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HPLC METHODS FORRECENTLY APPROVED

PHARMACEUTICALS

HPLC METHODS FORRECENTLY 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.

No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any formor by any means, electronic, mechanical, photocopying, recording, scanning, or otherwise, except aspermitted under Section 107 or 108 of the 1976 United States Copyright Act, without either the priorwritten permission of the Publisher, or authorization through payment of the appropriate per-copy feeto the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400, fax978-646-8600, or on the web at www.copyright.com. Requests to the Publisher for permission should beaddressed to the Permissions Department, John Wiley & Sons, Inc., 111 River Street, Hoboken, NJ07030, (201) 748-6011, fax (201) 748-6008.

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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 2005615′.1–dc22

2004016046

Printed in the United States of America.

10 9 8 7 6 5 4 3 2 1

CONTENTS

Preface / xiAcknowledgements / xiiiAbout This Book / xv

Abacavir / 1Acarbose / 5Acetyl sulfisoxazole / 6Acrivastine / 7Adapalene / 10Adefovir dipivoxil / 11Adrenocorticotropic hormone / 13Afloqualone / 15Alacepril / 16Alclometasone 17,21-dipropionate / 18Alitretinoin / 21Allethrin / 24Almotriptan / 27Alosetron / 29Amcinonide / 30Aminolevulinic acid / 33Amprenavir / 36Anagrelide / 42Anakinra / 43Apraclonidine / 45Aprepitant / 46Aranidipine / 48Arotinolol / 49Arteether / 52Articaine / 54Asparaginase / 57Atazanavir sulfate / 58Atipamezole / 60Atomoxetine hydrochloride / 62Atorvastatin / 64

Atosiban / 66Balofloxacin / 67Bambermycins / 69Befunolol / 70Benzalkonium chloride / 71Betaine / 72Bethanechol chloride / 74Bexarotene / 75Biapenem / 77Bimatoprost / 79Bioresmethrin / 80Bivalirudin / 81Boldenone / 82Bosentan / 83β-Boswellic acid / 86Brimonidine / 88Bromfenac / 90Brovincamine / 92Bucillamine / 93Budipine / 94Bulaquine / 95Butacaine / 97Butamben / 99Butoconazole / 100Butyl flufenamate / 101Cambendazole / 102Candesartan cilexetil / 104Capecitabine / 106Casanthranol / 108Caspofungin / 109

v

vi Contents

Castor oil / 112Cefbuperazone / 113Cefditoren / 114Cefoselis / 116Cefozopran / 117Cefuzonam / 118Celecoxib / 119Cerivastatin / 123Cetrorelix / 125Cetyl alcohol / 128Cevimeline hydrochloride / 130Chlorobutanol / 132Chloroprocaine / 133Chorionic gonadotropin / 134Cilnidipine / 135Cimetropium bromide / 136Cisatracurium besylate / 137Citric acid / 139Clioquinol / 142Clobetasol 17-propionate / 143Clopidogrel / 147Clopidol / 149Cloricromen / 151Clorsulon / 152Colistin / 153Cypermethrin / 155Dalfopristin / 156Dalteparin / 158Daptomycin / 159Deferiprone / 161Deflazacort / 162Desloratadine / 164Desogestrel / 166Desoximetasone / 167Desoxycorticosterone / 169Dexrazoxane / 172Dextran / 174Diacerein / 176Dichloroacetic acid / 177Dichlorophen / 178Diclazuril / 179Dihydrotachysterol / 181Dimethyl sulfoxide / 183Dinitolmide / 185Dipivefrin / 186Dithiazanine iodide / 187Docarpamine / 188Dofetilide / 189Dolasetron / 191Donepezil / 193

Doxefazepam / 195Doxercalciferol / 196Dropropizine / 198Drospirenone / 199Droxicam / 200Droxidopa / 201Ebrotidine / 202Edaravone / 204EDTA / 206Efavirenz / 208Efrotomycin / 212Egualen / 213Eletriptan / 214Emtricitabine / 215Enoxaparin sodium / 217Entacapone / 218Eperisone / 220Eplerenone / 222Epoprostenol / 224Eprosartan / 225Eptazocine / 227Eptifibatide / 228Erdosteine / 229Ergotamine / 230Ertapenem / 234Ethopabate / 236Ethyl icosapentate / 237Etonogestrel / 238Etoricoxib / 240Etorphine / 242Exemestane / 243Ezetimibe / 245Fadrozole / 247Falecalcitriol / 248Fenoxycarb / 250Fenticonazole / 251Fexofenadine / 253Flomoxef / 257Florfenicol / 258Fludrocortisone / 260Fluprostenol / 262Flurandrenolide / 264Flurithromycin / 267Flurogestone acetate / 268Fluticasone propionate / 270Flutrimazole / 273Fomepizole / 274Fomivirsen / 276Fondaparinux / 277Formestane / 278

Contents vii

Formoterol / 279Fosamprenavir calcium / 281Fosinopril / 283Fosphenytoin / 284Frovatriptan / 286Fumagillin / 288Galantamine / 290Ganirelix / 292Gatifloxacin / 293Gefitinib / 295Gemcitabine / 296Gemifloxacin / 298Gestodene / 300Gestrinone / 301Glycerin / 302Guanabenz / 304Guanadrel / 305Halobetasol propionate / 306Halofuginone / 308Hetastarch / 310Hydroquinone / 311Hygromycin B / 312Iloprost / 313Imatinib / 314Imidocarb / 316Iobenguane / 318Iodixanol / 320Iopanoic acid / 322Iopromide / 324Ioversol / 326Ipratropium bromide / 327Ipriflavone / 328Isoflupredone / 329Isopropamide iodide / 330Itopride / 332Kinetin / 333Lafutidine / 334Lamivudine / 335Latanoprost / 339Leflunomide / 341Lercanidipine / 343Letrozole / 345Levetiracetam / 346Levonordefrin / 348Levosimendan / 349Lidamidine / 351Lincomycin / 352Lindane / 354Linezolid / 355Liothyronine / 357

Lomerizine / 358Lopinavir / 359Loteprednol etabonate / 362Marbofloxacin / 364Masoprocol / 367Maxacalcitol / 368Medetomidine / 369Meglutol / 371Melatonin / 372Melengestrol acetate / 375Memantine / 378Menthol / 380Mepenzolate bromide / 381Mepixanox / 383Mequinol / 384Methenamine / 385Methoprene / 386Methoxychlor / 387Methyltestosterone / 392Metrizamide / 393Metyrosine / 394Micafungin / 396Milnacipran / 398Mirtazapine / 400Misoprostol / 405Mizolastine / 406Moexipril / 411Mofezolac / 412Mometasone furoate / 413Monensin / 415Morantel / 416Mosapride / 417Moxifloxacin / 420Moxonidine / 423Nadifloxacin / 424Naftopidil / 425Nandrolone / 427Narasin / 429Nartograstim / 430Nateglinide / 431Nebivolol / 433Nelfinavir / 435Nequinate / 440Neridronic acid / 441Nevirapine / 443Nicarbazin / 447Nilutamide / 448Nipradilol / 449Nitazoxanide / 450Nitenpyram / 452

viii Contents

Nomegestrol / 453Nonoxynol-9 / 454Nystatin / 455Octocrylene / 456Oleic acid / 457Olmesartan / 469Olopatadine / 470Orbifloxacin / 472Orlistat / 475Oseltamivir / 477Oxaliplatin / 479Oxiconazole / 481Panipenem / 483Parecoxib / 484Paricalcitol / 485Pazufloxacin / 487Penciclovir / 488Pentaerythritol tetranitrate / 490Pentosan polysulfate / 491Perflubron / 492Perospirone / 493Phenazopyridine hydrochloride / 494Phentermine / 497Phosphatidylcholine / 501Phosphatidylglycerol / 504Piketoprofen / 508Pilsicainide / 509Pioglitazone / 511Pipercuronium bromide / 514Pirlimycin / 515Poloxalene / 518Pramipexole / 519Pranlukast / 521Prednicarbate / 523Propionylpromazine / 524Propoxycaine hydrochloride / 527Propylene glycol / 528Propylhexedrine / 529Protirelin / 530Prulifloxacin / 532Pyrethrins / 533Quetiapine / 536Quinfamide / 539Quinupristin / 540Rabeprazole / 542Ractopamine / 544Raloxifene / 547Ramosetron / 549Rapacuronium bromide / 551Remifentanil / 553

