CRC Handbook of Chromatography: Drugs, Volume III 978-0-429-48740-8, 0429487401, 9780429945816, 0429945817, 9780429945823, 0429945825, 9780429945830, 0429945833, 978-1-138-59685-6

Table of contents :
Content: Cover
Title Page
Copyright Page
PREFACE
THE EDITOR
Table of Contents
Organization of Tables and Explanation of Abbreviations
Drug Tables
Index

Citation preview

CRC Series in Chromatography Editors-in-Chief

Gunter Zweig, Ph.D. and Joseph Sherma, Ph.D. General Data and Principles

Lipids

Gunter Zweig, Ph.D. and Joseph Sherma, Ph.D.

Helmut K. Mangold, Dr. rer. nat.

Hydrocarbons Walter L. Zielinski, Jr., Ph.D.

Carbohydrates Shirley C. Churms, Ph.D.

Inorganics M. Qureshi, Ph.D.

Drugs Ram Gupta, Ph.D.

Phenols and Organic Acids Toshihiko Hanai, Ph.D.

Terpenoids Carmine J. Coscia, Ph.D.

Amino Acids and Amines S. Blackburn, Ph.D.

Steroids Polymers

Joseph C. Touchstone, Ph.D.

Charles G. Smith, Norman E. Skelly, Ph.D., Carl D. Chow, and Richard A. Solomon

Pesticides and Related Organic Chemicals Plant Pigments

Joseph Sherma, Ph.D. and Joanne Follweiler, Ph.D.

Hans-Peter Kost, Ph.D.

Nucleic Acids and Related Compounds Ante M. Krstulovic, Ph.D.

CRC Handbook of Chromatography: Drugs Volume III Editor

Ram N. Gupta, Ph.D. Professor Department of Pathology McMaster University and Head, Toxicology Department of Laboratory Medicine St. Joseph’s Hospital Hamilton, Ontario, Canada Editors-in-Chief

Gunter Zweig, Ph.D.

Joseph P. Sherma, Ph.D.

President Zweig Associates Arlington, Virginia (Deceased)

Professor of Chemistry Lafayette College Easton, Pennsylvania

Boca Raton London New York

CRC Press is an imprint of the Taylor & Francis Group, an informa business

First published 1989 by CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742 Reissued 2018 by CRC Press © 1989 by Taylor & Francis Group. CRC Press is an imprint of Taylor & Francis Group, an Informa business

No claim to original U.S. Government works This book contains information obtained from authentic and highly regarded sources. Reasonable efforts have been made to publish reliable data and information, but the author and publisher cannot assume responsibility for the validity of all materials or the consequences of their use. The authors and publishers have attempted to trace the copyright holders of all material reproduced in this publication and apologize to copyright holders if permission to publish in this form has not been obtained. If any copyright material has not been acknowledged please write and let us know so we may rectify in any future reprint. Except as permitted under U.S. Copyright Law, no part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access www. copyright.com (http://www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC), 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organiza-tion that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe. Publisher's Note The publisher has gone to great lengths to ensure the quality of this reprint but points out that some imperfections in the original copies may be apparent. Disclaimer The publisher has made every effort to trace copyright holders and welcomes correspondence from those they have been unable to contact. ISBN 13: 978-l-138-59685-6 (hbk) ISBN 13: 978-0-429-48740-8 (ebk) Visit the Taylor & Francis Web site at http://www.taylorandfrancis.com and the CRC Press Web site at http://www.crcpress.com

CRC SERIES IN CHROMATOGRAPHY SERIES PREFACE The fat-soluble photosynthetic pigments present in plants and algae, including chloro­ phylls, carotenoids, and related pigments, comprise an important class of compounds with an extensive literature. Dr. K5st and his co-authors have done an admirable job in searching out and organizing much of the critical chromatographic data and methodology in the present volume. Because of the chemical nature of these prenyllipid compounds, liquid chromatography is preferred for their isolation, separation, and determination. The most widely used methods include low pressure column LC, paper chromatography, TLC, and, most recently, HPLC. All of these methods are covered by Dr. Kost. Chromatography was “ invented” in the early 1900s by Michael Tswett, a Russian botanist and plant physiologist who first applied liquid-solid chromatography on a column of chalk to resolution of the complex natural mixture of yellow and green chloroplast pigments in the extracts of leaves he was studying. On a personal note, I was fortunate to work with Dr. Harold Strain for five summers at the Argonne National Laboratory when I first began to teach. Dr. Strain was one of the first important American chromatography experts and used all variations of liquid chromatography extensively in his studies of photosynthetic pigments. My experience with Dr. Strain set the foundation for my lifelong career of research and writing in chromatography. Readers of this Handbook are asked to contact the Series Editor if they find errors or omissions in coverage as well as with suggestions for future volumes and authors within the Handbook of Chromatography series.

Joseph Sherma

PREFACE The phenomenal growth in the application of liquid chromatographic (LC) techniques for the determination of drugs in pharmaceutical preparations and in biological fluids has continued in the first half of this decade. In the mid to late 1970s, a large number of papers were published describing gas chromatographic (GC) procedures for the drug groups anticonvulsants and antidepressants. In the last few years, a large number of publications have appeared describing LC procedures for the same drugs. There have been a number of improvements in the LC instrumentation and column technology. Variable wavelength absorbance detectors are now available which match fixed wavelength detectors in sensitivity. Some of these detectors allow monitoring of absorbance at multiple wavelengths. A number of manufacturers now market photo diode array absorbance detectors which allow instant absorbance scanning over a wide wavelength range of any eluting peak, check peak purity, and complement component separation by mathematical manipulation of absorbance data of incomplete chromatographic separation. There are also improvements in the design of electrochemical detectors. Multielectrode detectors are now available which require little maintenance and allow ultra-high sensitivity. Fluorescence detectors with mono­ chromators and high energy power sources have also become available. However, use of lasers as power sources for fluorescence detectors is not yet common. A number of manufacturers market on-line sample preparation systems, samplers allowing precolumn derivatization, and efficient postcolumn reactors. Although instrumentation for narrow bore LC is commercially available, this technique has not yet been commonly applied for the determination of drugs. There is improved quality control in the manufacture of columns. For convenience, cartridge-type columns and fittings requiring no tools have become available. Good quality silica-based columns can now be purchased at economical proces from general suppliers. However, polymer-based columns have failed to gain popularity and are relatively more expensive than silica-based columns because of limited sales. In general, GC is now the preferred technique only when the required sensitivity is not available with an LC procedure for the determination of a particular drug. However, separation of widely different compounds is more efficiently accomplished with temperature programming GC analysis than by solvent programming LC analysis. Thus, considerable GC retention data of drugs with the use of capillary columns have been published for the identification of an unknown drug in a given matrix. However, the use of capillary columns for the determination of drugs has not been as widespread as was anticipated. The nitrogen detector is now the most widely used detector for the GC determination of drugs. Gas chromatography-mass spectrome­ try remains the ultimate standard to confirm the identification of an unknown drug. There has been a further decline in the popularity of thin-layer chromatography in the past few years. In the majority of the laboratories, drugs of abuse are now screened by immunoassay for improved sensitivity and convenience. The purpose of this handbook is to provide a reference source and summaries of different chromatographic techniques published in refereed journals during the past 6 years. When the number of publications of a given drug was numerous, only recent papers were selected, even if they described only the modification of the original key publications. Despite the size of this work, a number of publications or drugs might have been missed as the literature search was carried out manually. In some cases, copies of the required papers could not be obtained. A number of publications could not be included as they were either theoretical or did not provide information compatible with the format of this handbook. There is a significant difference between the present volumes and Volumes I and II of this handbook. For a number of drugs, e.g., cyclosporine, the chromatographic parameters of a number of publications are identical. However, they differ in the sample preparation techniques.

