
CH3
CH3
SAMPLE
Matrix: aqueous humor, tissue
Sample preparation: Inject a 50 µL aliquot of aqueous humor directly. Homogenize
eye tissue with MeCN:0.1% acetate 20:80 at 4◦ , centrifuge at 15000 rpm at 4◦ for 30 min.
Filter (Millipore Ultra-Free CL, 300 000 cut-off) while centrifuging at 15 000 rpm at 4◦
for 30 min, inject an aliquot of the ultrafiltrate
HPLC VARIABLES
Column: 150 × 4.6 C18-5A (Shodex)
Mobile phase: Gradient. MeCN:water:acetic acid from 20:80:0.1 to 100:0:0.1 over 40 min
Injection volume: 50
Detector: Radioactivity (3 H)
CHROMATOGRAM
Retention time: 30
OTHER SUBSTANCES
Extracted: metabolites
KEY WORDS
eye; rabbit
REFERENCE
Kashiwagi, K.; Iizuka, Y.; Tsukahara, S. Metabolites of isopropyl unoprostone as potential ophthalmic
solutions to reduce intraocular pressure in pigmented rabbits, Jpn.J.Pharmacol., 1999, 81, 56–62.
Valacyclovir
O
H
N
N
Molecular formula: C13 H20 N6 O4
Molecular weight: 324.33
CAS Registry No: 124832-26-4, 124832-27-5 (HCl)
Merck Index: 13, 9966
H2N
N
N
NH2
O
O
CH3
O
CH3
SAMPLE
Matrix: blood
Sample preparation: Acidify plasma with trichloroacetic acid to a final trichloroacetic
acid concentration of 3%, centrifuge at 9000 g at 4◦ for 10 min, inject an aliquot of the
ultrafiltrate.
HPLC VARIABLES
Column: 250 × 4.6 Adsorbosphere C18 (Alltech)
Mobile phase: MeCN:100 mM pH 3.5 ammonium formate buffer 10:90
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Retention time: 8
Limit of quantitation: 80 ng/mL
KEY WORDS
plasma; ultrafiltrate
REFERENCE
Weller, S.; Blum, M.R.; Doucette, M.; Burnette, T.; Cederberg, D.M.; de Miranda, P.; Smiley, M.L. Pharmacokinetics of the acyclovir pro-drug valaciclovir after escalating single- and multiple-dose administration to normal volunteers, Clin.Pharmacol.Ther., 1993, 54, 595–605.
SAMPLE
Matrix: urine
Sample preparation: Acidify urine with trichloroacetic acid to a final trichloroacetic
acid concentration of 1%, filter (Centrifree) while centrifuging at 2000 g at 4◦ for
10–20 min, inject an aliquot of the ultrafiltrate.
HPLC VARIABLES
Column: 250 × 4.6 Adsorbosphere C18 (Alltech)
Mobile phase: Gradient. A was 50 mM pH 3.5 ammonium formate buffer. B was
MeCN:50 mM pH 7.2 ammonium phosphate buffer 50:50. A:B ratio not given.
Flow rate: 1
Detector: UV 254, UV 280
CHROMATOGRAM
Retention time: 27
Limit of quantitation: 160 ng/mL
OTHER SUBSTANCES
Extracted: acyclovir (20), CMMG (14)
664
HPLC Methods for Recently Approved Pharmaceuticals. by George Lunn
Copyright 2005 John Wiley & Sons, Ltd. ISBN: 0-471-66941-5
Valacyclovir
665
KEY WORDS
ultrafiltrate
REFERENCE
Weller, S.; Blum, M.R.; Doucette, M.; Burnette, T.; Cederberg, D.M.; de Miranda, P.; Smiley, M.L. Pharmacokinetics of the acyclovir pro-drug valaciclovir after escalating single- and multiple-dose administration to normal volunteers, Clin.Pharmacol.Ther., 1993, 54, 595–605.
666
Valdecoxib
Valdecoxib
O
O
S
NH2
Molecular formula: C16 H14 N2 O3 S
Molecular weight: 314.36
CAS Registry No: 181695-72-7
N
O
CH3
SAMPLE
Matrix: blood
Sample preparation: Condition a 1 mL 100 mg Bond Elut C18 SPE cartridge with
2 mL MeOH and 2 mL water. Centrifuge plasma at 2000 g at 4◦ for 5 min, vortex 400 µL
of the supernatant with 400 µL 100 ng/mL IS in water, add to the SPE cartridge, wash
with 2 mL water, elute with 250 µL MeCN. Evaporate the eluate to dryness under a
stream of nitrogen, reconstitute the residue with 100 µL mobile phase, inject a 20 µL
aliquot.
HPLC VARIABLES
Column: 50 × 2.1 5 µm Zorbax XDB-C8
Mobile phase: MeCN:water 50:50 containing 10 mM ammonium acetate
Flow rate: 0.1
Injection volume: 20
Detector: MS, PE Sciex API-III Plus quadrupole, electrospray, negative ion mode,
electrospray interface – 3700 V, orifice – 62 V, nebulizer gas nitrogen at 50 psi, curtain
gas nitrogen at 1.8 L/min, collision gas argon, collision offset energy 25 eV, m/z 313–118
CHROMATOGRAM
Retention time: 3
Internal standard: 4-(5-methyl-3-(3-fluorophenyl)-isoxazol-4-yl)benzenesulfonamide
(m/z 331–118) (3.5)
Limit of quantitation: 0.5 ng/mL
OTHER SUBSTANCES
Extracted: metabolite (4-(5-hydroxymethyl-3-phenylisoxazol-4-yl)benzenesulfonamide)
(m/z 329–196) (2)
KEY WORDS
plasma; SPE
REFERENCE
Zhang, J.Y.; Fast, D.M.; Breau, A.P. Development and validation of an automated SPE-LC-MS/MS assay
for valdecoxib and its hydroxylated metabolite in human plasma, J.Pharm.Biomed.Anal., 2003, 33,
61–72.
SAMPLE
Matrix: blood, feces, urine
Sample preparation: Blood. Extract plasma or red blood cells three times with MeCN.
Combine the supernatants and evaporate to dryness under a stream of nitrogen,
reconstitute the residue with mobile phase A, inject an aliquot. Urine. Centrifuge urine
at 2000 g for 5 min, inject an aliquot. Feces. Homogenize feces three times with MeCN
and twice with mobile phase A.
HPLC VARIABLES
Column: 150 × 3.9 4 µm Novapak C8
Valdecoxib
667
Mobile phase: Gradient. A was MeCN:MeOH:25 mM pH 4 ammonium acetate buffer
3:6:81. B was MeCN:MeOH:25 mM pH 4 ammonium acetate buffer 20:40:60. A:B from
100:0 to 0:100 over 25 min, maintain at 0:100 for 5 min, return to initial conditions over
2 min, re-equilibrate for 8 min.
Flow rate: 1
Detector: UV 240; Radioactivity (14 C); MS, PE Sciex API-III Plus triple quadrupole,
negative electrospray, capillary – 3700 V, orifice – 65 V, nebulizer gas at 50 psi, curtain
gas nitrogen at 1.8 L/min, auxiliary gas nitrogen at 2 L/min, collision gas argon, collision
energy 30 eV, turbo ionspray 400◦ , 0.2 mL/min of column effluent entered the detector
CHROMATOGRAM
Retention time: 26
OTHER SUBSTANCES
Extracted: metabolites
KEY WORDS
mouse; pharmacokinetics; plasma; red blood cells
REFERENCE
Zhang, J.Y.; Yuan, J.J.; Wang, Y.-F.; Bible, R.H. Jr.; Breau, A.P. Pharmacokinetics and metabolism of a
COX-2 inhibitor, valdecoxib, in mice, Drug Metab.Dispos., 2003, 31, 491–501.
ANNOTATED BIBLIOGRAPHY
Yuan, J.J.; Yang, D.-C.; Zhang, J.Y.; Bible, R.; Karim, A.; Findlay, J.W.A. Disposition of a specific
cyclooxygenase-2 inhibitor, valdecoxib, in human, Drug Metab.Dispos., 2002, 30, 1013–1021. [radioactivity detection (14 C); LC-MS; metabolites]
Zhang, J.Y.; Fast, D.M.; Breau, A.P. Determination of valdecoxib and its metabolites in human urine by
automated solid-phase extraction-liquid chromatography-tandem mass spectrometry, J.Chromatogr.B,
2003, 785, 123–134. [LOQ 1 ng/mL]
668
Valganciclovir
Valganciclovir
Molecular formula: C14 H22 N6 O5
Molecular weight: 354.36
CAS Registry No: 175865-60-8
Merck Index: 13, 9973
O
H
N
N
H2N
N
OH
N
O
NH2
O
CH3
O
CH3
SAMPLE
Matrix: blood
Sample preparation: Vortex 250 µL plasma with 100 µL 1 M HCl and 100 µL cold
15% trichloroacetic acid, centrifuge, inject an aliquot of the supernatant. (A columnswitching system is used so that only the fraction of interest is diverted from the initial
column on to the analytical column. Details are not provided.)
HPLC VARIABLES
Column: BDS Hypersil C18
Mobile phase: MeCN:0.0425% phosphoric acid 5:95 (diastereomers are separated)
Detector: UV 254
CHROMATOGRAM
Limit of quantitation: 40 ng/mL
KEY WORDS
pharmacokinetics; plasma
REFERENCE
Brown, F.; Banken, L.; Saywell, K.; Arum, I. Pharmacokinetics of valganciclovir and ganciclovir following
multiple oral dosages of valganciclovir in HIV- and CMV-seropositive volunteers, Clin.Pharmacokinet.,
1999, 37, 167–176.
SAMPLE
Matrix: formulations
Sample preparation: Dilute oral solution with 25 mM pH 2.5 phosphate buffer containing 5 µg/mL IS, filter (0.2 µm), inject a 10 µL aliquot.
HPLC VARIABLES
Guard column: 15 × 4.6 5 µm MetaGuard Inertsil ODS-3
Column: 100 × 4.6 5 µm Inertsil ODS-3
Mobile phase: MeCN:25 mM pH 2.5 phosphate buffer 2.5:97.5
Flow rate: 1.6
Injection volume: 10
Detector: UV 254
CHROMATOGRAM
Retention time: 5.9, 6.9 (diastereomers)
Internal standard: hypoxanthine (1.8)
OTHER SUBSTANCES
Simultaneous: ganciclovir (2.5)
KEY WORDS
oral solution; stability-indicating
Valganciclovir
669
REFERENCE
Anaizi, N.H.; Dentinger, P.J.; Swenson, C.F. Stability of valganciclovir in an extemporaneously compounded oral liquid, Am.J.Health-Syst.Pharm., 2002, 59, 1267–1270.
ANNOTATED BIBLIOGRAPHY
Henkin, C.C.; Griener, J.C.; Ten Eick, A.P. Stability of valganciclovir in extemporaneously compounded
liquid formulations, Am.J.Health-Syst.Pharm., 2003, 60, 687–690. [stability-indicating]
670
Valrubicin
Valrubicin
O
OH
O
O
OH
Molecular formula: C34 H36 F3 NO13
Molecular weight: 723.64
CAS Registry No: 56124-62-0
Merck Index: 13, 9981
OCH3 O
OH
H3C
CH3
O
O
O
HN
OH
CF3
O
SAMPLE
Matrix: tissue
Sample preparation: Homogenize (Duall ground glass grinder) 1 g of tissue with 9 mL
50 mM pH 8.5 Tris HCl buffer containing 3% sodium dodecyl sulfate, extract three
times with 2 vol of ethyl acetate:n-propanol 90:10. Evaporate the combined extracts to
dryness at 45◦ , reconstitute the residue with MeOH, inject an aliquot.
HPLC VARIABLES
Column: µBondapak phenyl
Mobile phase: Gradient. MeCN:pH 4.00 ammonium formate buffer from 32:68 to 65:35
over 7 min.
Flow rate: 5
Detector: F ex 482 em Schoeffel no. 2–73 filter
CHROMATOGRAM
Retention time: 7
OTHER SUBSTANCES
Extracted: metabolites
KEY WORDS
liver; mouse; small intestine
REFERENCE
Israel, M.; Karkowsky, A.M.; Khetarpal, V.K. Distribution of radioactivity and anthracycline-fluorescence
in tissues of mice one hour after [14 C]-labeled AD 32 administration. Evidence for tissue aglycone
formation, Cancer Chemother.Pharmacol., 1981, 6, 25–30.
Valsartan
Valsartan
H 3C
O
H3C
Molecular formula: C24 H29 N5 O3
Molecular weight: 435.52
CAS Registry No: 137862-53-4
Merck Index: 13, 9982
N
671
CH3
COOH N N
N
N
H
SAMPLE
Matrix: blood
Sample preparation: Condition a 1 mL 100 mg Bond Elut C8 SPE cartridge with
2 mL MeOH and 1 mL 100 mM pH 2 phosphate buffer. Mix 250 µL plasma with IS,
add 250 µL 1 M phosphoric acid, shake, centrifuge at 10 000 g at 4◦ for 5 min, add the
supernatant to the SPE cartridge, wash with 500 µL MeOH:100 mM pH 2 phosphate
buffer 50:50, dry at full vacuum for 20 min, elute with 500 µL MeOH. Add 100 µL
MeOH:ethylene glycol 90:10 to the eluate (to prevent adsorption of the drug), vortex,
evaporate to dryness under a stream of nitrogen at 40◦ C, reconstitute the residue with
250 µL of the initial mobile phase, inject a 20 µL aliquot.
HPLC VARIABLES
Guard column: 20 × 3.9 4 µm Novapak C18 (Waters)
Column: 300 × 3.9 10 µm µBondapak C18
Mobile phase: Gradient. MeCN:5 mM pH 4 acetate buffer from 30:70 to 60:40 over
15 min, to 95:5 over 6 min, return to initial conditions over 3 min, re-equilibrate at
initial conditions for 1 min.
Flow rate: from 1 to 1.2 over 15 min, maintain at 1.2 for 6 min, to 1 over 3 min, maintain
at 1 for 1 min
Injection volume: 20
Detector: F ex 250 em 375
CHROMATOGRAM
Retention time: 14.4
Internal standard: bumetanide (13.5)
Limit of quantitation: 50 ng/mL
OTHER SUBSTANCES
Extracted: metabolites, candesartan (22.6, LOQ 3 ng/mL), irbesartan (12.6, LOQ
50 ng/mL), losartan (11.5, LOQ 16 ng/mL)
KEY WORDS
plasma; SPE
REFERENCE
González, L.; López, J.A.; Alonso, R.M.; Jiménez, R.M. Fast screening method for the determination of
angiotensin II receptor antagonists in human plasma by high-performance liquid chromatography
with fluorimetric detection, J.Chromatogr.A, 2002, 949, 49–60.
SAMPLE
Matrix: formulations
Sample preparation: Sonicate finely ground tablets in MeOH for 5 min, centrifuge,
dilute with MeOH containing 290 ng/mL IS, inject a 20 µL aliquot.
672
Valsartan
HPLC VARIABLES
Column: 150 × 4.6 5 µm Supelcosil LC 18
Mobile phase: MeCN:20 mM pH 3.2 phosphate buffer 45:55
Flow rate: 0.9
Injection volume: 20
Detector: UV 225
CHROMATOGRAM
Retention time: 6.99
Internal standard: trimethoprim (2.96)
Limit of detection: 17 ng/mL
Limit of quantitation: 58 ng/mL
OTHER SUBSTANCES
Extracted: hydrochlorothiazide (2.20)
KEY WORDS
comparison with derivative spectrophotometry; tablets
REFERENCE
Satana, E.; Altinay, S.; Göger, N.G.; Ozkan, S.A.; Sentürk, Z. Simultaneous determination of valsartan and hydrochlorothiazide in tablets by first-derivative ultraviolet spectrophotometry and LC,
J.Pharm.Biomed.Anal., 2001, 25, 1009–1013.
ANNOTATED BIBLIOGRAPHY
Carlucci, G.; Di Carlo, V.; Mazzeo, P. Simultaneous determination of valsartan and hydrochlorothiazide
in tablets by high-performance liquid chromatography, Anal.Lett., 2000, 33, 2491–2500.
Daneshtalab, N.; Lewanczuk, R.Z.; Jamali, F. High-performance liquid chromatographic analysis of
angiotensin II receptor antagonist valsartan using a liquid extraction method, J.Chromatogr.B, 2002,
766, 345–349. [fluorescence detection; LOQ 10 ng/mL; losartan is internal standard]
Francotte, E.; Davatz, A.; Richert, P. Development and validation of chiral high-performance liquid
chromatographic methods for the quantitation of valsartan and of the tosylate of valinebenzyl ester,
J.Chromatogr.B, 1996, 686, 77–83. [LOQ 0.1%; LOD 0.04%]
González, L.; Alonso, R.M.; Jiménez, R.M. A high-performance liquid chromatographic method for screening angiotensin II receptor antagonists in human urine, Chromatographia, 2000, 52, 735–740. [SPE;
LOQ 400 ng/mL; losartan; irbesartan; valsartan; candesartan]
Sioufi, A.; Marfil, F.; Jaouen, A.; Cardot, J.M.; Godbillon, J.; Ezzet, F.; Lloyd, P. The effect of age on the
pharmacokinetics of valsartan, Biopharm.Drug Dispos., 1998, 19, 237–244. [LOQ 5 ng/mL; fluorescence detection]
Tatar, S.; Saglik, S. Comparison of UV- and second derivative-spectrophotometric and LC methods for the
determination of valsartan in pharmaceutical formulation, J.Pharm.Biomed.Anal., 2002, 30, 371–375.
[capsules; losartan is internal standard; LOD 200 ng/mL; LOQ 1 µg/mL]
Waldmeier, F.; Flesch, G.; Müller, P.; Winkler, T.; Kriemler, H.-P.; Bühlmayer, P.; de Gasparo, M. Pharmacokinetics, disposition and biotransformation of [14 C]-radiolabelled valsartan in healthy male
volunteers after a single oral dose, Xenobiotica, 1997, 27, 59–71.
Zaleplon
CN
N
Molecular formula: C17 H15 N5 O
Molecular weight: 305.33
CAS Registry No: 151319-34-5
Merck Index: 13, 10165
N
N
O
N
CH3
CH3
SAMPLE
Matrix: blood
Sample preparation: Evaporate 100 µL 1 µg/mL IS in MeOH in the bottom of a tube
using a stream of nitrogen, add 1 mL whole blood, add 1 mL buffer, vortex for 1 min,
add 5 mL dichloromethane:hexane:ethyl acetate 50:40:10, shake horizontally at 200
cycles/min for 30 min, centrifuge at 2000 rpm for 30 min. Evaporate the organic layer
to dryness under a stream of nitrogen, reconstitute the residue with 100 µL initial
mobile phase, inject an aliquot. (Prepare the buffer by adjusting the pH of 1 L saturated
ammonium chloride solution to 9.5 with 25% ammonium hydroxide.)
HPLC VARIABLES
Guard column: Inertsil ODS-3
Column: 150 × 2 3 µm Inertsil ODS-3
Mobile phase: Gradient. MeCN:1 mM pH 4.0 ammonium formate buffer 10:90 for 2 min,
to 60:40 over 15 min, maintain at 60:40 for 3 min, return to initial conditions over 1 min,
re-equilibrate for 10 min.
Flow rate: 0.2
Detector: MS, PE Sciex API 150EX, single quadrupole, turbo ionspray, atmospheric
pressure ionization, positive mode, heater gas nitrogen, ionspray 5000 V, nebulizer gas
nitrogen at 1.2 L/min, curtain gas nitrogen at 1.0 L/min, turbo probe 475◦ , orifice 35 V,
ring 175 V, m/z 306.21
CHROMATOGRAM
Retention time: 14.35
Internal standard: methaqualone (m/z 251.11, orifice 20 V, ring 150 V) (16.12)
Limit of detection: 0.1 ng/mL (S/N 5)
Limit of quantitation: 1 ng/mL
OTHER SUBSTANCES
Extracted: zolpidem (m/z 308.21, orifice 40 V, ring 175 V) (9.78)
KEY WORDS
whole blood
REFERENCE
Giroud, C.; Augsburger, M.; Menetrey, A.; Mangin, P. Determination of zaleplon and zolpidem by liquid
chromatography-turbo-ionspray mass spectrometry: application to forensic cases, J.Chromatogr.B,
2003, 789, 131–138.
SAMPLE
Matrix: blood
Sample preparation: Add IS to 200 µL plasma, add 400 µL MeCN, vortex, centrifuge,
inject an aliquot of the supernatant.
HPLC Methods for Recently Approved Pharmaceuticals. by George Lunn
Copyright 2005 John Wiley & Sons, Ltd. ISBN: 0-471-66941-5
673
674
Zaleplon
HPLC VARIABLES
Column: 150 × 4.6 5 µm CSC-S-Octyl
Mobile phase: MeCN:100 mM sodium dihydrogen phosphate:100 mM disodium hydrogen phosphate 32:32.5:32.5
Flow rate: 1
Detector: F ex 345 em 460
CHROMATOGRAM
Internal standard: CL-218872
Limit of quantitation: 0.5 ng/mL
OTHER SUBSTANCES
Extracted: deethylzaleplon
KEY WORDS
pharmacokinetics; plasma
REFERENCE
Greenblatt, D.J.; Harmatz, J.S.; von Moltke, L.L.; Ehrenberg, B.L.; Harrel, L.; Corbett, K.; Counihan, M.;
Graf, J.A.; Darwish, M.; Mertzanis, P.; Martin, P.T.; Cevallos, W.H.; Shader, R.I. Comparative kinetics
and dynamics of zaleplon, zolpidem, and placebo, Clin.Pharmacol.Ther., 1998, 64, 553–561.
SAMPLE
Matrix: microsomal incubations
Sample preparation: Add 2 vol of ice-cold MeCN to the microsomal incubation, stir
vigorously, centrifuge at 1500 g for 15 min. Evaporate 500 µL of the supernatant to
dryness under a stream of nitrogen, reconstitute the residue with 200–400 µL initial
mobile phase, inject a 50 µL aliquot.
HPLC VARIABLES
Column: 150 × 4.6 Develosil ODS-UG-5
Mobile phase: Gradient. MeCN:50 mM pH 6.8 potassium phosphate buffer from 20:80
to 60:40 over 18 min.
Flow rate: 1
Injection volume: 50
Detector: UV 245
CHROMATOGRAM
Retention time: 17.6
Internal standard: CL 218,872 (20.8)
Limit of quantitation: 50 nM
OTHER SUBSTANCES
Extracted: N-desethyl-5-oxozaleplon (3.4), N-desethylzaleplon (14.5), 5-oxozaleplon (8.5)
KEY WORDS
liver; monkey; rat
REFERENCE
Kawashima, K.; Hosoi, K.; Naruke, T.; Shiba, T.; Kitamura, M.; Watabe, T. Aldehyde oxidase-dependent
marked species difference in hepatic metabolism of the sedative-hypnotic, zaleplon, between monkeys
and rats, Drug Metab.Dispos., 1999, 27, 422–428.
