HPLC METHODS FOR RECENTLY APPROVED PHARM

This book is a collection of procedures for the analysis of more than 390 ceuticals using high-performance liquid chromatography HPLC and covers the pharma-literature up to the end of 20

HPLC - METHODS FORRECENTLY APPROVED

PHARMACEUTICALS

A JOHN WILEY & SONS, INC., PUBLICATION

HPLC METHODS FOR RECENTLY APPROVED PHARMACEUTICALS

George Lunn

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 form

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to the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400, fax 978-646-8600, or on the web at www.copyright.com Requests to the Publisher for permission should be addressed to the Permissions Department, John Wiley & Sons, Inc., 111 River Street, Hoboken, NJ

07030, (201) 748-6011, fax (201) 748-6008.

Limit of Liability/Disclaimer of Warranty: While the publisher and author have used their best efforts

in preparing this book, they make no representations or warranties with respect to the accuracy or completeness of the contents of this book and specifically disclaim any implied warranties of

merchantability or fitness for a particular purpose No warranty may be created or extended by sales representatives or written sales materials The advice and strategies contained herein may not be suitable for your situation You should consult with a professional where appropriate Neither the publisher nor author shall be liable for any loss of profit or any other commercial damages, including but not limited to special, incidental, consequential, or other damages.

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Library of Congress Cataloging-in-Publication Data:

10 9 8 7 6 5 4 3 2 1

Bethanechol chloride / 74Bexarotene / 75

Biapenem / 77Bimatoprost / 79Bioresmethrin / 80Bivalirudin / 81Boldenone / 82Bosentan / 83

β-Boswellic acid / 86

Brimonidine / 88Bromfenac / 90Brovincamine / 92Bucillamine / 93Budipine / 94Bulaquine / 95Butacaine / 97Butamben / 99Butoconazole / 100Butyl flufenamate / 101Cambendazole / 102Candesartan cilexetil / 104Capecitabine / 106

Casanthranol / 108Caspofungin / 109

v

Fomepizole / 274Fomivirsen / 276Fondaparinux / 277Formestane / 278

Masoprocol / 367Maxacalcitol / 368Medetomidine / 369Meglutol / 371Melatonin / 372Melengestrol acetate / 375Memantine / 378

Menthol / 380Mepenzolate bromide / 381Mepixanox / 383

Mequinol / 384Methenamine / 385Methoprene / 386Methoxychlor / 387Methyltestosterone / 392Metrizamide / 393Metyrosine / 394Micafungin / 396Milnacipran / 398Mirtazapine / 400Misoprostol / 405Mizolastine / 406Moexipril / 411Mofezolac / 412Mometasone furoate / 413Monensin / 415

Morantel / 416Mosapride / 417Moxifloxacin / 420Moxonidine / 423Nadifloxacin / 424Naftopidil / 425Nandrolone / 427Narasin / 429Nartograstim / 430Nateglinide / 431Nebivolol / 433Nelfinavir / 435Nequinate / 440Neridronic acid / 441Nevirapine / 443Nicarbazin / 447Nilutamide / 448Nipradilol / 449Nitazoxanide / 450Nitenpyram / 452

Rofecoxib / 562Ropinirole / 565Rosiglitazone / 566Rosuvastatin calcium / 568Sarafloxacin / 569

Selamectin / 571Sermorelin / 572Sibutramine / 574Sildenafil / 576Simethicone / 579Sivelestat / 580Sodium oxybate / 582Somatropin / 583Squalane / 584Squalene / 585Stanozolol / 587Succimer / 588Succinylcholine chloride / 589Sulfabromomethazine / 591Sulfachlorpyridazine / 592Sulfaethoxypyridazine / 596Sulfamerazine / 598

Sulfanitran / 600Sultamicillin / 602Tacalcitol / 604Talipexole / 605Taltirelin / 607Technetium Tc 99m bicisate / 608Tegaserod / 609

Telithromycin / 610Telmesteine / 611Telmisartan / 612Temocapril / 614Temozolomide / 615Tenofovir disoproxil fumarate / 617Teprenone / 620