Repaglinide / 555Ricinoleic acid / 557Rifaximin / 558Rilmazafone / 559Risedronate sodium / 560Rizatriptan / 561Rofecoxib / 562Ropinirole / 565Rosiglitazone / 566Rosuvastatin calcium / 568Sarafloxacin / 569Selamectin / 571Sermorelin / 572Sibutramine / 574Sildenafil / 576Simethicone / 579Sivelestat / 580Sodium oxybate / 582Somatropin / 583Squalane / 584Squalene / 585Stanozolol / 587Succimer / 588Succinylcholine chloride / 589Sulfabromomethazine / 591Sulfachlorpyridazine / 592Sulfaethoxypyridazine / 596Sulfamerazine / 598Sulfanitran / 600Sultamicillin / 602Tacalcitol / 604Talipexole / 605Taltirelin / 607Technetium Tc 99m bicisate / 608Tegaserod / 609Telithromycin / 610Telmesteine / 611Telmisartan / 612Temocapril / 614Temozolomide / 615Tenofovir disoproxil fumarate / 617Teprenone / 620Teriparatide / 621Tetrachlorvinphos / 622Tetrahydrozoline / 623Thalidomide / 626Thialbarbital / 628Thyrotropin / 629Tiagabine / 630Tiletamine hydrochloride / 631

Contents ix

Tilmicosin / 633Tiludronic acid / 634Tirilazad / 635Tirofiban / 637Tiropramide / 639Tizanidine / 641Tolcapone / 643Topiramate / 645Topotecan / 647Tosufloxacin / 649Travoprost / 651Trenbolone / 652Treprostinil / 653Trichlorfon / 655Triethanolamine / 656Trifluridine / 657

Triptorelin / 658Troleandomycin / 660Troxipide / 661Ubenimex / 662Unoprostone isopropyl ester / 663Valacyclovir / 664Valdecoxib / 666Valganciclovir / 668Valrubicin / 670Valsartan / 671Zaleplon / 673Zaltoprofen / 676Zanamivir / 678Zinostatin / 680Zofenopril calcium / 681Zolazepam hydrochloride / 683

Cumulative Index / 685Cross-Index to Other Substances / 703

PREFACE

This book is a collection of procedures for the analysis of more than 390 pharma-ceuticals using high-performance liquid chromatography (HPLC) and covers theliterature up to the end of 2003. The current volume is a continuation of HPLCMethods for Pharmaceutical Analysis, published in four volumes from 1997 to 2000.The previous volumes described methods published in the literature through themiddle of 1998.

The current work lists procedures for the analysis of drugs in three broad categories:

ž Drugs that have been approved since the previous volumes were published.ž 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.

Please note that mention of a drug does not necessarily mean that it is currentlyapproved for use in the United States or indeed in any country.

Despite the ready availability of computer-aided literature, searching this resourceis 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 mostrelevant reference that can be turned into a practical analytical procedure in thesearcher’s own laboratory. The reference finally chosen will, naturally, depend onthe individual circumstances, such as the matrix in which the drug is present,availability of equipment, and so on. This book circumvents this lengthy process byproviding a number of abstracted and evaluated procedures for the analysis of eachdrug. 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 choosingan analytical procedure. Accordingly, we have noted such features of the analyticalprocedures as sensitivity, mode of detection, other compounds that interfere withthe analysis, other drugs that may be determined at the same time, and so on.

Readers familiar with our previous publications, HPLC Methods for Pharmaceu-tical Analysis, Volumes 1–4 (George Lunn and Norman R. Schmuff, John Wiley,New York, 1997–2000) and Handbook of Derivatization Reactions for HPLC (GeorgeLunn and Louise C. Hellwig, John Wiley, New York, 1998), will notice many similar-ities. 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 bedetermined using the same system have been added in parentheses after the drugname. Other data, such as the limit of detection (LOD), may also be added. Theretention time is the number without units. Unlike the previous volumes, this bookis not available on a CD in an electronic form.

At the end of the book a Cumulative Index and a Cross-Index to Other Substancesare provided. The Cumulative Index provides a comprehensive listing of the drugscovered in this book and the previous volumes. The Cross-Index lists the othercompounds that may also be chromatographed under the conditions described inthe monographs in this book. Using the information in the monographs it may bepossible to develop chromatographic procedures for these compounds.

GEORGE LUNN

ACKNOWLEDGEMENTS

I am grateful for the use of the National Institutes of Health Library, the FDAMedical Library, and the National Library of Medicine and I would like to expressmy appreciation for the hard work of the staff of these libraries, particularly thosediligent workers who reshelve the journal volumes after one of my forays. Althoughmany people have helped with the preparation of this work the mistakes are myown. I would appreciate hearing from anyone who has corrections, comments, orsuggestions. I can be reached at [email protected].

The content of this volume does not necessarily reflect the views or policiesof 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 foruse 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’sannual lists of drug approvals (available at www.fda.gov/cder) and Annual Reportsin Medicinal Chemistry published by Elsevier/Academic Press.

The journals routinely surveyed for relevant articles are:

American Journal of Health-System PharmacyAnalystAnalytica Chimica ActaAnalytical ChemistryAnalytical LettersAnalytical SciencesAntimicrobial Agents and ChemotherapyArzneimittelforschungBiological and Pharmaceutical BulletinBiomedical ChromatographyBiopharmaceutics and Drug DispositionChemical and Pharmaceutical BulletinChromatographiaClinical ChemistryClinical Pharmacology and TherapeuticsDrug Metabolism and DispositionFarmacoFood Additives and ContaminantsJournal of Analytical ToxicologyJournal of AOAC InternationalJournal of Chromatographic ScienceJournal of Chromatography, Part A and Part BJournal of Clinical PharmacologyJournal of Forensic Sciences

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xvi About This Book

Journal of Liquid Chromatography & Related TechnologyJournal of Pharmaceutical and Biomedical AnalysisJournal of Pharmaceutical SciencesJournal of Pharmacology and Experimental TherapeuticsPharmaceutical ResearchPharmazieTherapeutic Drug MonitoringXenobiotica

Other journals were consulted when relevant articles were identified by com-puter searches.

The literature was surveyed from 1998 through the end of 2003, although methodsfrom 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 asother useful data such as the CAS Registry Number, molecular formula, molecularweight, and Merck Index number (from the 13th edition).1 More useful informationsuch as melting point, solubility, optical rotation, references to reviews, and so oncan be found in the Merck Index.

In general, the United States Adopted Name (USAN)2 is used throughout toidentify each drug. Names of derivatives, such as esters, which would have differ-ent chromatographic properties, are identified by placing the derivative name inparentheses after the retention time.

Increasingly, drugs previously marketed as racemates are being marketed as asingle enantiomer with the name changed to reflect the enantiomer. For example,levofloxacin is the levorotatory form of ofloxacin. For an achiral HPLC method, thechromatography 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 proceduresunder the name of the racemate rather than the single enantiomer. The interestedreader is referred to the USP Dictionary2 (page 1208) for the naming conventionsused. Generally:

Levo rotatory S isomer Prefix lev/levo-Levo rotatory R isomer Prefix ar-Dextro rotatory R isomer Prefix dex/dextro-Dextro rotatory S isomer Prefix 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 volumeDextromethorphan in Volume 2Dextromoramide in Volume 2Dextrothyroxine in Volume 2

About This Book xvii

Levallorphan in Volume 3Levamisole in Volume 3Levobunolol in Volume 3Levodopa in Volume 3Levonordefrin in Volume 3 and this volumeLevorphanol in Volume 3Levosimendan in this volumeLevothyroxine 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 Levamisole 3Dexamphetamine Amphetamine 2Dexbrompheniramine Brompheniramine 2Dexbudesonide Budesonide 2Dexchlorpheniramine Chlorpheniramine 2Dexfenfluramine Fenfluramine 3Dexibuprofen Ibuprofen 1, 4Dexketoprofen Ketoprofen 1, 4Dexmedetomidine Medetomidine This volumeDexmethylphenidate Methylphenidate 1Dexpropranolol Propranolol 4Dexsotalol Sotalol 4Dextroamphetamine Amphetamine 2Dextropropoxyphene Propoxyphene 1, 4Dexverapamil Verapamil 1, 4