Therefore, detailed summaries of extraction procedures have now been provided for comparison of the different publications for the determinations of a given drug. I am grateful to Dr. Gillian Luxton, Head of the Clinical Chemistry Laboratory, St. Joseph’s Hospital for her encouragement to accept this project and for providing all the required facilities. Mrs. S. Rogers and Mrs. J. Maragno of this hospital library made a special effort to get copies of the published papers from different sources. Mrs. D. Thompson, Director of the hospital pharmacy, arranged to get information from the Drug Information Center in Toronto. Miss Maelly Lew went to different libraries to get the information in emergency situations when a paper under review would refer to earlier papers. I thank Miss Elisa Capretta, Mrs. Mary Bruce, Miss Rhita Gilners, and Miss Abha Gupta for preparing this manuscript. Mrs. Diane Kirshenblat provided moral support when there was a temptation to abandon the project. I am grateful to Ms. Sandy Pearlman, Director of Editing and Mrs. Amy Skallerup, Senior Editor, CRC Press for their help during the early phases of manuscript preparation. Mr. J. C. Richardson, Senior Coordinating Editor, had the difficult task of making this manuscript uniform within the constraints of space limitations. I thank him for his courteous response to my various suggestions and changes. Finally, I thank my family members, who tolerated my absence for more than a year.

Ram N. Gupta December, 1986

THE EDITORS-IN-CHIEF Gunter Zweig, Ph.D., received his undergraduate training at the University of Maryland, College Park, where he was awarded the Ph.D. in biochemistry in 1952. Two years following his graduation, Dr. Zweig was affiliated with the late R. J. Block, pioneer in paper chro­ matography of amino acids. Zweig, Block, and Le Strange wrote one of the first books on paper chromatography, which was published in 1952 by Academic Press and went into three editions, the last one authored by Gunter Zweig and Dr. Joe Sherma, the co-Editor-in-Chief of this series. Paper Chromatography (1952) was also translated into Russian. From 1953 to 1957, Dr. Zweig was research biochemist at the C. F. Kettering Foundation, Antioch College, Yellow Springs, Ohio, where he pursued research on the path of carbon and sulfur in plants, using the then newly developed techniques of autoradiography and paper chromatography. From 1957 to 1965, Dr. Zweig served as lecturer and chemist, University of California, Davis and worked on analytical methods for pesticide residues, mainly by chromatographic techniques. In 1965, Dr. Zweig became Director of Life Sci­ ences, Syracuse University Research Corporation, New York (research on environmental pollution), and in 1973 he became Chief, Environmental Fate Branch, Environmental Pro­ tection Agency (EPA) in Washington, D.C. From 1980 to 1984 Dr. Zweig was Visiting Research Chemist in the School of Public Health, University of California, Berkeley, where he was doing research on farmworker safety as related to pesticide exposure. During his government career, Dr. Zweig continued his scientific writing and editing. Among his works are (many in collaboration with Dr. Sherma) the now 11-volume series on Analytical Methods for Pesticides and Plant Growth Regulators (published by Academic Press); the pesticide book series for CRC Press; co-editor of Journal of Toxicology and Environmental Health; co-author of basic review on paper and thin-layer chromatography for Analytical Chemistry from 1968 to 1980; co-author of applied chromatography review on pesticide analysis for Analytical Chemistry, beginning in 1981. Among the scientific honors awarded to Dr. Zweig during his distinguished career were the Wiley Award in 1977, the Rothschild Fellowship to the Weizmann Institute in 1963/64; and the Bronze Medal by the EPA in 1980. Dr. Zweig authored or co-authored over 80 scientific papers on diverse subjects in chro­ matography and biochemistry, besides being the holder of three U.S. patents. In 1985, Dr. Zweig became president of Zweig Associates, Consultants in Arlington, Va. Following his death on January 27, 1987, the Agrochemicals Section of the American Chemical Society posthumously elected him a Fellow and established the Gunther Zweig Award for Young Chemists in his honor. Joseph Sherma, Ph.D., received a B.S. in Chemistry from Upsala College, East Orange, N .J., in 1955 and a Ph.D. in Analytical Chemistry from Rutgers University in 1958, carrying on his thesis research in ion exchange chromatography under the direction of the late William Rieman III. Dr. Sherma joined the faculty of Lafayette College in September, 1958, and is presently Charles A. Dana Professor and Head of the Chemistry Department. Dr. Sherma, independently and with others, has written over 300 research papers, chapters, books, and reviews involving chromatography and other analytical methodology. He is editor for residues and trace elements of the Journal of the Association of Official Analytical Chemists and a member of the advisory board of the Journal of Planar Chromatography. He is a consultant on analytical methodology for many companies and government agencies. Dr. Sherma has received two awards for superior teaching at Lafayette College and the 1979 Distinguished Alumnus Award from Upsala College for outstanding achievements as an educator, researcher, author, and editor. He is a member of the ACS, Sigma Xi, Phi Lambda Upsilon, SAS, AIC, and AO AC. Dr. Sherma’s current interests are in quantitative TLC, mainly applied to clinical analysis, pesticide residues, and food additives.

THE EDITOR Ram N. Gupta, Ph.D., is Head of Toxicology in the Department of Laboratory Medicine at St. Joseph’s Hospital and Professor in the Department of Pathology at McMaster University in Hamilton, Ontario, Canada. Dr. Gupta received his M.Sc. degree in 1962 and Ph.D. degree in 1963 in Organic Chemistry from McMaster University. He continued working in the Chemistry Department of McMaster University as a Research Associate until 1971 when he moved to the Department of Pathology at the same university. Dr. Gupta has been elected as a fellow of the Chemical Institute of Canada. He is a member of the American Chemical Society, American Association of Clinical Chemists, Canadian Society of Clinical Chemists, and the Association of Clinical Biochemists (U.K.). He is the author of more than 40 scientific publications. His present research interests are the development of chromatographic procedures for the assay of drugs and other biochemicals in biological fluids.

TABLE OF CONTENTS Organization of Tables and Explanation of Abbreviations..........................................................1 Drug Tables...................................................................................................................................... 3 In d e x .............................................................................................................................................231

Volume HI

1

ORGANIZATION OF TABLES AND EXPLANATION OF ABBREVIATIONS Gas Chromatography (GC)

Specimen: Cerebrospinal fluid (CSF); not available (NA). The number in parenthesis refers to milliliters of plasma or serum used for the preparation of sample extract unless stated otherwise. There is no indication when volumes of other specimens are different from that of plasma or serum. Extraction: In this column, the extraction procedure is given a number and the corresponding procedure is described at the end of the table for the extraction of plasma or serum unless indicated otherwise. Any difference in the extraction procedure of another type of specimen is not indicated. Column: Columns are made of glass or fused silica unless noted otherwise. Length is given in meters and inner diameter in millimeters. Packing: The number in the parenthesis shows the mesh size of the support. The film thickness of the capillary columns is given in μιη and indicated by a footnote. Gas: Gas flow, if given in units other than milliliters per minute, has been indicated by a footnote. DET: Detector. Flame ionization detector (FID); nitrogen phosphorous detector (NPD); also, alkali flame ionization detector; thermionic sensitive detector; or nitrogen specific detector; electron capture detector (ECD); electron-impact mass spectrometer (MS-EI); chemical ion­ ization mass spectrometer (MS-CI); negative ion chemical ionization mass spectrometer (MS-NCI). Any other detector used and the reagent gas used for chemical ionization, if different from the carrier gas, are indicated by footnotes. RT min: Retention time in minutes of the title drug. It may be the retention time of the parent drug or its derivative. A dash “ — ” indicates that the title drug is not determined in the procedure under review, whereas NA indicates that the retention time is not available. Internal Standard: The names of the compounds used as internal standards are given in full. Any abbreviation used to describe the internal standard is explained by a footnote. A dash “ — ” indicates that no internal standard was used in the procedure. The retention time in minutes is given in parenthesis as it appears in the chromatogram. It may be of the parent compound or its derivative. The retention time when the internal standard is an isotropically labelled drug is considered the same as of the drug itself. Deriv: Derivative. This column indicates the type of derivative formed at some stage of the sample preparation. The details of derivatization reagent and procedure are included in the corresponding extraction procedure. A dash “ — ” indicates that no derivative was prepared. Other Compounds: Metabolites of the parent drug or other similar or unrelated drugs when determined simultaneously with the title drug are listed in this column. Their retention times are given in parenthesis. Ref: Reference. Liquid Chromatography (LC) This includes column liquid chromatography, high pressure liquid chromatography, and high performance liquid chromatography (see under GC for the explanation of common columns). Column: Columns are made of steel unless noted otherwise. Length is in centimeters and inner diameter in millimeters. Packing: Packing is described by the trade names as used by the authors. Footnotes indicate if a precolumn, a guard column, or a temperature other than ambient were used. Elution: The eluting solvent is given a number and the corresponding solvent is described at the end of the table. The procedure is isocratic unless indicated as gradient. The conditions for gradient elution are described with the description of the elution solvent.