Zaleplon
675
ANNOTATED BIBLIOGRAPHY
Feng, F.; Jiang, J.; Dai, H.; Wu, J. Development and validation of a high-performance liquid chromatography-electrospray ionization-mass spectrometry assay for the determination of zaleplon in human
plasma, J.Chromatogr.Sci., 2003, 41, 17–21. [LOD 100 pg/mL; triazolam is internal standard]
676
Zaltoprofen
Zaltoprofen
S
CH3
COOH
Molecular formula: C17 H14 O3 S
Molecular weight: 298.36
CAS Registry No: 74711-43-6, 89482-01-9 ((S)-form)
Merck Index: 13, 10166
O
SAMPLE
Matrix: microsomal incubations
Sample preparation: Mix microsomal incubation with MeCN containing IS, centrifuge
at 9000 g for 10 min, inject an aliquot of the supernatant
HPLC VARIABLES
Guard column: 10 × 4 Capcell Pak C18 UG120 (Shiseido)
Column: 250 × 4.6 L-Column ODS (Chemicals Evaluation and Research Institute, Tokyo)
Column temperature: 35
Mobile phase: MeCN:water:acetic acid 60:40:1
Flow rate: 0.7
Detector: UV 330
CHROMATOGRAM
Internal standard: mefenamic acid
KEY WORDS
human; liver
REFERENCE
Furuta, S.; Akagawa, N.; Kamada, E.; Hiyama, A.; Kawabata, Y.; Kowata, N.; Inaba, A.; Matthews, A.;
Hall, M.; Kurimoto, T. Involvement of CYP2C9 and UGT2B7 in the metabolism of zaltoprofen, a
nonsteroidal anti-inflammatory drug, and its lack of clinically significant CYP inhibition potential,
Br.J.Clin.Pharmacol., 2002, 54, 295–303.
SAMPLE
Matrix: microsomal incubations
Sample preparation: Mix microsomal incubation with 1 M pH 4.5 acetate buffer and
dichloromethane, add IS, centrifuge at 2000 g for 10 min. Evaporate the organic layer to
dryness under a stream of nitrogen, reconstitute the residue with mobile phase, inject
an aliquot.
HPLC VARIABLES
Guard column: 10 × 4 Capcell Pak C18 UG120 (Shiseido)
Column: 250 × 4.6 Capcell Pak C18 UG120 (Shiseido)
Column temperature: 35
Mobile phase: MeCN:buffer 30:70 (The buffer was 65 mM sodium dihydrogen phosphate
containing 5 mM tetrabutylammonium chloride, adjusted to pH 6.0 with triethylamine.)
Flow rate: 1
Detector: UV 270
CHROMATOGRAM
Retention time: 24
Internal standard: methyl paraben (8)
Zaltoprofen
677
OTHER SUBSTANCES
Extracted: metabolites
KEY WORDS
human; liver
REFERENCE
Furuta, S.; Akagawa, N.; Kamada, E.; Hiyama, A.; Kawabata, Y.; Kowata, N.; Inaba, A.; Matthews, A.;
Hall, M.; Kurimoto, T. Involvement of CYP2C9 and UGT2B7 in the metabolism of zaltoprofen, a
nonsteroidal anti-inflammatory drug, and its lack of clinically significant CYP inhibition potential,
Br.J.Clin.Pharmacol., 2002, 54, 295–303.
678
Zanamivir
Zanamivir
Molecular formula: C12 H20 N4 O7
Molecular weight: 332.31
CAS Registry No: 139110-80-8
Merck Index: 13, 10167
OH
HO
H3C
O
HO
HN
OH
NH
O
O
H2N
NH
SAMPLE
Matrix: blood
Sample preparation: Condition a 1 mL 50 mg isolute SCX SPE cartridge with 500 µL
MeOH and 500 µL 10% acetic acid water. Vortex 200 µL serum with 100 µL 5 µg/mL
IS in water, 500 µL MeCN, and 100 µL 3% acetic acid in water, let stand at room
temperature for 5 min, centrifuge at 1400 g for 10 min, add the supernatant to the SPE
cartridge, elute with two 500 µL portions of 10% triethylamine in MeOH:water 50:50.
Evaporate the eluate to dryness under a stream of nitrogen at 60◦ , reconstitute the
residue with 200 µL mobile phase, vortex for 10 s, centrifuge at 5000 g for 5 min, inject
a 20–200 µL aliquot.
HPLC VARIABLES
Column: 100 × 4.6 5 µm Hypersil SAS C1
Mobile phase: MeCN:water:acetic acid 50:50:1
Flow rate: 1
Injection volume: 20–200
Detector: MS, PE Sciex API 300 triple quadrupole, TurboIonSpray, positive ion mode,
20% of column effluent entered the detector, collision gas nitrogen, collision energy
28 eV, m/z 333–60
CHROMATOGRAM
Retention time: 4
Internal standard: 13 C,15 N2 -zanamivir (m/z 336–63)
Limit of quantitation: 10 ng/mL
KEY WORDS
serum; SPE
REFERENCE
Allen, G.D.; Brookes, S.T.; Barrow, A.; Dunn, J.A.; Grosse, C.M. Liquid chromatographic-tandem mass
spectrometric method for the determination of the neuraminidase inhibitor zanamivir (GG167) in
human serum, J.Chromatogr.B, 1999, 732, 383–393.
SAMPLE
Matrix: blood
Sample preparation: Condition a Bond Elut SCX SPE cartridge with 2 mL MeOH
and 2 mL 10% formic acid. Mix 1 mL serum with 1 mL 10% formic acid, add to the
SPE cartridge, wash with 2 mL 1% trifluoroacetic acid in MeOH, wash with 2 mL
water, elute with four 500 µL portions of 10% triethylamine in MeOH:water 50:50.
Evaporate the eluate to dryness under a stream of nitrogen at 70◦ , reconstitute the
residue with 75 µL 4 mM benzoin in ethylene glycol, add 150 µL reagent, vortex, heat
at 100◦ for 3 min, cool, inject a 100 µL aliquot. (Prepare the reagent by diluting 750 µL
β-mercaptoethanol, 2.52 g sodium sulfite, and 10 mL 5 M KOH to 100 mL with water.)
HPLC VARIABLES
Column: 100 × 4.6 Hypersil ODS
Zanamivir
679
Mobile phase: Gradient. MeCN:buffer 20:80 for 12 min, to 80:20 (step gradient), maintain at 80:20 for 5 min, re-equilibrate at initial conditions for 10 min. (Prepare the buffer
by adding 100 mL 1 M tris(hydroxymethyl)methylamine (Tris) and 29.4 mL 1 M HCl to
water and making up to 2 L with water (50 mM pH 8.5).)
Flow rate: 1
Injection volume: 100
Detector: F ex 325 em 442
CHROMATOGRAM
Retention time: 14
Limit of quantitation: 10 ng/mL
KEY WORDS
derivatization; serum; SPE
REFERENCE
Stubbs, R.J.; Harker, A.J. Automated high-performance liquid chromatographic method for the determination of a neuraminidase inhibitor, J.Chromatogr.B, 1995, 670, 279–285.
680
Zinostatin
Zinostatin
CAS Registry No: 9014-02-2, 123760-07-6
Merck Index: 13, 10222
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: TSK G-3000SW
Mobile phase: 10 mM pH 7.9 ammonium bicarbonate containing 30 mM NaCl
Flow rate: 1
Detector: UV 254, UV 280
REFERENCE
Maeda, H.; Ueda, M.; Morinaga, T.; Matsumoto, T. Conjugation of poly(styrene-co-maleic acid) derivatives to the antitumor protein neocarzinostatin: pronounced improvements in pharmacological properties, J.Med.Chem., 1985, 28, 455–461.
Zofenopril calcium
Zofenopril calcium
O
H
O
H
S
Molecular formula: C44 H44 CaN2 O8 S4
Molecular weight: 897.17
CAS Registry No: 81938-43-4, 81872-10-8
(free acid)
681
Ca++
S
N
CH3
H
COO−
2
SAMPLE
Matrix: blood
Sample preparation: Collect 10 mL whole blood in a heparinized tube containing
20 mg N-ethylmaleimide, centrifuge. Shake 1 mL plasma with 20 µL MeOH containing
10 µg/mL IS1 and 15 µg/mL IS2, 1 mL 2 M phosphoric acid containing 2% tetrabutylammonium hydrogen sulfate, and 7 mL toluene in a PTFE-lined tube on a rotating
mixer at 32 rpm for 15 min, centrifuge at 1500 g for 5 min. Evaporate the organic layer
to dryness under a stream of nitrogen at 60◦ , reconstitute the residue with 200 µL
MeOH:water 50:50, inject an aliquot.
HPLC VARIABLES
Guard column: 10 × 3.2 5 µm C18 (Hichrom)
Column: 75 × 4.6 3 µm Luna C18
Mobile phase: Gradient. MeCN:26 mM pH 4.5 ammonium acetate buffer 20:80 for
0.5 min, to 80:20 over 2.5 min, maintain at 80:20 for 3 min, re-equilibrate at initial
conditions for 3 min.
Flow rate: 0.4
Detector: MS, PE Sciex API 365 triple quadrupole, TurboIonSpray, probe 450◦ , negative
ion mode, orifice – 35 V, ring – 200 V, nebulizer gas nitrogen at 10 units, curtain gas
nitrogen at 12 units, auxiliary gas air, collision gas nitrogen at 2 units, m/z 428–137
CHROMATOGRAM
Retention time: 6
Internal standard: IS1 (N-[3-mercapto-2-methylpropionyl]-4-(4-fluorophenylthio)-Lproline benzoate) (m/z 446–137) (6), IS2 (m/z 467–308) (Prepare a solution of IS2 by
reacting 7 mg N-[3-mercapto-2-methylpropionyl]-4-(4-fluorophenylthio)-L-proline with
1.75 mL of a 25 mg/mL solution of N-ethylmaleimide in 60 mM pH 7 buffer in the dark
at room temperature for 1 h, make up to 10 mL with acetone.) (5.3)
Limit of detection: 50 pg/mL (S/N 3)
Limit of quantitation: 1 ng/mL
OTHER SUBSTANCES
Extracted: zofenoprilat (N-[3-mercapto-2-methylpropionyl]-4-(phenylthio)-L-proline)
(m/z 449–290 (as derivative)) (LOQ 2 ng/mL, LOD 100 pg/mL) (5.3)
KEY WORDS
derivatization; pharmacokinetics; plasma; whole blood
REFERENCE
Dal Bo, L.; Mazzucchelli, P.; Marzo, A. Assay of zofenopril and its active metabolite zofenoprilat by liquid
chromatography coupled with tandem mass spectrometry, J.Chromatogr.B, 2000, 749, 287–294.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 × 4.6 5 µm Econosil
682
Zofenopril calcium
Mobile phase: MeCN:water:triethylamine:phosphoric acid 65:35:0.02:0.13, pH 2.4
Flow rate: 1
Detector: UV 220
CHROMATOGRAM
Internal standard: cephalothin
OTHER SUBSTANCES
Also analyzed: captopril, enalaprilat, fosinopril, quinapril, quinaprilat, ramipril
REFERENCE
Lin, C.J.; Akarawut, W.; Smith, D.E. Competitive inhibition of glycylsarcosine transport by enalapril
in rabbit renal brush border membrane vesicles: interaction of ACE inhibitors with high-affinity
H+ /peptide symporter, Pharm.Res., 1999, 16, 609–615.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 150 × 3.9 µBondapak phenyl
Column temperature: 30–40
Mobile phase: MeOH:water:85% phosphoric acid 68:32:0.2
Detector: UV 215–220
OTHER SUBSTANCES
Simultaneous: zofenoprilat
REFERENCE
Ranadive, S.A.; Chen, A.X.; Serajuddin, A.T. Relative lipophilicities and structural-pharmacological
considerations of various angiotensin-converting enzyme (ACE) inhibitors, Pharm.Res., 1992, 9,
1480–1486.
ANNOTATED BIBLIOGRAPHY
Marzo, A.; Dal Bo, L.; Mazzucchelli, P.; Monti, N.C.; Crivelli, F.; Ismaili, S.; Giusti, A.; Uhr, M.R. Pharmacokinetic and pharmacodynamic comparative study of zofenopril and enalapril in healthy volunteers,
Arzneimittelforschung, 2002, 52, 233–242. [LC-MS; LOQ 5 ng/mL]
683
Zolazepam hydrochloride
Zolazepam hydrochloride
Molecular formula: C15 H15 FN4 O.HCl
Molecular weight: 322.77
CAS Registry No: 33754-49-3, 31352-82-6 (free base)
H3C
H3C
O
N
N
N
HCl
N
H3C
F
SAMPLE
Matrix: blood, tissue
Sample preparation: Add IS to serum or tissue, make alkaline with pH 9.5 borate
buffer, extract with ethyl acetate. Extract the organic layer with 1 mL 100 mM HCl.
Basify the aqueous layer with 100 mg sodium borate, extract with ethyl acetate.
Evaporate the organic layer to dryness under a stream of nitrogen, reconstitute
the residue with MeCN:water 25:75 (serum) or mobile phase (tissue), inject a 70 µL
aliquot.
HPLC VARIABLES
Column: 250 × 4 5 µm LiChrospher 60 RP-select B C8
Mobile phase: MeCN:50 mM pH 6.8 phosphate buffer 26:74
Flow rate: 1
Injection volume: 70
Detector: UV 233
CHROMATOGRAM
Retention time: 12.5
Internal standard: pindolol (9.7)
Limit of quantitation: 2 ng/mL
OTHER SUBSTANCES
Extracted: tiletamine (LOQ 10 ng/mL) (18.9)
KEY WORDS
bear; muscle; serum
REFERENCE
Semple, H.A.; Gorecki, D.K.J.; Farley, S.D.; Ramsay, M.A. Pharmacokinetics and tissue residues of
Telazol in free-ranging polar bears, J.Wildlife Dis., 2000, 36, 653–662.
SAMPLE
Matrix: blood, urine
Sample preparation: Mix 1 mL pH 7 buffer with 5 mL whole blood or urine, add 1 µg
IS1, add 6 µg IS2, add 7 mL hexane:toluene:isoamyl alcohol 90:5:5, vortex, centrifuge,
remove the organic layer. Add 1 mL pH 9.5 buffer to the aqueous layer, extract with 6 mL
hexane:isoamyl alcohol 99:1. Combine the organic layers, evaporate to dryness under
a stream of nitrogen, reconstitute the residue with 250 µL MeCN:water:trifluoroacetic
acid 45:55:0.05, evaporate to half volume to remove MeCN, inject a 50 µL aliquot.
HPLC VARIABLES
Guard column: 5 µm Altima C18
Column: 20 × 2.1 5 µm Altima C18
Column temperature: 40
684
Zolazepam hydrochloride
Mobile phase: Gradient. A was MeCN:water:trifluoroacetic acid 10:90:0.05. B was
MeCN:water:trifluoroacetic acid 80:20:0.05. A:B 100:0 for 5 min, to 0:100 over 50 min,
re-equilibrate at initial conditions for 15 min.
Flow rate: 0.25
Injection volume: 50
Detector: MS, HP 5989B, electron impact; UV 205; UV 290
CHROMATOGRAM
Internal standard: SKF-525 A (IS1), 5-ethyl-5-p-tolylbarbituric acid (IS2)
OTHER SUBSTANCES
Extracted: tiletamine
KEY WORDS
whole blood
REFERENCE
Cording, C.J.; Deluca, R.; Camporese, T.; Spratt, E. A fatality related to the veterinary anesthetic Telazol,
J.Anal.Toxicol., 1999, 23, 552–555.
SAMPLE
Matrix: tissue
Sample preparation: Homogenize 1.5 g tissue with 300 µL 1 µg/mL IS in water and
6 mL 500 mM pH 9.5 sodium borate buffer, add 10 mL ethyl acetate, vortex for 20 min,
repeat the extraction. Combine the organic layers and extract twice with 2 mL portions
of 100 mM HCl. Combine the aqueous layers and make basic with 200 mg sodium
borate, extract with 12 mL ethyl acetate. Evaporate the organic layer to dryness under
a stream of nitrogen, reconstitute the residue with 200 µL MeCN:water 20:80, inject a
70 µL aliquot.
HPLC VARIABLES
Column: 250 × 4 5 µm LiChrospher 60 RP-select B C8
Mobile phase: MeCN:50 mM pH 5.5 sodium phosphate buffer 16:84
Flow rate: 1
Injection volume: 70
Detector: UV 233
CHROMATOGRAM
Retention time: 58
Internal standard: ripazapam (51.5)
OTHER SUBSTANCES
Extracted: tiletamine (45), metabolites
KEY WORDS
bear; fat; kidney; muscle
REFERENCE
Semple, H.A.; Gorecki, D.K.J.; Farley, S.D.; Ramsay, M.A. Pharmacokinetics and tissue residues of
Telazol in free-ranging polar bears, J.Wildlife Dis., 2000, 36, 653–662.
CUMULATIVE INDEX
This index provides a comprehensive listing of the drugs for which analytical methods have been
described in this volume and in earlier volumes in the series (available from John Wiley & Sons, Inc.).
These volumes are designated as follows:
I
II
III
IV
V
CE
HPLC Methods for Pharmaceutical Analysis
HPLC Methods for Pharmaceutical Analysis, Volume 2
HPLC Methods for Pharmaceutical Analysis, Volume 3
HPLC Methods for Pharmaceutical Analysis, Volume 4
HPLC Methods for Recently Approved Pharmaceuticals (this volume)
Capillary Electrophoresis Methods for Pharmaceutical Analysis
(For obvious reasons non-interfering compounds are not listed in this index.)