Esatenolol Atenolol 1, 2Escitalopram Citalopram 2Esflurbiprofen Flurbiprofen 3Esketamine Ketamine 3Esomeprazole Omeprazole 1, 3Esoxybutynin chloride Oxybutynin chloride 3Eszopiclone Zopiclone 4

Levalbuterol Albuterol 1, 2Levamfetamine Amphetamine 2Levamphetamine Amphetamine 2Levcycloserine Cycloserine 2Levdobutamine Dobutamine 2Levmetamfetamine Methamphetamine 3Levobetaxolol Betaxolol 2Levobupivacaine Bupivacaine 2Levocarnitine Carnitine 2Levocetirizine Cetirizine 2Levodropropizine Dropropizine 2, this volume

xviii About This Book

Levofenfluramine Fenfluramine 3Levofloxacin Ofloxacin 1, 3Levofuraltadone Furaltadone 3Levoleucovorin Leucovorin 3Levomenthol Menthol 3, this volumeLevomethadone Methadone 3Levomoprolol Moprolol 3Levonorgestrel Norgestrel 1Levopropoxyphene Propoxyphene 1, 4Levopropylhexedrine Propylhexedrine 4, this volumeLevosalbutamol Albuterol 1, 2Levosulpiride Sulpiride 4

Racementhol Menthol 3, this volumeRacemethorphan Dextromethorphan 2Racemetirosine Metyrosine This volumeRacemorphan Levorphanol 3Racephedrine Ephedrine 3Racepinephrine Epinephrine 3

BIBLIOGRAPHIES

For reasons of space, it is not possible to abstract every relevant paper, and so at theend of some chapters an Annotated Bibliography lists other relevant papers. Afterthe citation, a few features of the method that are not obvious from the title of thepaper 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 otherwisementioned, it may be assumed that a method involves liquid–liquid extraction of abiological fluid from a human and uses reversed-phase HPLC with UV detection.Thus, if a method uses solid-phase extraction (SPE) or fluorescence detection, thiswill 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.

MatrixSample preparationGuard columnColumnMobile phaseFlow rateInjection volumeRetention timeDetector

About This Book xix

Internal standardColumn temperatureExtractedSimultaneousAlsoNoninterferingInterferingLimit of detectionLimit of quantitationKey wordsReference

ABSTRACT CONVENTIONS

Also Compounds that can be analyzed at the same time. It is notspecified whether they interfere, but they can beextracted. See also Extracted, Simultaneous.

Column Dimensions are length (mm) × internal diameter (mm), andthe material is stainless steel unless otherwise indicated.

Column temperature If other than ambient (all temperatures are in degrees C).Derivatization Pre-column unless otherwise mentioned (in Key Words).Detector Wavelengths in nanometersExtracted Compounds that can be extracted from the matrix in

question and analyzed at the same time and do notinterfere. See also Also, Simultaneous.

Flow rates In milliliters per minute.Guard column Dimensions are length (mm) × internal diameter (mm).Injection volume In microliters (µL). Injection volume may be either the

volume actually injected or the volume of the injectionloop. If it is the volume actually injected, this value isalso given in the Sample preparation section. If theactual injection volume is not given in the Samplepreparation section, the Injection volume given is that ofthe injection loop.

Interfering Compounds that interfere with the analysis of the targetcompound. Compounds that interfere with thechromatography of the internal standard (IS) are listedunder simultaneous (another IS can always be selected oran external standard procedure can be used).

Matrix A controlled vocabulary is used (see below)Mobile phase Ratios are v/v and gradients are linear, unless otherwise

noted. Times given when describing gradient elution andother procedures such as column switching are the timesfor each step, e.g., ‘‘MeOH:water 15:85 for 4 min, to 50:50over 2 min, maintain at 50:50 for 4 min.’’ If we were toinclude the cumulative times (t) in the example above itwould read: ‘‘MeOH:water 15:85 for 4 min (t = 4), to 50:50over 2 min (t = 6), maintain at 50:50 for 4 min (t = 10).’’

xx About This Book

Noninterfering Compounds that do not interfere with the analysis forvarious reasons, e.g., they are not extracted, they are notdetected.

Retention time In minutes. This is frequently estimated from a reproducedchromatogram, and so the accuracy may not be great.When available, retention times are given for theanalyte, the internal standard, and other compounds thatmay be chromatographed under the same conditions. Forthe internal standard and other compounds that may bechromatographed under the same conditions, theretention times are given in parentheses after thecompound name.

Simultaneous Compounds that can be analyzed at the same time and donot interfere. Note that the compound cannot necessarilybe extracted from the matrix in question (although it maybe). See also Also, Extracted.

SPE For the sake of consistency, conditioning procedures forsolid-phase extraction (SPE) cartridges are alwaysdescribed at the beginning of the sample preparationsections. Bear in mind, however, that the conditioningprocedure should be carried out just prior to use. Thus, ifsample preparation is a lengthy procedure, it may benecessary to delay SPE cartridge conditioning until thestep requiring the cartridge.

Species 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 areanalyzed, both human and rat will be indicated (in KeyWords).

MATRIX

To help with searching, a controlled vocabulary is used to limit the number of termsin 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. Ina number of cases, the matrix is associated with various key words, which can beused to narrow the search. For example, the matrix term blood has the key wordsplasma, serum, and whole blood associated with it. Thus, if you are interested in thedetermination of the drug in blood in general, you should look in the matrix field forblood. If, however, you are specifically interested in finding the drug in plasma, youshould look in the key words field for plasma.

Matrix Associated Key Words

BileBlood Plasma, serum, whole bloodBulkCSFFormulations Capsules, creams, injections, ointment, tablets, etc.Microsomal incubations

About This Book xxi

MilkPerfusateReaction mixturesSalivaTissue Brain, heart, kidney, liver, muscle, spleen, etc.Urine

ABBREVIATIONS

BHT 2,6-Di-tert-butyl-4-methylphenol, butylated hydroxytolueneDMSO Dimethyl sulfoxideE Electrochemical detectionem Emission wavelengthEtOH Ethanolex Excitation wavelengthF Fluorescence detectionGPC Gel permeation chromatographyh HourHPLC High-performance liquid chromatographyID Internal diameterIS Internal standardL LiterLOD Limit of detection or some other description indicating that this is the

smallest concentration or quantity that can be detected or analyzed forLOQ Lower limit of quantitation, either given as such in the paper or taken as

the lower limit of the linear quantitation rangeM Molar (i.e., moles/L)MeCN AcetonitrileMeOH Methanolmin MinutesmL MillilitermM Millimolar (i.e., millimoles/L)MS Mass spectrometric detectionMSPD Matrix solid phase dispersionMTBE Methyl tert-butyl ethernM Nanomolar (i.e., nanomoles/L)psi Pounds/sq. in. (1 psi = 6.89476 kPa)s SecondsSEC Size Exclusion ChromatographySFC Supercritical fluid chromatographySFE Supercritical fluid extractionSPE Solid phase extractionTemp TemperatureU UnitsUV Ultraviolet detectionvol Volume

xxii About This Book

PIC REAGENTS

These reagents are offered by Waters as buffered solutions containing the followingcompounds:

PIC A is tetrabutylammonium sulfatePIC B5 is pentanesulfonic acidPIC B7 is heptanesulfonic acid.

WORKING PRACTICES

In general, good working practice, for example, using high-grade materials isassumed. Solutions should be protected from light, and silanized glassware shouldbe used unless you have good reason to believe that these precautions are notnecessary. Details of solution preparation are generally not given. It should beremembered that the preparation of a dilute aqueous solution of a relatively water-insoluble compound can frequently be made by dissolving the compound in a smallvolume of a water-miscible organic solvent and diluting this solution with water.A number of excellent texts3–9 discuss good working practices and procedures inHPLC. Please note that all the temperatures are in degrees C.

It is also assumed that safe working practices are observed. Organic solventsshould only be evaporated in a properly functioning chemical fume hood, correctprotective equipment should be worn when dealing with potentially hazardousbiological materials, and waste solutions should be disposed of in accordance withall applicable regulations.