2

CRC Handbook of Chromatography: Drugs

Flow Rate: Flow rate given in other units has been changed to milliliter per minute; a footnote indicates that only the pump pressure is given. Detector (DET); absorbance (ABS). Wavelength (nm) for absorbance detection is given. Two numbers are given when the absorbance is monitored simultaneously at two different wavelengths. A footnote indicates a programmed change of absorbance wavelength. Fluorescence (FL). The first number in the parenthesis is the excitation wavelength (nm), and the second, the emission wavelength. Other detectors are described without the use of abbreviations. Potentials for electrochemical detectors and procedures involving post-column reactors are indicated by footnotes. Thin-Layer Chromatography (TLC) See under GC and LC for the explanation of common columns.

Plate: Unless otherwise noted, plates are made of glass. Laboratory indicates that the plates have been coated by the authors in their laboratory. Layer: High performance thin-layer chromatography (HPTLC). Solvent: Developing solvent is given a number which is described at the end of the table. Post-Separation Treatment: (sp) The plate is spayed with the described reagent. (D) The plate is dipped in the described reagent. (E) The plate is exposed to the vapors of the described reagent. Det: Detection. Qualitative detection is indicated as visual. Wavelength (nm) for short or long wave UV lamp is given when fluorescence or quenching of fluorescence is observed under UV light. When the plate is scanned with the densitometer for quantitative determi­ nation of drug concentration, the mode of scanning is indicated as reflectance, transmission or reflectance/transmission for simultaneous mode. Wavelength (nm) for scanning and for fluorescence scanning, the excitation (first) and emission (second) are given.

1-1

2 x 4

Column Extraction (m x mm) 5% OV-17 Chromosorb W (100/120)

Packing (mesh) T.P*

Oven temp (°C) He (44)

Gas (m€/min) NPD

Det. 8.9

RT (min) b (10.2)

Internal standard (RT) Trimethylsilyl

Deriv. A515c (9.1)

Other compounds (RT)

1

Ref.

Plasma (0.1-1)

Specimen (m€)

1-1

4x3

Column Extraction (m x mm) 5% OV-225 Chromosorb W (100/120)

Packing (mesh)

Gas (ml/min) At (40)

Oven temp (°C) 210

ECD

Det.

Gas Chromatography

A643C

Internal standard (RT) A404C (15)

RT (min) 11

Pentafluoro propionyl

Deriv.

Other compounds (RT)

1

Ref.

1. Land, G., Shortman, N., and Ridout, G., The determination of 2,6-diamino-9-(2-hydroxyethoxymethyl)-9H-purine (A134U) and 2-amino-9-(2-hydroxyethoxymethyl)-9H-purine (A515U) in human plasma and urine by gas chromatography, Chromatographia, 19, 310, 1984.

REFERENCE

Extraction — 1-1. The sample was mixed with 50 μ€ of an aqueous solution of the internal standard (50 μg/m€), 500 μ€ of pH 8 phosphate buffer, and extracted twice with 8 m€ portions of butan-2-one-ethyl acetate (1:1). The combined organic extract was evaporated, the residue treated with 100 μ€ of N-methyl-N(trimethylsilyl)trifluoroacetamide containing 2% trimethylchlorosilane and 100 μ€ of tetrahydrofuran at room temperature for 8 hr, and aliquots of the reaction mixture were then injected with an autosampler.

a Initial temp = 230°C; initial time = 5 min; rate = 3°C/min; final temp = 320°C. b 2,6-Diamino-9-(3-hydroxypropoxymethyl)-9-H-purine. c Different chromatographic conditions are used for this compound.

Plasma (0.25)

Specimen (m€)

Gas Chromatography

A134U

Volume HI 3

Plasma (1)

Plasma (0.9) Plasma (1)

Feed (10 g) Feed (5 g) Plasma (1)

Specimen (m€)

Column (cm x mm)

25 X 4.6

NA

10 x 4.6

NA

25 X 4.6

25 X 4.6

Extraction

1-1

1-2

1-3

1-4

1-5

1-6

Spherisorb ODS (5)

μ-Bondapak C18 (10) Spherisorb ODS (5)

Partisil (5) Spherisorb ODS (10) Spherisorb ODS (5)

(μπι)

Packing

NA

E-3

E-6

E-5

1.2

1.0

1.3

1.5

E-2

E-4

2.0

E-l

Elution

Flow (ml/min)

Det. (nm)

FL (238, 450)

FL (235, 389) ABS (243)

FL (254, 460)

ABS (230) b

Liquid Chromatography

A CEBUTOLOL*

38, 40*

9.6

3.6

NA

16

9

RT (min)

M&B 17764 (22,23)*

(12.0)

M&B 17,764 (NA) LM 5008 (8)d

—

Diacetolol

_

Diacetolol (NA) Metabolite-le (6.6) Metabolite-2f Diacetolol (15, 16)*

Diacetolol (6) Diacetolol

(ID

Other compounds (RT) Internal standard (RT)

7

6

5C

4

3

2

Ref.

1. Parsons, D. N., Measurement of 2-amino-N-(l,l-dimethylhexyl)acetamide (A643C), an investigative antidepressant, in plasma by electron-capture gas chromatography, J. Chromatogr., 276, 197, 1983.

REFEREN CE

Extraction — I-1. To the sample 0.1 m£ of aqueous solution of internal standard (250 μg) and 1 m€ of 4 M sodium hydroxide were added and the mixture extracted with 10 m€ of π-hexane for 15 min. The organic layer was collected and extracted with 2 m€ of 2 M hydrochloric acid. The organic layer was discarded, the aqueous phase make alkaline with 0.8 m€ of 10 M sodium hydroxide, and extracted with 10 m€ of π-hexane for 10 min. The supernatant was collected in clean tubes, 30 μ€ of pentafluoropropionic anhydride added, mixed for 15 min and evaporated at room temperature in a current of nitrogen. The residue was dissolved in 0.4 m€ of cyclohexane and 5 μ€ injected.

4 CRC Handbook of Chromatography: Drugs

REFERENCE

1. Mehta, Anil C., HPLC determination of beta-adrenoceptor blocking drugs in biological fluids: a review, Pharm. J., 230, 191, 1983. 2. Bowker, M. J., Gladwin, R. P., Mills, D. J., and Stubbles, J. A., Semi-automated high-performance liquid chromatographic method for the determination of diacetolol hydrochloride in medicated animal diets, J. Chromatogr., 200, 261, 1980. 3. Schieffer, G. W., Reversed-phase high-performance liquid chromatography with differential pulse polarographic detection for assaying drugs in feed, J. Chromatogr., 202, 405, 1980.

Elution — E-l. Acetonitrile-methanol-0.1 M ammonia (3:1:1). E-2. Methanol-1% (W/V) ammonium acetate (35:65). E-3. 0.01 M Phosphate buffer (pH 4.0)-methanol-acetonitrile (7:3:1). E-4. Methanol-acetic acid-water (50:1:49). E-5. 0.1 M phosphate buffer (pH 4.0)-acetonitrile-water (6:55:39). E-6. Water-methanol-triethylamine (50:50:0.05).

