HPLC Methods recently approved Pharmaceuticals

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HPLC METHODS FOR

RECENTLY APPROVED

PHARMACEUTICALS

George Lunn

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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 or by any means, electronic, mechanical, photocopying, recording, scanning, or otherwise, except as permitted under Section 107 or 108 of the 1976 United States Copyright Act, without either the prior written permission of the Publisher, or authorization through payment of the appropriate per-copy fee 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.

For general information on our other products and services please contact our Customer Care Department within the U.S. at 877-762-2974, outside the U.S. at 317-572-3993 or fax 317-572-4002.

Wiley also publishes its books in a variety of electronic formats. Some content that appears in print, however, may not be available in electronic format.

Library of Congress Cataloging-in-Publication Data:

Lunn, George.

HPLC methods for recently approved pharmaceuticals / George Lunn. p. cm.

Includes index.

ISBN 0-471-66941-5 (cloth)

1. High performance liquid chromatography. 2. Drugs–Analysis. I. Title. RS189.5.H54L773 2005

615′_.1–dc22

2004016046

Printed in the United States of America.

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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 the literature up to the end of 2003. The current volume is a continuation of HPLC Methods for Pharmaceutical Analysis, published in four volumes from 1997 to 2000. The previous volumes described methods published in the literature through the middle of 1998.

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

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 currently approved for use in the United States or indeed in any country.

Despite the ready availability of computer-aided literature, searching this resource is not exploited as much as it might be. One reason for this reluctance is, of course, that a computer search merely produces a listing of possibly relevant references. Tedious and time-consuming searches in the library are necessary to find the most relevant reference that can be turned into a practical analytical procedure in the searcher’s own laboratory. The reference finally chosen will, naturally, depend on the individual circumstances, such as the matrix in which the drug is present, availability of equipment, and so on. This book circumvents this lengthy process by providing a number of abstracted and evaluated procedures for the analysis of each drug. The analyst can rapidly identify a relevant procedure and put it into practice. In addition to the analytical matrix, other factors may be important when choosing an analytical procedure. Accordingly, we have noted such features of the analytical procedures as sensitivity, mode of detection, other compounds that interfere with the analysis, other drugs that may be determined at the same time, and so on.

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xii Preface

should be reproducible without reference to the original literature is unchanged. A new feature is that the retention times (in minutes) of other drugs that may be determined using the same system have been added in parentheses after the drug name. Other data, such as the limit of detection (LOD), may also be added. The retention time is the number without units. Unlike the previous volumes, this book is not available on a CD in an electronic form.

At the end of the book a Cumulative Index and a Cross-Index to Other Substances are provided. The Cumulative Index provides a comprehensive listing of the drugs covered in this book and the previous volumes. The Cross-Index lists the other compounds that may also be chromatographed under the conditions described in the monographs in this book. Using the information in the monographs it may be possible to develop chromatographic procedures for these compounds.

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ACKNOWLEDGEMENTS

I am grateful for the use of the National Institutes of Health Library, the FDA Medical Library, and the National Library of Medicine and I would like to express my appreciation for the hard work of the staff of these libraries, particularly those diligent workers who reshelve the journal volumes after one of my forays. Although many people have helped with the preparation of this work the mistakes are my own. I would appreciate hearing from anyone who has corrections, comments, or suggestions. I can be reached at [email protected].

The content of this volume does not necessarily reflect the views or policies of the Food and Drug Administration, nor does the mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. Also, mention of a drug does not necessarily mean that it is currently approved for use in the United States or indeed in any country.

G.L.

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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) andAnnual Reports in Medicinal Chemistrypublished by Elsevier/Academic Press.

The journals routinely surveyed for relevant articles are:

American Journal of Health-System Pharmacy Analyst

Analytica Chimica Acta Analytical Chemistry Analytical Letters Analytical Sciences

Antimicrobial Agents and Chemotherapy Arzneimittelforschung

Biological and Pharmaceutical Bulletin Biomedical Chromatography

Biopharmaceutics and Drug Disposition Chemical and Pharmaceutical Bulletin Chromatographia

Clinical Chemistry

Clinical Pharmacology and Therapeutics Drug Metabolism and Disposition Farmaco

Food Additives and Contaminants Journal of Analytical Toxicology Journal of AOAC International Journal of Chromatographic Science

Journal of Chromatography, Part A and Part B Journal of Clinical Pharmacology

Journal of Forensic Sciences

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xvi About This Book

Journal of Liquid Chromatography & Related Technology Journal of Pharmaceutical and Biomedical Analysis Journal of Pharmaceutical Sciences

Journal of Pharmacology and Experimental Therapeutics Pharmaceutical Research

Pharmazie

Therapeutic Drug Monitoring Xenobiotica

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

The literature was surveyed from 1998 through the end of 2003, although methods from some older articles (and a few from 2004) are included.

NOMENCLATURE

Each chapter is headed by the name and structure of the target compound as well as other useful data such as the CAS Registry Number, molecular formula, molecular weight, and Merck Index number (from the 13th _edition).1_{More useful information} such as melting point, solubility, optical rotation, references to reviews, and so on can be found in the Merck Index.

In general, the United States Adopted Name (USAN)2 _{is used throughout to} identify each drug. Names of derivatives, such as esters, which would have differ-ent chromatographic properties, are iddiffer-entified by placing the derivative name in parentheses after the retention time.

Increasingly, drugs previously marketed as racemates are being marketed as a single enantiomer with the name changed to reflect the enantiomer. For example, levofloxacin is the levorotatory form of ofloxacin. For an achiral HPLC method, the chromatography of a single enantiomer is no different from that of the racemate. In general, in this work and the preceding works, we have listed HPLC procedures under the name of the racemate rather than the single enantiomer. The interested reader is referred to the USP Dictionary2 _{(page 1208) for the naming conventions} used. Generally:

Levo rotatory 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:

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About This Book xvii

Levallorphan in Volume 3 Levamisole in Volume 3 Levobunolol in Volume 3 Levodopa in Volume 3

Levonordefrin in Volume 3 and this volume Levorphanol in Volume 3

Levosimendan in this volume Levothyroxine in Volumes 1 and 3.