Abacavir, V 1
Acamprosate, II 1
Acarbose, CE 1, V 5
Acebutolol, CE 1, II 2
Aceclofenac, II 17
Acefylline, II 19
Acenocoumarol, CE 9, II 20
Acepromazine, CE 11, II 22
Aceprometazine, II 29
Acesulfame, II 31
Acetaminophen, CE 12, I 1, II 32
Acetazolamide, CE 27, II 37
Acetiamine, II 46
Acetohexamide, CE 28, II 47
Acetophenazine, II 49
Acetorphan, II 54
Acetyl sulfisoxazole, V 6
Acetylcholine, II 56
Acetylcysteine, CE 30, II 66
Acipimox, II 84
Acitretin, II 85
Aconitine, II 93
Acrivastine, II 95, V 7
Actinoquinol, II 96
Acyclovir, CE 33, I 27, II 97
Adapalene, V 10
Adefovir, II 101
Adefovir dipivoxil, V 11
Adenosine triphosphate, CE 39, II 111
Adenosine, CE 35, II 103
Adiphenine, I 33, II 118
Adrafinil, II 119
Adrenocorticotropic Hormone, V 13
Afloqualone, V 15
Ajmaline, II 120
Alacepril, V 16
Albendazole, II 122
Albuterol, CE 41, I 35, II 129
Alclometasone 17,21-Dipropionate, V 18
Aldesleukin, CE 51, II 131
Alendronate sodium, II 132
Alfentanil, II 136
Alfuzosin, II 138
Alitretinoin, V 21
Allantoin, CE 52, II 141
Allethrin, V 24
Allobarbital, CE 52
Allopurinol, CE 53, II 145
Alminoprofen, II 149
Almitrine, II 152
Almotriptan, V 27
Alosetron, CE 54, V 29
Alpidem, II 153
Alpiropride, II 156
Alprazolam, CE 55, I 48, II 158
Alprenolol, CE 57, II 160
Alprostadil, CE 62, II 166
Alteplase, I 56
HPLC Methods for Recently Approved Pharmaceuticals. by George Lunn
Copyright 2005 John Wiley & Sons, Ltd. ISBN: 0-471-66941-5
685
686
Cumulative Index
Altretamine, II 181
Amantadine, CE 62, II 183
Ambenonium chloride, II 187
Amcinonide, V 30
Amdinocillin, II 189
Amfenac, II 190
Amidox, II 191
Amifostine, II 193
Amikacin, CE 64, II 195
Amiloride, CE 67, II 198
Amineptine, II 205
Amino acids, CE 68, II 206
Aminobenzoic acid, CE 88, II 306
Aminocaproic acid, II 315
Aminoglutethimide, CE 91, II 318
Aminohippuric acid, CE 93, II 321
Aminolevulinic acid, V 33
Aminophylline, II 328
Aminopyrine, CE 94
Amiodarone, CE 95, II 331
Amiprilose, II 340
Amisulpride, II 341
Amitraz, II 348
Amitriptyline, CE 97, I 58, II 349
Amlexanox, II 355
Amlodipine, CE 101, I 87, II 356
Amobarbital, CE 103, II 359
Amodiaquin, II 368
Amorolfine, CE 107
Amosulalol, II 370
Amoxapine, II 372
Amoxicillin, CE 109, I 91, II 382
Amphetamine, CE 112, II 388
Amphotericin, II 424
Ampicillin, CE 124, I 107, II 434
Amprenavir, V 36
Amprolium, II 441
Amrinone, II 446
Amsacrine, II 449
Amylase, II 451
Amylocaine, II 452
Anagrelide, V 42
Anakinra, V 43
Angiotensin II, CE 127, II 454
Aniracetam, II 465
Anistreplase, II 467
Antazoline, II 468
Anthralin, II 470
Antipyrine, CE 130, II 472
Antitrypsin, II 487
Apigenin, II 489
Apomorphine, II 490
Apraclonidine, V 45
Apramycin, II 491
Aprepitant, V 46
Aprindine, II 493
Aprobarbital, CE 133, II 494
Aprotinin, CE 135, II 498
Aranidipine, V 48
Arbaprostil, II 500
Arbekacin, CE 136
Arbidol, II 502
Arecoline, II 503
Argatroban, II 504
Arotinolol, CE 137, V 49
Arteether, V 52
Artemisinin, II 505
Artesunate, CE 138
Articaine, V 54
Ascorbic acid, CE 139, II 509
Asparaginase, V 57
Aspartame, CE 147, II 526
Aspirin, CE 149, I 122, II 529
Aspoxicillin, CE 154, II 532
Astemizole, I 137, II 534
Atazanavir sulfate, V 58
Atenolol, CE 155, I 140, II 535
Atipamezole, V 60
Atomoxetine hydrochloride, V 62
Atorvastatin, V 64
Atosiban, CE 170, V 66
Atovaquone, II 543
Atracurium besylate, II 545
Atropine, CE 171, I 164, II 547
Auranofin, II 553
Avermectin, II 555
Avitriptan, II 557
Azacyclonol, II 558
Azaperone, CE 176, II 560
Azasetron, II 563
Azatadine, II 564
Azathioprine, CE 177, I 177, II 566
Azelaic acid, II 568
Azelastine, CE 177, II 572
Azithromycin, I 181, II 575
Azlocillin, II 578
Azosemide, II 580
Aztreonam, II 582
Bacampicillin, II 586
Bacitracin, CE 180, II 588
Baclofen, CE 180, II 590
Balofloxacin, V 67
Bambermycins, V 69
Bambuterol, CE 183, II 596
Bamethan, CE 185, II 598
Bamifylline, II 600
Barbital, CE 187, II 601
Barnidipine, II 604
Beclobrate, II 607
Beclomethasone, CE 190, II 609
Beclomethasone dipropionate, I 184
Befunolol, II 614, V 70
Benactyzine, II 615
Benazepril, I 188, II 617
Bendroflumethiazide, CE 191, II 619
Benfluorex, II 627
Benidipine, II 628
Benoxaprofen, II 630
Benperidol, II 632
Cumulative Index
Benproperine, CE 194
Benserazide, CE 196
Benzalkonium chloride, CE 197, II 635, V 71
Benzbromarone, II 640
Benzethidine, II 641
Benzethonium chloride, II 643
Benzocaine, CE 199, II 645
Benzoctamine, II 655
Benzoic acid, CE 201, II 657
Benzonatate, II 676
Benzoyl peroxide, II 677
Benzphetamine, CE 208, II 679
Benzquinamide, CE 208, II 682
Benzthiazide, CE 209, II 684
Benztropine mesylate, II 688
Benzydamine, II 692
Benzyl alcohol, II 693
Benzyl benzoate, II 694
Bepridil, II 698
Berberine, II 702
Besipirdine, II 704
Beta-carotene, II 705
Betahistine, II 717
Betaine, II 718, V 72
Betamethasone, CE 210, I 191, II 720
Betaxolol, CE 212, II 723
Bethanechol chloride, V 74
Betotastine, II 729
Bevantolol, II 730
Bexarotene, V 75
Bezafibrate, II 732
BHT, CE 213
Biapenem, V 77
Bicalutamide, II 733
Bidisomide, II 734
Bifonazole, CE 214, II 735
Bimatoprost, V 79
Bioresmethrin, II 737, V 80
Biotin, CE 214, II 738
Biperiden, CE 217, II 744
Bisacodyl, II 746
Bisantrene, II 748
Bisoprolol, CE 219, II 752
Bitolterol, II 757
Bivalirudin, V 81
Bleomycin sulfate, II 759
Boldenone, II 763, V 82
Boldine, II 767
Bopindolol, II 768
Bosentan, V 83
β-Boswellic acid, V 86
Bretylium tosylate, CE 221, II 771
Brimonidine, V 88
Brodifacoum, II 772
Brodimoprim, CE 222, II 773
Bromadiolone, II 775
Bromazepam, CE 223, II 776
Bromfenac, V 90
Bromhexine, II 781
Bromocriptine, I 201, II 783
Bromodiphenhydramine, II 784
Bromperidol, II 786
Brompheniramine, CE 226, II 788
Brotizolam, CE 230, II 798
Brovincamine, V 92
Bucillamine, II 801, V 93
Bucladesine, II 802
Buclizine, CE 230, II 803
Bucumolol, II 807
Budesonide, II 809
Budipine, V 94
Buflomedil, II 816
Bufotenine, II 817
Bufuralol, II 819
Bulaquine, V 95
Bumadizon, II 820
Bumetanide, CE 231, II 822
Bunazosin, II 832
Bupivacaine, CE 233, II 833
Bupranolol, CE 244, II 851
Buprenorphine, II 854
Bupropion, CE 246, II 865
Buserelin, CE 247, II 868
Buspirone, CE 247, I 204, II 871
Busulfan, II 873
Butabarbital, CE 248, II 877
Butacaine, II 884, V 97
Butalbital, CE 250, I 211
Butamben, II 887, V 99
Butamirate, CE 253
Butethal, II 888
Butethamate, CE 254, II 890
Buthionine sulfoximine, CE 256, II 892
Butibufen, II 894
Butoconazole, V 100
Butorphanol, II 895
Butyl flufenamate, V 101
Butylscopolammonium bromide, CE 257
Cabergoline, II 897
Cadralazine, II 899
Caffeine, CE 257, II 901
Calcifediol, II 943
Calcipotriene, II 946
Calcitonin, CE 274, II 948
Calcitriol, II 952
Cambendazole, V 102
Camostat, II 955
Camphor, II 956
Camptothecin, II 958
Canadine, CE 274
Candesartan cilexetil, V 104
Candoxatril, II 960
Capecitabine, V 106
Capreomycin, CE 275
Capsaicin, II 962
Captan, CE 276, II 964
Captodiamine, II 966
Captopril, CE 276, I 215, II 967
Carazolol, CE 278, II 976
687
688
Cumulative Index
Carbadox, II 977
Carbamazepine, CE 280, I 224, II 984
Carbaryl, CE 284, II 991
Carbenicillin, CE 285, II 1006
Carbetapentane, II 1010
Carbidopa, CE 286, II 1011
Carbimazole, II 1016
Carbinoxamine, CE 286, II 1017
Carbisocaine, II 1021
Carboplatin, CE 288, I 252, II 1022
Carbromal, II 1025
Carbutamide, II 1027
Carbuterol, CE 288
Carisoprodol, I 255
Carmustine, II 1028
Carnitine, CE 290, II 1031
Carpipramine, II 1042
Carprofen, CE 291, II 1044
Carteolol, CE 295, II 1046
Carumonam, II 1053
Carvedilol, CE 298, II 1055
Casanthranol, V 108
Caspofungin, V 109
Castor oil, V 112
Cefaclor, I 257, II 1061
Cefadroxil, I 265, II 1062
Cefamandole, CE 302, II 1065
Cefatrizine, II 1069
Cefazolin, II 1070
Cefbuperazone, II 1081, V 113
Cefdinir, II 1082
Cefditoren, V 114
Cefepime, II 1084
Cefetamet, II 1089
Cefixime, CE 303, I 274, II 1090
Cefmenoxime, CE 304, II 1091
Cefmetazole, II 1095
Cefminox, CE 304, II 1098
Cefoaxime, I 278
Cefodizime, CE 305, II 1099
Cefonicid, II 1102
Cefoperazone, CE 305, II 1104
Ceforanide, II 1115
Cefoselis, V 116
Cefotaxime, CE 306, II 1116
Cefotetan, II 1118
Cefotiam, II 1121
Cefoxitin, II 1125
Cefozopran, V 117
Cefpimizole, CE 308, II 1133
Cefpiramide, CE 309, II 1135
Cefpirome, CE 310, II 1139
Cefpodoxime proxetil, II 1142
Cefprozil, I 289, II 1146
Ceftazidime, CE 311, I 291, II 1147
Cefteram, II 1151
Ceftibuten, II 1153
Ceftiofur, CE 313, II 1160
Ceftizoxime, CE 313, II 1165
Ceftriaxone, CE 314, I 303, II 1170
Cefuroxime, CE 315, I 311, II 1173
Cefuzonam, V 118
Celecoxib, V 119
Celiprolol, CE 317, II 1176
Centchroman, II 1183
Cephalexin, CE 320, I 317, II 1185
Cephaloridine, II 1189
Cephalosporin C, II 1193
Cephalothin, CE 321, II 1194
Cephapirin sodium, CE 322, II 1200
Cephradine, CE 323, II 1208
Cerivastatin, V 123
Ceronapril, CE 324
Cetirizine, II 1214
Cetrorelix, V 125
Cetyl alcohol, V 128
Cevimeline hydrochloride, V 130
Chelidonine, II 1219
Chenodiol, CE 325, II 1221
Chlophedianol, CE 326
Chloral hydrate, II 1237
Chlorambucil, CE 327, II 1239
Chloramphenicol, CE 328, II 1244
Chlorcyclizine, CE 329, II 1257
Chlordiazepoxide, CE 331, II 1261
Chlorguanide, II 1276
Chlorhexidine, II 1278
Chlormadinone, II 1283
Chlormezanone, CE, II 1284
Chlorobutanol, V 132
Chloroprocaine, II 1288, V 133
Chloropyramine, II 1290
Chloroquine, CE 333, II 1291
Chlorothiazide, CE 338, II 1303
Chloroxine, II 1310
Chloroxylenol, II 1311
Chlorphenesin, II 1314
Chlorpheniramine, CE 340, II 1317
Chlorphenoxamine, CE 352, II 1334
Chlorpromazine, CE 353, II 1336
Chlorpropamide, CE 355, II 1356
Chlorprothixene, CE 357, II 1364
Chlorpyrifos, CE 358, II 1370
Chlortetracycline, CE 358, II 1375
Chlorthalidone, CE 362, II 1392
Chlorzoxazone, II 1402
Cholic acid, CE 365, II 1408
Choline, CE 366, II 1411
Chorionic gonadotropin, V 134
Chymopapain, II 1423
Chymotrypsin, CE 368, II 1424
Cicletanine, CE 368, II 1425
Ciclopirox, II 1429
Cicloprofen, CE 371
Cicloprolol, II 1431
Ciclotropium bromide, II 1432
Cidofovir, II 1434
Cifenline, II 1436
Cilastatin, I 330
Cilazapril, II 1441
Cumulative Index
Cilnidipine, V 135
Cilostazol, II 1443
Cimetidine, CE 372, I 334, II 1445
Cimetropium bromide, V 136
Cinchonidine, II 1449
Cinchonine, II 1452
Cinnarizine, II 1454
Cinolazepam, II 1457
Cinoxacin, II 1458
Ciprofibrate, II 1460
Ciprofloxacin, CE 375, I 347, II 1463
Ciprostene, II 1474
Cirazoline, II 1475
Cisapride, CE 376, I 364, II 1476
Cisatracurium besylate, V 137
Cisplatin, CE 376, II 1478
Citalopram, II 1487
Citric acid, V 139
Cladribine, II 1492
Clarithromycin, CE 377, I 368, II 1495
Clavulanic acid, CE 378, I 371, II 1497
Clemastine, II 1498
Clenbuterol, CE 378, II 1501
Clentiazem, CE 383
Clidinium bromide, CE 384, II 1508
Clinafloxacin, II 1510
Clindamycin, CE 388, I 379, II 1512
Clioquinol, II 1514, V 142
Clobazam, CE 388, II 1516
Clobenzorex, II 1519
Clobetasol, CE 390
Clobetasol 17-propionate, V 143
Clobetasone, CE 391
Clobutinol, CE 391
Clocinizine, II 1521
Clodronic acid, II 1522
Clofazimine, II 1525
Clofibrate, II 1527
Clofibric acid, II 1529
Clomethiazole, II 1532
Clomiphene, CE 393, II 1533
Clomipramine, CE 394, II 1536
Clonazepam, CE 395, I 383, II 1556
Clonidine, II 1559
Clonixin, II 1568
Clopenthixol, CE 399
Clopidogrel, V 147
Clopidol, V 149
Clorazepate, CE 399, II 1570
Cloricromen, V 151
Clorprenaline, CE 400
Clorsulon, V 152
Closantel, II 1577
Clothiapine, II 1578
Clotiazepam, CE 401, II 1579
Clotrimazole, I 395
Cloxacillin, CE 402, II 1580
Cloxazolam, CE 404
Cloxyquin, II 1596
Clozapine, CE 406, I 400, II 1597
Cocaine, CE 407, II 1602
Codeine, CE 413, I 412, II 1628
Colchicine, II 1633
Colistin, II 1640, V 153
Copovithane, II 1642
Corticosterone, CE 424, II 1643
Corticotropin, CE 426, II 1644
Cortisone, CE 427, II 1646
Cortivazol, II 1665
Cotinine, II 1666
Coumachlor, CE 433
Coumaphos, CE 434, II 1668
Coumarin, II 1671
Cromolyn, I 436, II 1673
Crotamiton, II 1675
Cyamemazine, II 1677
Cyclamic acid, II 1679
Cyclandelate, II 1680
Cyclazocine, II 1682
Cyclizine, CE 435, II 1685
Cyclobarbital, CE 436
Cyclobenzaprine, I 439, II 1691
Cyclodrine, CE 437
Cycloguanil, II 1692
Cyclopentamine, II 1694
Cyclopentolate, CE 438, II 1696
Cyclophosphamide, II 1697
Cycloserine, II 1702
Cyclosporine, I 446, II 1704
Cyclothiazide, II 1708
Cyfluthrin, II 1712
Cyheptamide, II 1714
Cymarin, II 1716
Cypermethrin, II 1718, V 155
Cyproheptadine, II 1723
Cyromazine, II 1729
Cysteamine, II 1731
Cytarabine, CE 439, II 1736
Dacarbazine, II 1741
Dactinomycin, II 1746
Dalfopristin, V 156
Dalteparin, V 158
Danazol, II 1747
Danofloxacin, II 1752
Danthron, II 1754
Dantrolene, II 1756
Dapsone, CE 440, II 1759
Daptomycin, V 159
Daunorubicin, CE 441, II 1768
Debrisoquin, CE 442, II 1776
Decoquinate, II 1780
Deferiprone, V 161
Deferoxamine, II 1781
Defibrotide, II 1786
Deflazacort, II 1787, V 162
Dehydrocholic acid, II 1788
Delapril, II 1790
Delavirdine, II 1791
Demecarium bromide, II 1793
689
690
Cumulative Index
Demeclocycline, CE 443, II 1794
Demexiptiline, II 1802
Demoxepam, II 1804
Denopamine, CE 445, II 1805
Deoxycholic acid, CE 447, II 1807
Deserpidine, CE 448, II 1821
Desipramine, CE 448, II 1823
Desloratadine, V 164
Deslorelin, II 1860
Desmopressin, II 1861
Desogestrel, I 457, V 166
Desonide, II 1862
Desoximetasone, V 167
Desoxycorticosterone, V 169
Detomidine, II 1863
Dexamethasone, CE 451, II 1865
Dexpanthenol, II 1887
Dexrazoxane, V 172
Dextran, II 1889, V 174
Dextromethorphan, CE 456, II 1890
Dextromoramide, II 1907
Dextrose, CE 457, II 1910
Dextrothyroxine, II 1942
Dezocine, II 1944
Diacerein, V 176
Diamorphine, CE 469, II 1945
Diatrizoate sodium, II 1951
Diaveridine, II 1953
Diazepam, CE 471, I 458, II 1954
Diazinon, II 1960
Diazoxide, II 1965
Dibekacin, II 1968
Dibucaine, CE 478, II 1970
Dichloroacetic acid, II 1973, V 177
Dichlorophen, V 178
Dichlorphenamide, CE 478, II 1974
Dichlorvos, CE 479, II 1976
Diclazuril, V 179
Diclofenac sodium, CE 480, I 486, II 1979
Dicloxacillin, CE 481, II 1993
Dicumarol, II 2006
Dicyclomine, I 500
Didanosine, CE 483, II 2007
Didox, II 2016
Dienestrol, II 2017
Diethylcarbamazine, II 2019
Diethylpropion, II 2021
Diethylstilbestrol, II 2026
Difemerine, II 2033
Difenoxin, II 2034
Diflorasone, II 2035
Difloxacin, II 2036
Diflunisal, CE 483, II 2038
Digitoxin, CE 484, II 2045
Digoxin, CE 485, I 503, II 2052
Dihydralazine, II 2055
Dihydroartemisinin, II 2057
Dihydrocodeine, CE 486, II 2058
Dihydroergotamine, II 2062
Dihydrostreptomycin, CE 488, II 2066
Dihydrotachysterol, V 181
Dihydroxyacetone, II 2071
Diitiazem, I 517
Dilevalol, CE 489, II 2073
Diltiazem, CE 490, II 2075
Dimenhydrinate, CE 495, II 2079
Dimethindene, CE 496, II 2080
Dimethyl sulfoxide, V 183
Dimethylaminobenzoic acid, II 2085
Dimetridazole, II 2087
Dinitolmide, V 185
Dinoprost, CE 500, II 2088
Dinoprostone, CE 501, II 2110
Diphenadione, II 2140
Diphenhydramine, CE 502, II 2141
Diphenidol, II 2159
Diphenoxylate, CE 503, II 2161
Dipipanone, II 2165
Dipivefrin, CE 504, V 186
Diprenorphine, II 2167
Dipyridamole, II 2170
Dipyrone, CE 506, II 2178
Dirithromycin, II 2184
Disopyramide, CE 507, II 2186
Disulfiram, II 2194
Dithiazanine iodide, V 187
Diuron, II 2198
Dobutamine, CE 513, II 2199
Docarpamine, V 188
Docetaxel, CE 516, II 2203
Docusate sodium, II 2205
Dofetilide, V 189
Dolasetron, V 191
Domperidone, II 2206
Donepezil, V 193
Dopamine, CE 517, II 2207
Dopexamine, II 2235
Doramectin, II 2236
Dorzolamide hydrochloride, II 2238
Dothiepin, II 2242
Doxacurium chloride, II 2248
Doxapram, CE 523, II 2249
Doxazosin, I 531, II 2254
Doxefazepam, V 195
Doxepin, CE 524, II 2255
Doxercalciferol, V 196
Doxifluridine, II 2276
Doxofylline, II 2277
Doxorubicin, CE 526, II 2279
Doxycycline, CE 527, I 534, II 2291
Doxylamine, CE 531, II 2295
Drofenine, II 2302
Droloxifene, II 2303
Dronabinol, II 2305
Droperidol, II 2312
Dropropizine, II 2320, V 198
Drospirenone, V 199
Droxicam, V 200
Droxidopa, V 201
Duloxetine, II 2321
Cumulative Index
Dyclonine, II 2323
Dyphylline, CE 537, II 2324
Ebastine, III 1
Ebiratide, III 2
Ebrotidine, V 202
Econazole, CE 537, III 4
Edaravone, V 204
Edrophonium chloride, III 6
EDTA, CE 538, III 7, V 206
Efavirenz, V 208
Eflornithine, CE 540, III 10
Efonidipine, III 12
Efrotomycin, V 212
Egualen, V 213
Eletriptan, V 214
Eliprodil, III 13
Emedastine, III 14
Emetine, III 14
Emtricitabine, V 215
Enalapril, CE 541, I 543, III 15
Enalaprilat, CE 542, III 17
Encainide, III 19
Enilconazole, III 23
Enocitabine, III 24
Enoxacin, CE 542, III 24
Enoxaparin sodium, V 217
Enoximone, III 33
Enprostil, III 35
Enrofloxacin, III 36
Entacapone, V 218
Eperisone, CE 543, V 220
Ephedrine, CE 544, III 42
Epicillin, III 57
Epidermal growth factor, III 57
Epinastine, CE 560, III 60
Epinephrine, CE 562, III 61
Epinine, III 83
Epirizole, III 84
Epirubicin, CE 572, III 84
Epithiazide, CE 572
Eplerenone, V 222
Epoetin, CE 573, I 546, III 90
Epoprostenol, CE 574, III 90, V 224
Eprinomectin, III 91
Eprosartan, V 225
Eptastigmine, III 92
Eptazocine, V 227
Eptifibatide, V 228
Equilenin, CE 575