Teriparatide / 621Tetrachlorvinphos / 622Tetrahydrozoline / 623Thalidomide / 626Thialbarbital / 628Thyrotropin / 629Tiagabine / 630Tiletamine hydrochloride / 631

Valdecoxib / 666Valganciclovir / 668Valrubicin / 670Valsartan / 671Zaleplon / 673Zaltoprofen / 676Zanamivir / 678Zinostatin / 680Zofenopril calcium / 681Zolazepam hydrochloride / 683Cumulative Index / 685

Cross-Index to Other Substances / 703

This book is a collection of procedures for the analysis of more than 390 ceuticals using high-performance liquid chromatography (HPLC) and covers the

pharma-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 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 forwhich analytical methods were not then available in the literature

ž Drugs for which procedures allowing determination in a blood matrix have onlybecome 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 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 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 choosing

an 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 (George

Lunn and Louise C Hellwig, John Wiley, New York, 1998), will notice many ities The abstract structure is very similar, and the philosophy that the procedures

similar-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 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 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

GEORGELUNN

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 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’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

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

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 Levo rotatory R isomer Prefix ar-Dextro rotatory R isomer Prefix dex/dextro-Dextro rotatory S isomer Prefix es-

lev/levo-For racemates, the rac- prefix is used

In some cases, the chiral prefix is used Thus, the following list shows the prefixesthat 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 xvii

Levosimendan in this volume

Levothyroxine in Volumes 1 and 3

More generally, the name of the racemic compound is used Thus,

xviii About This Book

About This Book xix

Also Compounds that can be analyzed at the same time It is not

specified whether they interfere, but they can beextracted See also Extracted, Simultaneous

Column Dimensions are length (mm)× internal diameter (mm), and

the 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)

Extracted 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 target

compound Compounds that interfere with thechromatography of the internal standard (IS) are listedunder simultaneous (another IS can always be selected or

an 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 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 Compounds that do not interfere with the analysis for

various reasons, e.g., they are not extracted, they are notdetected

Retention time 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 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 do

not interfere Note that the compound cannot necessarily

be extracted from the matrix in question (although it maybe) See also Also, Extracted

SPE For the sake of consistency, conditioning procedures for

solid-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 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 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

About This Book xxi

BHT 2,6-Di-tert-butyl-4-methylphenol, butylated hydroxytoluene

DMSO Dimethyl sulfoxide

LOD 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 range

M Molar (i.e., moles/L)

MeCN Acetonitrile

mL Milliliter

mM Millimolar (i.e., millimoles/L)

MS Mass spectrometric detection

MSPD Matrix solid phase dispersion

MTBE Methyl tert-butyl ether

nM Nanomolar (i.e., nanomoles/L)

psi Pounds/sq in (1 psi= 6.89476 kPa)

SEC Size Exclusion Chromatography

SFC Supercritical fluid chromatography

SFE Supercritical fluid extraction

SPE Solid phase extraction

UV Ultraviolet detection

xxii About This Book

PIC REAGENTS

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

PIC A is tetrabutylammonium sulfate

PIC B5 is pentanesulfonic acid

PIC B7 is heptanesulfonic acid

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;10chloroform,11dichloromethane,12dioxane,13and carbon tetra-chloride14 are carcinogenic in experimental animals; and DMF15 and MTBE16,17

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,18and 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, 13thedition, 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

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, 2nd

edition, John Wiley & Sons, New York, 1997.

7 Meyer, V.R Pitfalls and Errors of HPLC in Pictures, H ¨uthig, 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

gaso-line additive – causes testicular and lympho-haematopoietic cancers in rats,

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.

N H

to inactivate HIV Vortex 800µL 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 ammoniumacetate buffer, suck dry for 1 min, elute with 800µL MeOH Evaporate the eluate todryness under a stream of nitrogen at 40◦ and reconstitute the residue with 100µLmobile phase Vortex for 30 s, centrifuge at 18 000 g for 3 min, and inject an 80µLaliquot

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 36:64over 15 min, maintain at 36:64 for 3 min, re-equilibrate at initial conditions for 7 min

Sample preparation: Condition a 100 mg Dual Zone C18 SPE cartridge (Diazem) with

2 mL MeOH and 2 mL water Dilute 500µL serum with 1 mL water, add to the SPEcartridge, wash with 500µL water, elute with 1 mL MeOH Evaporate the eluate to