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

For Consult Volume

Arformoterol Formoterol 3, this volume

Dexamisole Levamisole 3

Dexamphetamine Amphetamine 2

Dexbrompheniramine Brompheniramine 2

Dexbudesonide Budesonide 2

Dexchlorpheniramine Chlorpheniramine 2 Dexfenfluramine Fenfluramine 3

Dexibuprofen Ibuprofen 1, 4

Dexketoprofen Ketoprofen 1, 4

Dexmedetomidine Medetomidine This volume Dexmethylphenidate Methylphenidate 1

Dexpropranolol Propranolol 4

Dexsotalol Sotalol 4

Dextroamphetamine Amphetamine 2 Dextropropoxyphene Propoxyphene 1, 4

Dexverapamil Verapamil 1, 4

Esatenolol Atenolol 1, 2

Escitalopram Citalopram 2

Esflurbiprofen Flurbiprofen 3

Esketamine Ketamine 3

Esomeprazole Omeprazole 1, 3

Esoxybutynin chloride Oxybutynin chloride 3

Eszopiclone Zopiclone 4

Levalbuterol Albuterol 1, 2

Levamfetamine Amphetamine 2

Levamphetamine Amphetamine 2

Levcycloserine Cycloserine 2

Levdobutamine Dobutamine 2

Levmetamfetamine Methamphetamine 3

Levobetaxolol Betaxolol 2

Levobupivacaine Bupivacaine 2

Levocarnitine Carnitine 2

Levocetirizine Cetirizine 2

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xviii About This Book

Levofenfluramine Fenfluramine 3

Levofloxacin Ofloxacin 1, 3

Levofuraltadone Furaltadone 3

Levoleucovorin Leucovorin 3

Levomenthol Menthol 3, this volume

Levomethadone Methadone 3

Levomoprolol Moprolol 3

Levonorgestrel Norgestrel 1

Levopropoxyphene Propoxyphene 1, 4

Levopropylhexedrine Propylhexedrine 4, this volume

Levosalbutamol Albuterol 1, 2

Levosulpiride Sulpiride 4

Racementhol Menthol 3, this volume

Racemethorphan Dextromethorphan 2

Racemetirosine Metyrosine This volume

Racemorphan Levorphanol 3

Racephedrine Ephedrine 3

Racepinephrine Epinephrine 3

BIBLIOGRAPHIES

For reasons of space, it is not possible to abstract every relevant paper, and so at the end of some chapters an Annotated Bibliography lists other relevant papers. After the citation, a few features of the method that are not obvious from the title of the paper may be briefly mentioned to help the reader decide if this paper may be of use. For example, the limit of quantitation of the method may be cited. Unless otherwise mentioned, it may be assumed that a method involves liquid–liquid extraction of a biological fluid from a human and uses reversed-phase HPLC with UV detection. Thus, if a method uses solid-phase extraction (SPE) or fluorescence detection, this will be mentioned.

ABSTRACT STRUCTURE

The detailed procedures given normally contain the following sections. Of course, not all papers give full details, so some sections may be missing.

Matrix

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About This Book xix

Also Compounds that can be analyzed at the same time. It is not specified whether they interfere, but they can be

extracted. See also Extracted, Simultaneous.

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). Detector Wavelengths in nanometers

Extracted Compounds that can be extracted from the matrix in question and analyzed at the same time and do not interfere. 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 injection loop. If it is the volume actually injected, this value is also given in the Sample preparation section. If the actual injection volume is not given in the Sample preparation section, the Injection volume given is that of the injection loop.

Interfering Compounds that interfere with the analysis of the target compound. Compounds that interfere with the

chromatography of the internal standard (IS) are listed under simultaneous (another IS can always be selected or an external standard procedure can be used).

Matrix A controlled vocabulary is used (see below)

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xx About This Book

Noninterfering Compounds that do not interfere with the analysis for various reasons, e.g., they are not extracted, they are not detected.

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 the

analyte, the internal standard, and other compounds that may be chromatographed under the same conditions. For the internal standard and other compounds that may be chromatographed under the same conditions, the retention times are given in parentheses after the compound name.

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 may be). See also Also, Extracted.

SPE For the sake of consistency, conditioning procedures for solid-phase extraction (SPE) cartridges are always described at the beginning of the sample preparation sections. Bear in mind, however, that the conditioning procedure should be carried out just prior to use. Thus, if sample preparation is a lengthy procedure, it may be necessary to delay SPE cartridge conditioning until the step requiring the cartridge.

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, ifbothhuman blood and rat blood are analyzed,bothhuman and rat will be indicated (in Key Words).

MATRIX

To help with searching, a controlled vocabulary is used to limit the number of terms in the matrix section. For example, the terms raw material, drug substance, or API (active pharmaceutical ingredient) are not used; the term bulk is used instead. In a number of cases, the matrix is associated with various key words, which can be used to narrow the search. For example, the matrix term blood has the key words plasma, serum, and whole blood associated with it. Thus, if you are interested in the determination of the drug in blood in general, you should look in the matrix field for blood. If, however, you are specifically interested in finding the drug in plasma, you should look in the key words field for plasma.

Matrix Associated Key Words

Bile

Blood Plasma, serum, whole blood Bulk

CSF

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About This Book xxi

Milk Perfusate

Reaction mixtures Saliva

Tissue Brain, heart, kidney, liver, muscle, spleen, etc. Urine

ABBREVIATIONS

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

E Electrochemical detection em Emission wavelength EtOH Ethanol

ex Excitation wavelength F Fluorescence detection

GPC Gel permeation chromatography

h Hour

HPLC High-performance liquid chromatography ID Internal diameter

IS Internal standard

L Liter

LOD Limit of detection or some other description indicating that this is the smallest concentration or quantity that can be detected or analyzed for LOQ 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 MeOH Methanol min Minutes 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)

s Seconds

SEC Size Exclusion Chromatography SFC Supercritical fluid chromatography SFE Supercritical fluid extraction SPE Solid phase extraction Temp Temperature

U Units

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xxii About This Book

PIC REAGENTS

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

PIC A is tetrabutylammonium sulfate PIC B5 is pentanesulfonic acid PIC B7 is heptanesulfonic acid.

WORKING PRACTICES

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

It is also assumed that safe working practices are observed. Organic solvents should only be evaporated in a properly functioning chemical fume hood, correct protective equipment should be worn when dealing with potentially hazardous biological materials, and waste solutions should be disposed of in accordance with all applicable regulations.

A number of solvents are particularly hazardous. For example, benzene is a human carcinogen;10_chloroform,11_{dichloromethane,}12_dioxane,13_{and carbon} tetra-chloride14 _{are carcinogenic in experimental animals; and DMF}15 _{and MTBE}16,17 may be carcinogenic. Organic solvents are, in general, flammable and toxic by inhalation, ingestion, and skin absorption. Sodium azide is carcinogenic and toxic and liberates explosive, volatile, toxic hydrazoic acid when mixed with acid. Sodium azide can form explosive heavy metal azides, for example, with plumbing fixtures, and so should not be discharged down the drain.18 Disposal procedures have been described for a number of hazardous drugs and reagents,18and a procedure for the hydrolysis of acetonitrile in waste solvent to the much less toxic acetic acid and ammonia19,20_{has been described.}_n_{-Hexane is surprisingly toxic.}21

REFERENCES

1. O’Neil, M.J., Ed.,The Merck Index, 13th_{edition, Merck & Co. Inc, Whitehouse Station,} NJ,2001.

2. United States Pharmacopeial Convention.USP Dictionary of USAN and International

Drug Names, United States Pharmacopeial Convention, Rockville, MD,2004.

3. Snyder, L.R.; Kirkland, J.J.Introduction to Modern Liquid Chromatography, 2nd edi-tion, John Wiley & Sons, New York,1979.