A number of solvents are particularly hazardous. For example, benzene is ahuman carcinogen;10 chloroform,11 dichloromethane,12 dioxane,13 and carbon tetra-chloride 14 are carcinogenic in experimental animals; and DMF 15 and MTBE 16,17

may be carcinogenic. Organic solvents are, in general, flammable and toxic byinhalation, ingestion, and skin absorption. Sodium azide is carcinogenic and toxicand liberates explosive, volatile, toxic hydrazoic acid when mixed with acid. Sodiumazide can form explosive heavy metal azides, for example, with plumbing fixtures,and so should not be discharged down the drain.18 Disposal procedures have beendescribed for a number of hazardous drugs and reagents,18 and a procedure for thehydrolysis of acetonitrile in waste solvent to the much less toxic acetic acid andammonia19,20 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 InternationalDrug Names, United States Pharmacopeial Convention, Rockville, MD, 2004.

3. Snyder, L.R.; Kirkland, J.J. Introduction to Modern Liquid Chromatography, 2nd edi-tion, 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, 2ndedition, 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, JohnWiley & 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, VanNostrand Reinhold, New York, 1992, pp. 356–358.





15. Lewis, R.J. Sr. Sax’s Dangerous Properties of Industrial Materials, 8th edition, VanNostrand Reinhold, New York, 1992, p. 1378.

16. Belpoggi, F.; Soffritti, M.; Maltoni, C. Methyl-tertiary-butyl ether (MTBE) – a gaso-line additive – causes testicular and lympho-haematopoietic cancers in rats, Toxi-col.Ind.Health., 1995, 11, 119–149.

17. Mehlman, M.A. Dangerous and cancer-causing properties of products and chemicals inthe oil refining and petrochemical industry: Part XV. Health hazards and health risksfrom 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, 2ndedition, John Wiley & Sons, New York, 1994.

19. Gilomen, K.; Stauffer, H.P.; Meyer, V.R. Detoxification of acetonitrile – water wastesfrom liquid chromatography, Chromatographia, 1995, 41, 488–491.

20. Gilomen, K.; Stauffer, H.P.; Meyer, V.R. Management and detoxification of acetonitrilewastes from liquid chromatography, LC.GC, 1996, 14, 56–58.

21. Meyer, V. A safer solvent, Anal.Chem., 1997, 69, 18A.

AbacavirMolecular formula: C14H18N6O

Molecular weight: 286.33

CAS Registry No: 136470-78-5 (base), 188062-50-2 (sulfate)

Merck Index: 13,1

N

NN

N

NH

NH2HO

SAMPLEMatrix: bloodSample 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 hto inactivate HIV. Vortex 800 µL plasma with 300 µL 2 µg/mL hexobarbital in 25 mMpH 7.0 ammonium acetate buffer for 30 s and centrifuge at 18 000 g for 5 min. Add 1 mLof the supernatant to the SPE cartridge, wash with 1 mL 100 mM pH 7.0 ammoniumacetate buffer, suck dry for 1 min, elute with 800 µL MeOH. Evaporate the eluate todryness under a stream of nitrogen at 40◦ and reconstitute the residue with 100 µLmobile phase. Vortex for 30 s, centrifuge at 18 000 g for 3 min, and inject an 80 µLaliquot.

HPLC VARIABLESGuard column: 20 × 3.9 5 µm Polarity dC18 (Waters)Column: 150 × 3.9 5 µm Polarity dC18 (Waters)Column temperature: 40Mobile 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 36:64over 15 min, maintain at 36:64 for 3 min, re-equilibrate at initial conditions for 7 min.

Flow rate: 1.1Injection volume: 80Detector: UV 269 for 11 min, UV 250 for 3 min, UV 271 for 10 min, UV 230 for 9 min

CHROMATOGRAMRetention time: 25.1Internal standard: hexobarbital (30.6)Limit of quantitation: 10.0 ng/mL

OTHER SUBSTANCESExtracted: didanosine (13.6), lamivudine (8.6), nevirapine (27.3), stavudine (15.7), zal-

citabine (5.9), zidovudine (23.8)Noninterfering: tenofovir

KEY WORDSplasma; SPE

REFERENCERezk, 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 absorbancedetection, J.Chromatogr.B, 2003, 791, 137–147.

SAMPLEMatrix: bloodSample 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 SPEcartridge, wash with 500 µL water, elute with 1 mL MeOH. Evaporate the eluate to

1HPLC Methods for Recently Approved Pharmaceuticals. by George LunnCopyright 2005 John Wiley & Sons, Ltd. ISBN: 0-471-66941-5

2 Abacavir

dryness with vortexing under reduced pressure at 40◦ and reconstitute the residue with300 µL MeOH, inject a 10 µL aliquot.

HPLC VARIABLESColumn: two 150 × 4.6 3 µm Luna C18 columns in seriesColumn temperature: 60Mobile phase: Gradient. MeCN:water from 5:95 to 45:55 over 20 min.Flow rate: 0.85Injection volume: 10Detector: UV 250

CHROMATOGRAMRetention time: 17Limit of detection: 75 ng/mL

OTHER SUBSTANCESExtracted: didanosine (10.5, LOD 120 ng/mL), lamivudine (9.5, LOD 260 ng/mL), stavu-

dine (11.5, LOD 40 ng/mL), zalcitabine (7.5, LOD 440 ng/mL), zidovudine (16, LOD30 ng/mL)

KEY WORDSSPE; serum

REFERENCESimon, V.A.; Thiam, M.D.; Lipford, L.C. Determination of serum levels of thirteen human immunodefi-

ciency virus-suppressing drugs by high-performance liquid chromatography, J.Chromatogr.A, 2001,913, 447–453.

SAMPLEMatrix: bloodSample 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 VARIABLESGuard column: 20 × 3.8 Symmetry C18 (Waters)Column: 100 × 4.6 3.5 µm Symmetry C18 (Waters)Column temperature: 41 ± 2Mobile phase: MeCN:25 mM pH 7.0 phosphate buffer 15:85Flow rate: 1Injection volume: 100Detector: UV 285

CHROMATOGRAMRetention time: 4.8Limit of quantitation: 20 ng/mL

OTHER SUBSTANCESSimultaneous: didanosine, folic acid, ganciclovir, lamivudine, nevirapine, pyrazinamide,

ranitidine, rifampin, stavudine, sulfamethoxazole, trimethoprim, zidovudineNoninterfering: adefovir, amprenavir, delavirdine, efavirenz, fluconazole, indinavir,

itraconazole, methadone, nelfinavir, oxazepam, pyrimethamine, rifampin, ritonavir,saquinavir, zalcitabine

KEY WORDSplasma

Abacavir 3

REFERENCEVeldkamp, A.I.; Sparidans, R.W.; Hoetelmans, R.M.W.; Beijnen, J.H. Quantitative determination of aba-

cavir (1592U89), a novel nucleoside reverse transcriptase inhibitor, in human plasma using isocraticreversed-phase high-performance liquid chromatography with ultraviolet detection, J.Chromatogr.B,1999, 736, 123–128.

SAMPLEMatrix: bloodSample preparation: Centrifuge plasma at 4000 g for 20 min using a Centrifree

micropartition device (Amicon), inject a 100 µL aliquot of the ultrafiltrate.

HPLC VARIABLESColumn: 250 × 4.6 Adsorbsphere C18Mobile 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: 1Injection volume: 100Detector: UV 260, UV 285

CHROMATOGRAMRetention time: 23

OTHER SUBSTANCESExtracted: carbovir (20)

KEY WORDSrat; pharmacokinetics; plasma

REFERENCEDaluge, 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 nucle-oside analog with potent, selective anti-human immunodeficiency virus activity, Antimicrob.AgentsChemother., 1997, 41, 1082–1093.

SAMPLEMatrix: CSF, urineSample 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 VARIABLESColumn: 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: 0.7Detector: UV 295

CHROMATOGRAMRetention time: 25.5Limit of quantitation: 62 ng/mL (CSF), 629 ng/mL (urine)

OTHER SUBSTANCESExtracted: metabolites, abacavir 5′-glucuronide, abacavir 5′-carboxylate

4 Abacavir

REFERENCERavitch, 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 BIBLIOGRAPHYFung, 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 massspectrometry, J.Chromatogr.B, 2001, 754, 285–295. [LC-MS]

Sparidans, R.W.; Hoetelmans, R.M.W.; Beijnen, J.H. Liquid chromatographic assay for simultaneousdetermination of abacavir and mycophenolic acid in human plasma using dual spectrophotometricdetection, J.Chromatogr.B, 2001, 750, 155–161.