1

HPLC Methods for Recently Approved Pharmaceuticals by George Lunn

Copyright  2005 John Wiley & Sons, Ltd ISBN: 0-471-66941-5

2 Abacavir

dryness with vortexing under reduced pressure at 40◦and reconstitute the residue with

300µL MeOH, inject a 10 µL aliquot

Simon, 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,

Guard column: 20× 3.8 Symmetry C18 (Waters)

Column: 100× 4.6 3.5 µm Symmetry C18 (Waters)

KEY WORDS

plasma

Column: 150× 3.2 5 µm Kromasil C18 (Phenomenex)

Mobile phase: Gradient MeOH:25 mM pH 4.0 ammonium acetate buffer from 5:95 to50:50 over 30 min, re-equilibrate at initial conditions for 10 min

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.

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,

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 namics of abacavir alone and in combination with zidovudine in human immunodeficiency virus-

pharmacody-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 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

H 3 C N HO

OH

OH

OH HO

HO

HO HO OH OH

SAMPLE

Matrix: formulations

Sample preparation: Powder tablet, extract 3 times with 5 mL aliquots of water withsonication 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 50µL aliquot

to 1 mL with MeOH, filter (0.2µM), inject a 20 µL aliquot

CH 3

CH 3

N O

of the filtrate to dryness under a stream of nitrogen, reconstitute with 5 mL 330µg/mLbenzanilide in MeCN, inject a 5µL aliquot

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;

sulfamer-azine; sulfamethsulfamer-azine; sulfapyridine; sulfisoxazole; N-acetylsulfisoxazole; sulfathiazole; in plasma]

5 min, keep the extract as B Add 20µL 1 µg/mL enalapril in MeOH:water 50:50 and

120 mg NaCl to the aqueous layer (A), mix, add 500µL pH 3 phosphate buffer, add

600µL 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 150µL initial mobile phase, sonicate, centrifuge,combine with extract B, inject a 30µL aliquot (Sample preparation from Gergov,M.;Robson,J.N.; Ojanper ¨a,I.; Heinonen,O.P.; Vuori,E Simultaneous screening and quanti-tation 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 4µm 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 at0:100 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: 0.2

Injection volume: 30

Detector: MS, PE Sciex API 365 triple stage quadrupole LC-MS-MS, PE Sciex Turbo IonSpray 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

<5 µg/mL), amiodarone (10.2, LOD 0.05 µg/mL), amitriptyline (6.6, LOD <0.02 µg/mL), astemizole (5.8, LOD <0.02µg/mL), atenolol (1.7, LOD 0.30 µg/mL), azacyclonol (5.1,LOD 0.02µg/mL), benzhexol (6.6, LOD <0.02 µg/mL), benzoylecgonine (3.3, LOD

0.01µg/mL), betaxolol (5.5, LOD 0.01 µg/mL), biperidine (6.2, LOD <0.02 µg/mL), bisoprolol (5.0, LOD <0.02µg/mL), brompheniramine (5.3, LOD 0.002 µg/mL),bupivacaine (5.1, LOD <0.02µg/mL), buprenorphine (5.9, LOD 0.01 µg/mL),buspirone (5.1, LOD 0.002µg/mL), caffeine (2.8, LOD 1 µg/mL), carbamazepine

8 Acrivastine

(6.1, LOD <0.02µg/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

0.05µg/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 0.02µg/mL), chlorpheniramine (5.1, LOD 0.002 µg/mL),chlorpromazine (7.0, LOD 0.02µg/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 <0.02µg/mL), clemastine (7.7, LOD 0.02 µg/mL), clobazam

(7.3, LOD <0.02µg/mL), clobutinol (5.3, LOD 0.02 µg/mL), clomethiazole (6.2,LOD 0.5µg/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 <0.02µg/mL), codeine (2.5, LOD 0.1 µg/mL), coumatetralyl(8.4, LOD 0.05µg/mL), cyclizine (5.8, LOD <0.02 µg/mL), dextropropoxyphene (6.6,