4. Lawrence, J.F.Organic Trace Analysis by Liquid Chromatography, Academic Press, New York,1981.

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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.

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,

Toxi-col.Ind.Health.,1995,11, 119–149.

17. Mehlman, M.A. Dangerous and cancer-causing properties of products and chemicals in the oil refining and petrochemical industry: Part XV. Health hazards and health risks from oxygenated automobile fuels (MTBE): lessons not heeded,Int.J.Occup.Med.Toxicol., 1995,4, 219–236.

18. Lunn, G.; Sansone, E.B.Destruction of Hazardous Chemicals in the Laboratory, 2nd edition, John Wiley & Sons, New York,1994.

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

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

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Abacavir

Molecular formula: C14H18N6O

Molecular weight:286.33

CAS Registry No: 136470-78-5 (base), 188062-50-2 (sulfate) Merck Index: 13,1

Sample preparation: Condition a 1 mL 100 mg Bond Elut-C SPE cartridge with 1 mL MeOH and 1 mL 100 mM pH 7.0 ammonium acetate buffer. Heat plasma at 58◦_{for 1 h}

to inactivate HIV. Vortex 800µL plasma with 300µL 2µg/mL hexobarbital in 25 mM pH 7.0 ammonium acetate buffer for 30 s and centrifuge at 18 000 g for 5 min. Add 1 mL of the supernatant to the SPE cartridge, wash with 1 mL 100 mM pH 7.0 ammonium acetate buffer, suck dry for 1 min, elute with 800µL MeOH. Evaporate the eluate to dryness under a stream of nitrogen at 40◦ _{and reconstitute the residue with 100}

µL mobile phase. Vortex for 30 s, centrifuge at 18 000 g for 3 min, and inject an 80µL aliquot.

HPLC VARIABLES

Guard column: 20×3.9 5µm Polarity dC18 (Waters) Column: 150×3.9 5µm Polarity dC18 (Waters)

Column temperature: 40

Mobile phase: Gradient. A was 10 mM pH 6.5 ammonium acetate buffer. B was 10 mM pH 6.5 ammonium acetate buffer:MeCN:MeOH 20:50:30. A:B 96:4 for 15 min, to 36:64 over 15 min, maintain at 36:64 for 3 min, re-equilibrate at initial conditions for 7 min.

Flow rate: 1.1

Injection volume: 80

Detector: UV 269 for 11 min, UV 250 for 3 min, UV 271 for 10 min, UV 230 for 9 min

CHROMATOGRAM

Retention time: 25.1

Internal standard: hexobarbital (30.6)

Limit of quantitation: 10.0 ng/mL

OTHER SUBSTANCES

Extracted: didanosine (13.6), lamivudine (8.6), nevirapine (27.3), stavudine (15.7), zal-citabine (5.9), zidovudine (23.8)

Noninterfering: tenofovir

KEY WORDS plasma; SPE

REFERENCE

Rezk, N.L.; Tidwell, R.R.; Kashuba, A.D.M. Simultaneous determination of six HIV nucleoside analogue reverse transcriptase inhibitors and nevirapine by liquid chromatography with ultraviolet absorbance detection,J.Chromatogr.B,2003,791, 137–147.

SAMPLE

Matrix: blood

Sample preparation: Condition a 100 mg Dual Zone C18 SPE cartridge (Diazem) with 2 mL MeOH and 2 mL water. Dilute 500µL serum with 1 mL water, add to the SPE cartridge, wash with 500µL water, elute with 1 mL MeOH. Evaporate the eluate to

1 HPLC Methods for Recently Approved Pharmaceuticals. by George Lunn

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2 Abacavir

dryness with vortexing under reduced pressure at 40◦and reconstitute the residue with 300µL MeOH, inject a 10µL aliquot.

HPLC VARIABLES

Column: two 150×4.6 3µm Luna C18 columns in series

Mobile phase: Gradient. MeCN:water from 5:95 to 45:55 over 20 min.

Flow rate: 0.85

Detector: UV 250

CHROMATOGRAM

Retention time: 17

Limit of detection: 75 ng/mL

Extracted: 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), zidovustavu-dine (16, LOD 30 ng/mL)

KEY WORDS SPE; serum

REFERENCE

Simon, V.A.; Thiam, M.D.; Lipford, L.C. Determination of serum levels of thirteen human immunodefi-ciency virus-suppressing drugs by high-performance liquid chromatography, J.Chromatogr.A, 2001,

913, 447–453.

SAMPLE

Matrix: blood

Sample preparation: Mix 300µL plasma with 75µL 20% perchloric acid for 30 s, centrifuge at 1300 g for 15 min, inject a 100µL aliquot.

HPLC VARIABLES

Guard column: 20×3.8 Symmetry C18 (Waters)

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

Column temperature: 41±2

Mobile phase: MeCN:25 mM pH 7.0 phosphate buffer 15:85

Flow rate: 1

CHROMATOGRAM

Limit of quantitation: 20 ng/mL

Simultaneous: didanosine, folic acid, ganciclovir, lamivudine, nevirapine, pyrazinamide, ranitidine, rifampin, stavudine, sulfamethoxazole, trimethoprim, zidovudine

Noninterfering: adefovir, amprenavir, delavirdine, efavirenz, fluconazole, indinavir, itraconazole, methadone, nelfinavir, oxazepam, pyrimethamine, rifampin, ritonavir, saquinavir, zalcitabine

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Abacavir 3

REFERENCE

Veldkamp, 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 isocratic reversed-phase high-performance liquid chromatography with ultraviolet detection,J.Chromatogr.B,

1999,736, 123–128.

SAMPLE

Matrix: blood

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

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

HPLC VARIABLES

Column: 250×4.6 Adsorbsphere C18

Mobile phase: Gradient. A was MeCN:water 80:20. B was 50 mM ammonium acetate

containing 0.1% triethylamine adjusted to pH 5.5. A:B from 0:100 to 50:50 over 30 min, re-equilibrate at initial conditions for 10 min.

Flow rate: 1

Detector: UV 260, UV 285

CHROMATOGRAM

Extracted: carbovir (20)

KEY WORDS

rat; pharmacokinetics; plasma

REFERENCE

Daluge, S.M.; Good, S.S.; Faletto, M.B.; Miller, W.H.; St.Clair, M.H.; Boone, L.R.; Tisdale, M.; Parry, N.R.; Reardon, J.E.; Dornsife, R.E.; Averett, D.R.; Krenitsky, T.A. 1592U89, a novel carbocyclic nucle-oside analog with potent, selective anti-human immunodeficiency virus activity,Antimicrob.Agents Chemother.,1997,41, 1082–1093.

SAMPLE

Matrix: CSF, urine

Sample preparation: Centrifuge CSF or urine at 12 000 g for 5 min, dilute a 75µL aliquot to 750µL with mobile phase, inject an aliquot.

HPLC VARIABLES

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

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

Flow rate: 0.7

CHROMATOGRAM

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

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4 Abacavir

REFERENCE

Ravitch, J.R.; Moseley, C.G. High-performance liquid chromatographic assay for abacavir and its two major metabolites in human urine and cerebrospinal fluid,J.Chromatogr.,2001,762, 165–173.