Extraction — 1-1. Feed (10 g) packed into a glass column (25 cm x 2.5 cm) was eluted with 100 m€ of methanol at a flow rate of 20 m€/min. 1-2. Feed (5 g) packed into a glass column of 2 cm ID was eluted with 100 m€ of methanol at a flow rate of 10 to 20 m€/min. 1-3. Sample after the addition of internal standard is made alkaline with 0.5 m€ of 1 M sodium hydroxide and extracted with 6 m€ of 4:1 mixture of diethyl ether/ chloroform. The organic layer is collected, evaporated and reconstituted in 100 μ€ of mobile phase. 1-4. Plasma after the addition of internal standard is made alkaline with 0.1 m€ of 1 N sodium hydroxide and extracted with 7 m€ of chloroformpentanol mixture (60/20) for 2 min. Organic layer was collected and extracted with 300 μ€ of 0.1 N sulfuric acid, 10 to 100 μ€ of which was injected. 1-5. Plasma after the addition of 1 m€ (1 μg) of internal standard dissolved in 0.01 M phosphate buffer, pH 6.0 made alkaline with 200 μ€ of 2 M sodium hydroxide and extracted with 10 m€ of ethyl acetate. The organic layer is collected and extracted with 150 μ€ of 0.005 M sulfuric acid. The aqueous phase is injected with an auto-injector. 1-6. Plasma after the addition of internal standard was made alkaline with 0.5 m€ of 1 N sodium hydroxide and extracted with 5 m€ of diethyl ether. Ether layer was collected and evaporated. The residue was dissolved in dry dichloromethane (100 μ€), phenylethyl isocyanate in dichloromethane (20 μ€ of 100 μg/m€) was added and reacted for 30 min at room temperature. Diethyl ether (100 μ€) was added, mixed and evaporated at 50°C in oxygen-free nitrogen. The residue was dissolved in 100 μ€ of mobile phase for injection.

a HPLC determination of beta-adrenoreceptor blocking drugs in biological fluids has been reviewed.1 b Model 310 DME polarographic detector in differential mode with a modulation amplitude of 50 mV. c Extraction and chromatographic conditions for a number of other B-blocking drugs are described. d ±1 -(2-propy1-4-w-butyramindophenoxy)-2-hydroxy-3-isopropylaminopropane. e ± l-(2-acetyl-4-w-butyramidophenoxy)-2-hydroxy-3-isopropyl aminopropane. f ± l-(2-acetyl-4-acetamidophenoxy)-2-hydroxy-3-isopropyl aminopropane. g Retention times of S-( —) and R-( + ) diasteromers, respectively.

Volume III 5

1-1

Extraction

12.5 x 4.6

Column (cm x mm) NucleosilRP18 (5)a

Packing (μπι)

Flow ml/min 1.4

Elution E-l

ABS (254)

Det. (nm) 6.9

RT min

—

Internal standard (RT)

Indomethacin (8.7)

Other compounds (RT)

1

Ref.

REFERENCE

1. Schollnhammer, G., Dell, H. D., Doersing, K., and Kamp, R., Quantitative determination of acemetacin and its metabolite indometacin in blood and plasma by column liquid chromatography, J. Chromatogr., 375, 331, 1986.

Elution — E-l. 0.02 M phosphate buffer (pH 4.5)-methanol (45:55).

Extraction — 1-1. The sample was mixed with 0.15 M phosphate buffer (pH 3) and extracted twice with 5 m l portions of diethylether by shaking for 15 min each time. The combined ether extract was evaporated at 37°C. The residue was dissolved in 1 m l of methanol to wash the walls of the tube and evaporated with a current of nitrogen. The residue was dissolved in 150 μ ΐ of mobile phase and 25 μ ΐ injected with an auto-injector.

■ Column at 40°C.

Plasma (1)

Specimen (ml)

Liquid Chromatography

ACEMETACIN

4. Holt, J. E., Kaye, C. M., and Samtey, M. G., Use of reversed-phase high-pressure liquid chromatography for the assay of acebutolol, practolol and propranolol in plasma, Br. J. Clin. Pharmacol., 12, 282, 1981. 5. Lefebvre, M. A., Girault, J., and Fourtillan, J. B., β-blocking agents: determination of biological levels using high-performance liquid chromatography, J. Liq. Chromatogr., 4, 483, 1981. 6. Buskin, J. N., Upton, R. A., Jones, R. M., and Williams, R. L., High-performance liquid chromatography assay of acebutolol and two of its metabolites in plasma and urine, J. Chromatogr., 230, 438, 1982. 7. Gulaid, A. A. and Houghton, G. W., Separation of acebutolol and diacetolol diastereomers by reversed-phase high-performance liquid chromatography, J. Chromatogr., 318, 393, 1985.

ACEBUTOLOL (continued)

6 CRC Handbook of Chromatography: Drugs

Lichrosorb RP-2 (10)a LichrosoibRP-18 (5) μ-Bondapak C18 (10)a LichrosorbRP-8 (5)a

25 x 2.2

10 x 2

30 X 6

15 X 4.6

1-2

1-3

1-4

Packing (μπι)

1-1

Extraction

Column (cm x mm)

E-4

E-3

E-2

E-l

Elution

1.5

1.4

0.8

0.7

Flow (ml/min)

Det. (nm)

ABS (313) ABS (303)

ABS (305) ABS (308)

4.2

5

4.2

5.5

RT (min)

Warfarin (5)

Methylwarfarin (9) 5-Methoxypsoralen (2.2) —

Internal standard (RT)

Acetamido metabolite (2) Aminometabolite (3.5)b

—

—

—

Other compounds (RT)

4

3

2

1

Ref.

Elution — E-l. Acetonitrile-water-acetic acid (37:62:1) containing 0.75 g ammonium acetate/100 m l. E-2. Acetonitrile-0.1% acetic acid (35:65).

Extraction — ]1-1. To the sample, 1 m l of 3 N HC1 and 5 m l of n-butyl chloride containing methyl warfarin (240 ng/ml) were added. The mixture was agitated at slpw speed for 10 min. After centrifugation organic layer was collected and evaporated in a current of dry nitrogen at room temperature. The residue was dissolved in 30 μ ΐ of acetonitrile and 5 to 10 μ ΐ were injected. 1-2. To 1 m l of sample, 25 μ ΐ on the internal standard solution (4 mg/l 5 methoxypsoralen in ethanol) and 1 m l of 0.1 Af sodium acetate buffer (pH 4) and 7 m l of petroleum ether (40 to 60°) + dischloromethane (1:1) were added. After mixing (15 min) and centrifugation (10 min) the organic layer was evaporated in a current of nitrogen at 40°C. The residue was dissolved in 50 μ ΐ of mobile phase and 20 μ ΐ were injected. 1-3. The sample at pH 6 was applied to a column (50 cm x 10 mm) packed with Tenax GC (5 g). The column was washed with water (50 m l) at a flow rate of 2 ml/min. The column was eluted with 50 m l of ethanol which was. evaporated in vacuo. The residue was dissolved in 500 μ ΐ of ethanol. 1-4. To the sample, 10 μ ΐ of 50 μg/m l solution of warfarin in water, 1 m l of concentrated citric acid-phosphate buffer (pH 4.4) were added. The mixture was extracted twice with 5 m l portions of dichloromethane-n heptane (1:1). The combined organic layer was extracted with 0.2 m l of 0.5 N HC1 and evaporated to dryness at 35°C under a stream of nitrogen. The residue was dissolved in 100 μ ΐ of mobile phase.

* A precolumn (5 cm x 2.2 mm) packed with Vydac-RP (30 to 40 μπι) was used. b Amino metabolite is separated by extraction into acid and diazotized prior to chromatography.

Plasma (NA) Plasma (1)

Plasma (0.5-1) Plasma (1)

Specimen (m€)

Liquid Chromatography

ACENOCOUMAROL

Volume III 1

1-1

2x3

Column Extraction (m x mm) 3% OV-17 Gas Chrom Q (100/120)

Packing (mesh) 255

Oven temp (°C) N2 (40)

Gas (ml/min) FID

Det. 5.7

RT (min)

N-7 Theophylline propionic acid (7)

Internal standard (RT)

Ethyl

Deriv.

—

Other compounds (RT)

1

Ref.

1. Zuidema, J. and Hilbers, H., Gas-liquid chromatographic determination of acephylline in urine, J. Chromatogr., 182, 445, 1980.

REFERENCE

Extraction — 1-1. To 1 m l of sample of urine, 60 μ ΐ of 8 M HC1, 0.5 m l of the internal standard solution (150 μg/ml) were added and the mixture extracted twice with 5 m l portions of chloroform-isopropanol (20:1). The combined organic extract was evaporated at 70°C under nitrogen to 500 μ ΐ. This was transferred to a small ampoule and evaporated to dryness. The residue was treated with 100 μ ΐ of a 10% ethyliodide solution in acetonitrile and 5 to 10 mg of K2C 0 3 were added. The ampoule was sealed under nitrogen, heated at 80°C for 1 hr, and 1 to 2 μ ΐ were injected.