Equilin, III 93
Erdosteine, V 229
Ergocornine, III 94
Ergocristine, III 95
Ergocristinine, III 96
Ergocryptine, III 97
Ergoloid mesylates, III 98
Ergonovine, CE 576, III 99
Ergosine, III 104
Ergotamine, CE 576, III 105, V 230
691
Ertapenem, V 234
Erythrityl tetranitrate, III 108
Erythromycin, CE 578, I 549, III 109
Esculin, III 111
Esmolol, III 112
Estazolam, CE 580, III 114
Estradiol, CE 583, I 557, III 118
Estramustine, III 121
Estriol, III 123
Estrogens, conjugated, CE 586, I 569, III 124
Estrone, CE 587, III 128
Ethacrynic acid, CE 589, III 135
Ethambutol, III 141
Ethaverine, III 144
Ethchlorvynol, III 146
Ethenzamide, CE 590
Ethinyl estradiol, CE 591, I 579, III 147
Ethionamide, III 150
Ethoheptazine, CE 592
Ethopabate, III 151, V 236
Ethopropazine, CE 593, III 152
Ethosuximide, III 155, CE 594
Ethotoin, III 164
Ethyl icosapentate, V 237
Ethylmorphine, III 166
Ethynodiol diacetate, I 588
Ethynodiol, CE 596, III 166
Etidocaine, III 167
Etidronic acid, III 169
Etilefrin, CE 597
Etizolam, CE 599, III 170
Etodolac, CE 601, I 590, III 172
Etonitazene, III 173
Etonogestrel, V 238
Etoposide, I 597, III 174
Etoricoxib, V 240
Etorphine, III 177, V 242
Etoxeridine, III 179
Etretinate, III 181
Eugenol, III 184
Exemestane, V 243
Exifone, III 185
Ezetimibe, V 245
Factor VIII, III 186
Fadrozole, V 247
Falecalcitriol, V 248
Famciclovir, III 187
Famotidine, CE 603, I 605, III 189
Febantel, III 190
Felbamate, CE 603, III 191
Felbinac, III 196
Felodipine, III 199
Felypressin, III 203
Fenbendazole, III 204
Fenbufen, III 212
Fenbutrazate, III 216
Fencamfamine, III 217
Fenchone, III 219
Fendiline, CE 604
692
Cumulative Index
Fenethazine, III 219
Fenfluramine, CE 606, III 220
Fenofibrate, III 228
Fenoldopam, III 229
Fenoprofen, CE 608, III 230
Fenoterol, CE 614, III 242
Fenoverine, III 243
Fenoxycarb, V 250
Fenozolone, III 244
Fenproporex, III 245
Fenprostalene, III 246
Fentanyl, CE 615, III 248
Fenthion, III 256
Fentiazac, III 258
Fenticonazole, III 259, V 251
Feprazone, III 260
Fexofenadine, V 253
Fibrinolysin, III 261
Finasteride, I 611, III 262
Flavoxate, III 263
Flecainide, CE 616, III 265
Fleroxacin, CE 618, III 271
Flezelastine, III 278
Floctafenine, III 278
Flomoxef, V 257
Florfenicol, V 258
Flosequinan, III 280
Floxacillin, III 282
Floxuridine, III 283
Flubendazole, III 289
Fluconazole, CE 618, I 615, III 291
Flucytosine, CE 619, III 293
Fludarabine, III 297
Fludeoxyglucose F18, III 299
Fludiazepam, CE 620
Fludrocortisone, CE 622, III 301, V 260
Flufenamic acid, CE 623, III 303
Fluindione, III 305
Flumazenil, III 306
Flumequine, CE 624, III 310
Flumethasone, CE 624, III 312
Flunarizine, III 315
Flunisolide, III 316
Flunitrazepam, CE 625, III 317
Flunixin, CE 628, III 321
Flunoxaprofen, III 325
Fluocinolone acetonide, CE 628, III 325
Fluocinonide, CE 630, III 329
Fluocortolone, CE 631, III 330
Fluorometholone, III 330
Fluorouracil, III 333
Fluoxetine, CE 632, I 621, III 347
Fluoxymesterone, III 351
Flupentixol, CE 633, III 357
Fluphenazine, CE 634, III 358
Flupirtine, III 366
Fluprostenol, V 262
Flurandrenolide, V 264
Flurazepam, CE 634, III 367
Flurbiprofen, CE 638, III 375
Flurithromycin, V 267
Flurogestone acetate, V 268
Flutamide, I 633, III 395
Flutazolam, III 396
Fluticasone propionate, CE 648, V 270
Flutoprazepam, III 397
Flutrimazole, III 398, V 273
Fluvastatin, I 634, III 398
Fluvoxamine, CE 649, III 399
Folic acid, CE 650, III 409
Fomepizole, V 274
Fomivirsen, V 276
Fonazine, III 414
Fondaparinux, V 277
Formestane, V 278
Formoterol, CE 650, III 415, V 279
Fosamprenavir calcium, V 281
Foscarnet sodium, III 417
Fosfosal, III 418
Fosinopril, CE 652, III 419, V 283
Fosphenytoin, III 420, V 284
Fotemustine, III 421
Frovatriptan, V 286
Fructose, III 422
Fumagillin, V 288
Furaltadone, III 423
Furazolidone, III 424
Furosemide, CE 652, I 637, III 434
Furprofen, III 440
Fusidic acid, III 440
Gabapentin, CE 655, III 442
Gadodiamide, III 446
Gadopentetic acid, III 447
Galantamine, V 290
Gallamine triethiodide, III 447
Gallopamil, CE 656, III 449
Ganciclovir, III 452
Ganirelix, V 292
Gatifloxacin, V 293
Gefitinib, V 295
Gemcitabine, V 296
Gemeprost, III 454
Gemfibrozil, I 654
Gemifloxacin, V 298
Gentamicin, CE 659, III 455
Gentian violet, III 462
Gentisic acid, III 466
Gestodene, V 300
Gestrinone, V 301
Glafenine, III 467
Glibornuride, III 468
Gliclazide, III 469
Glimepiride, III 470
Glipizide, CE 660, I 659, III 471
Glucagon, III 472
Gluconolactone, III 472
Glutethimide, CE 663, III 473
Glyburide, CE 664, I 665, III 480
Glycerin, CE 667, III 483, V 302
Cumulative Index
Glycopyrrolate, III 487
Gonadorelin, CE 668, III 488
Gonadotropin, III 494
Goserelin, CE 669, I 672, III 495
Gossypol, III 495
Gramicidin, III 496
Granisetron, III 497
Grepafloxacin, III 503
Griseofulvin, III 503
Guaiacol, III 506
Guaifenesin, CE 669, I 673, III 508
Guanabenz, III 509, V 304
Guanadrel, V 305
Guanethidine, III 511
Guanfacine, III 512
Gusperimus, III 513
Halazepam, CE 672, III 515
Halcinonide, III 518
Halobetasol propionate, V 306
Halofantrine, CE 673, III 518
Halofuginone, V 308
Haloperidol, CE 674, III 525
Halothane, III 543
Haloxazolam, CE 677, III 543
Heparin, CE 678, III 544
Heptaminol, CE 680
Hesperidin, III 547
Hetacillin, III 551
Hetastarch, V 310
Hexachlorophene, III 553
Hexobarbital, CE 681, III 553
Hirudin, CE 682
Histamine, CE 683, III 554
Histrelin, III 571
Homatropine, CE 686, III 572
Hyaluronic acid, CE 691, III 575
Hydralazine, III 577
Hydrochiorothiazide, I 682, CE 693, III 583
Hydrocodone, I 699, III 586
Hydrocortisone, CE 695, I 705, III 587
Hydroflumethiazide, CE 707, III 592
Hydrogen peroxide, III 597
Hydromorphone, CE 708, III 598
Hydroquinone, CE 708, III 605, V 311
Hydroxychloroquine, CE 709, III 609
Hydroxyprogesterone, CE 710, III 615
Hydroxypropyl methylcellulose, III 623
Hydroxyquinoline, III 624
Hydroxyurea, III 625
Hydroxyzine, CE 713, III 626
Hygromycin B, V 312
Ibafloxacin, III 633
Ibogaine, III 634
Ibopamine, III 636
Ibufenac, III 638
Ibuprofen, CE 714, I 741, III 638
Ibutilide, III 646
Idarubicin, CE 724, III 647
Idazoxan, CE 724
Idebenone, III 648
Idoxuridine, III 649
Ifosfamide, III 652
Iloprost, III 654, V 313
Imatinib, V 314
Imidapril, III 655
Imidocarb, V 316
Imipenem, I 769, III 657
Imipramine, CE 725, III 658
Indalpine, III 689
Indapamide, CE 729, I 774, III 691
Indeloxazine hydrochloride, III 692
Indinavir, III 692
Indobufen, III 695
Indocyanine green, III 698
Indomethacin, CE 730, III 699
Indoprofen, CE 734
Indoramin, III 718
Inositol, III 719
Insulin, CE 738, I 778, III 719
Interferon, CE 741, I 787, III 721
Iobenguane, V 318
Iodinated glycerol, III 723
Iodixanol, V 320
Iodochlorhydroxyquin, III 723
Iodoquinol, III 726
Iofratol, III 727
Iohexol, CE 743, III 728
Iopamidol, CE 744, III 729
Iopanoic acid, CE 746, V 322
Iopromide, V 324
Iothalamate, III 730
Iothalamic acid, CE 746
Ioversol, V 326
Ipecac, III 735
Ipratropium bromide, CE 749, I 791, V 327
Ipriflavone, III 738, V 328
Iprindole, III 739
Iproniazid, III 740
Irbesartan, III 741
Irinotecan, III 742
Isepamicin, III 747
Isocarboxazid, CE 751, III 750
Isoetharine, III 752
Isofezolac, III 753
Isoflupredone, III 753, V 329
Isoflurane, III 754
Isoniazid, CE 752, III 755
Isopropamide iodide, V 330
Isoproterenol, CE 753, III 763
Isosorbide dinitrate, III 772
Isosorbide mononitrate, III 776
Isothipendyl, CE 763, III 778
Isoxicam, III 780
Isoxsuprine, CE 765, III 785
Isradipine, III 789
Itopride, V 332
Itraconazole, CE 767, III 792
Ivermectin, III 799
693
694
Cumulative Index
Josamycin, CE 768, III 809
Kanamycin, CE 768, III 812
Ketamine, CE 769, III 815
Ketanserin, III 822
Ketoconazole, CE 776, I 792, III 824
Ketoprofen, CE 777, I 798, III 826
Ketorolac, I 819, III 833
Ketotifen, III 834
Kinetin, V 333
Labetalol, CE 787, III 835
Lacidipine, III 843
Lactulose, III 844
Lafutidine, V 334
Lamivudine, CE 792, III 848, V 335
Lamotrigine, CE 794, III 849
Lansoprazole, CE 794, III 855
Lasalocid A, III 858
Latanoprost, V 339
Lazabemide, III 862
Leflunomide, V 341
Lenampicillin, III 863
Lercanidipine, V 343
Letrozole, III 864, V 345
Leucovorin, CE 795, III 864
Leuprolide, I 829
Levallorphan, CE 795, III 869
Levamisole, CE 796, III 870
Levetiracetam, V 346
Levobunolol, CE 797, III 874
Levodopa, CE 798, III 875
Levonordefrin, III 887, V 348
Levorphanol, CE 802, III 888
Levosimendan, V 349
Levothyroxine sodium, CE 804, III 896
Levothyroxine, I 831
Lidamidine, V 351
Lidocaine, CE 805, III 901
Lidoflazine, III 920
Limaprost, III 921
Lincomycin, CE 809, III 922, V 352
Lindane, V 354
Linezolid, III 924, V 355
Linoleic acid, III 924
Linuron, III 955
Liothyronine, CE 810, III 956, V 357
Lisinopril, CE 811, I 836, III 961
Lisofylline, III 962
Lisuride, III 962
Litracen, CE 812
Lobeline, III 963
Lobenzarit, III 964
Lodamide, III 1722
Lodoxamide, III 965
Lofepramine, III 966
Lomefloxacin, CE 813, III 967
Lomerizine, V 358
Lometrexol, III 971
Lomustine, III 972
Lonidamine, III 973
Loperamide, III 975
Lopinavir, V 359
Loprazolam, III 977
Loracarbef, I 839
Loratadine, I 841, III 979
Lorazepam, CE 813, I 844, III 979
Lormetazepam, III 982
Lornoxicam, III 983
Losartan, III 984
Loteprednol etabonate, V 362
Lovastatin, I 858
Loxapine, III 990
Loxoprofen, III 999
Lufenuron, III 1004
Lypressin, CE 816, III 1005
Lysozyme, CE 816, III 1007
Mabuterol, III 1013
Maduramicin, III 1014
Mafenide, III 1014
Malachite green, III 1015
Malotilate, III 1017
Manidipine, III 1018
Mannitol, CE 821, III 1019
Maprotiline, CE 821, III 1025
Marbofloxacin, V 364
Masoprocol, V 367
Maxacalcitol, V 368
Mazindol, III 1039
Mebendazole, III 1042
Mecamylamine, III 1050
Mechlorethamine, III 1051
Meclizine, CE 823, III 1053
Meclocycline, CE 825, III 1057
Meclofenamic acid, CE 825, III 1058
Meclofenoxate, III 1061
Medazepam, CE 826, III 1063
Medetomidine, V 369
Medifoxamine, III 1066
Medroxyprogesterone, III 1069
Medroxyprogesterone acetate, I 862
Mefenamic acid, CE 828, III 1072
Mefenorex, III 1083
Mefloquine hydrochloride, CE 828, III 1083
Megazol, III 1089
Megestrol acetate, III 1090
Meglutol, III 1093, V 371
Melatonin, V 372
Melengestrol acetate, V 375
Melitracen, III 1094
Meloxicam, III 1094
Melphalan, CE 833, III 1095
Memantine, V 378
Menadione, III 1102
Menotropins, III 1106
Menthol, III 1107, V 380
Mepazine, III 1108
Mepenzolate bromide, CE 833, III 1109, V 381
Cumulative Index
Meperidine, CE 835, III 1110
Mephenesin, III 1119
Mephentermine, III 1121
Mephenytoin, CE 837, III 1125
Mephobarbital, CE 837, III 1131
Mepindolol, CE 841
Mepivacaine, CE 843, III 1138
Mepixanox, V 383
Meprobamate, III 1145
Meptazinol, III 1146
Mequinol, V 384
Mequitazine, CE 848
Mercaptobenzothiazole, CE 850, III 1149
Mercaptopurine, III 1151
Meropenem, III 1159
Mesalamine, CE 851, III 1163
Mescaline, III 1168
Mesna, III 1169
Mesoridazine, CE 853, III 1172
Mestranol, CE 854, III 1181
Metaclazepam, CE 854
Metapramine, III 1184
Metaproterenol, CE 856, III 1188
Metaraminol, III 1192
Metformin, CE 861, III 1194
Methacholine chloride, CE 862, III 1200
Methacycline, CE 863, III 1201
Methadone, CE 863, III 1201
Methamphetamine, CE 868, III 1216
Methaphenilene, CE 876
Methapyrilene, III 1241
Methaqualone, CE 877, III 1243
Metharbital, CE 877
Methazolamide, III 1245
Methdilazine, III 1247
Methenamine, V 385
Methicillin sodium, III 1249
Methimazole, III 1255
Methocarbamol, III 1256
Methohexital, III 1262
Methoprene, III 1266, V 386
Methotrexate, CE 879, III 1266
Methotrimeprazine, CE 881, III 1278
Methoxamine, CE 882, III 1285
Methoxsalen, III 1290
Methoxychlor, V 387
Methoxyphenamine, CE 882, III 1292
Methoxypromazine, III 1293
Methscopolamine bromide, III 1294
Methsuximide, III 1295
Methyclothiazide, CE 883, III 1299
Methyl salicylate, III 1301
Methyldopa, CE 884, III 1304
Methyldopate, III 1309
Methylene blue, CE 884, III 1310
Methylephedrine, III 1313
Methylergonovine, III 1314
Methylphenidate, CE 885, I 867
Methylprednisolone, CE 886, I 874, III 1317
Methyltestosterone, CE 888, III 1319, V 392
Methylthiouracil, CE 889
Methyprylon, III 1327
Methysergide, III 1328
Metiamide, III 1330
Metipranolol, CE 890, III 1331
Metoclopramide, III 1332
Metocurine iodide, III 1341
Metolazone, CE 892, III 1342
Metopimazine, III 1346
Metoprolol, CE 893, I 887, III 1347
Metrizamide, V 393
Metronidazole, III 1354
Metyrapone, CE 904, III 1363
Metyrosine, V 394
Mexiletine, CE 904, III 1365
Mezlocillin, III 1376
Mianserin, CE 906, III 1380
Mibefradil, III 1384
Mibolerone, III 1384
Micafungin, V 396
Miconazole, CE 909, III 1386
Midazolam, I 911, III 1390
Midodrine, CE 909
Mifepristone, III 1395
Milnacipran, V 398
Miloxacin, III 1396
Milrinone, III 1397
Miltefosine, III 1399
Minaprine, III 1400
Minocycline, CE 910, III 1401
Minoxidil, III 1407
Miokamycin, III 1409
Mirtazapine, V 400, III 1410
Misoprostol, III 1411, V 405
Mitoguazone, III 1412
Mitolactol, III 1412
Mitomycin, III 1413
Mitotane, III 1419
Mitoxantrone, III 1421
Mivacurium chloride, III 1424
Mizolastine, V 406
Mizoribine, III 1426
Moclobemide, CE 912, III 1427
Modafinil, III 1432
Moexipril, V 411
Mofezolac, V 412
Molindone, CE 913, III 1433
Molsidomine, III 1433
Mometasone furoate, I 924, V 413
Monensin, III 1434, V 415
Montelukast, III 1441
Moperone, III 1443
Moprolol, III 1445
Morantel, III 1445, V 416
Morazone, III 1447
Moricizine, III 1448
Moroxydine, III 1449
Morphine, CE 913, III 1450
Morsuximide, III 1483
Mosapride, V 417
695
696
Cumulative Index
Moxidectin, III 1484
Moxifloxacin, V 420
Moxonidine, III 1485, V 423
Mupirocin, I 925
Muzolimine, III 1485
Mycophenolic acid, III 1486
Nabumetone, I 927, III 1488
Nadifloxacin, V 424
Nadolol, CE 923, III 1488
Nadoxolol, III 1501
Nafarelin, CE 928, III 1502
Nafcillin, CE 929, III 1503
Nafronyl, CE 930, III 1511
Naftifine, III 1512
Naftopidil, V 425
Nalbuphine, CE 932, III 1513
Nalidixic acid, CE 933, III 1519
Nalmefene, III 1525
Nalorphine, CE 933, III 1526
Naloxone, III 1537
Naltrexone, III 1546
Nandrolone, CE 936, III 1552, V 427
Naphazoline, CE 937, III 1557
Naproxen, CE 938, I 929, III 1565
Narasin, III 1570, V 429
Naratriptan, III 1573
Nartograstim, V 430
Natamycin, III 1574
Nateglinide, V 431
Nebivolol, V 433
Nedocromil, CE 947, III 1576
Nefazodone, III 1577
Nefopam, CE 948, III 1579
Nelfinavir, III 1581, V 435
Nemonapride, III 1582
Neomycin, CE 951, I 951, III 1583
Neostigmine bromide, CE 952, III 1584
Nequinate, V 440
Neridronic acid, V 441
Netilmicin, CE 953, III 1587
Nevirapine, V 443
Niacin, CE 954, III 1591
Niacinamide, CE 959, III 1598
Nialamide, III 1605
Niaprazine, III 1606
Nicarbazin, III 1607, V 447
Nicardipine, CE 965, III 1610
Nicergoline, III 1616
Niclosamide, III 1616
Nicorandil, III 1618
Nicotine, CE 968, III 1620
Nifedipine, CE 971, I 958, III 1630
Nifenalol, CE 972, III 1633
Niflumic acid, III 1634
Nifuroxazide, III 1637
Nikethamide, CE 973, III 1637
Nilutamide, III 1638, V 448
Nilvadipine, III 1639
Nimesulide, III 1640
Nimetazepam, CE 947
Nimodipine, CE 975, III 1641
Nipradilol, V 449
Nisin, CE 976
Nisoldipine, CE 976, III 1646
Nitazoxanide, V 450
Nitenpyram, V 452
Nitrazepam, CE 977, III 1649
Nitrendipine, CE 981, III 1654
Nitrofurantoin, I 968, III 1660
Nitrofurazone, III 1661
Nitroglycerin, I 973
Nizatidine, I 979, III 1667
Nomegestrol, V 453
Nomifensine, III 1668
Nonoxynol-9, III 1671, V 454
Nordazepam, CE 982
Norepinephrine, CE 982, III 1672
Norethindrone, CE 992, I 984, III 1697
Norethynodrel, CE 994, III 1698
Norfenefrine, CE 994
Norfloxacin, CE 996, III 1698
Norgestimate, CE 997, III 1711
Norgestrel, CE 998, I 993, III 1713
Norpseudoephedrine, CE 999
Nortriptyline, CE 1000, I 1000, III 1715
Noscapine, CE 1003, III 1720
Novobiocin, III 1723
Nylidrin, CE 1004, III 1726
Nystatin, CE 1005, III 1728, V 455
Octhilinone, III 1730
Octocrylene, V 456
Octopamine, CE 1006, III 1731
Octreotide, III 1732
Octyl methoxycinnamate, III 1734
Odapipam, III 1735
Ofloxacin, CE 1007, I 1029, III 1735
Olanzapine, III 1741
Olaquindox, III 1743
Oleandomycin, CE 1012, III 1744
Oleic acid, V 457
Olmesartan, V 469
Olopatadine, V 470
Olsalazine, III 1744
Omeprazole, CE 1012, I 1044, III 1745
Ondansetron, CE 1013, I 1052, III 1747
Ontazolast, III 1749
Opipramol, CE 1014, III 1749
Orbifloxacin, V 472
Orlistat, V 475
Ormetoprim, CE 1015, III 1752
Ornidazole, CE 1016
Orphenadrine, CE 1018, III 1753
Oryzalin, III 1756
Oseltamivir, V 477
Oxacillin, CE 1021, III 1757
Oxaliplatin, V 479
Oxamniquine, CE 1023, III 1768
Oxandrolone, III 1769
Cumulative Index
Oxaprozin, I 1062
Oxatomide, III 1770
Oxazepam, CE 1023, III 1771
Oxazolam, III 1793
Oxcarbazepine, III 1793
Oxeladin, III 1798
Oxfendazole, III 1799
Oxibendazole, III 1805
Oxiconazole, V 481
Oxiracetam, III 1806
Oxolinic acid, CE 1027, III 1808
Oxomemazine, CE 1028
Oxprenolol, CE 1029, III 1811
Oxybenzone, III 1817
Oxybutynin chloride, CE 1034, III 1819
Oxyclozanide, III 1819
Oxycodone, I 1065, III 1820
Oxymetazoline, CE 1035, III 1821
Oxymetholone, III 1825
Oxymorphone, III 1826