Thomas, S.A.; Bye, E.; Segal, M.B. Transport characteristics of the anti-human immunodeficiency virusnucleoside 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 witheach 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 agentsin 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 pharmacody-namics of abacavir alone and in combination with zidovudine in human immunodeficiency virus-infected 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. Safetyand pharmacokinetics of abacavir (1592U89) following oral administration of escalating single dosesin 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. Pharmacokineticsof [14C]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.AgentsChemother., 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 humanimmunodeficiency virus infection, Antimicrob.Agents Chemother., 1999, 43, 1708–1715.

Acarbose 5

AcarboseMolecular formula: C25H43NO18Molecular weight: 645.60

CAS Registry No: 56180-94-0

Merck Index: 13, 18

O

O O

O O

OH

OH

H3C

NHO

H OH

OH

OH

OHHO

HO

HOHO

OH

OH

SAMPLEMatrix: formulationsSample 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 gfor 5 min, combine supernatants, make up to 20 mL with water. Dilute a 50 µL aliquotto 1 mL with MeOH, filter (0.2 µM), inject a 20 µL aliquot.

HPLC VARIABLESColumn: 250 × 4.6 5 µm Nucleosil-NH2Mobile phase: MeOH:dichloromethane 65:35Flow rate: 1Injection volume: 20Detector: ELSD, nebulizing gas air at 2.5 bar and 4 L/min, solvent evaporated at 40◦

CHROMATOGRAMRetention time: 4.1Limit of detection: 5 µg/mLLimit of quantitation: 15 µg/mL

OTHER SUBSTANCESSimultaneous: sucrose (3.5)

KEY WORDScomparison with capillary electrophoresis; tablets

REFERENCECherkaoui, S.; Daali, Y.; Christen, P.; Veuthey, J.-L. Development and validation of liquid chromatog-

raphy and capillary electrophoresis methods for acarbose determination in pharmaceutical tablets,J.Pharm.Biomed.Anal., 1998, 18, 729–735.

6 Acetyl sulfisoxazole

Acetyl sulfisoxazoleMolecular formula: C13H15N3O4S



Merck Index: 13, 9041

NS

CH3CH3

NO

CH3

O O

H2N O

SAMPLEMatrix: formulationsSample 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 mLwith chloroform, filter (0.45 µm silver membrane, Selas Corp.). Evaporate a 2 mL aliquotof the filtrate to dryness under a stream of nitrogen, reconstitute with 5 mL 330 µg/mLbenzanilide in MeCN, inject a 5 µL aliquot.

HPLC VARIABLESColumn: 300 × 4 10 µm µBondapak C18Mobile phase: MeCN:water 40:60Flow rate: 1.5Injection volume: 5Detector: UV 254

CHROMATOGRAMRetention time: 7Internal standard: benzanilide (11)

OTHER SUBSTANCESSimultaneous: sulfanilamide (2.5), sulfisoxazole (3)Noninterfering: erythromycin ethylsuccinate

KEY WORDSoral suspensions

REFERENCEElrod, 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 BIBLIOGRAPHYSuber, 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; sulfamer-azine; sulfamethazine; sulfapyridine; sulfisoxazole; N-acetylsulfisoxazole; sulfathiazole; in plasma]

Acrivastine 7

AcrivastineMolecular formula: C22H24N2O2Molecular weight: 348.44



N COOH

N

H3C

SAMPLEMatrix: bloodSample 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 layerand add it to 75 µL 10 mM ammonium acetate buffer containing 0.1% formic acid (pH3.2), evaporate (?). Add 75 µL MeCN, sonicate for 5 min, centrifuge at 5000 rpm for5 min, keep the extract as B. Add 20 µL 1 µg/mL enalapril in MeOH:water 50:50 and120 mg NaCl to the aqueous layer (A), mix, add 500 µL pH 3 phosphate buffer, add600 µL 8.5% phosphoric acid, add 5 mL dichloromethane:isopropanol 95:5, shake at250 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 at45◦. 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 quanti-tation of 18 antihistamine drugs in blood by liquid chromatography ionspray tandemmass spectrometry. Forensic Sci.Inter. 2001, 121, 108–115.)

HPLC VARIABLESGuard column: 40 mm long 4 µm Purospher RP-18 LiChro Cart 4-4Column: 100 × 2.1 4 µm Genesis C18 (Jones Chromatography)Column temperature: 35Mobile 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 mMammonium acetate containing 0.1% formic acid (pH 3.2).

Flow rate: 0.2Injection volume: 30Detector: 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 heater375◦, heater gas flow 7 L/min

CHROMATOGRAMRetention time: 5.7Internal standard: dibenzepin, enalaprilLimit of detection:

8 Acrivastine

(6.1, 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

10 Adapalene

AdapaleneMolecular formula: C28H28O3Molecular weight: 412.52



COOH

H3CO

SAMPLEMatrix: formulationsSample preparation: Inject an aliquot of a 0.1% gel.

HPLC VARIABLESColumn: 250 × 4 ODS-RP18 (Merck)Mobile phase: MeCN:THF:water:trifluoroacetic acid 43:36:21:0.02Flow rate: 1Detector: UV 270

CHROMATOGRAMRetention time: 6.1

OTHER SUBSTANCESNoninterfering: tretinoin

KEY WORDSgel

REFERENCEMartin, 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.

Adefovir dipivoxil 11

Adefovir dipivoxilMolecular formula: C20H32N5O8P




N

N N

N

O P

OO

O

O

O

CH3

CH3CH3

O

O

CH3CH3

CH3

NH2

SAMPLEMatrix: bloodSample preparation: Mix 100 µL plasma with 200 µL 0.1% trifluoroacetic acid in

MeCN. Evaporate the supernatant to dryness under reduced pressure at room temper-ature. 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 mMtetrabutylammonium hydrogen phosphate, inject a 50 µL aliquot.

HPLC VARIABLESGuard column: 15 × 3.2 Brownlee RP-18 NewguardColumn: 150 × 4.6 Zorbax RX-C18Column temperature: 35Mobile 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 mMpH 6.0 potassium phosphate buffer containing 5 mM tetrabutylammonium hydrogenphosphate 65:35. A:B 100:0 for 2 min, to 0:100 over 13 min, re-equilibrate at initialconditions for 10 min. (Only adefovir is detected in blood. However, the method isreported to distinguish between adefovir and adefovir dipivoxil.)

Flow rate: 1.5Injection volume: 50Detector: F ex 236 em 420

KEY WORDSderivatization; dog; pharmacokinetics; plasma

REFERENCECundy, K.C.; Sue, I.-L.; Visor, G.C.; Marshburn, J.; Nakamura, C.; Lee, W.A.; Shaw, J.P. Oral formula-

tions of adefovir dipivoxil: In vitro dissolution and in vivo bioavailability in dogs, J.Pharm.Sci., 1997,86, 1334–1338.

SAMPLEMatrix: bloodSample 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 itwith 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 VARIABLESGuard column: 10 × 3 R3 (Chrompack)Column: 150 × 4.6 5 µm Chromspher C8Column temperature: 40 ± 2Mobile 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: 20Detector: F ex 254 em 425

CHROMATOGRAMRetention time: 4.5Limit of quantitation: 10 ng/mL

OTHER SUBSTANCESExtracted: adefovir (4)

KEY WORDSderivatization; plasma

REFERENCESparidans, R.W.; Veldkamp, A.; Hoetelmans, R.M.W.; Beijnen, J.H. Improved and simplified liquid chro-

matographic assay for adefovir, a novel antiviral drug, in human plasma using derivatization withchloroacetaldehyde, J.Chromatogr.B, 1999, 736, 115–121.