ANNOTATED BIBLIOGRAPHY

Fung, E.N.; Cai, Z.; Burnette, T.C.; Sinhababu, A.K. Simultaneous determination of Ziagen and its phosphorylated metabolites by ion-pairing high-performance liquid chromatography-tandem mass spectrometry,J.Chromatogr.B,2001,754, 285–295. [LC-MS]

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

Thomas, S.A.; Bye, E.; Segal, M.B. Transport characteristics of the anti-human immunodeficiency virus nucleoside analog, abacavir, into brain and cerebrospinal fluid, J.Pharmacol.Exp.Ther., 2001, 298, 947–953.

Yuen, G.J.; Lou, Y.; Thompson, N.F.; Otto, V.R.; Allsup, T.L.; Mahony, W.B.; Hutman, H.W. Abacavir/lamivudine/zidovudine as a combined formulation tablet: Bioequivalence compared with each component administered concurrently and the effect of food on absorption,J.Clin.Pharmacol.,

2001,41, 277–288.

Aymard, G.; Legrand, M.; Trichereau, N.; Diquet, B. Determination of twelve antiretroviral agents in human plasma sample using reversed-phase high-performance liquid chromatography,

J.Chromatogr.B, 2000, 744, 227–240. [for amprenavir; efavirenz; indinavir; nelfinavir; ritonavir; saquinavir; abacavir; didanosine; lamivudine; stavudine; nevirapine; zidovudine]

McDowell, J.A.; Lou, Y.; Symonds, W.S.; Stein, D.S. Multiple-dose pharmacokinetics and 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. Safety and pharmacokinetics of abacavir (1592U89) following oral administration of escalating single doses in human immunodeficiency virus type 1-infected adults,Antimicrob.Agents Chemother., 1999, 43, 603–608.

McDowell, J.A.; Chittick, G.E.; Ravitch, J.R.; Polk, R.E.; Kerkering, T.M.; Stein, D.S. Pharmacokinetics of [14_{C]abacavir, a human immunodeficiency virus type 1 (HIV-1) reverse transcriptase inhibitor,}

administered in a single oral dose to HIV-1-infected adults: a mass balance study,Antimicrob.Agents Chemother.,1999,43, 2855–2861.

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Acarbose 5

Acarbose

Molecular formula: C25H43NO18

Molecular weight:645.60 CAS Registry No: 56180-94-0 Merck Index: 13, 18

O

Sample preparation: Powder tablet, extract 3 times with 5 mL aliquots of water with sonication for 15 min with vortexing at 5 min intervals each time, centrifuge at 2750 g for 5 min, combine supernatants, make up to 20 mL with water. Dilute a 50µL aliquot to 1 mL with MeOH, filter (0.2µM), inject a 20µL aliquot.

HPLC VARIABLES

Column: 250×4.6 5µm Nucleosil-NH2

Mobile phase: MeOH:dichloromethane 65:35

Flow rate: 1

Detector: ELSD, nebulizing gas air at 2.5 bar and 4 L/min, solvent evaporated at 40◦

CHROMATOGRAM

Limit of detection: 5µg/mL Limit of quantitation: 15µg/mL

Simultaneous: sucrose (3.5)

KEY WORDS

comparison with capillary electrophoresis; tablets

REFERENCE

Cherkaoui, S.; Daali, Y.; Christen, P.; Veuthey, J.-L. Development and validation of liquid chromatog-raphy and capillary electrophoresis methods for acarbose determination in pharmaceutical tablets,

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6 Acetyl sulfisoxazole

Acetyl sulfisoxazole

Molecular formula: C13H15N3O4S

Molecular weight:309.35 CAS Registry No: 80-74-0 Merck Index:13, 9041

N S

CH3 CH3

N O

CH3

O O

H2N O

SAMPLE

Matrix: formulations

Sample preparation: Extract 1 mL suspension with three 15 mL aliquots of chloroform (Caution! Chloroform is a carcinogen!), combine the organic layers and make up to 50 mL with chloroform, filter (0.45µm silver membrane, Selas Corp.). Evaporate a 2 mL aliquot of the filtrate to dryness under a stream of nitrogen, reconstitute with 5 mL 330µg/mL benzanilide in MeCN, inject a 5µL aliquot.

HPLC VARIABLES

Column: 300×4 10µmµBondapak C18

Mobile phase: MeCN:water 40:60

Flow rate: 1.5

CHROMATOGRAM

Internal standard: benzanilide (11)

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.

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Acrivastine 7

Acrivastine

Molecular formula: C22H24N2O2

N COOH

N

H3C

SAMPLE

Matrix: blood

Sample preparation: Mix 1 mL whole blood with 20µL 1µg/mL dibenzepin in

MeOH:water 50:50, add 300µL pH 11 tris buffer, mix, add 500µL butyl acetate, vortex for 2 min, centrifuge. Preserve the aqueous layer (A). Remove the organic layer and add it to 75µL 10 mM ammonium acetate buffer containing 0.1% formic acid (pH 3.2), evaporate (?). Add 75µL MeCN, sonicate for 5 min, centrifuge at 5000 rpm for 5 min, keep the extract as B. Add 20µL 1µg/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 4µm Genesis C18 (Jones Chromatography)

Mobile phase: Gradient. MeCN:buffer from 20:80 to 100:0 over 10 min, maintain at 0:100 for 3 min, re-equilibrate at initial conditions for 5 min. (Buffer was 10 mM ammonium acetate containing 0.1% formic acid (pH 3.2).

Flow rate: 0.2

Detector: MS, PE Sciex API 365 triple stage quadrupole LC-MS-MS, PE Sciex Turbo Ion Spray interface, positive ion mode, needle voltage 5.2 kV, nebulizer gas air at 60 psi, curtain gas nitrogen at 40 psi, collision cell gas nitrogen at 40 psi, turbo ionspray heater 375◦, heater gas flow 7 L/min

CHROMATOGRAM

Internal standard: dibenzepin, enalapril

Limit of detection: <20 ng/mL OTHER SUBSTANCES

Extracted: acebutolol (3.8, LOD 0.1µg/mL), acetaminophen (2.5, LOD <5µg/mL), alprazolam (6.1, LOD <0.02µg/mL), alprenolol (5.4, LOD 0.01µg/mL), amantadine (3.4, LOD 0.1µg/mL), amiloride (2.0, LOD 0.1µg/mL), aminophenazone (2.8, LOD