LOD 0.05µg/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 <0.02µg/mL), dipyridamole (5.4, LOD 0.005 µg/mL),

disopyramine (4.4, LOD <0.02µg/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

0.05µg/mL), ebastine (9.6, LOD 0.005 µg/mL), embutramide (6.7, LOD 0.005 µg/mL),ergotamine (5.5, LOD 0.005µg/mL), ethenzamide (5.0, LOD 0.05 µg/mL), ethylmorphine(3.2, LOD 0.05µg/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 <0.02µg/mL), fluconazole (4.0, LOD 0.1 µg/mL), flumazenil (5.2, LOD

<0.02µg/mL), flunitrazepam (7.1, LOD 0.002 µg/mL), fluoxetine (6.8, LOD 0.1 µg/mL),flupentixol (7.5, LOD 0.18µg/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 <0.02µg/mL), histapyrrodine (6.3, LOD 0.02 µg/mL), hydrocodone (3.0, LOD0.05µg/mL), hydroxychloroquine (2.4, LOD <0.3 µg/mL), hydroxyzine (6.3, LOD

<0.02µg/mL), imipramine (6.4, LOD 0.05 µg/mL), indomethacin (8.6, LOD 0.05 µg/mL),isoniazid (2.2, LOD 3µg/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 0.05µg/mL), labetalol (4.9, LOD 0.05 µg/mL), lamotrigine (4.0,LOD 0.1µg/mL), levocabastine (5.8, LOD 0.01 µg/mL), levomepromazine (6.5, LOD0.02µg/mL), lidocaine (3.7, LOD <0.05 µg/mL), loratadine (9.3, LOD 0.002 µg/mL),

lorazepam (6.6, LOD 0.02µg/mL), lormetazepam (7.4, LOD <0.02 µg/mL), LSD (4.7, LOD <0.02µg/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 <0.02µg/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 <0.02µg/mL), meprobamate (4.9, LOD 0.1 µg/mL), mesoridazine (5.4, LOD

<0.02µg/mL), methamphetamine (3.3, LOD 0.05 µg/mL), methadone (6.7, LOD

<0.02µg/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

0.02µg/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 <0.02µg/mL), mizolastine (5.5, LOD 0.01 µg/mL), moclobemide (3.7,LOD 0.05µg/mL), molindone (4.0, LOD <0.02 µg/mL), monoacetylmorphine (2.7,

LOD 0.1µg/mL), morphine (2.0, LOD 0.1 µg/mL), nicotine (2.2, LOD 0.05 µg/mL),nifedipine (7.5, LOD 0.02µg/mL), nikethamide (3.6, LOD <0.02 µg/mL), nitrazepam (6.5, LOD <0.02µg/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 <0.02µg/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 <0.02µg/mL), oxcarbazepine (5.3, LOD 0.02 µg/mL), oxprenolol (4.7, LOD0.02µg/mL), oxycodone (2.8, LOD 0.05 µg/mL), papaverine (4.8, LOD <0.02 µg/mL),

paroxetine (6.2, LOD 0.02µg/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 <0.02µg/mL), perphenazine (6.9, LOD 0.002 µg/mL), phenazone (3.9, LOD

Acrivastine 9

0.05µg/mL), phencyclidine (5.3, LOD 0.05 µg/mL), pheniramine (4.1, LOD 0.02 µg/mL),

phenylbutazone (9.0, LOD <5µg/mL), phenylpropanolamine (2.5, LOD 0.3 µg/mL),phenytoin (6.1, LOD 0.05µg/mL), pindolol (3.3, LOD 0.05 µg/mL), piroxicam (6.6, LOD0.02µg/mL), pitofenone (5.4, LOD <0.02 µg/mL), pizotifen (6.5, LOD <0.02 µg/mL),

practolol (1.8, LOD 0.1µg/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 0.02µg/mL), promazine (6.2, LOD <0.02 µg/mL),

promethazine (6.0, LOD 0.05µg/mL), propafenone (6.3, LOD <0.02 µg/mL), propranolol

(5.4, LOD 0.02µg/mL), propyphenazone (6.6, LOD 0.50 µg/mL), pseudoephedrine (2.6,LOD 1µg/mL), quinine (4.2, LOD 0.02 µg/mL), ranitidine (1.8, LOD 0.1 µg/mL),

risperidone (4.9, LOD <0.02µg/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