Oxyphenbutazone, III 1828
Oxyphencyclimine, CE 1036
Oxyquinoline, III 1829
Oxytetracycline, CE 1037, III 1830
Oxytocin, CE 1043, III 1850
Ozagrel, III 1855
Paclitaxel, CE 1045, I 1072, IV 1
Padimate O, IV 6
Palinavir, IV 6
Pamidronate, IV 7
Pamoic acid, CE 1047
Pancreatin, IV 12
Pancuronium bromide, IV 13
Panipenem, V 483
Panomifene, IV 14
Pantoprazole, CE 1047, IV 15
Pantothenic acid, CE 1048, IV 17
Papaverine, CE 1050, IV 20
Paramethadione, IV 29
Paramethasone, IV 30
Parathyroid hormone, IV 31
Parbendazole, IV 32
Parconazole, IV 33
Parecoxib, V 484
Pargyline, IV 34
Paricalcitol, V 485
Paromomycin, CE 1054, IV 36
Paroxetine, IV 39
Pazufloxacin, V 487
Pefloxacin, CE 1056, IV 44
Pemirolast, IV 51
Pemoline, CE 1056, IV 51
Penbutolol, CE 1057, IV 55
Penciclovir, CE 1057, IV 62, V 488
Penclomedine, IV 63
Penfluridol, IV 64
Penicillamine, IV 66
Penicillin G, CE 1058, IV 76
Penicillin V, CE 1061, I 1085, IV 95
697
Pentaerythritol tetranitrate, IV 98, V 490
Pentamidine, CE 1062, IV 100
Pentazocine, CE 1062, IV 104
Penthienate bromide, IV 115
Pentobarbital, CE 1064, IV 116
Pentosan polysulfate, V 491
Pentostatin, IV 124
Pentoxifylline, CE 1071, I 1096, IV 130
Perflubron, V 492
Pergolide, IV 131
Perhexiline, IV 132
Pericyazine, IV 133
Perindopril, IV 134
Permethrin, IV 135
Perospirone, V 493
Perphenazine, CE 1071, IV 139
Phenacemide, IV 146
Phenacetin, CE 1073, IV 147
Phenadoxone, IV 149
Phenampromide, IV 150
Phenazocine, IV 151
Phenazopyridine hydrochloride, CE 1075, IV
154, V 494
Phencyclidine, IV 156
Phendimetrazine, CE 1075, IV 159
Phenelzine, IV 161
Phenformin, CE 1076, IV 166
Phenglutarimide, IV 166
Phenindamine, IV 167
Phenindione, IV 169
Pheniramine, CE 1077, IV 170
Phenmetrazine, CE 1080, IV 178
Phenobarbital, CE 1081, IV 181
Phenol, CE 1091, IV 205
Phenolphthalein, IV 224
Phenomorphan, IV 228
Phenoperidine, IV 229
Phenothiazine, CE 1094, IV 230
Phenoxybenzamine, CE 1095, IV 235
Phenprocoumon, CE 1098
Phensuximide, CE 1098, IV 237
Phentermine, CE 1099, IV 239, V 497
Phentolamine, IV 245
Phenyl salicylate, IV 248
Phenylbutazone, IV 249
Phenylephrine, CE 1100, IV 258
Phenylpropanolamine, CE 1102, I 1103, IV 266
Phenyltoloxamine, IV 267
Phenytoin, CE 1108, I 1117, IV 270
Phloroglucinol, IV 274
Pholcodine, IV 275
Pholedrine, CE 1111
Phosmet, IV 276
Phosphatidylcholine, IV 277, V 501
Phosphatidylglycerol, V 504
Physostigmine, CE 1114, IV 284
Picotamide, IV 289
Picroside, IV 290
Picumeterol, CE 1114
Pidotimod, IV 290
698
Cumulative Index
Piketoprofen, V 508
Pilocarpine, CE 1115, IV 291
Pilsicainide, V 509
Piminodine, IV 295
Pimobendan, IV 296
Pimozide, CE 1116, IV 298
Pinacidil, IV 303
Pinaverium bromide, IV 306
Pindolol, CE 1117, IV 307
Pioglitazone, V 511
Pipamazine, IV 323
Pipamperone, IV 324
Pipazethate, IV 326
Pipecuronium bromide, IV 327
Pipemidic acid, CE 1134, IV 328
Piperacetazine, IV 330
Piperacillin, CE 1134, IV 331
Piperazine, IV 340
Pipercuronium bromide, V 514
Piperidolate, IV 341
Piperine, IV 342
Piperocaine, IV 343
Piperonyl butoxide, IV 344
Piperoxan, CE 1136
Pipotiazine, IV 347
Pipradrol, IV 348
Piracetam, CE 1137, IV 349
Pirarubicin, IV 350
Pirbuterol, CE 1137
Pirenzepine, IV 352
Piretanide, CE 1140, IV 353
Piritramide, IV 354
Pirlimycin, V 515
Pirmenol, IV 355
Piromidic acid, CE 1141, IV 358
Piroxicam, CE 1141, I 1140, IV 359
Pivampicillin, IV 361
Pizotyline, IV 362
Podofilox, IV 364
Poloxalene, V 518
Polyethylene glycol, CE 1144
Polymyxin, I 1150, IV 365
Polythiazide, IV 367
Porfiromycin, IV 369
Practolol, IV 370
Pralidoxime chloride, IV 371
Pramipexole, V 519
Pramiracetam, IV 373
Pramlintide, IV 374
Pramoxine, IV 375
Pranlukast, V 521
Pranoprofen, CE 1144
Pravastatin sodium, I 1153, IV 378
Prazepam, CE 1145, IV 380
Praziquantel, CE 1146, IV 390
Prazosin, CE 1147, IV 393
Prednicarbate, IV 402, V 523
Prednisolone, CE 1147, IV 403
Prednisone, CE 1150, I 1156, IV 421
Premafloxacin, IV 424
Prenalterol, CE 1151
Prenylamine, IV 424
Prifinium bromide, IV 425
Prilocaine, CE 1152, IV 426
Primaquine, CE 1157, IV 432
Primidone, CE 1161, IV 437
Pristinamycin, IV 448
Proadifen, IV 449
Probenecid, CE 1164, IV 450
Probucol, IV 459
Procainamide, CE 1165, IV 460
Procaine, CE 1168, IV 469
Procarbazine, IV 478
Prochlorperazine, CE 1172, IV 481
Procyclidine, CE 1173, IV 488
Progabide, IV 490
Progesterone, CE 1175, IV 492
Proglumide, CE 1179
Proheptazine, IV 505
Prolintane, CE 1180, IV 506
Promazine, CE 1180, IV 507
Promethazine, CE 1182, I 1171, IV 515
Pronethalol, IV 517
Propafenone, CE 1187, IV 518
Propantheline bromide, IV 529
Proparacaine, IV 532
Propentofylline, CE 1190, IV 533
Properidine, IV 534
Propicillin, IV 535
Propiomazine, CE 1190, IV 536
Propionylpromazine, V 524
Propiverine, IV 541
Propofol, I 1185, IV 542
Propoxur, CE 1191, IV 545
Propoxycaine hydrochloride, V 527
Propoxyphene, CE 1191, I 1189, IV 551
Propranolol, CE 1193, IV 555
Propylene glycol, IV 602, V 528
Propylhexedrine, IV 605, V 529
Propylparaben, IV 606
Propylthiouracil, CE 1213, IV 607
Propyphenazone, CE 1214
Proscillaridin, IV 609
Prospidium chloride, CE 1214
Protamine sulfate, IV 609
Prothipendyl, IV 611
Protionamide, IV 612
Protirelin, CE 1215, IV 613, V 530
Protriptyline, CE 1216, IV 614
Prulifloxacin, V 532
Pseudoephedrine, CE 1219, IV 627
Psilocybin, IV 636
Pumactant, CE 1226, IV 637
Puromycin, IV 639
Pyrantel, IV 640
Pyrazinamide, IV 644
Pyrazoloacridine, IV 647
Pyrethrins, V 533
Pyridostigmine bromide, IV 648
Pyridoxal, CE 1227, IV 651
Cumulative Index
Pyridoxamine dihydrochloride, CE 1228, IV 656
Pyridoxine, CE 1230, IV 658
Pyrilamine, CE 1235, IV 667
Pyrimethamine, CE 1236, IV 675
Pyrisuccideanol, IV 683
Pyrithione, IV 684
Pyrithyldione, IV 684
Pyrocatechol, IV 685
Pyrrobutamine, CE 1238
Quazepam, IV 686
Quercetin, IV 689
Quetiapine, V 536
Quinacrine, CE 1238, IV 690
Quinagolide, CE 1239
Quinaldic acid, IV 692
Quinapril, I 1202, IV 693
Quinestrol, IV 694
Quinethazone, IV 695
Quinfamide, V 539
Quinidine, CE 1240, IV 696
Quinine, CE 1243, IV 710
Quinupramine, IV 723
Quinupristin, V 540
Rabeprazole, V 542
Ractopamine, V 544
Raloxifene, V 547
Ramatroban, IV 725
Ramipril, I 1205, IV 726
Ramosetron, V 549
Ranitidine, CE 1246, I 1209, IV 727
Rapacuronium bromide, V 551
Rebamipide, IV 731
Reboxetine, IV 731
Remifentanil, V 553
Remikiren, IV 733
Remoxipride, CE 1251, IV 733
Repaglinide, V 555
Repirinast, IV 736
Reproterol, CE 1253, IV 737
Rescinnamine, IV 738
Reserpine, CE 1255, IV 740
Resorcinol, CE 1256, IV 748
Retinoic acid, CE 1256, I 1223, IV 753
Ribavirin, IV 757
Riboflavin, CE 1258, IV 760
Ricinoleic acid, IV 769, V 557
Rifabutin, IV 769
Rifampin, IV 773
Rifapentine, IV 782
Rifaximin, IV 784, V 558
Rilmazafone, IV 785, V 559
Rilmenidine, IV 786
Riluzole, IV 786
Rimantadine, CE 1262, IV 787
Risedronate sodium, V 560
Risperidone, IV 789
Ritodrine, CE 1263, IV 793
Ritonavir, IV 795
Rizatriptan, IV 798, V 561
Robenidine, IV 799
Rocuronium bromide, IV 800
Rofecoxib, V 562
Rokitamycin, IV 802
Romurtide, IV 803
Ronidazole, IV 803
Ropinirole, IV 804, V 565
Ropivacaine, CE 1264, IV 805
Rosiglitazone, V 566
Rosoxacin, CE 1265, IV 806
Rosuvastatin calcium, V 568
Rotenone, IV 807
Roxarsone, IV 809
Roxatidine acetate, IV 810
Roxithromycin, IV 811
Rufloxacin, IV 814
Rutin, CE 1266, IV 817
Saccharin, CE 1267, IV 818
Salicylamide, CE 1268, IV 825
Salicylic acid, CE 1270, IV 830
Salinomycin, IV 856
Salmeterol, I 1234
Salsalate, IV 859
Sampatrilat, IV 860
Saperconazole, IV 861
Saquinavir, IV 862
Sarafloxacin, IV 863, V 569
Scopolamine, CE 1279, I 1235, IV 865
Scopoletin, IV 867
Secnidazole, IV 868
Secobarbital, CE 1281, IV 869
Secretin, IV 880
Selamectin, V 571
Selegiline, CE 1286, IV 881
Selfotel, IV 883
Semduramicin, IV 884
Seratrodast, IV 885
Sermorelin, V 572
Sertaconazole, IV 886
Sertindole, IV 887
Sertraline, I 1244, IV 888
Sibutramine, V 574
Sildenafil, V 576
Simazine, IV 888
Simethicone, V 579
Simvastatin, I 1249, IV 889
Sincalide, IV 890
Sirolimus, IV 892
Sisomicin, IV 894
Sivelestat, V 580
Sobrerol, IV 895
Sodium nitroprusside, IV 895
Sodium oxybate, V 582
Somatomedin, IV 897
Somatrem, CE 1288, IV 899
Somatropin, CE 1289, I 1252, IV 900, V 583
Sorivudine, IV 902
699
700
Cumulative Index
Sotalol, CE 1291, IV 904
Sparfloxacin, CE 1295, IV 917
Spectinomycin, IV 922
Spiperone, IV 926
Spiramycin, IV 927
Spirapril, CE 1296, IV 928
Spironolactone, CE 1296, IV 929
Squalane, V 584
Squalene, V 585
Stanozolol, IV 935, V 587
Stavudine, CE 1297, IV 937
Stearic acid, IV 941
Stiripentol, IV 942
Streptokinase, IV 943
Streptomycin, CE 1298, IV 944
Streptozocin, IV 948
Strychnine, IV 950
Succimer, IV 951, V 588
Succinylcholine chloride, IV 953, V 589
Sucralfate, I 1256
Sucrose, IV 954
Sufentanil, CE 1299, IV 954
Sulbactam, I 1257
Sulbenicillin, IV 956
Sulbentine, IV 957
Sulconazole, CE 1300, IV 958
Sulfabenzamide, CE 1301, IV 959
Sulfabromomethazine, V 591
Sulfacetamide, CE 1302, IV 962
Sulfachlorpyridazine, CE 1303, V 592
Sulfadiazine, CE 1305, IV 970
Sulfadimethoxine, CE 1309, IV 991
Sulfadoxine, CE 1312, IV 1010
Sulfaethidole, IV 1018
Sulfaethoxypyridazine, V 596
Sulfaguanidine, CE 1313, IV 1019
Sulfalene, CE 1314, IV 1020
Sulfamerazine, CE 1316, IV 1020, V 598
Sulfameter, CE 1318
Sulfamethazine, CE 1320, IV 1022
Sulfamethizole, CE 1324, IV 1047
Sulfamethoxazole, CE 1324, I 1263, IV 1054
Sulfamethoxypyridazine, IV 1059
Sulfamonomethoxine, CE 1330, IV 1059
Sulfanilamide, CE 1331, IV 1061
Sulfanilic acid, CE 1333, IV 1075
Sulfanitran, V 600
Sulfapyridine, CE 1334, IV 1076
Sulfaquinoxaline, CE 1335, IV 1079
Sulfasalazine, IV 1087
Sulfathiazole, CE 1339, IV 1090
Sulfinpyrazone, IV 1105
Sulfisomidine, CE 1342
Sulfisoxazole, CE 1344, IV 1109
Sulfur, IV 1114
Sulindac, CE 1347, IV 1115
Sulpiride, CE 1349, IV 1128
Sultamicillin, V 602
Sultopride, IV 1130
Sumatriptan, CE 1352, I 1284, IV 1132
Suprofen, CE 1353, IV 1134
Suramin, CE 1357
Suriclone, IV 1136
Synephrine, CE 1358, IV 1138
Tacalcitol, V 604
Tacrine, CE 1362, IV 1138
Tacrolimus, IV 1145
Talinolol, CE 1362
Talipexole, V 605
Taltirelin, V 607
Tamoxifen, CE 1364, IV 1150
Tamsulosin, IV 1160
Taurine, CE 1366, IV 1161
Tazarotene, IV 1176
Tazobactam, IV 1176
Tazofelone, IV 1180
Technetium Tc 99m bicisate, V 608
Tegafur, IV 1181
Tegaserod, V 609
Teicoplanin, IV 1182
Telithromycin, V 610
Telmesteine, V 611
Telmisartan, V 612
Temafloxacin, IV 1185
Temazepam, CE 1368, I 1286, IV 1189
Temocapril, V 614
Temocillin, IV 1191
Temozolomide, V 615
Teniposide, IV 1192
Tenofovir disoproxil fumarate, V 617
Tenoxicam, CE 1369, IV 1198
Teprenone, V 620
Terazosin, I 1302, IV 1203
Terbinafine, I 1306, IV 1204
Terbutaline, CE 1370, IV 1205
Terconazole, I 1310
Terfenadine, I 1312, IV 1214
Teriparatide, V 621
Teroxirone, IV 1217
Terpin, IV 1218
Tertatolol, IV 1218
Testolactone, IV 1220
Testosterone, CE 1383, IV 1222
Tetracaine, CE 1387, IV 1240
Tetrachlorvinphos, V 622
Tetracycline, CE 1390, I 1321, IV 1250
Tetrahydrozoline, CE 1395, IV 1256, V 623
Tetramethrin, IV 1259
Tetrazepam, IV 1262
Thalidomide, CE 1399, IV 1264, V 626
Thebaine, CE 1401, IV 1264
Thenyldiamine, IV 1265
Theobromine, IV 1266
Theodrenaline, CE 1402
Theophylline, CE 1404, I 1336, IV 1268
Thiabendazole, IV 1272
Thiacetarsamide, IV 1282
Thialbarbital, V 628
Thiambutene, IV 1282
Cumulative Index
Thiamine, CE 1413, IV 1283
Thiamphenicol, CE 1418, IV 1296
Thiamylal, IV 1296
Thiethylperazine, IV 1301
Thimerosal, IV 1304
Thiobutabarbital, IV 1307
Thioctic acid, IV 1308
Thioguanine, IV 1309
Thiopental, CE 1419, IV 1313
Thiopropazate, IV 1329
Thioproperazine, IV 1330
Thioridazine, CE 1422, IV 1333
Thiosalicylic acid, IV 1346
Thiotepa, IV 1349
Thiothixene, CE 1424, IV 1351
Thonzylamine, IV 1360
Thymol, IV 1361
Thymopentin, IV 1363
Thyrotropin, V 629
Tiagabine, IV 1363, V 630
Tiamenidine, IV 1364
Tiamulin, IV 1364
Tianeptine, IV 1366
Tiapride, IV 1368
Tiaprofenic acid, CE 1425, IV 1370
Ticarcillin, CE 1427, IV 1373
Ticlopidine, CE 1428, IV 1377
Tiemonium iodide, IV 1380
Tiletamine hydrochloride, V 631
Tilisolol, IV 1381
Tilmicosin, IV 1383, V 633
Tiludronic acid, V 634
Timepidium bromide, CE 1429
Timiperone, IV 1384
Timolol, CE 1429, I 1371, IV 1384
Tinidazole, IV 1388
Tioclomarol, IV 1389
Tioconazole, CE 1434, IV 1391
Tiopronin, CE 1437, IV 1393
Tiracizine, IV 1397
Tirilazad, V 635
Tirofiban, V 637
Tiropramide, V 639
Tizanidine, V 641
Toborinone, IV 1398
Tobramycin, CE 1437, IV 1399
Tocainide, CE 1438, IV 1404
Tofisopam, IV 1411
Tolazamide, CE 1440, IV 1414
Tolazoline, IV 1416
Tolbutamide, CE 1443, IV 1419
Tolcapone, V 643
Tolfenamic acid, IV 1429
Tolmetin, CE 1446, IV 1430
Tolnaftate, IV 1435
Toloxatone, IV 1436
Tolperisone, CE 1447
Tolpropamine, IV 1439
Tolrestat, IV 1440
Tolterodine, IV 1442
Tolycaine, IV 1443
Topiramate, V 645
Topotecan, IV 1445, V 647
Toremifene, IV 1446
Torsemide, IV 1449
Tosufloxacin, V 649
Tramadol, CE 1448, I 1383, IV 1453
Tramazoline, IV 1455
Trandolapril, IV 1455
Tranexamic acid, IV 1456
Tranilast, IV 1458
Tranylcypromine, CE 1449, IV 1459
Travoprost, V 651
Trazodone, IV 1467
Trenbolone, IV 1476, V 652
Treprostinil, V 653
Tretoquinol, CE 1450
Triamcinolone, CE 1453, I 1385, IV 1483
Triamterene, CE 1456, I 1398, IV 1486
Triazolam, CE 1457, IV 1489
Trichlorfon, IV 1499, V 655
Trichlormethiazide, CE 1460, IV 1501
Triclocarban, IV 1505
Triclosan, IV 1506
Trientine, IV 1507
Triethanolamine, V 656
Trifluoperazine, CE 1461, IV 1507
Trifluperidol, IV 1518
Triflupromazine, CE 1463, IV 1521
Trifluridine, IV 1527, V 657
Trihexyphenidyl, CE 1463, IV 1528
Trilostane, IV 1532
Trimazosin, IV 1533
Trimebutine, CE 1468, IV 1534
Trimeperidine, IV 1534
Trimeprazine, CE 1469, IV 1535
Trimetazidine, IV 1545
Trimethadione, IV 1546
Trimethobenzamide, IV 1546
Trimethoprim, CE 1472, I 1409, IV 1548
Trimetrexate, IV 1551
Trimidox, IV 1554
Trimipramine, CE 1475, IV 1554
Trioxsalen, IV 1568
Tripelennamine, CE 1480, IV 1569
Triprolidine, CE 1481, IV 1574
Triptorelin, IV 1583, V 658
Tritoqualine, IV 1584
Troglitazone, CE 1482, IV 1584
Troleandomycin, CE 1483, V 660
Tromethamine, CE 1484, IV 1585
Tropacocaine, IV 1589
Tropic acid, IV 1590
Tropicamide, CE 1484, IV 1590
Tropisetron, IV 1591
Trospium chloride, IV 1593
Trovafloxacin, IV 1594
Troxipide, V 661
Trypsin, IV 1594
Tryptophan, IV 1596
701
702
Cumulative Index
Tubocurarine chloride, IV 1596
Tylosin, IV 1600
Tyloxapol, IV 1606
Tymazoline, IV 1607
Ubenimex, V 662
Undecylenic acid, IV 1607
Unoprostone isopropyl ester, V 663
Urea, IV 1609
Urokinase, IV 1613
Ursodiol, CE 1489, IV 1614
Valacyclovir, IV 1626, V 664
Valdecoxib, V 666
Valganciclovir, V 668
Valproic acid, CE 1490, I 1429, IV 1627
Valrubicin, V 670
Valsartan, V 671
Valspodar, IV 1629
Vancomycin, CE 1491, I 1438, IV 1630
Vanillin, IV 1632
Vasopressin, IV 1633
Vecuronium bromide, IV 1636
Venlafaxine, CE 1491, I 1445, IV 1639
Verapamil, CE 1492, I 1448, IV 1640
Vesnarinone, IV 1647
Vidarabine, IV 1648
Vigabatrin, IV 1654
Viloxazine, IV 1657
Vinblastine, CE 1502, IV 1659
Vincamine, IV 1665
Vincristine, CE 1503, IV 1667
Vindesine, IV 1673
Vinorelbine, IV 1674
Vinylbital, IV 1678
Virginiamycin, IV 1679
Vitamin A, CE 1504, IV 1680
Vitamin B12 , CE 1506, IV 1705
Vitamin D2 , CE 1508, IV 1710
Vitamin D3 , CE 1509, IV 1714
Vitamin E, CE 1511, IV 1719
Vitamin K, IV 1741
Vitamin K1 , IV 1742
Voriconazole, IV 1747
Warfarin, CE 1512, I 1466, IV 1748
Xamoterol, IV 1752
Xanomeline, IV 1754
Xipamide, IV 1755
Xylazine, IV 1757
Xylometazoline, CE 1520, IV 1760
Xylose, CE 1521, IV 1764
Yohimbine, IV 1777
Zafirlukast, IV 1783
Zalcitabine, CE 1529, IV 1784
Zaleplon, V 673
Zaltoprofen, V 676
Zanamivir, V 678
Zearalenone, IV 1788
Zeranol, IV 1789
Zidovudine, CE 1529, I 1480, IV 1794
Zileuton, IV 1795
Zinostatin, IV 1797, V 680
Zipeprol, IV 1798
Ziprasidone, IV 1799
Zofenopril calcium, CE 1530, V 681
Zolazepam hydrochloride, V683
Zolpidem, CE 1531, I 1491,
IV 1799
Zonisamide, IV 1801
Zopiclone, CE 1532, IV 1804
Zorubicin, IV 1812
Zoxazolamine, IV 1813
Zuclopenthixol, IV 1815
CROSS-INDEX TO OTHER
SUBSTANCES
In many cases other compounds may be chromatographed under the conditions described in the
monographs in this book. Using the information in the monographs it may be possible to develop
chromatographic procedures for these compounds. The compounds fall into the following categories:
AL
Also chromatographed
Compounds which can be analyzed at the same time. It is not specified
whether or not they interfere but they can be extracted.
EX
Extracted
Compounds which can be extracted from the matrix in question and
analyzed at the same time and do not interfere.
IN
Interfering
Compounds which interfere with the analysis of the target compound.
IS
Internal Standard
A compound added at some time during the sample preparation
procedure to act as a standard.
SI
Simultaneous
Compounds which can be analyzed at the same time and do not
interfere. Note that the compound may or may not be extracted from
the matrix in question.
(For obvious reasons non-interfering compounds are not listed in this index.)