Urine (1)

Specimen (ml)

Gas Chromatography

ACEPHYLLINE

1. Wong, L. T. and Solomonny, G., Quantitation of acenocoumarol in plasma by reversed-phase high-performance liquid chromatography, J. Chromatogr., 163, 103, 1979. 2. deWolff, F. A., Tetteroo-Tempelman, C. A. M., and Edelbroek, P. M., Determination of nanogram levels of the anticoagulant acenocoumarin in serum by high-performance liquid chromatography, J. Anal. Toxicol., 4, 156, 1980. 3. Thonnart, N. and Reuse, J., Quantitative determination of acenocoumarin in anticoagulated patients, J. Liq. Chromatogr., 4, 833, 1981. 4. Thijssen, Η. H. W., Bears, L. G., and Reminders, M. J., Analysis of acenocoumarin and its amino and acetamido metabolites in body fluids by highperformance liquid chromatography, J. Chromatogr., 274, 231, 1983.

REFERENCE

E-3. Ethanol-0.1% acetic acid (50:50). E-4. Acetonitrile-0.1% acetic acid-ethyl acetate (90:100:1), adjust to pH 4.9 with 1 N NH4OH, ethyl acetate.

8 CRC Handbook of Chromatography: Drugs

30 x 4

30 x 4.6

1-4

1-5

Plasma (1) Urine (1)

30 x 3.9

1-3

Column (cm x mm)

1.5 x 4

0.9 x 2

Packing (mesh)

μ-Bondapak-Cig (10)c μ-Bondapak-Qg (10)

μ-Bondapak-Cig (10)

Packing (μπι)

3.2% OV-1 Chromosorb W (80/100) 3% Apolane 87a Chromosorb W (100/120)

Column (m x mm)

Serum (0.05)

Extraction

1-2

Plasma (0.1)

Specimen (m€)

1-1

Extraction

Plasma (0.5)

Specimen (m€)

(40)

n2

(40)

n2

Gas (m€/min)

FID

FID

Det.

E-3

E-2

E-l

Elutioni

2.0

1.0

NA

Flow (ml/min) Det. (nm)

1.5

1.9

RT (min)

ABS (240) ABSd (248)

ABS (248)

Liquid Chromatography

235

120

Oven temp (°C)

Gas Chromatography

A C E T A M IN O P H E N

7.5

7

2.5

Other compounds (RT)

—

3-Hydroxyacetaminophen (5) Acetaminophen glucuronide (3.5) Acetaminosulfate (6)

—

5

4

3

Ref.

2

1

_ b

Ref.

Other compounds (RT)

N,N-DieSalicylic acid thyl(3.8) m-toluamide (5.5)

Internal standard (RT)

Acetyl

N-Butyryl-paminophenol (2.5)

RT (min)

Trifluoro acetyl

Deriv.

2-Acetaminophenol (1)

Internal standard (RT)

Volume III 9

E-6; 4.0 grad

Radial Pak-C19 (10)

10 X 8

1-8

Urine

1.0

E-5

1-7

Plasma, urine (0.1)

μ-Bondapak-C,,, 00)

10 X 5

1-6

1.0

Gas (m€/min)

30 X 3.9

Oven temp (°C)

E-4

Packing (mesh)

Radial Pak C18 (10)

Column Extraction (m x mm)

Serum (0.2)

Specimen (m€)

RT (min)

ABS (254, 280)

ABS (237)

ABS (280)

Det.

Gas Chromatography

ACETAMINOPHEN (continued)

3.7

9.7

3

Internal standard (RT)

8-Chlorotheophylline (8.7)

Deriv.

3-Cysteinylacetaminophen (7) 3-Methoxyacetaminophen (13) 3-Methylthioacetaminophen (28) Theophylline (4.6) Salicylic acid (6.2) Salicylic acid (5.7) Salicyluric acid (7.4) Acetaminophen cysteine conjugate (3) 3-Methoxyacetaminophen (4.8) Acetaminophen glucuronide (6) Acetaminophen mercapturic acid (9.4)

Other compounds (RT)

8

7

6

Ref.

10 CRC Handbook of Chromatography: Drugs

25 X 4.5

1-10

1-11

1-12

1-13

Serum (0.5)

Plasma, urine (0.05)

Serum

Serum (0.1)

Serum (1)

Specimen (m€)

1-14

Extraction

20 x 20 cm (Whatman)

Plate (Manu­ facturer)

10 x 4.5

15 x 4.6

25 x 4.5

25 x 4.6

Dosage

4

ABS (254) ABS (249)

1.5

1.0 NA

E -ll

E-12 E-13

S-l

Solvent

Visual

Det. (nm)

5.7

ABS (254)

1.0

E-10

0.41

Rf

2.5

2.5

ABS (234)

1.8

E-9; grad­ ient

Thin-Layer Chromatography

Silica gel F254 (LK6DF) (0.25)

Layer (mm)

Supelcosil LC-CN (5) Hypersil-C18 (5)g

IBM RP-8

Spherisorb-ODS (5)

4.5

ABS (256) ABS (270)

1.5

E-8e

μ-Bondapak Phenyl (10) Perkin-Elmer RP-18 (10)

30 x 4

Dosage 4

5.4

ABS (254)

1.0

E-7

Spherisorb-ODS (5)

1-9

25 x 4.6

Plasma (0.1)

j

(Rf) (Rf) ___

Other compounds

i

Caffeine (6.5) Phenacetin (10) Propyphenazone (12) Acetylsalicylic acid (3.5) Salicylic acid (4.5) Acetaminophen glucuronide (3.2) Acetaminophen sulfate (4.2) f

e

Internal standard

Benzoic acidh (8.2)

Theophyl­ line (7.3)

—

N-Propionylaminophenol (6.9)

Acetaminophen sulfate (10) 3-Thiomethylacetaminophen sulfate (12)

16

Ref.

15

14

13

11

10

9

Volume III 11

10 x 10 cm (Merck)

Silica gel F254 (HPTLC)

5.2

Reflectance (250)

k

—

—

17

C87 hydrocarbon. Retention times of a number of potentially interfering drugs are given. A precolumn packed with C18 corasil was used. An electrochemical detector ( + 0.60 V) was also used. Different mobile phases, flow rates and detection wavelength are described for the assay of components of cold medicines. Different columns, mobile phases, flow rates are described for the assay of different drugs in serum. A fluorescence detector was used for the assay of quinidine. Protected by a 3 x 0.45 cm guard column packed with Hypersil-C18 (5 μηι). This compound is added to a variety of foods as a preservative. Retention times of a number of potentially interfering drugs are given. Rf values of a number of potentially interfering drugs are given. Retention time of the spot obtained by the integrator during scanning of the plate is shown as 1.9 min.

1-15

Extraction — 1-1. The sample was extracted with 2.5 m€ of ethyl acetate containing 15 m g/l of the internal standard. An aliquot of 1 m l of the organic extract was evaporated at 50°C in a stream of nitrogen. Toluene (25 μ ΐ) and trifluoroacetic anhydride (25 μ ΐ) were added to the residue. After vigorous mixing, 1 μ ΐ was injected. 1-2. The sample was mixed with 100 μ ΐ of the internal standard solution (200 mg/l in chloroform); 50 μ ΐ of phosphate buffer (0.067 M, pH 7.4) and 20 μ ΐ of acetylating reagent (acetic anhydride-N-methylimidazole-chloroform 5:1:30) for 30 sec and centrifuged. Aliquots of 3 to 5 μ ΐ of the chloroform layer were injected. 1-3. An aliquot of 200 μ ΐ of acetonitrile containing 80 m g/l of the internal standard was mixed with the sample. After centrifugation 20 μ ΐ of the clear supernatant was injected. 1-4. The sample was treated with 1 m l of 0.3 N Ba(OH)2 solution. After thorough mixing 1 m l of 5%, ZnS04 was added, vortexed at high speed for 1 min. After centrifugation, aliquots of the clear supernatant were injected with an autosampler. 1-5. Urine (1 m€) was mixed with 4 m l of 2 M acetate buffer, pH 5. After centrifugation, aliquots of this diluted urine were injected. Alternatively, the diluted urine sample was incubated overnight at 37°C with 50 μ ΐ of β-glucuronidase-sulfatase prior to injection into the liquid chromatograph. 1-6. The sample was mixed 20 μ ΐ of the internal standard solution in methanol (15 mg/100 m l) and 200 μ ΐ of 1 M HC1. The mixture was extracted with 2 m l of dichloromethane-isopropanol (9:1). The organic layer was evaporated to dryness at 50°C with a gentle stream of air. The residue was dissolved in 100 μ ΐ of the mobile phase, centrifuged and a 20-μ€ aliquot of the clear supernatant was injected. 1-7. The pH of plasma and urine samples was adjusted to 2 and 5, respectively. The samples were diluted with 1 m l of water, 4 g sodium chloride added, and extracted with 15 m l of ethyl acetate. Ten milliliters of the organic layer was evaporated at 45°C under a gentle stream of nitrogen. The residue was dissolved in 100 μ ΐ of methanol, mixed, and an aliquot of 20 μ ΐ was injected.