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8 Acrivastine

(6.1, LOD <0.02µg/mL), carbinoxamine (5.1, LOD 0.002µg/mL), carisoprodol (6.7, LOD <5µg/mL), carvedilol (6.2, LOD <0.02µg/mL), celiprolol (4.3, LOD 0.05µg/mL), cetirizine (6.3, LOD 0.05µg/mL), chlorcyclizine (6.6, LOD<0.02µg/mL), chlordiazepoxide (5.7, LOD <0.02µg/mL), chlormezanone (5.8, LOD <5µg/mL), chloroquine (2.7, LOD 0.02µg/mL), chlorpheniramine (5.1, LOD 0.002µg/mL), chlorpromazine (7.0, LOD 0.02µg/mL), chlorpropamide (6.7, LOD <5µg/mL), chlorprothixene (7.0, LOD <0.02µg/mL), cinnarizine (7.9, LOD <0.02µg/mL), citalopram (5.7, LOD <0.02µg/mL), clemastine (7.7, LOD 0.02µg/mL), clobazam (7.3, LOD <0.02µg/mL), clobutinol (5.3, LOD 0.02µg/mL), clomethiazole (6.2, LOD 0.5µg/mL), clomipramine (7.1, LOD <0.02µg/mL), clonazepam (6.6, LOD

<0.02µg/mL), clonidine (2.8, LOD 0.1µg/mL), clozapine (5.6, LOD <0.02µg/mL), cocaine (4.6, LOD <0.02µg/mL), codeine (2.5, LOD 0.1µg/mL), coumatetralyl (8.4, LOD 0.05µg/mL), cyclizine (5.8, LOD <0.02µg/mL), dextropropoxyphene (6.6, LOD 0.05µg/mL), demoxepam (5.8, LOD 0.02µg/mL), dextromethorphan (5.5, LOD

<0.02µg/mL), diazepam (8.1, LOD 0.02µg/mL), diltiazem (5.8, LOD <0.02µg/mL), diphenhydramine (5.7, LOD <0.02µg/mL), dipyridamole (5.4, LOD 0.005µg/mL), disopyramine (4.4, LOD <0.02µg/mL), dixyrazine (6.8, LOD 0.005µg/mL), doxapram (4.8, LOD <0.02µg/mL), doxepin (5.9, LOD <0.02µg/mL), dronabinol (12.3, LOD 0.05µg/mL), ebastine (9.6, LOD 0.005µg/mL), embutramide (6.7, LOD 0.005µg/mL), ergotamine (5.5, LOD 0.005µg/mL), ethenzamide (5.0, LOD 0.05µg/mL), ethylmorphine (3.2, LOD 0.05µg/mL), ethylparathion (9.7, LOD<5µg/mL), etodroxizine (6.4, LOD

<0.02µg/mL), felodipine (9.6, LOD 0.02µg/mL), fenazepam (7.5, LOD<0.02µg/mL), fenfluramine (5.3, LOD <0.02µg/mL), fenkamfamine (5.1, LOD <0.02µg/mL), fentanyl (5.5, LOD <0.02µg/mL), fexofenadine (6.3, LOD <0.02µg/mL), flecainide (5.9, LOD <0.02µg/mL), fluconazole (4.0, LOD 0.1µg/mL), flumazenil (5.2, LOD

<0.02µg/mL), flunitrazepam (7.1, LOD 0.002µg/mL), fluoxetine (6.8, LOD 0.1µg/mL), flupentixol (7.5, LOD 0.18µg/mL), fluvoxamine (6.3, LOD 0.02µg/mL), glibenclamide (8.5, LOD <0.02µg/mL), glipizide (6.8, LOD <0.05µg/mL), haloperidol (6.1, LOD <0.02µg/mL), histapyrrodine (6.3, LOD 0.02µg/mL), hydrocodone (3.0, LOD 0.05µg/mL), hydroxychloroquine (2.4, LOD <0.3µg/mL), hydroxyzine (6.3, LOD

<0.02µg/mL), imipramine (6.4, LOD 0.05µg/mL), indomethacin (8.6, LOD 0.05µg/mL), isoniazid (2.2, LOD 3µg/mL), isradipine (8.6, LOD 0.05µg/mL), ketamine (3.6, LOD

<0.05µg/mL), ketobemidone (3.3, LOD<0.05µg/mL), ketoprofen (7.3, LOD 0.1µg/mL), ketorolac (6.2, LOD 0.05µg/mL), labetalol (4.9, LOD 0.05µg/mL), lamotrigine (4.0, LOD 0.1µg/mL), levocabastine (5.8, LOD 0.01µg/mL), levomepromazine (6.5, LOD 0.02µg/mL), lidocaine (3.7, LOD <0.05µg/mL), loratadine (9.3, LOD 0.002µg/mL), lorazepam (6.6, LOD 0.02µg/mL), lormetazepam (7.4, LOD <0.02µg/mL), LSD (4.7, LOD <0.02µg/mL), malathion (8.9, LOD 10µg/mL), maprotiline (6.4, LOD

<0.02µg/mL), MDMA (3.3, LOD 0.02µg/mL), meclozine (8.5, LOD <0.02µg/mL), medazepam (6.3, LOD <0.02µg/mL), meloxicam (7.1, LOD 0.01µg/mL), melperone (5.0, LOD <0.02µg/mL), meperidine (4.7, LOD <0.02µg/mL), mepivacaine (3.7, LOD <0.02µg/mL), meprobamate (4.9, LOD 0.1µg/mL), mesoridazine (5.4, LOD

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

<0.02µg/mL), methylparathion (8.6, LOD 10µg/mL), methylphenidate (4.2, LOD

<0.02µg/mL), metoclopramide (3.8, LOD <0.02µg/mL), metoprolol (4.1, LOD 0.02µg/mL), metronidazole (2.6, LOD 1µg/mL), mexiletine (4.4, LOD 0.05µg/mL), mianserin (5.7, LOD<0.02µg/mL), midazolam (5.9, LOD <0.02µg/mL), mirtazapine (4.4, LOD <0.02µg/mL), mizolastine (5.5, LOD 0.01µg/mL), moclobemide (3.7, LOD 0.05µg/mL), molindone (4.0, LOD <0.02µg/mL), monoacetylmorphine (2.7, LOD 0.1µg/mL), morphine (2.0, LOD 0.1µg/mL), nicotine (2.2, LOD 0.05µg/mL), nifedipine (7.5, LOD 0.02µg/mL), nikethamide (3.6, LOD <0.02µg/mL), nitrazepam (6.5, LOD <0.02µg/mL), nizatidine (1.7, LOD 1µg/mL), nomifensine (4.6, LOD

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Acrivastine 9

0.05µg/mL), phencyclidine (5.3, LOD 0.05µg/mL), pheniramine (4.1, LOD 0.02µg/mL), phenylbutazone (9.0, LOD <5µg/mL), phenylpropanolamine (2.5, LOD 0.3µg/mL), phenytoin (6.1, LOD 0.05µg/mL), pindolol (3.3, LOD 0.05µg/mL), piroxicam (6.6, LOD 0.02µg/mL), pitofenone (5.4, LOD <0.02µg/mL), pizotifen (6.5, LOD <0.02µg/mL), practolol (1.8, LOD 0.1µg/mL), prazosin (4.1, LOD 0.05µg/mL), prilocaine (3.8, LOD