Abacavir (EX) 335, 336
Abacavir 5’-carboxylate (EX) 3
Abacavir 5’-glucuronide (EX) 3
Acamprosate (EX) 387
Acebutolol (EX) 7, 230, 253, 387, 400, 406, 434,
495, 623; (IN) 499
Acefylline heptaminol (EX) 387
Acenocoumarol (EX) 387
Acepromazine (EX) 387, 525
Aceprometazine (EX) 387
Acetaminophen (EX) 7, 232, 254, 384, 387, 401,
407, 495, 498, 623; (IS) 315; (SI) 16, 173,
178, 202, 603, 614
Acetamiprid (EX) 452
Acetazolamide (EX) 387; (SI) 188, 220, 522
Acetiamin (EX) 387
Acetophenetidine (SI) 178
Acetorphan (EX) 387
Acetylcodeine (AL) 98
Acetylleucine (EX) 387
Acetylmethionine (EX) 387
6-Acetylmorphine (EX) 387
Acetylsulfabromomethazine (EX) 591, 594, 597
Acetylsulfachlorpyridazine (EX) 591, 594, 597
Acetylsulfadiazine (EX) 591, 594, 597
Acetylsulfadimethoxine (EX) 591, 594, 597
Acetylsulfaethoxypridazine (EX) 591, 594, 597
Acetylsulfamethazine (EX) 591, 594, 597
Acetylsulfisoxazole (EX) 599
N-Acetylprocainamide (EX) 495, 498
N-Acetyl-L-tyrosine ethyl ester (EX) 389
Acrivastine (EX) 230, 253, 400, 406, 623
Actinoquinol (EX) 387
Acyclovir (EX) 387, 488, 664; (SI) 188, 220, 522
Adefovir (EX) 12; (IS) 617
Adenosine (EX) 387; (IS) 372
Adipic acid (EX) 139, 371
Adrafinil (EX) 387
Aklomide (EX) 601
Alacepril (SI) 603, 614
HPLC Methods for Recently Approved Pharmaceuticals. by George Lunn
Copyright 2005 John Wiley & Sons, Ltd. ISBN: 0-471-66941-5
703
704
Cross-Index to Other Substances
Albendazole (EX) 102, 103, 387
Alclometasone 17,21-dipropionate (SI) 30, 32,
143, 145, 167, 170, 264, 271, 306
Aldicarb (EX) 387
Alendronate (SI) 441
Alfuzosin (EX) 387
Alimemazine (EX) 387
Alizapride (IN) 552
Allethrin (EX) 80, 535
Allopurinol (SI) 188, 220, 522
Alminoprofen (EX) 387
Almitrine (EX) 387
Alpiropride (IS) 50
Alprazolam (EX) 7, 230, 253, 387, 400, 406, 498,
623; (IN) 496; (SI) 194
Alprenolol (EX) 7, 230, 253, 400, 406, 434, 623
Alprostadil (EX) 460
Altazide (EX) 387
Altretamine (EX) 387
Amantadine (EX) 7, 230, 253, 400, 406, 623; (IS)
378
Ambemonium (EX) 387; (IS) 137
Amcinonide (SI) 18, 20, 143, 145, 167, 170, 264,
271, 306
Amfepramone (EX) 387
Amiloride (EX) 7, 230, 253, 387, 400, 406, 623
Amineptine (EX) 387, 495, 498
p-Aminobenzoic acid (EX) 97; (SI) 456
4-Amino-3-nitroanisole (IS) 334
Aminophenazone (EX) 7, 230, 253, 400, 406, 623
p-Aminosalicylic acid (EX) 390
Aminophylline (IN) 204; (SI) 178
Amiodarone (EX) 7, 230, 253, 387, 400, 406, 623;
(IS) 343
Amisulpride (EX) 387, 537
Amitriptyline (EX) 7, 230, 253, 387, 400, 406,
495, 498, 537, 623; (SI) 47, 383, 512, 547
Amlodipine (EX) 387
Ammonium (SI) 656
Amobarbital (EX) 387, 495, 498; (SI) 178
Amoxapine (EX) 378, 387, 495, 498
Amoxicillin (EX) 387; (SI) 202
Amphetamine (AL) 98; (EX) 387, 495, 497, 498,
529
Amphotericin B (EX) 387
Ampicillin (EX) 387; (SI) 16, 603, 614
Amprenavir (EX) 209, 281, 282, 359, 360, 436,
444
Androstenedione (EX) 169
Anethole (EX) 387
Angiotensin I (SI) 13
Angiotensin II (SI) 13
Angiotensin III (SI) 13
Anhydrodaptomycin (SI) 159
Anhydroglucitol (EX) 302
Aniracetam (SI) 188, 220, 522
Anthraquinone (IS) 357
Antipyrine (EX) 204; (SI) 178
Antrenyl (SI) 331, 382
Apomorphine (EX) 387
Aprepitant (SI) 512, 547
Aprindine (EX) 387
Aprobarbital (EX) 495, 498; (SI) 178
Arachidonic acid (EX) 457, 458, 459, 460, 461,
462; (SI) 557
Arecoline (EX) 387
Artemisinin (IS) 52
Articaine (AL) 98
Articainic acid (EX) 55
Ascorbic acid (SI) 140
Aspartame (EX) 387
β-Asp daptomycin (SI) 159
Aspirin (SI) 16, 178, 202, 603, 614
Astemizole (AL) 38; (EX) 7, 231, 253, 387, 400,
406, 623
Atenolol (EX) 7, 231, 253, 387, 400, 406, 434,
495, 498, 623
Atipamazole (EX) 369, 370
Atracurium (IN) 552
Atrazine (EX) 387
Atrial natriuretic peptide (SI) 13
Atropine (EX) 327, 387, 495, 498
Azacyclonol (EX) 7, 231, 253, 400, 406, 623
Azaperol (EX) 525
Azaperone (EX) 525
Azithromycin (SI) 267
Bacampicillin (EX) 387
Baclofen (SI) 642
Bamethan (EX) 387
Bamifylline (EX) 387
Barbital (EX) 387, 495, 499; (SI) 178
Baythroid (cyfluthrin) (AL) 25; (EX) 26, 80, 535
Beclomethasone (EX) 168, 265
Beclomethasone 17,21-dipropionate (SI) 20, 32,
145, 167, 170, 264, 271, 306, 414
Befunolol (EX) 434
Benazepril (EX) 387; (SI) 104, 188, 220, 522
Bendroflumethiazide (EX) 387
Benfluorex (EX) 387
Benperidol (SI) 537
Bentazone (EX) 387
Benzanilide (IS) 6
Benzbromarone (EX) 387
Benzene (EX) 387
Benzhexol (EX) 7, 231, 253, 400, 406, 495, 499,
623
Benzoic acid (SI) 178
Benzophenone (EX) 387
Benzophenone-3 (SI) 456
Benzophenone-4 (SI) 456
Benzoylecgonine (EX) 7, 231, 253, 387, 400, 406,
495, 499, 623
Benztropine (EX) 495, 499
Benzydamine (EX) 387
Benzyl alcohol (IS) 136; (SI) 516
3-Benzylidene-d, l-camphor (SI) 456
Benzylidene camphorsulfonic acid (SI) 456
1-Benzylimidazole (IS) 100
Bepridil (EX) 387
Cross-Index to Other Substances
Betahistine (EX) 388
Betamethasone (EX) 168, 265, 388; (IS) 523; (SI)
18, 20, 30, 32, 143, 145, 167, 170, 188, 220,
264, 271, 306, 522
Betamethasone-17-acetate (SI) 18, 30, 143
Betamethasone-21-acetate (SI) 18, 20, 30, 32,
143, 145, 167, 170, 264, 271, 306
Betamethasone 17-benzoate (SI) 18, 20, 30, 32,
143, 145, 167, 170, 271, 306
Betamethasone-17,21-diacetate (SI) 18, 30, 143
Betamethasone 17,21-dipropionate (IS) 18, 20,
30, 32, 143, 145, 167, 170, 264, 271, 306,
413
Betamethasone-17,21-divalerate (SI) 18, 30, 143
Betamethasone propionate (SI) 18, 30, 143
Betamethasone-17-propionate-21-butyrate (SI)
18, 30, 143
Betamethasone-17-propionate-21-stearate (SI)
18, 30, 143
Betamethasone 17-valerate (SI) 18, 20, 30, 32,
143, 145, 167, 170, 264, 271, 306
Betamethasone-17-valerate-21-acetate (SI) 18,
30, 143
Betaxolol (EX) 7, 231, 253, 388, 400, 406, 434,
623
Bezafibrate (EX) 388
BHT (EX) 388
Bidesethylchloroquine (EX) 388
Bifenthrin (EX) 26, 80, 535
Bifonazole (SI) 482
Bioallethrin (SI) 534
Biotin (Vitamin H) (EX) 391
Biperiden (EX) 388
Biperidine (EX) 7, 231, 253, 400, 406, 623
Biscoumacetate (EX) 388
Bisoprolol (EX) 7, 231, 253, 388, 400, 406, 434,
623
Boldine (EX) 388
Bombesin (SI) 13
Bradykinin (SI) 13
Bromadiolone (EX) 388
Bromazepam (EX) 388, 398, 495, 499
Bromocriptine (EX) 233, 388
4-Bromo-2,5-dimethoxyphenylethylamine (EX)
498
3-Bromoprimaquine diphosphate (IS) 96
5-Bromouracil (SI) 173
Brompheniramine (EX) 7, 231, 253, 388, 400,
406, 499, 623; (IN) 496
Buclizide (EX) 388
Budesonide (SI) 20, 32, 145, 167, 170, 264, 271,
306, 414
Buflomedil (EX) 388
Bumetanide (EX) 388; (IS) 105, 671
Bunitrolol (EX) 434
Bupivacaine (AL) 98; (EX) 7, 231, 254, 400, 406,
623
Bupranolol (IS) 433
Buprenorphine (EX) 7, 231, 254, 388, 400, 406,
495, 499, 623
705
Buspirone (EX) 7, 231, 254, 388, 400, 406, 495,
499, 623
Butabarbital (EX) 495, 499; (IN) 178
1,4-Butanediol (IS) 528
Butethal (SI) 178
Butobarbital (EX) 388
Butofilolol (EX) 434
2-n-Butylmercaptobenzimidazole (IS) 103
Butyl methoxydibenzoylmethane (SI) 456
Butyl paraben (EX) 388; (IS) 577
N-Butylscopolammonium bromide (EX) 388
Cafedrine (EX) 388
Caffeic acid (EX) 388
Caffeine (AL) 98; (EX) 7, 231, 254, 384, 388, 400,
406, 495, 499, 623; (SI) 16, 173, 178, 202,
603, 614, 656
Camostat mesylate (SI) 188, 220, 522
Campesterol (SI) 585
Camphor benzalkonium methosulfate (SI) 456
Candesartan (EX) 671
Canrenoate (EX) 388
Capric acid (EX) 461; (SI) 463
Caproic acid (EX) 461
Caprylic acid (EX) 461
Captodiamine (EX) 388
Captopril (AL) 682; (EX) 388; (IS) 229
Carazolol (EX) 434, 525
Carbamazepine (EX) 7, 231, 254, 388, 400, 406,
495, 499, 623; (IN) 349; (IS) 431; (SI) 188,
220, 522
Carbaryl (EX) 250, 388
Carbenicillin (SI) 16, 603, 614
Carbimazole (EX) 388
Carbinoxamine (EX) 8, 231, 254, 388, 401, 407,
624
Carbofuran (EX) 250; (SI) 354
Carboplatin (EX) 480
Carbovir (EX) 3
Carbutamide (EX) 388
Carisoprodol (EX) 8, 231, 254, 401, 407, 624
Carnitine (EX) 388
β-Carotene (EX) 22
Carpipramine (EX) 388
Carteolol (EX) 388, 434
Carvedilol (EX) 8, 231, 254, 401, 407, 426, 434,
624; (IS) 425
Cathinone (EX) 497, 529
Cefaclor (EX) 113, 118, 257
Cefadroxil (EX) 388
Cefamandole (EX) 113, 118, 257; (SI) 552
Cefatrizine (EX) 388
Cefazolin (EX) 113, 118, 257, 388
Cefbuperazone (EX) 118, 257
Cefixime (EX) 113, 118, 257, 388
Cefmenoxime (EX) 113, 118, 257
Cefmetazole (EX) 113, 118, 257
Cefoperazone (EX) 113, 118, 257
Cefotaxime (EX) 113, 118, 257; (IS) 114; (SI) 552
Cefotetan (EX) 113, 118, 257
706
Cross-Index to Other Substances
Cefotiam (EX) 113, 118, 257; (SI) 16, 603, 614
Cefpiramide (EX) 113, 118, 257
Cefpirome sulfate (IS) 116
Cefpodoxime (EX) 388
Cefsulodin (EX) 113, 118, 257
Ceftazidime (EX) 113, 118, 257; (SI) 552
Ceftizoxime (EX) 113, 118, 257
Ceftriaxone (EX) 113, 118, 257, 388; (SI) 552
Cefuroxime (EX) 113, 118, 257, 388; (SI) 552
Cefuzonam (EX) 113, 257
Celecoxib (IS) 567
Celiprolol (EX) 8, 231, 254, 388, 401, 407, 434,
624
Cephalexin (EX) 113, 118, 257, 388
Cephaloglycine (EX) 113, 118, 257
Cephaloridine (EX) 113, 118, 257
Cephalothin (EX) 113, 118, 257; (IS) 682
Cephradine (SI) 552
Cetirizine (EX) 8, 231, 254, 388, 401, 407, 624
Chlorambucil (EX) 388
Chloramphenicol (EX) 388, 495, 499; (IS) 258;
(SI) 178
Chlorcyclizine (EX) 8, 231, 254, 401, 407, 624
Chlordiazepoxide (EX) 8, 231, 254, 388, 401, 407,
495, 499, 624; (SI) 383
Chlorhexidine (EX) 388
Chloridazone (EX) 388
Chlormadinone (EX) 375, 388
Chlormethiazole (EX) 495; (IN) 499
Chlormezanone (EX) 8, 231, 254, 388, 401, 407,
624
Chlorobenzoic acid (EX) 388
Chloromadinone acetate (EX) 269, 376
Chlorophacinone (EX) 388
4-Chlorophenylbiguanide (EX) 388
m-Chlorophenylpiperazine (EX) 378, 537
Chloropicrin (EX) 388
Chloroquine (EX) 8, 231, 254, 388, 401, 407, 624;
(SI) 95
Chlorotestosterone acetate (EX) 269, 376
8-Chlorotheophylline (IS) 173
Chlorothiazide (SI) 178
5-Chlorouracil (SI) 173
Chlorpheniramine (EX) 8, 231, 254, 388, 401,
407, 499, 624; (IN) 496; (SI) 331
Chlorpromazine (EX) 8, 231, 254, 388, 401, 407,
495, 499, 525, 624; (IS) 383; (SI) 16, 194,
603, 614
Chlorpropamide (EX) 8, 231, 254, 388, 401, 407,
624; (SI) 178
Chlorprothixene (EX) 8, 231, 254, 401, 407, 624;
(SI) 537
Chlorpyrifos (SI) 354
Chlortetracycline (EX) 592
Chlortoluron (EX) 388
Chlorzoxazone (SI) 642
Cholesterol (SI) 585
Cibenzoline (EX) 388
Cicletanine (EX) 388
Cilazapril (EX) 388; (SI) 104, 188, 220, 522
Cimetidine (EX) 388, 495, 499; (SI) 188, 202,
220, 522
Cinchonine (EX) 388
Cinerin (SI) 534
Cinerin I (SI) 533
Cinerin II (SI) 533
Cinnarizine (EX) 8, 231, 254, 388, 401, 407, 624;
(IS) 358
Cinoxacin (EX) 365
Ciprofibrate (EX) 388
Ciprofloxacin (EX) 365, 388, 473, 650; (IN) 364;
(IS) 294; (SI) 293, 472
Cisapride (EX) 388; (IS) 194, 640
Citalopram (EX) 8, 231, 254, 401, 407, 495, 499,
537, 624; (SI) 399
Citric acid (EX) 371
Clarithromycin (SI) 267
Clemastine (EX) 8, 231, 254, 401, 407, 624
Clenbuterol (EX) 388; (IS) 304, 473
Clidinium bromide (EX) 330, 381, 388; (SI) 331,
382
Clindamycin (EX) 352, 353, 388; (SI) 251, 252,
516
Clobazam (EX) 8, 231, 254, 388, 401, 407, 495,
499, 624
Clobenzorex (EX) 388
Clobetasol 17-propionate (SI) 18, 20, 30, 32, 167,
170, 264, 271, 306
Clobetasone butyrate (SI) 18, 30, 143
Clobutinol (EX) 8, 231, 254, 401, 407, 624
Clocinizine (EX) 388
Clodronate (SI) 441
Clofazimine (SI) 627
Clofibrate (EX) 388; (SI) 188, 220, 522
Clomethiazole (EX) 8, 231, 254, 388, 401, 407,
624
Clomipramine (EX) 8, 231, 254, 388, 401, 407,
495, 499, 537, 624
Clonazepam (EX) 8, 231, 254, 388, 401, 407, 495,
499, 624; (SI) 188, 220, 522
Clonidine (EX) 8, 231, 254, 388, 401, 407, 495,
499, 624; (IS) 423; (SI) 45
Cloperastine (EX) 495, 499
Cloranolol (EX) 434
Clorazepate (EX) 388
Clotiazepam (EX) 388
Clotrimazole (EX) 273
Clovoxamine (EX) 378
Cloxacillin (EX) 388
Cloxazolam (EX) 495, 499
Clozapine (EX) 8, 231, 254, 401, 407, 499, 537,
624; (IN) 496
Cocaine (EX) 8, 231, 254, 388, 401, 407, 495,
499, 624
Codeine (AL) 98; (EX) 8, 231, 254, 388, 401, 407,
495, 499, 624; (IS) 290; (SI) 202
Codethyline (EX) 388
Colchicine (EX) 388; (SI) 178
Colchicoside (EX) 388
Colchicosine (EX) 388
Cross-Index to Other Substances
Corticosterone (EX) 169, 171
Cortisone (EX) 169; (SI) 18, 20, 30, 32, 143, 145,
163, 167, 170, 178, 261, 264, 271, 306
Cortisone 21-acetate (SI) 19, 20, 30, 32, 143, 145,
167, 170, 264, 271, 306
Cortivazol (EX) 388
Cotinine (EX) 388
Coumachlor (EX) 388
Coumafen (EX) 388
Coumarin (SI) 178
Coumatetralyl (EX) 8, 231, 254, 388, 401, 407,
624
m-Cresol (SI) 654
Crimidine (EX) 388
Cromolyn (SI) 16, 603, 614
Cyamemazine (EX) 388
Cyclandelate (EX) 388
Cyclizine (EX) 8, 231, 254, 401, 407, 624
Cycloguanil (EX) 388
Cyclohexymethylguanidine (IS) 305
Cyclophosphamide (SI) 188, 220, 522
Cyclothiazide (SI) 178
Cyfluthrin (AL) 25; (EX) 26, 80, 535
Cyhalothrin (EX) 155
Cyheptamide (SI) 178
Cypermethrin (AL) 26; (SI) 24
Cyproheptadine (EX) 388
Cyromazine (EX) 388
Dacarbazine (EX) 388
Dalfopristin (EX) 540; (SI) 541
Danazol (SI) 178
Danofloxacin (EX) 365, 473, 650; (SI) 364; 472
Danthron (SI) 178
Dapsone (EX) 388; (IN) 304; (SI) 178, 627
Deacetylalacepril (EX) 17
Decanol (EX) 380
Deethylzaleplon 674
Deflazacort (EX) 261
Dehydrocorticosterone (SI) 19, 30, 143
Delapril (SI) 188, 220, 522
Delavirdine (EX) 208, 435, 443; (IN) 36
Delmadinone acetate (EX) 269, 376
Deltamethrin (EX) 155
Demoxepam (EX) 8, 231, 254, 401, 407, 624
3′ -Deoxy-2′ ,3′ -didehydrothymidine (IS) 215
11-Deoxycortisol (EX) 169, 260
Deoxycortone (EX) 168, 265
21-Deoxydexamethasone (EX) 168, 265
Deoxymethasone (SI) 19, 30, 144
Desethylatrazine (EX) 388
N-Desethyl-5-oxozaleplon (EX) 674
N-Desethylzaleplon (EX) 674
Desfluoroaprepitant (IS) 46, 47
Desipramine (EX) 378, 388, 495, 499, 537
Desmethylcitalopram (EX) 378; (SI) 399
Desmethylclomipramine (EX) 378
Desmethyldoxepin (EX) 378
Desmethylmaprotiline (EX) 378
Desmethylmianserin (EX) 378
707
Desmethylmirtazapine (SI) 399
Desmethylsertraline (EX) 378
Desmethyltrimipramine (EX) 378
Desmethylvenlafaxine (EX) 537; (SI) 399
Desonide (SI) 20, 32, 145, 167, 170, 265, 271, 306
Desoximetasone (EX) 265; (SI) 20, 32, 145, 167,
170, 264, 271, 306
Desoxy-2-phenobarbital (EX) 388
Desoxycorticosterone acetate (SI) 20, 32, 145,
167, 264, 271, 306
Desoxycorticosterone pivalate (SI) 20, 32, 145,
167, 264, 271, 306
Detomidine (IS) 60, 369
Dexamethasone (EX) 168, 260, 265, 388; (SI) 19,
20, 30, 32, 144, 145, 167, 170, 188, 220, 264,
271, 306, 522
Dexamethasone 21-acetate (EX) 169; (SI) 19, 20,
31, 32, 144, 145, 167, 170; 264, 271, 306;
(IS) 414
Dexamethasone isonicotinate (SI) 19, 31, 144
Dexamethasone pivalate (SI) 19, 31, 144
Dexamethasone valerate (SI) 19, 31, 144
Dextromethorphan (EX) 8, 231, 254, 401, 407,
388, 624
Dextromoramide (EX) 388
Dextropropoxyphene (EX) 8, 231, 254, 388, 401,
407, 624; see also propoxyphene (EX) 495,
499
Dextrose (EX) 302, 303
DFdU (2′ ,2′ -difluorodeoxyuridine) (EX) 296
Diamorphine (AL) 98; (EX) 388
Diarachidoylphosphatidylcholine (EX) 502, 505
Diatrizoate (EX) 322, 325
Diaveridine (EX) 388
Diazepam (EX) 8, 231, 254, 388, 401, 407, 495,
499, 537, 624; (IS) 195, 539; (SI) 188, 194,
202, 220, 522
Diazinon (EX) 388
Dibekacin (EX) 388
Dibenzepin (IN) 537; (IS) 7, 230, 253, 400, 406,
623
Dibucaine (AL) 98
Dichlorisone (EX) 168, 265
Dichlorprop (EX) 388
Diclofenac (EX) 388; (SI) 47, 512, 547
Didanosine (EX) 1, 2, 335, 336; (SI) 2
Diethofencarb (EX) 250
Diethylstilbestrol (EX) 388; (SI) 178
Difemerine (EX) 388
Diflorasone 17,21-diacetate (SI) 20, 32, 145, 167,
170, 264, 271, 306
Difloxacin (EX) 569, 650; (SI) 364, 472
Diflucortolone valerate (SI) 19, 31, 144
2′ ,2′ -Difluorodeoxyuridine (dFdU) (EX) 296
Digitoxin (EX) 388
Digoxin (EX) 388; (SI) 188, 220, 522
Dihomo-gamma-linolenic acid (EX) 458
Dihydralazine (EX) 388
Dihydrobefunolol (EX) 70
Dihydrocodeine (EX) 388, 495, 499
708
Cross-Index to Other Substances
Dihydrostreptomycin (EX) 312
3,4-Dihydroxybenzylamine (IS) 348
Dilazep (SI) 188, 220, 522
Dilinoleylphosphatidylcholine (EX) 502, 505
Diltiazem (EX) 8, 231, 254, 388, 401, 407, 495,
499, 624; (IS) 494; (SI) 188, 220, 522
Dimefline (SI) 383
Dimerazole (EX) 388
3-Dimethylaminomethyleneimino-2,4,6triiodobenzoic acid
(IS) 393
N,N-Dimethylglycine (EX) 72
Dimethyl phthalate (EX) 388
2,2′ -Dimethylpropiophenone (IS) 533
Dimetridazole (EX) 388
Diminazene (EX) 316
2,4-Dinitrochlorobenzene (IS) 237
Dinoprost (EX) 460
Dinoprostone (EX) 460
Dinoseb (EX) 388
Dinsed (EX) 600
Dipalmitolyphosphatidylcholine 506
Diphacinone (EX) 388
Diphenhydramine (EX) 8, 231, 254, 401, 407,
495, 499, 624
Dipheniramine (EX) 388
Diphenylphosphate (IS) 284
Diphosphatidylglycerol (EX) 502, 505
Diprophylline (EX) 388