a b c d e f g h 'l j k

Serum (1)

Post-separation treatment Sp: Equal vols of A + B mixed just prior to use. A. 2,4,6,-Tris-(2-pyridyl)-s-triazine (3.75 g) in methanol (40 m l) B. 110 mM ferric chloride in 0.1 M HC1

ACETAMINOPHEN (continued)

12 CRC Handbook of Chromatography: Drugs

REFERENCE

1. Kaa, E., Rapid gas chromatographic method for emergency determination of paracetamol in human serum, J. Chromatogr., 221, 414, 1980. 2. Huggett, A., Andrews, P ., and Flanagan, R. J., Rapid micro-method for the measurement of paracetamol in blood plasma or serum using gas-liquid chromatography with flame-ionisation detection, J. Chromatogr., 209, 67, 1981.

Solvent — S-l. Ethyl acetate-toluene-methanol-water (180:160:54:2). S-2. n-Hexane-ethanol-acetone (60:1030).

Elution — E -l. Methanol-5% acetic acid (60:40, V/V). E-2. Methanol-water (15:85). E-3. Methanol-acetic acid-0.1 Af KH2P 0 4 (7:0.75:92.25). E-4. Methanol-0.033 Af acetate buffer (pH 3.6) (28:72). E-5. Acetonitrile-4 mAf phosphate buffer, pH 5.7 (6:94). E-6. (A) Methanol; (B) 0.01 Af tetrabutylammonium hydroxide -I- 0.01 Af tris-hydroxymethylaminomethylaminomethane, pH 5 (H3P 0 4). Gradient from 10% (A) to 50% (A) over 12 min. E-7. Methanol-water (1:3, V/V). E-8. Methanol-0.02 Af ammonium acetate buffer, pH 7 (15:85). E-9. (A) Methanol; (B) water. Linear gradient from 30% (A) to 95% (A) over 13 min. E-10. Acetonitrile-methanol-water (25:10:65) to pH 3 with orthophosphoric acid. E -ll. Acetonitrile-0.05 Af sodium sulfate buffer, pH 2 with orthophosphoric acid (7:93). E-12. 0.05 Af sodium dodecyl sulfate in distilled water. E-13. Methanol-0.05 Af sodium acetate buffer, pH 4.6 (15:85).

1-8. Urine samples were diluted (5:1) with methanol, filtered through 0.5 μπι filter, aliquots of which were injected. 1-9. The sample was treated with an aqueous solution of the internal standard (2 μg/ml) and extracted with 5 m l of acetonitrile. The organic layer was evaporated under a nitrogen stream at 40°C. The residue was reconstituted with 2 m l of water and aliquots of 50 μ ΐ were injected. I-10. The sample was acidified with 0.1 m l of hydrochloric acid and extracted three times with 2 m l aliquots of chloroform-acetonitrile (6:4). The combined organic extract was evaporated to dryness under a stream of nitrogen and redissolved in 100 μ ΐ of the mobile phase. Aliquots of 20 μ ΐ were injected. 1-11. To 50 μ ΐ plasma were added 100 μ ΐ of a 20 mg/l solution of the internal standard in 6% perchloric acid. After centrifugation 5 μ ΐ of the supernatant was injected. 1-12. Untreated serum samples are injected directly. 1-13. The sample was treated with 200 μ ΐ of 1% ZnS04,7H20 containing 12 mg/100 (or 150 mg/100 ml) of the internal standard. After vortexing and centrifugation, aliquots of 20 μ ΐ of the clear supernatant were injected. 1-14. To the sample was added 0.5 m l of 3 Af phosphate buffer, pH 5.5 and was extracted with 10 m l of chloroform-methanol (95:5). The organic extract was evaporated under a gentle stream of nitrogen at 60°C. The residue was dissolved in 100 μ ΐ of methanol and 10 μ ΐ of this solution was spotted. 1-15. The sample was treated with 70 mg of lead nitrate. After centrifugation, 0.5 m l of the supernatant was freeze dried. The residue was treated with 100 μ ΐ of ethanol in an ultrasonic bath, centrifuged and 5 μ ΐ of the supernatant was applied to the plate.

Volume III 13

3. Ferrell, W. J. and Goyette, G. W., Analysis of acetaminophen and salicylate by reverse phase HPLC, J. Liq. Chromatogr., 5, 93, 1982. 4. O’Connell, S. E. and Zurzola, F. J., Rapid quantitative determination of acetaminophen in plasma, J. Pharm. Sci., 71, 1291, 1982. 5. Wilson, J. M., Slattery, J. T., Forte, A. J., and Nelson, S. D., Analysis of acetaminophen metabolites in urine by high-performance liquid chromatography with UV and amperometric detection, J. Chromatogr., 227, 453, 1982. 6. Kinberger, B. and Holmen, A., Simultaneous determination of acetaminophen, theophylline and salicylate in serum by high-performance liquid chromatography, J. Chromatogr., 229, 492, 1982. 7. Douidar, S. M. and Ahmed, A. E., Studies on simultaneous determination of acetaminophen, salicylic acid and salicyluric acid in biological fluids by high performance liquid chromatography, J. Clin. Chem. Clin. Biochem., 20, 791, 1982. 8. Hart, S. J., Aguilar, Μ. I., Healey, K., Smail, M. C., and Calder, I. C., Improved high-performance liquid chromatographic separation of urinary paracetamol metabolites using radially compressed columns, J. Chromatogr., 306, 215, 1984. 9. Korduba, C. A. and Petruzzi, R. F., High-performance liquid chromatographic method for the determination of trace amounts of acetaminophen in plasma, J. Pharm. Sci., 73, 117, 1984. 10. Gupta, V. D. and Heble, A. R., Quantitation of acetaminophen, chlorpheniramine maleate, dextromethorphan hydrobromide, and phenylpropanolamine hydrochloride in combination using high-performance liquid chromatography, J. Pharm. Sci., 73, 1553, 1984. 11. Mamolo, M. G., Vio, L., and Maurich, V., Simultaneous quantitation of paracetamol, caffeine and propyphenazone by high-performance liquid chromatography, J. Pharm. Biomed. Anal., 3, 157, 1985. 12. Tebbett, I. R., Omile, C. I., and Danesh, B., Determination of paracetamol, salicylic acid and acetyl salicylic acid in serum by high-performance liquid chromatography, J. Chromatogr., 329, 196, 1985. 13. Jung, D. and Ulzafar, N., Micro high-performance liquid chromatographic assay of acetaminophen and its major metabolites in plasma and urine, J. Chromatogr., 339, 198, 1985. 14. DeLuccia, F. J., Arunyanart, M., and Love, L. J. C ., Direct serum injection with micellar liquid chromatography for therapeutic drug monitoring, Anal. Chem., 57, 1564, 1985. 15. Starkey, B. J., Loscombe, S. M., and Smith, J. M., Paracetamol (acetaminophen) analysis by high performance liquid chromatography: interference studies and comparison with an enzymatic procedure, Ther. Drug Monit., 8, 78, 1986. 16. Kelly, R. C., Doshier, L. A., and Rubin, H. R., A convenient thin-layer chromatographic screening method for acetaminophen in serum, J. Anal. Toxicol., 8, 54, 1984. 17. Berner, G., Staab, R., and Wagener, Η. H., HPTLC-Bestimmung von Paracetamol im Serum, Fresenius Z. Anal. Chem., 321, 601, 1985.