<0.02µg/mL), primidone (4.0, LOD<5µg/mL), procainamide (2.2, LOD 0.05µg/mL), prochlorperazine (7.5, LOD 0.02µg/mL), promazine (6.2, LOD <0.02µg/mL), promethazine (6.0, LOD 0.05µg/mL), propafenone (6.3, LOD<0.02µg/mL), propranolol (5.4, LOD 0.02µg/mL), propyphenazone (6.6, LOD 0.50µg/mL), pseudoephedrine (2.6, LOD 1µg/mL), quinine (4.2, LOD 0.02µg/mL), ranitidine (1.8, LOD 0.1µg/mL), risperidone (4.9, LOD <0.02µg/mL), rocurone (3.8, LOD 0.1µg/mL), ropivacaine (4.6, LOD <0.02µg/mL), salicylamide (4.2, LOD <5µg/mL), selegiline (4.1, LOD 0.05µg/mL), sertindole (7.2, LOD <0.02µg/mL), sertraline (6.8, LOD 0.02µg/mL), sulindac (6.5, LOD 0.02µg/mL), simazine (6.0, LOD 0.1µg/mL), sincocaine (6.5, LOD<0.02µg/mL), sisapride (5.9, LOD <0.02µg/mL), sotalol (2.1, LOD 0.1µg/mL), strychnine (5.3, LOD 0.05µg/mL), sulpiride (1.9, LOD 0.1µg/mL), sulthiame (4.1, LOD 0.05µg/mL), temazepam (7.2, LOD<0.02µg/mL), terbutaline (2.3, LOD 0.1µg/mL), terfenadine (8.1, LOD 0.002µg/mL), terodiline (6.7, LOD<0.02µg/mL), tetracaine (5.7, LOD <0.02µg/mL), tetrahydrozoline (3.6, LOD 0.1µg/mL), theobromine (2.3, LOD

<5µg/mL), theophylline (2.4, LOD <5µg/mL), thioridazine (7.5, LOD 0.02µg/mL), timolol (3.8, LOD 0.05µg/mL), thiothixene (6.7, LOD 0.02µg/mL), tolbutamide (7.1, LOD<5µg/mL), toremifene (8.7, LOD 0.02µg/mL), tramadol (4.2, LOD 0.02µg/mL), trazodone (5.2, LOD<0.02µg/mL), triamterene (3.2, LOD 0.1µg/mL), triazolam (6.7, LOD 0.002µg/mL), trimeprazine (6.4, LOD <0.02µg/mL), trimethoprim (3.1, LOD 0.05µg/mL), trimipramine (6.7, LOD<0.02µg/mL), venlafaxine (4.9, LOD 0.02µg/mL), verapamil (6.5, LOD<0.02µg/mL), warfarin (7.9, LOD<0.02µg/mL), yohimbine (4.5, LOD<0.02µg/mL), zolpidem (4.7, LOD<0.02µg/mL), zopiclone (4.0, LOD 0.1µg/mL) KEY WORDS

whole blood

REFERENCE

Gergov, M.; Ojanper ¨a, I.; Vuori, E. Simultaneous screening for 238 drugs in blood by liquid chromatography-ionspray tandem mass spectrometry with multiple-reaction monitoring,

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10 Adapalene

Adapalene

Molecular formula: C28H28O3

COOH

H3CO

SAMPLE

Sample preparation: Inject an aliquot of a 0.1% gel.

HPLC VARIABLES

Column: 250×4 ODS-RP18 (Merck)

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

Flow rate: 1

CHROMATOGRAM

Noninterfering: tretinoin

KEY WORDS gel

REFERENCE

Martin, B.; Meunier, C.; Montels, D.; Watts, O. Chemical stability of adapalene and tretinoin when combined with benzoyl peroxide in presence and in absence of visible light and ultraviolet radiation,

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Adefovir dipivoxil 11

Adefovir dipivoxil

Molecular formula: C20H32N5O8P

N

Sample preparation: Mix 100µL plasma with 200µL 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 100µL 25 mM pH 6.0 potassium phosphate buffer containing 5 mM tetrabutylammonium hydrogen phosphate, inject a 50µL aliquot.

HPLC VARIABLES

Guard column: 15×3.2 Brownlee RP-18 Newguard

Column: 150×4.6 Zorbax RX-C18

Mobile phase: Gradient. A was MeCN:25 mM pH 6.0 potassium phosphate buffer

containing 5 mM tetrabutylammonium hydrogen phosphate 2:98. B was MeCN:25 mM pH 6.0 potassium phosphate buffer containing 5 mM tetrabutylammonium hydrogen phosphate 65:35. A:B 100:0 for 2 min, to 0:100 over 13 min, re-equilibrate at initial conditions for 10 min. (Only adefovir is detected in blood. However, the method is reported to distinguish between adefovir and adefovir dipivoxil.)

Flow rate: 1.5

Detector: F ex 236 em 420

KEY WORDS

derivatization; dog; pharmacokinetics; plasma

REFERENCE

Cundy, K.C.; Sue, I.-L.; Visor, G.C.; Marshburn, J.; Nakamura, C.; Lee, W.A.; Shaw, J.P. Oral formula-tions of adefovir dipivoxil: In vitro dissolution and in vivo bioavailability in dogs,J.Pharm.Sci.,1997,

86, 1334–1338.

SAMPLE

Matrix: blood

Sample preparation: Vortex 200µL plasma with 50µL 20% trichloroacetic acid in water, centrifuge at 1300 g for 15 min. Remove 150µL of the supernatant and mix it with 50µL 160 mM chloroacetaldehyde in water containing 2 M sodium acetate, vortex, close the tube, heat at 98◦_{for 30 min, cool to 2}◦_{, vortex, inject a 20}

µL aliquot.

HPLC VARIABLES

Guard column: 10×3 R3 (Chrompack)

Column: 150×4.6 5µm Chromspher C8

Column temperature: 40±2

Mobile phase: MeCN:buffer 10:90 (Buffer was 10 mM pH 7.0 sodium phosphate buffer containing 2 mM tetrabutylammonium hydrogen sulfate.)

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12 Adefovir dipivoxil

Detector: F ex 254 em 425

CHROMATOGRAM

Limit of quantitation: 10 ng/mL

Extracted: adefovir (4)

KEY WORDS

derivatization; plasma

REFERENCE

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Adrenocorticotropic hormone 13

Adrenocorticotropic hormone

CAS Registry No: 9002-60-2 Merck Index: 13, 136

SAMPLE

Matrix: blood

Sample preparation: Condition a 1 mL Analytichem weak cation-exchange

(car-boxymethylhydrogen form, CBA) SPE cartridge with 1 mL 1% trifluoroacetic acid in MeOH, 1 mL MeOH, and 2 mL water. Add 1 mL plasma to the SPE cartridge, rinse the tube with 1 mL water, add the rinse to the SPE cartridge, wash with 1 mL 1% trifluoroacetic acid in water, wash with 2 mL water, wash with 2 mL MeOH, elute with 2 mL 1% trifluoroacetic acid in MeOH. Evaporate the eluate to dryness under a stream of nitrogen, reconstitute the residue in 100µL MeOH:buffer 50:50, inject a 5–75µL aliquot. (Buffer was 5.7 g monochloroacetic acid, 2.0 g NaOH, and 0.2 g disodium EDTA in 1 L water, pH 3.2.) (The procedure was not necessarily validated for this compound.)