Dipropyline (EX) 388
Dipyridamole (EX) 8, 231, 254, 388, 401, 407,
624
Dipyrone (SI) 383
Disopyramine (EX) 8, 231, 254, 388, 401, 407,
495, 499, 624
Disulfiram (EX) 388
Di-syston (EX) 388
Dithiothreitol (EX) 588
Diuron (EX) 388
Dixyrazine (EX) 8, 231, 254, 401, 407, 624; (SI)
552
Docarpamine (SI) 220, 522
Docosahexaenoic acid (EX) 458, 459
Docosanoic acid (EX) 457
Dodecyl alcohol (SI) 129
Domperidone (EX) 388
Dopamine (IS) 394
Dosulepin (EX) 388
Dothiepin (EX) 495, 499
Doxapram (EX) 8, 231, 254, 401, 407, 624; (SI)
383
Doxazosin (EX) 189
Doxepin (EX) 8, 231, 254, 388, 401, 407, 495,
499, 537, 624
Doxorubicin (EX) 388
Doxylamine (EX) 388
Dronabinol (EX) 8, 232, 255, 402, 408, 624; (SI)
178
Droperidol (EX) 388
Dropropizine (EX) 388
Ebastine (EX) 8, 231, 254, 401, 407, 624
Econazole (EX) 388; (IS) 251
EDDP (EX) 388
Efavirenz (EX) 36, 38, 360, 435, 436, 443, 444
Eicosanoic acid (EX) 457
Eicosapentaenoic acid (EX) 458, 459
Eicosapentanoic acid (EX) 457
Eicosatrienoic acid (EX) 457
Eicosyl alcohol (SI) 129
Elaidic acid (EX) 459
Embutramide (EX) 8, 231, 254, 388, 401, 407,
624
Emetine (EX) 388
Emtricitabine diphosphate (EX) 216
Emtricitabine monophosphate (EX) 216
Emtricitabine triphosphate (EX) 216
Enalapril (EX) 388; (IS) 7, 230, 253, 400, 406,
623; (SI) 16, 603, 614
Enalaprilat (AL) 682
Endorphins (SI) 14
Enoxacin (EX) 365, 388; (SI) 47, 512, 547
Enrofloxacin (EX) 365, 473, 650; (SI) 364, 421,
472, 570
Eperisone (SI) 188, 522
Ephedrine (AL) 98; (EX) 388, 495, 497, 499, 529
Epigalantamine (EX) 291
Epinephrine (AL) 98; (EX) 348, 389, 498
Ergotamine (EX) 8, 254, 401, 407, 624
Erythritol (EX) 302
Erythromycin (EX) 495, 499; (SI) 267
Esculin (EX) 389
Eserine (EX) 389
Esmolol (EX) 434
Estazolam (EX) 389
Estradiol (EX) 169; (SI) 178
17α-Estradiol (IS) 558
β-Estradiol (EX) 389
Estriol (EX) 169, 389; (SI) 178
Estrone (EX) 169; (SI) 178
Ethanolamine (SI) 656
Ethazolastone (IS) 615, 616
Ethenzamide (EX) 8, 231, 254, 401, 407, 624
Ethinyl estradiol (EX) 300; (SI) 239
Ethiofencarb (EX) 250
Ethosuximide (EX) 389; (SI) 178, 188, 220, 522
4-Ethoxyphenol (IS) 384
3-(4′ -Ethylbenzylidene)-d,l-camphor (SI) 456
Ethylene glycol (EX) 302
Ethylmorphine (AL) 98; (EX) 8, 231, 254, 401,
407, 624
Ethyl paraben (EX) 389; (IS) 160
Ethylparathion (EX) 8, 231, 254, 401, 407, 624
Etidocaine (AL) 98; (IS) 54
Etidronate (SI) 441
Etodolac (EX) 389; (IS) 162
Etodroxizine (EX) 8, 231, 254, 401, 407, 624
Eugenol (SI) 178
Famciclovir (EX) 489
Famotidine (EX) 389
Cross-Index to Other Substances
Febantel (EX) 103
Felodipine (EX) 8, 231, 254, 389, 401, 407, 624
Fenazepam (EX) 8, 231, 254, 401, 407, 624
Fenbendazole (EX) 102, 103
Fenbufen (EX) 389; (SI) 188, 220, 522
Fenfluramine (EX) 8, 231, 254, 378, 389, 401,
407, 497, 498, 499, 529, 624; (IN) 496
Fenkamfamine (EX) 8, 231, 254, 401, 407, 624
Fenobucarb (EX) 250
Fenofibrate (EX) 389; (SI) 47, 512, 548
Fenoprofen (EX) 389; (SI) 178
Fenoverine (EX) 389
Fenozolone (EX) 389
Fenpiverinium bromide (IS) 331
Fenpropathrin (AL) 26
Fenproporex (EX) 389
Fentanyl (EX) 8, 231, 254, 389, 401, 407, 495,
499, 624
Fenvalerate (AL) 26; (EX) 26, 80, 535
Fexofenadine (EX) 8, 231, 401, 407, 624
Finasteride (SI) 47, 512, 548
Flavone (EX) 389; (IS) 480
Flecaine (EX) 389
Flecainide (EX) 8, 231, 254, 401, 407, 495, 499,
624
Fleroxacin (EX) 650
Floctafenine (EX) 389
Flomoxef (EX) 113, 118
Flubendazole (EX) 103, 389
Fluclorolone acetonide (EX) 168, 265
Fluconazole (EX) 8, 231, 254, 389, 401, 407, 624;
(SI) 188, 220, 522
Flucytosine (EX) 389
Fludrocortisone (EX) 168, 265; (IS) 260
Fludrocortisone 21-acetate (IS) 163, 261; (SI) 19,
20, 31, 32, 144, 145, 167, 170, 264, 271, 306
Flumazenil (EX) 8, 231, 254, 401, 407, 495, 499,
624
Flumequine (EX) 365, 473, 592
Flumethasone (EX) 168, 265
Flumethasone 21-pivalate (SI) 19, 20, 31, 32,
144, 145, 167, 170, 264, 271, 306
Flumethrin (EX) 155
Flunarizine (EX) 358, 389
Flunindione (EX) 389
Flunisolide (SI) 20, 32, 145, 167, 170, 264, 271,
306
Flunitrazepam (EX) 8, 231, 254, 389, 401, 407,
499, 624; (IN) 36, 496
Fluocinolone acetonide (EX) 168, 265; (SI) 19,
20, 31, 32, 144, 145, 167, 170, 264, 271, 306
Fluocinonide (EX) 168, 265; (SI) 19, 20, 31, 32,
144, 145, 167, 170, 264, 271, 306
Fluocortin butyl ester (SI) 19, 31, 144
Fluocortolone (EX) 168, 265
Fluocortolone caproate (SI) 19, 31, 144
Fluocortolone pivalate (SI) 19, 31, 144
6-Fluoroalosetron (IS) 29
5-Fluorocytosine (SI) 173
709
Fluorometholone (EX) 168, 265; (SI) 19, 20, 31,
32, 144, 145, 167, 170, 264, 271, 306
9-α-Fluoroprednisolone (SI) 19, 31, 144
9-α-Fluoroprednisolone acetate (SI) 19, 31, 144
5-Fluorouracil (EX) 389; (SI) 173, 178
Fluoxetine (EX) 8, 231, 254, 378, 389, 401, 407,
495, 499, 537, 624; (IS) 498; (SI) 399
Fluoxymesterone (EX) 427, 587; (SI) 178
Flupentixol (EX) 8, 231, 254, 389, 401, 407, 495,
499, 624
Fluphenazine (EX) 389, 495, 499; (SI) 383, 537
Fluprednisolone (EX) 168, 265; (SI) 20, 32, 145,
167, 170, 264, 271, 306
Flurandrenolide (EX) 168; (SI) 19, 20, 31, 32,
144, 145, 167, 170, 271, 306
Flurazepam (EX) 495, 499
Flurbiprofen (EX) 389
Flurogestone acetate (EX) 376
Flutamide (EX) 389; (SI) 188, 220, 522
Fluticasone 17-propionate (SI) 20, 32, 145, 167,
170, 264, 306, 414
Fluvoxamine (EX) 8, 231, 254, 378, 389, 401,
407, 495, 499, 624; (IN) 537; (SI) 399
Folic acid (EX) 389; (SI) 2
Fominoben (SI) 188, 220, 522
Fosinopril (AL) 682
Fosinoprilat (EX) 283
FTU (EX) 215
Fumaric acid (EX) 389
Furaltadone (EX) 389
Furazolidone (EX) 389, 601
Furosemide (EX) 389, 495, 499; (SI) 178
Fusidic acid (EX) 389
Ganciclovir (SI) 2, 668
Gentamicin (EX) 312, 389
Gitoxigenin (SI) 178
Glibenclamide (EX) 8, 231, 254, 389, 401, 407,
624
Gliclazide (EX) 389
Glipizide (EX) 8, 231, 254, 389, 401, 407, 624
Glutathione (EX) 389
Glutethimide (EX) 495, 499; (SI) 178
Glycopyrrolate (IS) 136; (SI) 331
Gonadorelin (LHRH) (SI) 13
Grepafloxacin (SI) 293
Griseofulvin (EX) 389
Guaiacol (EX) 389; (SI) 178
Guaifenesin (EX) 389, 495, 499; (IS) 641
Guanfacine (EX) 389
Halcinonide (SI) 19, 20, 31, 32, 144, 145, 167,
170, 264, 271, 306
Halobetasol propionate (SI) 20, 32, 145, 167,
170, 264, 271
Halofantrine (EX) 389
Haloperidol (EX) 8, 231, 254, 389, 401, 407, 495,
499, 525, 624; (SI) 537
Harpagoside (EX) 389
710
Cross-Index to Other Substances
Hematoporphyrin (EX) 389
Heptadecanoic acid (IS) 462
Heptanoic acid (EX) 461
Hexachlorobenzene (SI) 354
Hexobarbital (IS) 335; (SI) 178
Hippuric acid (SI) 178
Histapyrrodine (EX) 8, 231, 254, 401, 407, 624
Histidine (EX) 389
Homosalate (SI) 456
Hydralazine (EX) 495, 499
Hydrochlorothiazide (EX) 104, 389, 672
Hydrocodone (EX) 8, 231, 254, 401, 407, 624
Hydrocortisone (EX) 168, 169, 260, 265, 389; (SI)
19, 20, 31, 32, 144, 145, 163, 167, 170, 178,
261, 264, 271, 306, 414
Hydrocortisone 21-acetate (SI) 19, 20, 31, 32,
144, 145, 167, 170, 188, 264, 271, 306, 522
Hydrocortisone-17-butyrate (SI) 19, 20, 31, 32,
144, 145, 167, 170, 264, 271, 306
Hydrocortisone 21-cypionate (SI) 20, 32, 145,
167, 170, 264, 271, 306
Hydrocortisone pivalate (SI) 19, 31, 144
Hydrocortisone 17-valerate (SI) 20, 32, 145, 167,
170, 264, 272, 306
Hydroflumethiazide (SI) 104
Hydroquinone (SI) 178
p-Hydroxybestatin (EX) 662
4-Hydroxybutyrate (EX) 389
Hydroxychloroquine (EX) 8, 231, 254, 401, 407,
624
21-Hydroxydeflazacort (EX) 163, 261
18-Hydroxydeoxycorticosterone (EX) 171
5-Hydroxyindole-3-acetic acid (IS) 77
10-Hydroxynortriptyline (EX) 378
Hydroxyprogesterone (EX) 169
9-Hydroxyrisperidone (EX) 537
3-Hydroxytyramine (EX) 389
Hydroxyzine (EX) 8, 231, 254, 389, 401, 407,
495, 499, 624
Hyoscyamine (EX) 389
Hypoxanthine (IS) 668
Ibuprofen (EX) 389, 495, 499; (SI) 178
Iloprost (EX) 405
Imidacloprid (EX) 452
Imidapril (SI) 188, 220, 522
Imidazole (EX) 273, 389
Imipramine (EX) 8, 231, 254, 389, 401, 407, 495,
499, 537, 624; (IN) 202; (IS) 403; (SI) 383
Inchonidine (EX) 389
Indenolol (EX) 434
Indinavir (AL) 38; (EX) 36, 37, 38, 208, 209, 359,
360, 435, 436, 443, 444; (SI) 47, 512, 548,
627
Indomethacin (EX) 8, 231, 254, 389, 401, 407,
495, 499, 624; (IS) 341; (SI) 178, 188, 221,
522
Indoramine (EX) 389
Iodoform (EX) 389
Iohexol (EX) 324, 325, 326
Iothalamic acid (EX) 322
Iotrolan (EX) 325
Ioxaglic acid (EX) 322
Irbesartan (EX) 105, 671
Irinotecan (SI) 188, 221, 522
Isoamyl p-methoxycinnamate (SI) 456
Isoflupredone (EX) 168, 265
Isoniazid (EX) 8, 231, 254, 401, 407, 624; (SI) 627
Iso-pirlimycin (IS) 516
Isoprocarb (EX) 250
Isopropamide iodide (EX) 381; (SI) 382
Isopropyl dibenzoylmethane (SI) 456
Isoquercitrin (EX) 389
Isosorbide (EX) 389
Isosorbide dinitrate (EX) 490
Isothipendyl (EX) 389
Isotretinoin (EX) 21, 22; (SI) 23
Isoxicam (SI) 200
Isoxsuprine (IS) 544
Isradipine (EX) 8, 231, 254, 389, 401, 407, 624
Jasmolin (SI) 534
Jasmolin I (SI) 533
Jasmolin II (SI) 533
Josamycine (EX) 389
Ketamine (EX) 8, 231, 254, 389, 401, 407, 495,
624; (IN) 499, 552
Ketobemidone (EX) 8, 231, 254, 401, 407, 624
Ketoconazole (AL) 38; (EX) 389, 495, 499
Ketogan (IN) 552
Ketoprofen (EX) 8, 231, 254, 389, 401, 407, 624
Ketorolac (EX) 8, 231, 254, 401, 407, 624
Labetalol (EX) 8, 231, 254, 401, 407, 434, 495,
499, 624; (IS) 49, 626
Lacidipine (EX) 389
Lactic acid (EX) 389
Lamivudine (EX) 1, 2; (SI) 2, 627
Lamotrigine (EX) 8, 231, 254, 401, 407, 624
Lanolin (SI) 112
Lanzoprazole (EX) 389
Lasalocid (EX) 415, 429
Latamoxef (EX) 113, 118, 257
Laudanosine (EX) 137; (SI) 138
Lauric acid (EX) 457, 458, 459, 460, 461; (SI) 463
Levamisole (EX) 389
Levobunolol (EX) 434; (IS) 70
Levocabastine (EX) 8, 231, 254, 401, 407, 624
Levodopa (EX) 389
Levomepromazine (EX) 8, 231, 254, 389, 401,
407, 624; (SI) 194, 383
Levonordefrin (SI) 527
Levopenbutolol (EX) 389
Levothyroxine (EX) 357
Levrazoxane (EX) 172
LHRH (gonadorelin) (SI) 13
Lidocaine (AL) 98; (EX) 8, 231, 254, 389, 401,
407, 495, 499, 624; (IN) 552; (IS) 133
Cross-Index to Other Substances
Lincomycin (SI) 516
Linoleic acid (EX) 457, 458, 459, 460, 461, 462;
(SI) 463, 557
Linolenic acid (EX) 457, 458, 459, 460, 461, 462;
(SI) 463, 557
Gamma-linolenic acid (EX) 457
Linuron (EX) 389
Lipotropic hormone (SI) 14
Lisinopril (SI) 104
Lisuride (EX) 233, 389
Lithium (SI) 656
Lobeline (EX) 389
Loflazepate (EX) 389
Lomefloxacin (EX) 650; (IS) 420, 487
Lomustine (EX) 389
Loperamide (SI) 16, 603, 614
Lopinavir (EX) 37, 38, 209, 436, 444
Loprazolam (EX) 389
Loratadine (EX) 8, 164, 165, 231, 254, 389, 401,
407, 624
Lorazepam (EX) 8, 231, 254, 389, 401, 407, 495,
499, 624
Lormetazepam (EX) 8, 231, 254, 401, 407, 495,
499, 624
Losartan (EX) 105, 671; (IS) 124
Loxapine (EX) 389, 495, 499
LSD (EX) 8, 231, 254, 389, 401, 407, 624
Lufenuron (EX) 389
Luteinizing hormone (SI) 134
Lysophosphatidylcholine (EX) 502, 505
Magnesium (SI) 656
Malathion (EX) 8, 231, 254, 401, 407, 624
Malic acid (SI) 140
Maprotiline (EX) 8, 231, 254, 378, 389, 401, 407,
495, 499, 537, 624; (SI) 188, 221, 522
Marbofloxacin (SI) 472
Margaric acid (EX) 458, 460; (IS) 457, 459, 461,
557
MCPA (EX) 389
MDA (EX) 389, 497, 529; (IN) 499
MDEA (EX) 497, 529
MDMA (EX) 8, 231, 254, 389, 401, 407, 495, 499,
529, 624; (IN) 499
MDPA (EX) 389
Mebendazole (EX) 102, 103, 389
Mebezonium (EX) 389
Meclozine (EX) 8, 231, 254, 389, 401, 407, 624
Mecoprop (EX) 389
Medazepam (EX) 8, 231, 254, 389, 401, 407, 624
Medetomidine (EX) 60, 61, 389
Medroxyprogesterone (EX) 389
Medroxyprogesterone acetate (EX) 269, 376
Mefenamic acid (IS) 676; (SI) 178
Mefenorex (EX) 389
Mefloquine (EX) 389
Megasol (SI) 456
Megestrol (EX) 375, 389
Megestrol acetate (EX) 269, 376
Meglutol (EX) 139
711
Melanotropin (SI) 14
Melatonin (EX) 389
Melengestrol acetate (EX) 269
Meloxicam (EX) 8, 231, 254, 401, 407, 624
Melperone (EX) 8, 231, 254, 401, 407, 537, 624;
(IS) 537
Melphalan (EX) 389
Menotropins (SI) 14
Mepenzolate bromide (EX) 330; (SI) 331
Meperidine (pethidine) (EX) 8, 231, 254, 389,
401, 407, 495, 499, 624; (SI) 552
Mepindolol (EX) 434
Mepivacaine (EX) 8, 54, 231, 254, 401, 407, 624
Meprednisone (SI) 20, 32, 145, 167, 170, 264,
272, 306
Meprobamate (EX) 8, 231, 254, 401, 407, 624
Meropenem (IS) 234
Mesalamine (EX) 389
Mesoridazine (EX) 8, 231, 254, 401, 407, 496,
499, 624
Mestranol (SI) 239
Metamitron (EX) 389
Metaproterenol (EX) 389
Metformin (EX) 389, 512
Methacycline (EX) 389
Methadone (AL) 38; (EX) 8, 231, 254, 389, 401,
407, 496, 499, 624
Methamphetamine (EX) 8, 231, 254, 389, 401,
407, 495, 497, 529, 624; (IN) 499
Methandienone (IS) 82
Methandrostenolone (EX) 427, 587
Methaqualone (EX) 495, 499; (IS) 673
Methiocarb (EX) 250
Methionine (EX) 389
Methocarbamal (SI) 178, 642
Metholcarb (EX) 250
Methoprene (SI) 533
Methoprene acid (EX) 386
Methotrexate (SI) 188, 221, 522
p-Methoxylevodropropizine (IS) 198
Methoxyselamectin (IS) 571
Methyclothiazide (EX) 389
Methylbenacyzine (SI) 331
Methyldopa (SI) 178, 389, 394
3-Methyldopamine (EX) 389
4-Methyldopamine (EX) 389
Methylene blue (SI) 187
Methylephedrine (EX) 495, 499
7-Methyleptazocine (IS) 227
Methylhydroxyprogesterone (EX) 389
3-Methylleflunomide (EX) 341
5-Methyl-2-nitrophenol (IS) 255
Methyl paraben (EX) 389; (IS) 512, 676; (SI) 178,
331
Methylparathion (EX) 8, 231, 254, 401, 407, 624
Methylphenidate (EX) 8, 231, 254, 401, 407, 496,
499, 624
Methylprednisolone (EX) 168, 260, 265, 389; (SI)
19, 20, 31, 32, 144, 145, 167, 170, 264, 270,
306
712
Cross-Index to Other Substances
Methylprednisolone 21-acetate (SI) 20, 32, 145,
167, 170, 264, 272, 306
3-Methylpyrazole (IS) 274
Methyl salicylate (SI) 178
Methyltestosterone (EX) 427, 587; (IS) 652; (SI)
178
Metipranolol (EX) 434
Metoclopramide (EX) 8, 231, 254, 318, 384, 389,
401, 407, 495, 624; (IN) 499, 552
Metocurine (SI) 552
Metoprolol (EX) 8, 231, 254, 389, 401, 407, 434,
495, 499, 624; (SI) 552
Metronidazole (EX) 8, 231, 254, 389, 401, 407,
624
Mexiletine (EX) 8, 231, 254, 389, 401, 407, 624;
(IS) 509
Mianserin (EX) 8, 231, 254, 389, 401, 407, 495,
499, 537, 624
Miconazole (EX) 389; (IS) 252
Midazolam (EX) 8, 60, 231, 254, 369, 389, 401,
407, 495, 499, 624
Mifepristone (IS) 301
Minaprin (EX) 389
Minocycline (EX) 389
Minoxidil (EX) 389
Mirtazapine (EX) 8, 231, 254, 407, 537, 624; (SI)
399
Misoprostol (EX) 313
Mitomycin C (EX) 89
Mivacurium (EX) 137; (SI) 589
Mizolastine (EX) 8, 231, 254, 401, 624
Moclobemide (EX) 8, 231, 254, 389, 401, 407,
495, 499, 537, 624
Molindone (EX) 8, 231, 254, 401, 407, 495, 499,
624
Molsidomine (EX) 389
Mometasone (EX) 413
Mometasone 17-furoate (SI) 19, 20, 31, 32, 144,
145, 167, 170, 264, 272, 306
Monensin (EX) 429
Monoacetylmorphine (AL) 98; (EX) 8, 231, 254,
401, 407, 624
Monodesbutylhalofantrine (EX) 389
Monodesethylchloroquine (EX) 389
Moprolol (EX) 434
Moroxydine (EX) 389
Morphine (AL) 98; (EX) 8, 231, 254, 389, 401,
407, 495, 499, 624
Moxifloxacin (SI) 293
Myristic acid (EX) 457, 458, 459, 460, 461, 462;
(SI) 463, 557
Myristoleic acid (EX) 458, 459, 460, 461
Nadolol (EX) 389, 434, 495, 499
Nadoxolol (EX) 389
Naftidrofuryl (EX) 389
Naftopidil (IS) 426
Nalidixic acid (EX) 365, 389, 473
Nalorphine (EX) 389
Naloxone (EX) 389, 495, 499
Naltrexone (EX) 389
Nandrolone (EX) 587
2-Naphthoic acid (IS) 313, 405
Naphthol (SI) 354
Naproxen (EX) 495, 499; (IS) 147
Narasin (EX) 415
Nefazodone (EX) 537
Nefiracetam (SI) 188, 221, 522
Nefopam (EX) 389
Nelfinavir (AL) 38; (EX) 36, 37, 38, 208, 209,
281, 359, 360, 435, 443, 444
Nemonapride (EX) 389
Neomycin (EX) 312
Neopynamine (EX) 389
Neostigmine (EX) 389; (SI) 331
Netilmicin (EX) 389; (IS) 153
Nevirapine (EX) 1, 38, 208, 209, 335, 360, 435,
436; (SI) 2, 627
Niacin (EX) 389; (SI) 178
Niacinamide (EX) 389
Niaprazine (EX) 389
Nicarbazin (EX) 149
Nicardipine (EX) 390
Nicergoline (EX) 390
Niclosamide (EX) 390
Nicotine (EX) 8, 231, 254, 401, 407, 624
Nifedipine (EX) 8, 99, 231, 254, 390, 401, 407,
495, 499, 624; (IS) 48; (SI) 188, 221, 522
Nifenalol (EX) 434
Niflumic acid (EX) 390
Nifuroxazide (EX) 390; (IS) 450
Nikethamide (EX) 8, 231, 254, 390, 401, 407,
624; (SI) 383
Nimesulide (IS) 556
Nimodipine (IN) 552
Nitrazepam (EX) 8, 231, 254, 390, 401, 407, 495,
499, 624; (SI) 188, 194, 221, 522
Nitrendipine (EX) 390
o-Nitroacetanilide (IS) 77
Nitrofural (EX) 390
Nitrofurantoin (SI) 178
Nitrofurazone (EX) 601
Nitroglycerin (EX) 490
Nitromide (EX) 601
Nizatidine (EX) 8, 231, 254, 390, 401, 407, 624
Nomifensine (EX) 8, 231, 254, 401, 407, 624
Nonanoic acid (EX) 461
Nonanol (EX) 380
Noramidopyrine (EX) 390
Norclomipramine (EX) 390, 537
Norclozapine (EX) 378, 537
Nordiazepam (EX) 398, 495, 499
Nordoxepin (EX) 537
Norephedrine (EX) 390