ACETAMINOPHEN (continued)

14 CRC Handbook of Chromatography: Drugs

1-5

Plasma (0.25)

30 x 3.9

30 x 3.9

μ-Bondapak-Qg (10)d

μ-Bondapak-Qg (10)d

μ-Bondapak-Qg (10) UltrasphereODS (5)b

Silica gel (5)

Packing (pm)

E-5

E-4

E-3

E-2

E-l

Elution

2

2

1

1.5

3

Flow (ml/min)

ABS (265)

ABS (254)

ABS (254) ABS (254)

ABS (280)

Det. (nm)

6

7.6

10

3.9

2

RT (min) Chloro­ thiazide (4) a (5.3) Chloro­ thiazide (12) Sulfadi­ azine (9.8) Chloro­ thiazide (8)

Internal standard (RT)

—

—

—

—

—

Other compounds (RT)

5

4

3

2

1

Ref.

Extraction — 1-1. To 1 m l of the sample, 0.5 m l of internal standard (20 μg of chlorothiazide/ml of water), 2 m l of sodium acetate buffer (0.05 M, pH 4.8) and 10 m l of ethyl acetate were added. After mixing (10 min) and centrifugation (10 min), the organic layer was collected and the aqueous layer re-extracted with another 10 m l portion of ethyl acetate. The combined organic layer was evaporated at 60°C under a current of nitrogen. The residue was dissolved in 10 μ ΐ of 0.45 N NaOH and 1 m l ethyl acetate and the contents were vortex mixed. Aliquots of 200 μ ΐ of this solution were injected. 1-2. To 100 μ ΐ of sample, 100 μ ΐ of internal standard (25 μg/m l in water) and 50 μ ΐ of 0.5 M ammonium acetate buffer, pH 4.5 were added. The solution was extracted with 3 m l of ethyl acetate. The organic layer was collected, evaporated in a current of nitrogen, dissolved in 50 μ ΐ of methanol and 2.4 μ ΐ injected. 1-3. The extraction tubes were treated with a 10% solution of surfasil in acetone. Serum (1 m l), water (0.1 m l), 0.05 M citrate buffer, pH 4.6 (1 m l), sodium chloride (700 mg) and 10 m l of ethyl acetate containing 2.5 μg/ml of chlorothiazide were placed in the extraction tubes. The tubes were vortex mixed for 5 min and centrifuged. The ethyl acetate layer was collected and back extracted with 0.4 m l of phosphate buffer (0.1 M, pH 11.9) and 35 μ ΐ of aqueous phase was injected with an auto-injector.

a The internal standard, 2-acetamido-4-methyl-5-thiazolisulfonamide was prepared from 2-acetamido-4-methyl-5-thiazolesulfonyl chloride. b The column was protected by a precolumn (40 x 4.6 mm) packed with CO:Pell ODS. c Urine is diluted 1:10. d The column is protected by a (2.3 x 3.9 mm) guard column packed with corasil.

1-4

25 x 4.6

1-3

Blood, plasma, urinec (0.2)

25 x 4

1-2

Plasma (0.1) Serum (1)

25 x 3.2

1-1

Extraction

Column (cm x mm)

Plasma (1)

Specimen (ml)

Liquid Chromatography

ACETAZOLAMIDE

Volume III 15

Plasma (3)

Specimen (m€)

1-1

1.8x2

Column Extraction (m x mm) 3% XE-60 GasChromQ (80/100)

Packing (mesh)

Gas (ml/min) 200 N2 (30)

Oven temp (°C)

FID

Det.

Gas Chromatography

ACETYLMETHADOL

3.1

RT (min)

Triacontane8 (10.1)

Internal standard (RT)

—

Deriv.

b

Other compounds (RT)

1

Ref.

1. Hossie, R. D., Mousseau, N., Sved, S., and Brien, R., Quantitation of acetazolamide in plasma by high-performance liquid chromatography, J. Pharm. Sci., 69, 348, 1980. 2. Gal, J., Ellis, P. P., and Rendi, M., Determination of acetazolamide in biological fluids by high-performance liquid chromatography, Curr. Eye Res., 1, 361, 1981. 3. Chambers, D. M., White, Μ. H., and Kostenbauder, Η. B., Efficient extraction and reversed-phase high-performance liquid chromatography-ultraviolet quantittion of acetazolamide in serum, J. Chromatogr., 225, 231, 1981. 4. Chapron, D. J. and White, L. B., Determination of acetazolamide in biological fluids by reversed-phase high-performance liquid chromatography, J. Pharm. Sci., 73, 985, 1984. 5. Hwang, P. T. R., Lang, J. R., Wood, G. C., and Meyer, M. C., High-performance liquid chromatographic determination of acetazolamide in human plasma, J. Liq. Chromatogr., 8, 1465, 1985.

REFERENCE

Elution — E-l. Hexane-methanol-chloroform-acetic acid (65:25:10:0.25). E-2. Methanol-water (500:1600), +2.3 g monobasic ammonium phosphate and 400 μ ΐ phosphoric acid. E-3. Acetonitrile-0.05 M acetate buffer, pH 4.5 (10:90). E-4. Acetonitrile-methanol-0.05 M sodium acetate buffer, pH 4.0 (3:2:95). E-5. Acetonitrile-0.05 M acetate buffer, pH 4.5 (6:94).

1-4. Whole blood was hemolyzed by freezing and thawing. The samples were extracted with ethyl acetate (3 m€). The organic layer was washed with 1.5 m€ of phosphate buffer (pH 8), and then back extracted into 20 μ€ of glycine buffer, pH 10. The aqueous layer was washed with ether and 25 to 50 μ€ of aqueous phase was injected. 1-5. The sample (0.25 m€) after the addition of 50 μ€ aliquot of the internal standard and 1 m€ of acetonitrile was vortexed for 20 sec and centrifuged at 3000 rpm at 5°C for 10 min. About 0.5 m€ of the supernatant was transferred to the autosampler vials and 25 μ€ was injected.

ACETAZOLAMIDE (continued)

16 CRC Handbook of Chromatography: Drugs

1-3

Extraction

1-2

30 x 4

Column (cm x mm)

Packing (μπι) LiChrosorb Si-60 (5)f

25 x 0.2 OV-1

He (0.5)

E-l

Elution 1.5

Flow (ml/min)

Liquid Chromatography

T.P.C

ABS (218)

Det. (nm)

NPD

4.5

RT (min)

5.6

d

—

Propranolol (15)

Internal standard (RT)

(7.2)

Methadol (5.5) Noracetylmethadol (7.5) Dinoracetyl methadol (10)

Other compounds (RT)

Noracetyl-6 methadol Dinoracetylmethadol (9.1)e

3

Ref.

2

Extraction — 1-1. The sample (3 m€ plasma) after the addition of 1 m€ of 0.2 M phosphate buffer, pH 7.4, 1 drop of 1-octanal was extracted with 9 m€ of 1chlorobutane by horizontally shaking for 10 min. After centrifugation 8 m€ of organic layer was collected and back extracted into 5 m€ of 0.2 M HC1. The organic phase was discarded and the aqueous phase washed with 5 m€ of hexane. The aqueous phase was made alkaline (pH 13) with 50% NaOH and extracted with 5 m€ of dichloromethane. An aliquot of 2 m€ of the organic extract was evaporated and the residue dissolved in 20 μ€ of carbon disulfide containing 0.3 mg of triaconate as external standard. Another aliquot of 2 m€ was used for the preparation of heptaflurobutyryl derivatives of the metabolites. 1-2. To 0.5 m€ of sample, 25 μ€ of internal standard (150 ng in methanol), 0.5 m€ of water and 100 μ€ of 50% NaOH were added, mixed and placed in a heating block at 70°C for 15 min. The samples were coated and extracted with 5 m€ of n-butyl chloride for 10 min and centrifuged. The organic layer was collected and evaporated on a vortex evaporator. The residue was reconstituted with 25 μ€ of ethyl acetate and 5 μ€ were injected. 1-3. To 1 m€ of sample, 0.1 m€ of 1 μg/m€ solution of propranolol in methanol, 0.5 m€ of bicarbonate buffer (1 M, pH 9.6) were added and extracted twice with 5 m€ of n-butyl chloride for 7 min and centrifuged. The combined organic layer was collected and evaporated to dryness and the residue reconstituted in 5 m€ of nhexane which was back extracted with 2.5 m€ of 0.2 N HC1. The aqueous phase was washed with n-hexane, made alkaline (pH 9.6) and extracted twice with 5 m€

a External standard, added just prior to injection. b Determination of metabolites of acetyl methadol after derivatization with heptafluorobutyric anhydride is carried out separately with the use of an electron capture detector. c Initial temp = 190°C; hold time = 38 sec; rate = 30°C/min to 220°C, hold time = 5.8 min; rate = 18°C/min to final temp = 240°C; Hold time = 2 min. d Diethylaminoethyldiphenylpropyl acetate. e The metabolites are converted to corresponding amides during heat treatment at alkaline pH. f Column temp = 30°C. A guard column (20 x 4 mm) packed with Vydac silica (40 μπι) was used.