HPLC VARIABLES

Column: 250×2 5µm Ultrasphere octyl

Mobile phase: Gradient. A was MeOH containing 10 mM sodium octanesulfonate. B

was buffer containing 10 mM sodium octanesulfonate. A:B from 45:55 to 70:30 over 30 min, maintain at 70:30 for 1 h. (The buffer was 5.7 g monochloroacetic acid, 2.0 g NaOH, and 0.2 g disodium EDTA in 1 L water, pH 3.2.)

Flow rate: 0.3

Injection volume: 5–75

Detector: F ex 390 em 470 following post-column reaction. The column effluent

mixed with 400 mM NaOH pumped at 0.15 mL/min and 0.05% ninhydrin pumped at 0.05 mL/min and the mixture flowed through a 12 m×0.33 mm ID reaction coil at 70◦to the detector.

CHROMATOGRAM

Limit of detection: 100 fmole

Simultaneous: angiotensin I, angiotensin II, angiotensin III, atrial natriuretic peptide, bombesin, bradykinin, gonadorelin (LHRH), somatoliberin, vasopressin

KEY WORDS

plasma; post-column reaction; SPE

REFERENCE

Rhodes, G.R.; Boppana, V.K. High-performance liquid chromatographic analysis of arginine-containing peptides in biological fluids by means of a selective post-column reaction with fluorescence detection,

J.Chromatogr.,1988,444, 123–131.

SAMPLE

Matrix: solutions

HPLC VARIABLES

Column: 300×3.9 10µmµBondapak C18

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14 Adrenocorticotropic hormone

Flow rate: 1

CHROMATOGRAM

Simultaneous: adrenocorticotropic hormone fragments, melanotropin

KEY WORDS human

REFERENCE

McDermott, J.R.; Smith, A.I.; Biggins, J.A.; Al-Noaemi, M.C.; Edwardson, J.A. Characterization and determination of neuropeptides by high-performance liquid chromatography and radioimmunoassay,

J.Chromatogr.,1981,222, 371–379.

SAMPLE

Sample preparation: Dissolve in 100 mM NaH2PO4adjusted to pH 2.1 with orthophos-phoric acid, inject a 100µL aliquot.

HPLC VARIABLES

Column: 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: 1

CHROMATOGRAM

Simultaneous: adrenocorticotropin hormone fragments, lipotropic hormone and frag-ments, melanotropin, endorphins, prolactin, somatropin, menotropins

KEY WORDS pig

REFERENCE

Richter, W.O.; Schwandt, P. Separation of neuropeptides by HPLC: evaluation of different supports, with analytical and preparative applications to human and porcine neurophysins,β-lipotropin, adrenocor-ticotropic hormone, andβ-endorphin,J.Neurochem.,1985,44, 1697–1703.

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.

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Afloqualone 15

Afloqualone

Molecular formula: C16H14FN3O

N N

F

O H2N

H3C

SAMPLE

HPLC VARIABLES

Column: Chiralpak AS

Mobile phase: Hexane:EtOH 95:5

Flow rate: 1.3

CHROMATOGRAM

Retention time: 30, 35 (enantiomers)

KEY WORDS chiral

REFERENCE

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16 Alacepril

Alacepril

Molecular formula: C20H26N2O5S

H3C S N

Mobile phase: Gradient. MeCN:10 mM pH 2.5 potassium phosphate buffer 0:100 for 2 min, to 75:25 over 7.5 min, maintain at 75:25 for 5.5 min. Isocratic. MeCN:buffer 40:60

Flow rate: 1.5

Detector: UV (wavelength not specified)

CHROMATOGRAM

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

Simultaneous: 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)

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 200µL MeCN, add

200µL of a 20µg/mL solution ofn-propyl paraben, centrifuge, inject a 30µL aliquot of the supernatant.

HPLC VARIABLES

Column: 250×4.6 Cosmosil 5-C18 MS

Mobile phase: MeCN:10 mM pH 2.5 potassium phosphate buffer 40:60

Flow rate: 1.5

CHROMATOGRAM

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Alacepril 17

Extracted: deacetylalacepril

REFERENCE

Usui, 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.,

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18 Alclometasone 17,21-dipropionate

Alclometasone 17,21-dipropionate

Molecular formula: C28H37ClO7

O

Sample preparation: Condition a 3 mL 500 mg Megabond MF C18 SPE cartridge

(Varian) with 3 mL MeOH and 3 mL water. Sonicate 1 g cosmetic with 10 mL MeOH or MeOH:dichloromethane 10:90 (depending on what appears visually to give best solubility) at 40◦_{for 10 min, centrifuge, collect the clear supernatant. Add 5 mL of the}

supernatant to the SPE cartridge, wash with 4 mL acetone:water 20:80, wash with 1 mLn-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 10µL aliquot.

HPLC VARIABLES

Column: 250×4.6 5µm endcapped Purospher RP-18

Mobile phase: Isocratic. MeCN:water 60:40. Gradient. MeCN:water from 25:75 to 90:10 over 30 min, maintain at 90:10 for 10 min.

Flow rate: 1

CHROMATOGRAM

Retention time: k′_{2.55 (isocratic); 21.0 min (gradient)}

Limit of detection: 300 ng/mL

Simultaneous: amcinonide (isocratic k′_{3.18; gradient retention time (min) 22.6; LOD}

0.1µg/mL), betamethasone (isocratic k′0.18; gradient retention time (min) 11.8; LOD 0.1µg/mL), betamethasone-17-acetate (isocratic k′0.73; gradient retention time (min) 15.4; LOD 0.3µg/mL), betamethasone-17-benzoate (isocratic k′2.04; 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), 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 0.4µg/mL), betamethasone-17-valerate (isocratic k′2.32; gradient retention time (min) 21.4; LOD 0.3µg/mL), betamethasone-17,21-dipropionate (isocratic k′4.00; gradient retention time (min) 24.2; LOD 0.4µg/mL), betamethasone-17,21-diacetate (isocratic k′_{1.81; gradient retention time (min) 20.5; LOD 0.3}

µg/mL), betamethasone-17,21-divalerate (isocratic k′_{10.82; gradient retention time (min) 28.0; LOD 0.4}

µg/mL), betamethasone-21-acetate (isocratic k′ _{0.77; gradient retention time (min) 15.6; LOD}

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Alclometasone 17,21-dipropionate 19

(min) 11.1; LOD 0.6µg/mL), cortisone acetate (isocratic k′0.73; gradient retention time (min) 15.2; LOD 0.6µg/mL), dehydrocorticosterone (isocratic k′4.27; gradient retention time (min) 22.3; LOD 0.5µg/mL), deoxymethasone (isocratic k′0.64; gradient retention time (min) 14.2; LOD 0.2µg/mL), dexamethasone (isocratic k′0.27; 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 k′_{1.05; gradient retention time (min) 17.7; LOD 0.4}