Norepinephrine (EX) 348, 390, 498; (SI) 527
Norethisterone (EX) 390
Norfenfluramine (EX) 378, 497, 529; (S) 529
Norfloxacin (EX) 365, 650; (IN) 364; (IS) 472, 569
Norfluoxetine (EX) 378, 495, 499, 537; (SI) 399
Norgalantamine (EX) 290
Cross-Index to Other Substances
Norgestrel (EX) 390; (IS) 243
Normaprotiline (IN) 537
Normethsuximide (SI) 178
Normirtazapine (SI) 537
Norpropoxyphene (EX) 390
17α-Nortestosterone (EX) 428
Nortriptyline (EX) 8, 231, 254, 378, 390, 401,
407, 495, 499, 537, 624
Norverapamil (EX) 8, 231, 254, 401, 407, 624
Noscapine (EX) 8, 231, 254, 390, 401, 407, 624
Octanol (EX) 380
Octocrylene (SI) 456
Octyl dimethyl PABA (SI) 456
Octyl methoxycinnamate (SI) 456
Octyl salicylate (SI) 456
Octyl triazone (SI) 456
Ofloxacin (EX) 365, 390, 495, 499, 650; (SI) 16,
293, 603, 614
Olanzapine (EX) 8, 231, 254, 401, 407, 624; (SI)
537
Oleandomycin (SI) 267
Oleic acid (SI) 557
Olpadronate (SI) 441
Omeprazole (EX) 390; (SI) 202
Ondansetron (EX) 8, 231, 254, 401, 407, 624
Opiclone (EX) 390
Opipramol (EX) 390; (IN) 537
Orbifloxacin (SI) 364
Orphenadrine (EX) 8, 231, 254, 401, 407, 624
Oxacillin (EX) 390
Oxalic acid (SI) 140
Oxamyl (EX) 250
Oxatomide (EX) 390
Oxazepam (EX) 8, 231, 254, 390, 401, 407, 499,
624; (IN) 496
Oxazolam (EX) 499; (IN) 496
Oxcarbazepine (EX) 8, 232, 254, 401, 407, 624
Oxfendazole (EX) 102, 103
Oxibendazole (EX) 102, 103
2-Oxoglutaric acid (EX) 139, 371
Oxolinic acid (EX) 365, 473
5-Oxozaleplon (EX) 674
Oxprenolol (EX) 8, 232, 254, 390, 401, 407, 434,
624
Oxycodone (EX) 8, 232, 254, 401, 407, 495, 499,
624
Oxymorphone (EX) 495, 499
Oxyphenbutazone (SI) 178
Oxytetracycline (EX) 592
Palmitic acid (EX) 457, 458, 459, 460, 461, 462;
(SI) 463, 557
Palmitoleic acid (EX) 457, 458, 459, 460, 461,
462; (SI) 463, 557
Pamidronate (SI) 441
Pancuronium (EX) 137, 390
Pantothenic acid (Vitamin B5 ) (EX) 391
Papaverine (EX) 8, 232, 254, 390, 401, 407, 624
713
Paramethasone 21-acetate (SI) 20, 32, 145, 167,
170, 264, 272, 306
Paraxanthine (SI) 178
Parbendazole (EX) 102, 390
Parconazole (EX) 390
Paroxetine (EX) 8, 232, 254, 378, 390, 401, 407,
495, 499, 537, 624; (SI) 399
Pefloxacin (EX) 390
Pemoline (EX) 8, 232, 254, 401, 407, 624
Penbutolol (EX) 434; (IS) 473
Penfluridol (EX) 390
Pentaerythritol (EX) 390
Pentazocine (EX) 8, 232, 254, 390, 401, 407, 499,
624; (IN) 496; (IS) 242
Pentienate bromide (SI) 331
Pentifylline (EX) 8, 232, 254, 401, 407, 624
Pentobarbital (EX) 390, 495, 499; (SI) 178, 188,
221, 522
Pentoxifylline (EX) 390
Pentoxyverine (EX) 8, 232, 255, 390, 401, 407,
624
1-(4-Perfluorobutylphenyl)-1-hexanone (IN) 492
Pericyazine (EX) 495, 499
Perindopril (EX) 390
Permethrin (AL) 25; (EX) 26, 80, 386, 535; (SI)
24
Perphenazine (EX) 8, 232, 255, 390, 401, 407,
495, 499, 624
Pethidine (meperidine) (EX) 8, 231, 254, 389,
401, 407, 495, 499, 624; (SI) 552
Phenacetin (IS) 120, 204, 627
Phenazone (EX) 8, 232, 255, 401, 407, 624; (IS)
240
Phenazopyridine (EX) 495, 499
Phencyclidine (EX) 9, 232, 255, 401, 407, 495,
499, 624
Phenindione (EX) 390
Pheniramine (EX) 9, 232, 255, 401, 407, 495,
499, 624
Phenobarbital (EX) 390, 495, 499; (SI) 188, 221,
522
Phenol (EX) 390; (SI) 492
Phenolphthalein (EX) 499; (IN) 496
Phenoprolamine (IS) 574
Phenothrin (EX) 26, 80, 535
m-Phenoxybenzoic acid (EX) 386
m-Phenoxybenzyl alcohol (EX) 26, 80, 386, 535
Phenoxyethanol (EX) 390
Phentermine (EX) 495, 499, 529
Phenylbenzimidazole sulfonic acid (SI) 456
Phenylbutazone (EX) 9, 232, 255, 390, 402, 408,
624; (SI) 178
Phenylephrine (EX) 495, 499
2-Phenylethylamine (EX) 498, 529
Phenylethyleneglycol (SI) 173
Phenylpropanolamine (EX) 9, 232, 255, 402, 408,
495, 497, 499, 529, 624; (SI) 331
Phenyltoloxamine (EX) 495, 499
Phenytoin (EX) 9, 232, 255, 285, 390, 402, 408,
495, 499, 624; (SI) 188, 221, 522
714
Cross-Index to Other Substances
Phloroglucinol (EX) 390
Pholcodine (EX) 390, 495, 499
Phorate (EX) 390
Phosdrin (EX) 390
Phosphatidic acid (EX) 502, 505
Phosphatidylcholine (EX) 504, 505; (SI) 505
Phosphatidylethanolamine (EX) 501, 502, 504,
505
Phosphatidylglycerol (EX) 502
Phosphatidylinositol (EX) 502, 504, 505
Phosphatidylserine (EX) 505, 504, 505
9-[2-(Phosphonomethoxy)ethyl]adenine (PMEA)
(IS) 618
Phthalic acid (EX) 390
Pilocarpine (EX) 390
Pimaricin (EX) 390
Pimozide (EX) 390, 495, 499; (SI) 537
Pinaverium (EX) 390
Pinazepam (EX) 495, 499
Pindolol (EX) 9, 232, 255, 390, 402, 408, 434,
625; (IS) 631, 683; (SI) 188, 221, 522
Pioglitazone (IS) 566; (SI) 47, 548
Pipamperone (EX) 390
Pipecuronium (IS) 589
Pipemidic acid (EX) 650
Pipenzolate (SI) 331, 382
Piperacillin (EX) 390
Piperine (EX) 390
Piperonyl butoxide (EX) 390; (SI) 178, 533
Pipothiazine (EX) 390
Piracetam (EX) 390
Piretanide (EX) 390
Pirimicarb (EX) 250
Pirisudanol (EX) 390
Pirlimycin sulfone (EX) 516
Pirlimycin sulfoxide (EX) 516
Piromidic acid (EX) 473
Piroxicam (EX) 9, 232, 255, 390, 402, 408, 495,
499, 625; (IS) 577; (SI) 200
Pitofenone (EX) 9, 232, 255, 402, 408, 625
Pivampicillin (EX) 390
Pizotifen (EX) 9, 232, 255, 390, 402, 408, 625
PMEA (9-[2-(phosphonomethoxy)ethyl]adenine)
(IS) 618
Porcine C-peptide (EX) 530
Porcine glucagon (EX) 530
Porcine monocomponent insulin (EX) 530
Porcine pancreatic polypeptide (EX) 530
Porcine proinsulin (EX) 530
Potassium (SI) 656
Practolol (EX) 9, 232, 255, 402, 408, 625
Pralidoxime (EX) 390
Pramipexole (EX) 605, 606
Pranlukast (SI) 188, 221
Pranoprofen (SI) 188, 221, 522
Prazosin (EX) 9, 232, 255, 390, 402, 408, 495,
499, 625
Prednicarbate (SI) 20, 32, 145, 167, 170, 264,
272, 306
Prednisolone (EX) 168, 260, 265, 390, 523; (SI)
20, 32, 145, 167, 170, 178, 188, 221, 264,
272, 306, 522
Prednisolone-21-acetate (SI) 19, 20, 31, 32, 144,
145, 170, 264, 272, 306
Prednisolone acetonide (SI) 19, 31, 144
Prednisolone 17-ethylcarbonate (EX) 523
Prednisolone 21-ethylcarbonate (EX) 523
Prednisolone pivalate (SI) 19, 31, 144
Prednisolone 21-tebuate (SI) 20, 32, 145, 167,
170, 264, 272, 306
Prednisone (EX) 168, 265, 390; (SI) 20, 32, 145,
167, 170, 178, 264, 272, 306, 414
Prednisone acetate (EX) 169
Prifinium (EX) 390
Prilocaine (AL) 98, (EX) 9, 232, 255, 402, 408,
625
Primaquine (EX) 96; (SI) 95
Primidone (EX) 9, 232, 255, 390, 402, 408, 495,
499, 625; (SI) 178, 188, 221, 522
Pristinamycin (EX) 390
Pristinamycin II A (EX) 156, 540
Probenecid (SI) 178
Procainamide (EX) 9, 232, 255, 402, 408, 495,
499, 625; (SI) 16, 603, 614
Procaine (EX) 97, 390; (SI) 16, 527, 603, 614
Prochlorperazine (EX) 9, 232, 255, 402, 408, 625;
(IN) 552
Progesterone (EX) 169, 171, 390, 428; (IS) 239;
(SI) 178
Proguanil (EX) 390
Prolactin (SI) 14
Promazine (EX) 9, 232, 255, 402, 408, 625; (SI)
383
Promethazine (EX) 9, 232, 255, 390, 402, 408,
495, 499, 625; (SI) 383, 537
Prometrine (EX) 390
Propafenone (EX) 9, 232, 255, 390, 402, 408, 625
Propantheline (SI) 331, 382
Propazine (EX) 390
Propericyazine (EX) 390
Propofol (SI) 138
Propoxur (EX) 250, 390
Propoxyphene (EX) 495, 499; see also
Dextropropoxyphene (EX) 8, 231, 254, 401,
407, 388, 624
Propranolol (EX) 9, 232, 255, 378, 390, 402, 408,
434, 499, 625; (IN) 496; (SI) 16, 165, 603,
614
Propyl paraben (EX) 390; (IS) 16, 145; (SI) 112,
178
Propyphenazone (EX) 9, 232, 255, 402, 408, 499,
625; (IN) 496
Proscillaridine a (EX) 390
Protopine (EX) 390
Protriptyline (EX) 378, 495, 499
Pseudoephedrine (EX) 9, 232, 255, 402, 408, 495,
499, 625
Psilocybin (EX) 390
Pyramidon (IS) 205
Cross-Index to Other Substances
Pyrantel tartrate (IS) 416
Pyrazinamide (EX) 390; (SI) 2
Pyrethrin I (EX) 26, 80
Pyrethrin II (EX) 26, 80
Pyridostigmine (EX) 390
Pyridoxine (Vitamin B6 ) (EX) 391
Pyrimethamine (EX) 390
Pyrithydione (SI) 178
Pyrocatechol (SI) 178
Pyroglutamic acid (EX) 390
Quercetin (EX) 390
Quetiapine (EX) 537
Quinapril (AL) 682; (EX) 390; (SI) 188, 221, 522
Quinaprilat (AL) 682
Quinidine (EX) 390, 495, 499
Quinine (EX) 9, 232, 255, 390, 402, 408, 625; (IS)
365
Quinupramine (EX) 390
Quinupristin (EX) 156; (SI) 157
Raloxifene (SI) 47, 512
Ramipril (AL) 682; (EX) 390
Ranitidine (EX) 9, 232, 255, 390, 402, 408, 495,
499, 625; (SI) 2, 202
Reboxetine (EX) 537
Reserpine (EX) 390; (SI) 178
Resmethrin (AL) 25; (EX) 26, 535
Resorcinol (EX) 390; (SI) 178
all-trans Retinal (EX) 22
Retinyl acetate (IS) 22
all-trans Retinyl stearate (EX) 22
all-trans Retinyl palmitate (EX) 22
Rhein (EX) 108
Riboflavin (Vitamin B2 ) (EX) 391
Rifabutin (AL) 38; (EX) 390
Rifampin (AL) 38; (EX) 390; (SI) 2, 627
Rifamycin (EX) 390
Rilmenidine (EX) 390
Rimantadine (EX) 378
Ripazapam (IS) 632, 684
Risperidone (EX) 9, 232, 255, 402, 408, 499, 537,
625; (IN) 496; (IS) 418
Ritodrine (IS) 545
Ritonavir (AL) 38; (EX) 36, 37, 38, 208, 209, 359,
360, 435, 436, 443, 444; (SI) 627
Rocurone (EX) 9, 232, 255, 402, 408, 625
Rocuronium (EX) 137; (SI) 589
Ronidazole (EX) 390
Ropivacaine (EX) 9, 232, 255, 402, 408, 625
Rosmarinic acid (EX) 390
Rosoxacin (EX) 390
Roxithromycin (EX) 390; (SI) 267
Rutine (EX) 390
Saccharin (EX) 390; (SI) 178
Salicylamide (EX) 9, 232, 255, 402, 408, 625; (SI)
178
715
Salicylic acid (EX) 384, 390, 495, 499; (SI) 178,
293
Salinomycin (EX) 415, 429
Saquinavir (AL) 38; (EX) 36, 37, 38, 208, 209,
359, 360, 435, 436, 443, 444, 627
Sarafloxacin (EX) 473; (SI) 364, 472
Scopolamine (EX) 390
Secnidazole (EX) 390
Secobarbital (EX) 390, 495, 499; (SI) 178
Selegiline (EX) 9, 232, 255, 390, 402, 408, 625
Sennidin A (EX) 108
Sennidin B (EX) 108
Sertindole (EX) 9, 232, 255, 402, 408, 625
Sertraline (EX) 9, 232, 255, 378, 402, 408, 495,
499, 537, 625; (SI) 399
Simazine (EX) 9, 232, 255, 390, 402, 408, 625
Sincocaine (EX) 9, 232, 255, 402, 408, 625
Sisapride (EX) 9, 232, 255, 402, 408, 625
Sisomicin (EX) 390
β-Sitosterol (SI) 585
Sodium (SI) 656
Somatoliberin (SI) 13
Somatostatin (EX) 530
Somatropin (SI) 14
Sotalol (EX) 9, 232, 255, 390, 402, 408, 434, 625
Spectinomycin (EX) 312
Sphingomyelin (EX) 501, 502, 505
Spirenone (EX) 199
Spironolactone (EX) 390, 495, 499; (IS) 427, 587;
(SI) 188, 221, 522
Stanozolol (EX) 427; (SI) 178
Stavudine (EX) 1, 2, 335, 336; (SI) 2, 627
Stearic acid (EX) 457, 458, 459, 460, 461, 462;
(SI) 463, 557
Stearyl alcohol (SI) 129
Streptomycin (EX) 312
Strychnine (EX) 9, 232, 255, 390, 402, 408, 625
Succinic acid (SI) 140
Sucrose (EX) 303; (SI) 5
Sulbutiamine (EX) 390
Sulfabenzamide (SI) 593
Sulfabromomethazine (EX) 594, 597
Sulfacetamide (EX) 592; (SI) 178, 593
Sulfachloropyrazine (EX) 390, 592
Sulfachlorpyridazine (EX) 591, 597; (SI) 596
Sulfadiazine (EX) 390, 591, 592, 594, 597, 598;
(SI) 593, 596
Sulfadimethoxine (EX) 390, 591, 592, 594, 597;
(SI) 178, 593, 596
Sulfadoxine (EX) 390, 592, 594; (SI) 596
Sulfaethidole (SI) 178
Sulfaethoxypridazine (EX) 591, 594
Sulfaguanidine (EX) 390, 592, 594, 599
Sulfamerazine (EX) 592, 594; (SI) 178, 593, 596
Sulfamethazine (EX) 591, 592, 593, 594, 597,
599; (IS) 598; (SI) 178, 593
Sulfamethizole (EX) 592, 594; (SI) 178, 593
Sulfamethoxazole (EX) 390, 495, 499, 592; (SI) 2,
178, 552, 593, 596
716
Cross-Index to Other Substances
Sulfamethoxypyridazine (EX) 390, 592, 594; (SI)
596
Sulfanilamide (EX) 390, 592, 594, 599; (SI) 6,
178, 593, 596
Sulfapyridine (EX) 592, 594, 598, 599; (SI) 178,
593
Sulfaquinoxaline (IN) 185
Sulfathiazole (EX) 390, 592, 594, 599; (SI) 596
Sulfisoxazole (EX) 592, 599; (S) 6, 178, 593
Sulindac (EX) 9, 232, 255, 390, 402, 408, 625;
(SI) 178
Sulpiride (EX) 9, 232, 255, 390, 402, 408, 495,
499, 537, 625; (SI) 188, 221, 522
Sultamicillin tosylate (SI) 16, 614
Sulthiame (EX) 9, 232, 255, 402, 408, 625; (SI)
188, 221, 522
Sultopride (EX) 390
Synephrine (EX) 390
Talinolol (EX) 434
Tamoxifen (EX) 390
Tartaric acid (SI) 140
Tegafur (SI) 16, 603, 614
Temazepam (EX) 9, 232, 255, 390, 402, 408, 495,
499, 625
Temocapril (SI) 16, 603
Tenoxicam (EX) 390
Terbutaline (EX) 9, 232, 255, 390, 402, 408, 625
Terephthalydiene dicamphor sulfonic acid (SI)
456
Terfenadine (AL) 38; (EX) 9, 232, 255, 390, 402,
408, 495, 499, 625
Terodiline (EX) 9, 232, 255, 402, 408, 625
Terpine (EX) 390
Testosterone (EX) 82, 169, 427, 428, 587; (IS)
268, 428
Testosterone acetate (SI) 178
Testosterone 17β-cypionate (SI) 178
Testosterone enanthate (SI) 178
Testosterone propionate (IS) 392; (SI) 178
Tetracaine (EX) 9, 232, 255, 390, 402, 408, 625;
(IS) 97
Tetracosanoic acid (EX) 457
Tetracycline (EX) 390, 592
Tetradecyl alcohol (SI) 129
Tetrahydrozoline (EX) 9, 232, 255, 402, 408
Tetramethrin (AL) 25, 26; (EX) 26, 80, 535; (SI)
24
Tetramisole (EX) 390
Tetraplatin (EX) 480
Tetrazepam (EX) 390; (IS) 398
Theobromine (EX) 9, 232, 255, 390, 402, 408,
625; (SI) 178
Theodrenaline (EX) 390
Theophylline (EX) 9, 232, 255, 384, 390, 402,
408, 495, 499, 625; (SI) 16, 173, 202, 603,
614
Thiabendazole (EX) 102, 103
Thiamine (Vitamin B1 ) (EX) 391
Thiamphenicol (EX) 390; (SI) 259
Thiobarbituric acid (SI) 178
Thiobencarb (EX) 250
Thiopental (thiopentone) (EX) 390, 495, 499
Thioproperazine (EX) 390
Thioridazine (EX) 9, 232, 255, 390, 402, 408,
495, 499, 625; (SI) 383, 537
Thiosalicylic acid (SI) 178
Thiothixene (EX) 9, 232, 255, 402, 408, 495, 499,
625
Thyroid-stimulating hormone (SI) 134
Tianeptine (EX) 390
Tiapride (EX) 390
Tiaprofenic acid (EX) 391
Ticlopidine (EX) 391
Tiemonium iodide (EX) 391; (SI) 331
Tiletamine (EX) 683, 684
Timolol (EX) 9, 89, 232, 255, 391, 402, 408, 434,
625; (IS) 151
Tinidazole (EX) 391
Tioclomarol (EX) 391
Tiospirone (IS) 493
Tobramycin (EX) 312, 391
Tocainide (EX) 495, 499
Tocopherol (EX) 22; (SI) 585
Tofisopam (EX) 391
Tolbutamide (EX) 9, 232, 255, 402, 408, 495,
499, 625; (SI) 178, 188, 221, 522
Toliprolol (EX) 434
Tolmetin (SI) 178
Toloxatone (EX) 391
Tolperisone (IS) 220
Toluene (EX) 391
p-Toluenesulfonic acid (EX) 602
o-Toluidine (AL) 98
Toremifene (EX) 9, 232, 255, 402, 408, 625
Tramadol (EX) 9, 232, 255, 402, 408, 625
Trandolapril (EX) 391
Tranilast (SI) 188, 221, 522
Trazodone (EX) 9, 232, 255, 391, 402, 408, 495,
499, 625
Tretinoin (EX) 21, 22; (SI) 23
Triamcinolone (EX) 168, 265; (SI) 19, 20, 31, 32,
144, 145, 167, 170, 178, 188, 221, 264, 272,
306, 522
Triamcinolone acetonide (EX) 168, 265; (SI) 19,
20, 31, 32, 144, 145, 167, 170, 178, 264, 272,
306
Triamcinolone 16,21-diacetate (SI) 19, 20, 31,
32, 144, 145, 167, 170, 264, 272, 306
Triamcinolone hexacetonide (SI) 20, 32, 145,
167, 170, 264, 272, 306
Triamterene (EX) 9, 232, 255, 391, 402, 408, 625
Triazolam (EX) 9, 232, 255, 391, 402, 408, 495,
499, 625
Trichlorocarbanilide (EX) 391
Trichloromethiazide (EX) 391
Triclabendazole (EX) 103
Tridecanoic acid (EX) 461
Trifluoperazine (EX) 391
Trifluperidol (EX) 391; (SI) 537
Cross-Index to Other Substances
Triflupromazine (EX) 495, 499
Trihexyphenidyl (EX) 391
2,4,6-Triiodobenzoic acid (IS) 322
Trimebutine (EX) 391
Trimeprazine (EX) 9, 232, 255, 402, 408, 496,
499, 625
Trimetazidine (EX) 391
Trimethoprim (AL) 38; (EX) 9, 232, 255, 391,
402, 408, 496, 499, 625; (IS) 672; (SI) 2,
552, 627
Trimipramine (EX) 9, 232, 255, 391, 402, 408,
496, 499, 537, 625
Triprolidine (EX) 391
Tritoqualine (EX) 391
Tropatepine (EX) 391
Trovafloxacin (IS) 293
Tryptophan (SI) 293
Tubocurarine (IN) 552
Tulobuterol (SI) 16, 603, 614
Undecanoic acid (EX) 461
Urocanic acid (SI) 456
Valdecoxib (SI) 484
Valethamate (SI) 331, 382
Valsartan (EX) 105
Vanillin (EX) 391
Vasoactive intestinal polypeptide (EX) 530
Vasopressin (SI) 13
Vecuronium (EX) 137, 514; (SI) 589
Venlafaxine (EX) 9, 232, 255, 402, 408, 537, 625;
(SI) 399
Verapamil (EX) 9, 232, 255, 391, 402, 408, 496,
499, 625
Veratrine (EX) 391
Veratrole (EX) 391
Viloxazine (EX) 391
Vinblastine (EX) 391
Vincamine (EX) 391; (IS) 92
717
Vincristine (EX) 391
Vinylbital (EX) 391
Virginiamycin (EX) 391
Vitamin A (EX) 21, 22
Vitamin B1 (thiamine) (EX) 391
Vitamin B2 (riboflavin) (EX) 391
Vitamin B5 (pantothenic acid) (EX) 391
Vitamin B6 (pyridoxine) (EX) 391
Vitamin B12 (EX) 391
Vitamin C (EX) 391
Vitamin D2 (IS) 181
Vitamin E (EX) 22; (SI) 585
Vitamin H (biotin) (EX) 391
Warfarin (EX) 9, 232, 255, 391, 402, 408, 625;
(IS) 342; (SI) 178, 188, 221, 522
Xanthydrol (EX) 391
Xipamide (EX) 391
Xylazine (EX) 525
Xylene (EX) 391
Yohimbine (EX) 9, 232, 255, 391, 402, 408, 625
Zalcitabine (EX) 1, 2, 335, 336
Zeranol (EX) 427, 587
Zidovudine (EX) 1, 2, 335, 336; (SI) 2, 627
Zipeprol (EX) 391
Ziprasidone (EX) 537
Zoalene (EX) 601
Zofenoprilat (EX) 681; (SI) 682
Zolazepam (EX) 631, 632
Zolpidem (EX) 9, 232, 255, 391, 402, 408, 625,
673; (SI) 537
Zonisamide (SI) 188, 221, 522
Zopiclone (EX) 9, 232, 255, 402, 408, 496, 499,
625
Zuclopenthixol (EX) 391, 496, 499