Adrenocorticotropic hormone 13

Adrenocorticotropic hormoneCAS Registry No: 9002-60-2


SAMPLEMatrix: bloodSample preparation: Condition a 1 mL Analytichem weak cation-exchange (car-

boxymethylhydrogen form, CBA) SPE cartridge with 1 mL 1% trifluoroacetic acidin MeOH, 1 mL MeOH, and 2 mL water. Add 1 mL plasma to the SPE cartridge, rinsethe 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 with2 mL 1% trifluoroacetic acid in MeOH. Evaporate the eluate to dryness under a streamof nitrogen, reconstitute the residue in 100 µL MeOH:buffer 50:50, inject a 5–75 µLaliquot. (Buffer was 5.7 g monochloroacetic acid, 2.0 g NaOH, and 0.2 g disodium EDTAin 1 L water, pH 3.2.) (The procedure was not necessarily validated for this compound.)

HPLC VARIABLESColumn: 250 × 2 5 µm Ultrasphere octylColumn temperature: 60Mobile 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 over30 min, maintain at 70:30 for 1 h. (The buffer was 5.7 g monochloroacetic acid, 2.0 gNaOH, and 0.2 g disodium EDTA in 1 L water, pH 3.2.)

Flow rate: 0.3Injection volume: 5–75Detector: 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 pumpedat 0.05 mL/min and the mixture flowed through a 12 m ×0.33 mm ID reaction coil at70◦ to the detector.

CHROMATOGRAMRetention time: 45Limit of detection: 100 fmole

OTHER SUBSTANCESSimultaneous: angiotensin I, angiotensin II, angiotensin III, atrial natriuretic peptide,

bombesin, bradykinin, gonadorelin (LHRH), somatoliberin, vasopressin

KEY WORDSplasma; post-column reaction; SPE

REFERENCERhodes, 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.

SAMPLEMatrix: solutions

HPLC VARIABLESColumn: 300 × 3.9 10 µm µBondapak C18Mobile phase: Gradient. A was 0.08% trifluoroacetic acid. B was MeCN:0.08% trifluo-

roacetic acid 70:30. A:B from 70:30 to 50:50 over 30 min.

14 Adrenocorticotropic hormone

Flow rate: 1Detector: UV 206


OTHER SUBSTANCESSimultaneous: adrenocorticotropic hormone fragments, melanotropin

KEY WORDShuman

REFERENCEMcDermott, 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.

SAMPLEMatrix: solutionsSample preparation: Dissolve in 100 mM NaH2PO4 adjusted to pH 2.1 with orthophos-

phoric acid, inject a 100 µL aliquot.

HPLC VARIABLESColumn: 250 × 4 Aquapore RP 300 (Kontron)Mobile phase: Gradient. A was 100 mM NaH2PO4 adjusted to pH 2.1 with orthophos-

phoric acid. B was MeOH. A:B from 90:10 to 35:65 over 180 min.Flow rate: 1Injection volume: 100Detector: UV 225


OTHER SUBSTANCESSimultaneous: adrenocorticotropin hormone fragments, lipotropic hormone and frag-

ments, melanotropin, endorphins, prolactin, somatropin, menotropins

KEY WORDSpig

REFERENCERichter, 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, adrenocor-ticotropic hormone, and β-endorphin, J.Neurochem., 1985, 44, 1697–1703.

ANNOTATED BIBLIOGRAPHYCapp, M.W.; Simonian, M.H. Separation of the major adrenal steroids by reversed-phase high-

performance 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 reversed-

phase 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.

Afloqualone 15

AfloqualoneMolecular formula: C16H14FN3O




N

NF

O

H2N

H3C


HPLC VARIABLESColumn: Chiralpak ASColumn temperature: 50Mobile phase: Hexane:EtOH 95:5Flow rate: 1.3Detector: UV 254

CHROMATOGRAMRetention time: 30, 35 (enantiomers)

KEY WORDSchiral

REFERENCEApplication Guide for Chiral Column Selection, Second Edition; Chiral Technologies: Exton PA, 1995,

p. 43.

16 Alacepril

AlaceprilMolecular formula: C20H26N2O5S




H3C S N

O

CH3

OO N

H

COOH


HPLC VARIABLESColumn: 250 × 4.6 10 µm Cosmosil 5C18-MSColumn temperature: 50Mobile 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 40:60Flow rate: 1.5Detector: UV (wavelength not specified)

CHROMATOGRAMRetention time: 10.9 (gradient) or 4.1 (isocratic)

OTHER SUBSTANCESSimultaneous: acetaminophen (7.9), ampicillin (7.9), aspirin (10.0), caffeine (8.5), car-

benicillin (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)

REFERENCESugiyama, T.; Matsuyama, R.; Usui, S.; Katagiri, Y.; Hirano, K. Selection of mobile phases in high-

performance liquid chromatographic determination for medicines, Biol.Pharm.Bull., 2000, 23,274–278.

SAMPLEMatrix: enzyme reactionsSample 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 ofthe supernatant.

HPLC VARIABLESColumn: 250 × 4.6 Cosmosil 5-C18 MSColumn temperature: 50Mobile phase: MeCN:10 mM pH 2.5 potassium phosphate buffer 40:60Flow rate: 1.5Injection volume: 30Detector: UV 220

CHROMATOGRAMInternal standard: n-propyl paraben

Alacepril 17

OTHER SUBSTANCESExtracted: deacetylalacepril

REFERENCEUsui, S.; Kubota, M.; Iguchi, K.; Kiho, T.; Sugiyama, T.; Katagiri, Y.; Hirano, K. Sialic acid 9-O-

acetylesterase catalyzes the hydrolyzing reaction from alacepril to deacetylalacepril, Pharm.Res.,2003, 20, 1309–1316.

18 Alclometasone 17,21-dipropionate

Alclometasone 17,21-dipropionateMolecular formula: C28H37ClO7Molecular weight: 521.05CAS Registry No: 66734-13-2Merck Index: 13, 219

O

H

CH3 H

H

HO

O

O

CH3O

CH3

O CH3

O

Cl

CH3

SAMPLEMatrix: formulationsSample 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 MeOHor MeOH:dichloromethane 10:90 (depending on what appears visually to give bestsolubility) at 40◦ for 10 min, centrifuge, collect the clear supernatant. Add 5 mL of thesupernatant to the SPE cartridge, wash with 4 mL acetone:water 20:80, wash with1 mL n-hexane, elute with 4 mL diethyl ether. Evaporate the eluate to dryness underreduced pressure, reconstitute the residue with 5 mL (or more) MeOH, inject a 10 µLaliquot.

HPLC VARIABLESColumn: 250 × 4.6 5 µm endcapped Purospher RP-18Column temperature: 25Mobile 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: 1Injection volume: 10Detector: UV 239

CHROMATOGRAMRetention time: k′ 2.55 (isocratic); 21.0 min (gradient)Limit of detection: 300 ng/mL

OTHER SUBSTANCESSimultaneous: 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; LOD0.1 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 retentiontime (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-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 retentiontime (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), 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; LOD0.3 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 retentiontime (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 retentiontime (min) 22.3; LOD 0.5 µg/mL), deoxymethasone (isocratic k′ 0.64; gradient retentiontime (min) 14.2; LOD 0.2 µg/mL), dexamethasone (isocratic k′ 0.27; gradient retentiontime (min) 11.9; LOD 0.1 µg/mL), dexamethasone-21-acetate (isocratic k′ 0.91; gradi-ent retention time (min) 16.1; LOD 0.2 µg/mL), dexamethasone isonicotinate (isocratick′ 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), dexamethasonevalerate (isocratic k′ 3.00; gradient retention time (min) 21.6; LOD 0.3 µg/mL), diflucor-tolone 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; LOD0.3 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 retentiontime (min) 25.1; LOD 0.3 µg/mL), fluocortolone pivalate (isocratic k′ 4.50; gradientretention time (min) 23.6; LOD 0.3 µg/mL), fluorometholone (isocratic k′ 0.59; gradientretention 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; LOD0.6 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 retentiontime (min) 22.0; LOD 0.2 µg/mL), prednisolone-21-acetate (isocratic k′ 0.60; gradientretention 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 (isocratick′ 2.05; gradient retention time (min) 19.7; LOD 0.3 µg/mL), triamcinolone (isocratick′ 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), triamcinolonediacetate (isocratic k′ 0.45; gradient retention time (min) 13.9; LOD 0.3 µg/mL)

KEY WORDScosmetics; SPE

REFERENCEGagliardi, 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 theidentification and determination of corticosteroids in cosmetic products, Anal.Chim.Acta, 2002, 457,187–198.