µg/mL), dexamethasone pivalate (isocratic k′_{3.45; gradient retention time (min) 24.1; LOD 0.3}

µg/mL), dexamethasone valerate (isocratic k′_{3.00; gradient retention time (min) 21.6; LOD 0.3}

µg/mL), 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; LOD}

0.3µg/mL), flumethasone pivalate (isocratic k′2.68; gradient retention time (min) 21.2; LOD 0.3µg/mL), fluocinolone acetonide (isocratic k′0.91; 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 k′5.59; gradient retention time (min) 24.6; LOD 0.3µg/mL), fluocortolone caproate (isocratic k′6.59; 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 k′0.59; 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 k′_{0.50; gradient retention time (min) 13.9; LOD 0.2}

µg/mL), flurandrenolide (isocratic k′ _{0.50; gradient retention time (min) 13.5; LOD 0.1}

µg/mL), halcinonide (isocratic k′_{1.64; gradient retention time (min) 20.6; LOD 0.1}

µg/mL), hydrocortisone (isocratic k′_{0.18; gradient retention time (min) 10.0; LOD 0.4}

µg/mL), hydrocortisone-17-butyrate (isocratic k′ _{1.09; gradient retention time (min) 17.7; LOD 0.6}

µg/mL), hydrocortisone-21-acetate (isocratic k′ _{0.77; gradient retention time (min) 15.3; LOD}

0.6µg/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 k′3.05; 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 0.3}

µg/mL), triamcinolone (isocratic k′ _{0.14; gradient retention time (min) 7.2; LOD 0.1}

µg/mL), triamcinolone acetonide (isocratic k′ _{0.50; gradient retention time (min) 13.9; LOD 0.2}

µg/mL), triamcinolone diacetate (isocratic k′_{0.45; gradient retention time (min) 13.9; LOD 0.3}

µg/mL)

KEY WORDS cosmetics; SPE

REFERENCE

Gagliardi, L.; De Orsi, D.; Del Giudice, M.R.; Gatta, F.; 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, 187–198.

SAMPLE

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20 Alclometasone 17,21-dipropionate

HPLC VARIABLES

Guard column: 15×3.2 7µm Brownlee NewGuard C18 Column: 75×4.6 3.5µm Symmetry C18 (Waters)

Mobile phase: Gradient. MeCN:water 18:82 for 2 min, to 82:18 over 12 min, maintain at 82:18 for 3 min, re-equilibrate at initial conditions for 12 min.

Flow rate: 1

CHROMATOGRAM

Limit of detection: 0.001%

Simultaneous: amcinonide (10.90), beclomethasone 17,21-dipropionate (11.90),

betamethasone (6.52), betamethasone 21-acetate (8.47), betamethasone 17-benzoate (10.28), betamethasone 17,21-dipropionate (11.42), betamethasone 17-valerate (10.25), budesonide (8.55, 8.69 (epimers)), clobetasol 17-propionate (11.06), cortisone (5.62), cortisone 21-acetate (8.07), dexamethasone (6.57), dexamethasone 21-acetate (8.68), desonide (6.99), desoximetasone (7.60), desoxycorticosterone acetate (10.90), desoxycorticosterone pivalate (14.45), diflorasone 17,21-diacetate (9.81), fluocinolone acetonide (7.38), fluocinonide (9.79), flurandrenolide (7.36), fludrocortisone 21-acetate (7.77), fluorometholone (7.67), flumethasone 21-pivalate (11.20), flunisolide (7.14), fluprednisolone (5.46), fluticasone 17-propionate (11.19), halcinonide (10.72), halobetasol propionate (10.98), hydrocortisone (5.50), hydrocortisone 21-acetate (7.65), hydrocortisone 17-butyrate (8.66), hydrocortisone 21-cypionate (12.54), hydrocortisone 17-valerate (9.53), mometasone 17-furoate (11.24), methylprednisolone (6.31), methylprednisolone 21-acetate (8.34), meprednisone (6.55), prednisolone (5.37), prednisolone 21-acetate (7.47), prednisolone 21-tebuate (11.01), paramethasone 21-acetate (8.64), prednicarbate (10.72), prednisone (5.46), triamcinolone (4.15), triamcinolone acetonide (7.04), triamcinolone 16,21-diacetate (7.49), triamcinolone hexacetonide (13.12)

KEY WORDS

body wash, cream, gel, lotion, shampoo, spray

REFERENCE

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Alitretinoin 21

Alitretinoin

Molecular formula: C20H28O2

Molecular weight:300.43 CAS Registry No: 5300-03-8 Merck Index: 13, 244 [9-cis-retinoic acid]

Sample preparation: 1 mL plasma+50µL 500µg/mL IS in MeOH:MeCN 50:50 +

1 mL 1 M pH 6.0 phosphate buffer, mix, add 6 mL MTBE, shake on a horizontal shaker for 10 min, freeze the aqueous layer in a dry ice/acetone bath. Decant the organic layer and evaporate it to dryness under nitrogen at 25◦_{, reconstitute the residue with 200}

µL MeOH, add 100µL 5 mM ammonium acetate, centrifuge at 13 000 g for 3 min, inject a 100µL aliquot. (Use silanized glassware. Process under yellow light.)

HPLC VARIABLES

Guard column: 10×2 5µm Hypersil BDS C18

Column: 100×4.6 3µm Microsorb Short One C18 (Rainin)

Mobile phase: Gradient. A was 5 mM pH 2.7 ammonium acetate/acetic acid buffer. B was 1% acetic acid in MeOH. A:B 30:70 for 6.5 min, to 20:80 over 0.5 min, to 11:80 over 14.4 min, to 30:70 over 0.5 min, maintain at 30:70 for 10 min.

Flow rate: 1

CHROMATOGRAM

Internal 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

Extracted: isotretinoin (19.5), tretinoin (21.5), vitamin A (20.5)

KEY WORDS plasma

REFERENCE

Dzerk, A.M.; Carlson, A.; Loewen, G.R.; Shirley, M.A.; Lee, J.W. A HPLC method for the determination of 9-cis retinoic acid (ALRT1057) and its 4-oxo metabolite in human plasma,J.Pharm.Biomed.Anal.,

1998,16, 1013–1019.

SAMPLE

Matrix: blood, food, formulations, tissue

HPLC Methods recently approved Pharmaceuticals

HPLC METHODS FOR

RECENTLY APPROVED

PHARMACEUTICALS

George Lunn

CONTENTS

PREFACE

ACKNOWLEDGEMENTS

ABOUT THIS BOOK

Abacavir

Acarbose

Acetyl sulfisoxazole

Acrivastine

Adapalene

Adefovir dipivoxil

Adrenocorticotropic hormone

Afloqualone

Alacepril

Alclometasone 17,21-dipropionate

Alitretinoin