0.05µg/mL), sertindole (7.2, LOD <0.02 µg/mL), sertraline (6.8, LOD 0.02 µg/mL),

sulindac (6.5, LOD 0.02µg/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 0.05µg/mL), sulpiride (1.9, LOD 0.1 µg/mL), sulthiame (4.1, LOD0.05µg/mL), temazepam (7.2, LOD <0.02 µg/mL), terbutaline (2.3, LOD 0.1 µg/mL),

terfenadine (8.1, LOD 0.002µg/mL), terodiline (6.7, LOD <0.02 µg/mL), tetracaine (5.7, LOD <0.02µg/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 0.05µg/mL), thiothixene (6.7, LOD 0.02 µg/mL), tolbutamide (7.1,

LOD <5µg/mL), toremifene (8.7, LOD 0.02 µg/mL), tramadol (4.2, LOD 0.02 µg/mL),

trazodone (5.2, LOD <0.02µg/mL), triamterene (3.2, LOD 0.1 µg/mL), triazolam (6.7,LOD 0.002µg/mL), trimeprazine (6.4, LOD <0.02 µg/mL), trimethoprim (3.1, LOD

0.05µg/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)

Column: 250× 4 ODS-RP18 (Merck)

Mobile phase: MeCN:THF:water:trifluoroacetic acid 43:36:21:0.02

Cundy, 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,

2 mL 1% trifluoroacetic acid in MeOH Evaporate the eluate to dryness under a stream

of nitrogen, reconstitute the residue in 100µL 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 EDTA

in 1 L water, pH 3.2.) (The procedure was not necessarily validated for this compound.)

Column: 250× 4 Aquapore RP 300 (Kontron)

Mobile phase: Gradient A was 100 mM NaH2PO4 adjusted to pH 2.1 with phoric acid B was MeOH A:B from 90:10 to 35:65 over 180 min

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,

adrenocor-ticotropic 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

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.

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

car-REFERENCE

Sugiyama, 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.

SAMPLE

Matrix: enzyme reactions

Sample preparation: Mix 40µL enzyme reaction mixture with 200 µL MeCN, add

200µL of a 20 µg/mL solution of n-propyl paraben, centrifuge, inject a 30 µL aliquot of

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 10µLaliquot

0.1µg/mL), betamethasone-17-acetate (isocratic k0.73; gradient retention time (min)

15.4; LOD 0.3µg/mL), betamethasone-17-benzoate (isocratic k2.04; gradient retention

time (min) 20.6; LOD 0.3µg/mL), betamethasone-17-propionate-21-stearate (isocratic k

>13; gradient retention time (min) >35; LOD 0.5µg/mL), 21-butyrate (isocratic k 5.91; gradient retention time (min) 26.1; LOD 0.4µg/mL),betamethasone-17-valerate-21-acetate (isocratic k 4.41; gradient retention time (min)23.1; LOD 0.4µg/mL), betamethasone-17-valerate (isocratic k2.32; gradient retention

betamethasone-17-propionate-time (min) 21.4; LOD 0.3µg/mL), betamethasone-17,21-dipropionate (isocratic k4.00;

gradient retention time (min) 24.2; LOD 0.4µg/mL), betamethasone-17,21-diacetate(isocratic k1.81; gradient retention time (min) 20.5; LOD 0.3µg/mL), betamethasone-17,21-divalerate (isocratic k10.82; gradient retention time (min) 28.0; LOD 0.4µg/mL),betamethasone-21-acetate (isocratic k0.77; gradient retention time (min) 15.6; LOD0.3µg/mL), betamethasone propionate (isocratic k0.82; gradient retention time (min)

17.1; LOD 0.3µg/mL), clobetasol propionate (isocratic k3.41; gradient retention time

(min) 23.4; LOD 0.1µg/mL), clobetasone butyrate (isocratic k5.45; gradient retention

time (min) 26.3; LOD 0.1µg/mL), cortisone (isocratic k 0.18; gradient retention time

Alclometasone 17,21-dipropionate 19

(min) 11.1; LOD 0.6µg/mL), cortisone acetate (isocratic k0.73; gradient retention time