SAMPLEMatrix: formulationsSample 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 ensurethat no large clumps stick to the tube, mix with the tube on its side on an oscillatingshaker for 15 min. Remove the upper ethyl acetate layer and wash with citrate buffer asbefore. 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. Combinethe MeCN/water layers and evaporate them to dryness, dissolve the residue in 300 µLMeOH, filter (0.45 µm nylon), inject a 5 µL aliquot.

20 Alclometasone 17,21-dipropionate

HPLC VARIABLESGuard column: 15 × 3.2 7 µm Brownlee NewGuard C18Column: 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: 1Injection volume: 5Detector: UV 240

CHROMATOGRAMRetention time: 10.93Limit of detection: 0.001%

OTHER SUBSTANCESSimultaneous: 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), fluocinoloneacetonide (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), hydrocortisone17-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), paramethasone21-acetate (8.64), prednicarbate (10.72), prednisone (5.46), triamcinolone (4.15),triamcinolone acetonide (7.04), triamcinolone 16,21-diacetate (7.49), triamcinolonehexacetonide (13.12)

KEY WORDSbody wash, cream, gel, lotion, shampoo, spray

REFERENCEReepmeyer, 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 21

AlitretinoinMolecular formula: C20H28O2Molecular weight: 300.43


Merck Index: 13, 244[9-cis-retinoic acid]

CH3

H3C

COOH

CH3H3CCH3

SAMPLEMatrix: bloodSample 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 shakerfor 10 min, freeze the aqueous layer in a dry ice/acetone bath. Decant the organic layerand evaporate it to dryness under nitrogen at 25◦, reconstitute the residue with 200 µLMeOH, add 100 µL 5 mM ammonium acetate, centrifuge at 13 000 g for 3 min, inject a100 µL aliquot. (Use silanized glassware. Process under yellow light.)

HPLC VARIABLESGuard column: 10 × 2 5 µm Hypersil BDS C18Column: 100 × 4.6 3 µm Microsorb Short One C18 (Rainin)Column temperature: 36Mobile 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 over14.4 min, to 30:70 over 0.5 min, maintain at 30:70 for 10 min.

Flow rate: 1Injection volume: 100Detector: UV 348

CHROMATOGRAMRetention time: 21Internal standard: all-trans-3,7-dimethyl-9-(2,4,6-trimethylphenyl)-2,4,6,8-nonatetra-

enoic acid (Ro 11–5036) (19)Limit of detection: 2.5 ng/mL

OTHER SUBSTANCESExtracted: isotretinoin (19.5), tretinoin (21.5), vitamin A (20.5)

KEY WORDSplasma

REFERENCEDzerk, 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.

SAMPLEMatrix: blood, food, formulations, tissueSample preparation: Serum. Extract one volume (20–100 µL) serum with three vol-

umes isopropanol:dichloromethane 2:1 containing about 6 nM IS and 1 mM butylatedhydroxytoluene (BHT, antioxidant), add glacial acetic acid (1 µL/20 µL serum). Vortexfor 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 pulpof fruits and fresh vegetables with 3–5 mL isopropanol:dichloromethane 2:1, make upto 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, vortexthe mixture, centrifuge or filter. Evaporate the supernatant or filtrate to dryness ina 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 mLisopropanol: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, centrifugeabout 500 µL solution, inject a 50 µL aliquot of the supernatant.

HPLC VARIABLESGuard column: C18 (Upchurch)Column: 100 × 4.6 3 µm Microsorb MVMobile 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 conditionsfor 10 min.

Flow rate: 0.8Injection volume: 50Detector: UV 340

CHROMATOGRAMRetention time: 10.2Internal standard: retinyl acetate (13.8)

OTHER SUBSTANCESExtracted: 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 WORDShuman, ketchup, liver, mango, multivitamin tablets, papaya, rat, serum, spinach, tomato

REFERENCEBarua, 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 animaland plant samples, J.Chromatogr.B, 1998, 707, 69–79.

SAMPLEMatrix: formulationsSample 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 at3500 rpm for 6 min. Filter the supernatants, combine, make up to 250 mL. Dilute a1 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 rpmfor 10 min. Filter the supernatant and make up to 10 mL. Dilute a 1 mL aliquot to5 mL with mobile phase, filter (nylon 0.45 µm), inject an aliquot. Cream. Sonicate analiquot 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 mLwith MeCN:EtOH:1% acetic acid 70:20:10. Dilute a 2 mL aliquot to 5 mL with mobilephase, filter (nylon 0.45 µm), inject an aliquot.

HPLC VARIABLESColumn: 250 × 3.2 Phenomenex Prodigy 5ODSColumn temperature: 32 ± 2Mobile phase: MeCN:EtOH:1% acetic acid 68:8:24Flow rate: 0.4Injection volume: 20Detector: F ex 350 em 520

Alitretinoin 23

CHROMATOGRAMRetention time: 31Limit of detection: 11.09 pmole (S/N = 3)

OTHER SUBSTANCESSimultaneous: isotretinoin (28.5), tretinoin (33)

KEY WORDSavoid exposure to light, use amber-colored glassware, capsules, cream, gel

REFERENCEGatti, 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 BIBLIOGRAPHYDisdier, 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-cis-

retinoic 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 liquidchromatography 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 threemajor 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 forretinoid 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 cat-alyzed by sulfhydryl compounds, Drug Metab.Dispos., 1997, 25, 27–32.

Van Merris, V.; Meyer, E.; De Wasch, K.; Burvenich, C. Simple quantification of endogenous retinoidsin 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-trans-retinoic acid (tretinoin) and their 4-oxo metabolites in human and animal plasma by high-performanceliquid 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. Deter-mination of endogenous levels of retinoic acid isomers in type II diabetes mellitus patients. Possiblecorrelation with HbA1c values, Biol.Pharm.Bull., 2002, 25, 1268–1271.

24 Allethrin

AllethrinMolecular formula: C19H26O3Molecular weight: 302.41



H3C

O

OH3C

O

CH2

CH3H3C

H3C

SAMPLEMatrix: fruit, vegetablesSample 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 col-umn, wash with 40 mL toluene, wash with 40 mL toluene:MeCN 99:1. Homogenize25 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 themto 60 mL toluene and 300 mL 10% NaCl in water, shake well for 3 min, let lay-ers separate. Dry the organic layer by passing it through 20 g anhydrous sodiumsulfate in a 20 mm diameter column, concentrate to about 5 mL under reducedpressure 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◦, reconstitutewith 1 mL MeOH, inject an aliquot. (Reflux activated charcoal (20–40 mesh) with1 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 VARIABLESColumn: 300 × 3.9 10 µm µBondapak C18Column temperature: 50Mobile phase: Gradient. MeCN:water from 62:38 to 78:22 over 32 min (Waters curve 6).Flow rate: 1.5Detector: UV 206

CHROMATOGRAMRetention time: 11.55Limit of detection: 50 ng/g

OTHER SUBSTANCESSimultaneous: cypermethrin (21.05–22.08), permethrin (24.60, 27.01), tetramethrin

(13.08)

KEY WORDSapple, cabbage, cucumber; peach, pear, tomato, SPE

REFERENCEPang, G.-F.; Chao, Y.-Z.; Liu, X.-S.; Fan, C.-L. Multiresidue liquid chromatographic method for simul-

taneous determination of pyrethroid insecticides in fruits and vegetables, J.AOAC Int., 1995, 78,1474–1480.


HPLC VARIABLESColumn: Two 250 × 4 Phase 3019 columns in series (Phenomenex)Mobile phase: Hexane:1,2-dichloroethane:EtOH 500:30:0.15

Allethrin 25

Flow rate: 0.8Detector: UV 230

CHROMATOGRAMRetention time: 34, 36, 37, 39, 40, 42, 44, 46 (isomers)

REFERENCEPhenomenex Catalogue, Phenomenex: Torrance CA, 1994, p. 1035.