(min) 15.2; LOD 0.6µg/mL), dehydrocorticosterone (isocratic k4.27; gradient retention

time (min) 22.3; LOD 0.5µg/mL), deoxymethasone (isocratic k0.64; gradient retention

time (min) 14.2; LOD 0.2µg/mL), dexamethasone (isocratic k0.27; gradient retention

time (min) 11.9; LOD 0.1µg/mL), dexamethasone-21-acetate (isocratic k 0.91;

gradi-ent retgradi-ention time (min) 16.1; LOD 0.2µg/mL), dexamethasone isonicotinate (isocratic

k1.05; gradient retention time (min) 17.7; LOD 0.4µg/mL), dexamethasone pivalate(isocratic k3.45; gradient retention time (min) 24.1; LOD 0.3µg/mL), dexamethasonevalerate (isocratic k3.00; gradient retention time (min) 21.6; LOD 0.3µg/mL), diflucor-tolone valerate (isocratic k4.73; gradient retention time (min) 23.3; LOD 0.3µg/mL),fludrocortisone acetate (isocratic k 0.59; gradient retention time (min) 14.1; LOD0.3µg/mL), flumethasone pivalate (isocratic k2.68; gradient retention time (min) 21.2;

LOD 0.3µg/mL), fluocinolone acetonide (isocratic k0.91; gradient retention time (min)

13.4; LOD 0.3µg/mL), fluocinonide (isocratic k 1.45; gradient retention time (min)

20.5; LOD 0.1µg/mL), fluocortin butyl ester (isocratic k5.59; gradient retention time

(min) 24.6; LOD 0.3µg/mL), fluocortolone caproate (isocratic k6.59; gradient retention

time (min) 25.1; LOD 0.3µg/mL), fluocortolone pivalate (isocratic k 4.50; gradient

retention time (min) 23.6; LOD 0.3µg/mL), fluorometholone (isocratic k0.59; gradient

retention time (min) 14.4; LOD 0.1µg/mL), 9-α-fluoroprednisolone (isocratic k 0.18;

gradient retention time (min) 10.0; LOD 0.1µg/mL), 9-α-fluoroprednisolone acetate

(isocratic k0.50; gradient retention time (min) 13.9; LOD 0.2µg/mL), flurandrenolide(isocratic k 0.50; gradient retention time (min) 13.5; LOD 0.1µg/mL), halcinonide(isocratic k1.64; gradient retention time (min) 20.6; LOD 0.1µg/mL), hydrocortisone(isocratic k0.18; gradient retention time (min) 10.0; LOD 0.4µg/mL), hydrocortisone-17-butyrate (isocratic k 1.09; gradient retention time (min) 17.7; LOD 0.6µg/mL),hydrocortisone-21-acetate (isocratic k 0.77; gradient retention time (min) 15.3; LOD0.6µg/mL), hydrocortisone pivalate (isocratic k 2.27; gradient retention time (min)

20.4; LOD 0.8µg/mL), methylprednisolone (isocratic k 0.55; gradient retention time

(min) 13.5; LOD 0.1µg/mL), mometasone furoate (isocratic k3.05; gradient retention

time (min) 22.0; LOD 0.2µg/mL), prednisolone-21-acetate (isocratic k 0.60; gradient

retention time (min) 13.6; LOD 0.2µg/mL), prednisolone acetonide (isocratic k 0.50;

gradient retention time (min) 13.0; LOD 0.3µg/mL), prednisolone pivalate (isocratic

k 2.05; gradient retention time (min) 19.7; LOD 0.3µg/mL), triamcinolone (isocratic

k 0.14; gradient retention time (min) 7.2; LOD 0.1µg/mL), triamcinolone acetonide(isocratic k 0.50; gradient retention time (min) 13.9; LOD 0.2µg/mL), triamcinolonediacetate (isocratic k0.45; gradient retention time (min) 13.9; LOD 0.3µg/mL)

KEY WORDS

cosmetics; SPE

REFERENCE

Gagliardi, L.; De Orsi, D.; Del Giudice, M.R.; Gatta, F.; Porr `a, 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,

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

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.