HPLC VARIABLESGuard column: 50 × 4 40 µm pellicular materialColumn: 250 × 4.6 5 µm Ultrasphere octadecylsilicaMobile phase: MeOH:water 80:20Flow rate: 1Injection volume: 10Detector: UV 254

CHROMATOGRAMRetention time: k′ 4.68 (cis), k′ 5.32 (trans)

OTHER SUBSTANCESAlso 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))

REFERENCEAbidi, S.L. Column selectivity in high-performance liquid chromatography of substituted gem-dimethyl-

cyclopropanes, J.Chromatogr., 1986, 368, 59–76.


HPLC VARIABLESColumn: 250 × 4.6 5 µm Cyclobond I cyclodextrin-modified silica (Astec)Mobile phase: MeCN:water 22:78Flow rate: 1Detector: UV 220

CHROMATOGRAMRetention time: 7 (cis isomers), 9.5 (1R,trans, αS), 10.5 (1S,trans, αR), 13 (1R,trans,

αR), 15 (1S,trans, αS)

KEY WORDScomparison with GC

REFERENCEKutter, J.P.; Class, T.J. Diastereoselective and enantioselective chromatography of the pyrethroid insec-

ticides allethrin and cypermethrin, Chromatographia, 1992, 33, 103–112.

SAMPLEMatrix: solutionsSample preparation: Inject an aliquot of a 0.1–1 mg/mL solution in hexane.

26 Allethrin

HPLC VARIABLESGuard column: 5 µm Spherisorb NH2Column: 250 × 4.6 Pirkle ionic type 1-A column (Technicol)Mobile phase: Hexane:isopropanol 99.85:0.15Flow rate: 0.8Detector: UV 230

OTHER SUBSTANCESAlso analyzed: cypermethrin, fenpropathrin, fenvalerate, tetramethrin

KEY WORDSchiral

REFERENCELisseter, S.G.; Hambling, S.G. Chiral high-performance liquid chromatography of synthetic pyrethroid

insecticides, J.Chromatogr., 1991, 539, 207–210.

SAMPLEMatrix: urineSample 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 theaqueous layer with 1 mL MeCN. Combine the MeCN layers and adjust to a knownvolume (0.5–1 mL), mix, filter (0.45 µm), inject a 40 µL aliquot.

HPLC VARIABLESColumn: 150 × 3 3 µm Luna C18(2) (Phenomenex)Column temperature: 30Mobile 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 initialconditions over 1 min.

Flow rate: 0.5Injection volume: 40Detector: UV 235

CHROMATOGRAMRetention time: 31.8Limit of detection: 5 ng/mL

OTHER SUBSTANCESExtracted: 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, LOD5 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)

REFERENCELoper, 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 27

AlmotriptanMolecular formula: C17H25N3O2S




N

NS

N

O O

H

SAMPLEMatrix: blood, urineSample 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 µLwater, elute with mobile phase over 1 min (straight onto column (?)).

HPLC VARIABLESGuard column: Guardpak µBondapak CNColumn: 150 × 4 5 µm Spherisorb ODS-2Mobile phase: MeCN:50 mM pH 4.0 sodium phosphate buffer:triethylamine 20:80:0.2Flow rate: 1Detector: UV 227

CHROMATOGRAMRetention time: 6.5Internal standard: 4-[3-(2-aminoethyl)-1H-indol-5-ylmethylsulfonyl]piperazine-1-car-

boxylic acid ethyl ester (10)Limit of quantitation: 1 ng/mL (plasma), 50 ng/mL urine

KEY WORDSplasma, SPE

REFERENCEJansat, J.M.; Costa, J.; Salvà, P.; Fernandez, F.J.; Martinez-Tobed, A. Absolute bioavailability, pharma-cokinetics, and urinary excretion of the novel antimigraine agent almotriptan in healthy male volunteers,J.Clin.Pharmacol., 2002, 42, 1303–1310.

SAMPLEMatrix: microsomal incubationsSample preparation: Mix 500 µL microsomal incubation with 1 mL 200 mM pH 4

sodium acetate buffer, centrifuge, inject an aliquot.

HPLC VARIABLESGuard column: GuardPak µBondapak CNColumn: 300 × 3.9 10 µm µBondapakMobile 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% triethy-lamine, adjusted to pH 6.5 with NaOH.

Flow rate: 1Detector: UV 227


OTHER SUBSTANCESExtracted: metabolites

28 Almotriptan

KEY WORDShuman, liver

REFERENCESalva, 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.

Alosetron 29

AlosetronMolecular formula: C17H18N4O


CAS Registry No: 122852-42-0, 122852-69-1 (HCl)

Merck Index: 13, 305N

N

H3CN

N

CH3

O

H

SAMPLEMatrix: bloodSample 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 buffercontaining 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 ofMeCN: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. (Thebuffer was 10 mM ammonium acetate adjusted to pH 4.0 with glacial acetic acid.)

HPLC VARIABLESGuard column: 15 × 4.6 7 µm Spherisorb cyanopropylColumn: 100 × 4.6 5 µm Spheri cyanopropyl (Brownlee)Column temperature: 45Mobile phase: MeOH:THF:10 mM pH 4.0 ammonium acetate buffer 24:6:70Flow rate: 0.5Injection volume: 200Detector: F ex 295 em 370

CHROMATOGRAMRetention time: 10.1Internal standard: GR87442, 6-fluoroalosetron (Glaxo) (13.7)Limit of quantitation: 0.1 ng/mL

OTHER SUBSTANCESNoninterfering: amitriptyline, carbamazepine, carmustine, chlorpromazine, cimeti-

dine, cisplatin, cyclophosphamide, dexamethasone, diazepam, digoxin, etoposide, furo-semide, haloperidol, ibuprofen, imipramine, indomethacin, methotrexate, phenobarbi-tal, phenytoin, propranolol, ranitidine, theophylline, triazolam, warfarin

KEY WORDSplasma; serum; SPE

REFERENCELloyd, 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

AmcinonideMolecular formula: C28H35FO7Molecular weight: 502.57


Merck Index: 13, 387O

CH3 H

CH3

F H

OO

OOH3C

O

HO

SAMPLEMatrix: formulationsSample 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 MeOHor MeOH:dichloromethane 10:90 (depending on what appears visually to give the bestsolubility) at 40◦ for 10 min, centrifuge, collect the clear supernatant. Add 5 mL of thesupernatant to the SPE cartridge, wash with 4 mL acetone:water 20:80, wash with1 mL n-hexane, elute with 4 mL diethyl ether. Evaporate the eluate to dryness underreduced pressure, reconstitute the residue with 5 mL (or more) MeOH, inject a 10 µLaliquot.

HPLC VARIABLESColumn: 250 × 4.6 5 µm endcapped Purospher RP-18Column temperature: 25Mobile 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: 1Injection volume: 10Detector: UV 239

CHROMATOGRAMRetention time: k′ 3.18 (isocratic); 22.6 min (gradient)Limit of detection: 100 ng/mL

OTHER SUBSTANCESSimultaneous: 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 reten-tion 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-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 (iso-cratic 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), betamethasone-17,21-divalerate (isocratic k′ 10.82; gradient retentiontime (min) 28.0; LOD 0.4 µg/mL), betamethasone-21-acetate (isocratic k′ 0.77; gradientretention 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 (iso-cratic k′ 5.45; gradient retention time (min) 26.3; LOD 0.1 µg/mL), cortisone (isocratick′ 0.18; gradient retention time (min) 11.1; LOD 0.6 µg/mL), cortisone acetate (isocratick′ 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), 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; LOD0.4 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 retentiontime (min) 21.6; LOD 0.3 µg/mL), diflucortolone valerate (isocratic k′ 4.73; gradientretention 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 (isocratick′ 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 (iso-cratic 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), fluocortolonecaproate (isocratic k′ 6.59; gradient retention time (min) 25.1; LOD 0.3 µg/mL), fluocor-tolone 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; LOD0.1 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 retentiontime (min) 17.7; LOD 0.6 µg/mL), hydrocortisone-21-acetate (isocratic k′ 0.77; gradientretention time (min) 15.3; LOD 0.6 µg/mL), hydrocortisone pivalate (isocratic k′ 2.27;gradient retenti

hplc methods for pharmaceuticals methods for... · 2018. 11. 24. · levosimendan / 349 lidamidine...

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