Plasma, urine (1)

Specimen (m€)

Serum (0.5)

Volume III 17

1-1

1-2

Plasma, urinea (1)

Extraction

Plasma (0.2)

Specimen (m€)

25 x 4.6

30 x 3.9

Column (cm x mm) E-l

E-2

SpherisorbODS (5)

Elution

μ-Bondapak-Qg (10)

Packing (pm)

1.0

1.0

Flow (m€/min)

Liquid Chromatography

8.5

8

ABS (237)

ABS (234)

RT (min)

Det. (nm)

ACETYLSALICYLIC ACID

Other compounds (RT) Gentisic acid (5.2) Salicyluric acid (6.2) Salicylic acid (11.2) m-Hy droxy- Salicyluric acid benzoic (6) acid Salicylic acid (3.5) (ID p-Toluic acid (13.6)

Internal standard (RT)

2

1

Ref.

1. Tse, F. L. S. and Welling, P. G., Pharmacokinetics of acetylmethadols I: gas-liquid chromatographic determination of L-acetylmethadolol and its major metabolites in plasma, Biopharm. Drug Dispos., 1, 203, 1980. 2. Verebey, K., DePace, A., and Mule, S. J.,, Quantitation of 1-a-acetyl-methadol and its metabolites in human serum by capillary gas-liquid chromatography and nitrogen detection, J. Chromatogr., 343, 339, 1985. 3. Kiang, C., Campos-Flor, S., and Inturrisi., C. E., Determination of acetylmethadol and metabolites by use of high-performance liquid chromatography, J. Chromatogr., 222, 81, 1982.

REFERENCE

Elution — E -l. Methanol-acetonitrile (75:25) with 0.008% ammonium hydroxide.

portions of ethyl acetate. The combined layer was evaporated, this residue reconstituted in 100 μ€ of methanol-acetonitrile (20:80) and volumes up to 100 μ€ were injected.

18 CRC Handbook of Chromatography: Drugs

Plasma (0.1)

Specimen (m€)

1-8

Extraction

20 x 20 cm Aluminum sheets (E. Merck)

Plate (Manu­ facturer) Silica gel 60 (0.2)

Layer (mm) S-l

Solvent

Heat®

Post-separation treatment

FI (d, 433)

Det. (nm)

ABS (280)

Thin-Layer Chromatography

0.7

E-7

UltrasphereODS (5)

15 X 4.6

1-7

Blood (0.1)

ABS (237)

1.3

E-6

Nucleosil-C8 (5)

25 X 4.6

1-6

Plasma, urine* (1)

NA

Rf

m-Anisic acid (30.8)

18.9

(Rf)

Internal standard

Mephenytoin (11.6)

3,4,5-Trimethoxybenzaldehyde (8.7)

o-Toluic acid (10.2)

7.4

5.5

ABS (235)

2.0

E-5

Alltech-C,k (10)

25 X 4.6

1-5

Plasma, urine* (0.2)

4

ABS (280)

1.5

E-4

LiChrosorb RP-18 (5)

15 X 4

1-4

Plasma (0.2)

4.8

ABS (238)

2.0

μ-Bondapak-C,, E-3 (10)

30 x 3.9

1-3

Plasma, urine (0.1)

(Rf)

Other compounds

Gentisic acid (3.0) Salicyluric acid (3.7) Salicylic acid (7.1) Salicyluric acid (3) Salicylic acid (7) Gentisic acid (3.3) Salicluric acid (4.1) Salicylic acid (6.7) Gentisic acid (4.5) Salicyluric acid (5.7) Salicylic acid GO) Salicylic acid (23.4)

8

Ref.

7

6

5

4

3

Volume III 19

Modified conditions are described for the analysis of urine. Column was protected by a guard column (40 x 2 mm) packed with Co:Pell ODS (30 to 40 μιη). The plate is heated after development to convert acetylsalicylic acid to salicylic acid. A UVB filter is used for selecting exitation wavelength.

Solvent — S-l. Dioxane-benzene-acetic acid-methanol (65:30:4:5:0.5).

Elution — E -l. Acetonitrile-0.03% phorphoric acid, pH 2.5 (30:70). E-2. Water-0.2 M phosphate buffer, pH 2.5, acetonitrile (35:40:25). E-3. Water-methanol-acetic acid (64:35:1). E-4. Methanol-water (40:60), adjusted to pH 3.00 with 0.005 M phosphoric acid. E-5. Methanol-0.1% phosphate buffer, pH 3.9 (35:65). E-6. Methanol-acetonitrile-5 mM phosphate buffer, pH 2.5 (16:16:68). E-7. Acetonitrile-acetic acid-water (10:5:85).

Extraction — 1-1. To plasma sample (200 μ ΐ), 20 μ ΐ of 30% HC104, containing p-toluic acid (0.02%) and 200 μ ΐ of methanol were added. After vortexing the tubes were centrifuged and 20 μ ΐ of the supernatant was injected. 1-2. To 1 m l of the sample, 1 m l of aqueous solution of m-hydroxy-benzoic acid (0.7 μg/ml), and 1 m l of 1 mol/l oxalic acid were added. The mixture was extracted with 10 m l of an equivolume solution of ether/hexane by vortex mixing for 90 sec. After centrifugation the organic layer was collected and back extracted with 300 μ ΐ of 0.5 mol/l phosphate buffer, pH 7.0, aqueous phase (200 μ ΐ) was acidified with 200 μ ΐ of 1.0 mol/l phosphate buffer, pH 2.0 and 20 to 150 μ ΐ were injected. 1-3. To 100 μ ΐ of sample, internal standard dissolved in acetonitrile-water (1:2) (100 μg/ml) was added and the protein precipitated with acetonitrile. After vortexing and centrifugation 20 μ ΐ were injected. 1-4. To 200 μ ΐ of sample, 50 μ ΐ of concentrated phosphoric acid and 600 μ ΐ of ethyl acetate are added. After vortex mixing and centrifugation, 400 μ ΐ of organic layer is collected under a stream of nitrogen on an ice bath. The residue is dissolved in 200 μ ΐ of mobile phase and 100 μ ΐ are injected. 1-5. To the sample (200 μ ΐ), 20 μ ΐ of 30% perchloric acid solution containing trimethoxybenzaldehyde (0.02%) and 200 μ ΐ of methanol were added. The mixture was vortex mixed, centrifuged and 20 μ ΐ of the clear supernatant was injected. 1-6. To the sample (1 m l), 150 μ ΐ of 2 M orthophosphoric acid, 400 mg of sodium chloride and 12 m l of dichloromethane containing 3 μg/ml of mephenytoin were added. After mixing (10 min) and centrifugation, upper aqueous layer was discarded and 8 m l of organic layer was evporated under reduced pressure at room temperature. The residue was dissolved in 200 μ ΐ of mobile phase and 10 μ ΐ were injected. 1-7. Blood (100 μ ΐ) was collected in chilled tubes, 0.1 m l of 50/50 phosphoric acid containing 0.25 g/ml of ammonium sulfate and 2.5 mg/ml of sodium fluoride was added. The sample was extracted with 6 m l of 50/50 ethylacetate in butyl chloride containing 0.2 μg/ml of m-tanisic acid. After vortex mixing (2 min) and centrifugation (10 min), the organic layer was transferred to a new set of tubes and evaporated in a vortex evaporator at 35°C. The residue was dissolved in 0.6 m l of the mobile phase and 100 μ ΐ of this solution was injected. 1-8. To the sample (100 μ ΐ), 20 μ ΐ of 0.5 N HC1 and 1 m l of ethyl acetate were added. After mixing (5 min) and centrifugation, 500 μ ΐ of the supernatant was evaporated at 55°C under a stream of nitrogen. The residue was dissolved in 25 μ ΐ of ethylacetate and 5-μΙ aliquot was applicated on a TLC plate.

* b c d

ACETYLSALICYLIC ACID (continued)

20 CRC Handbook of Chromatography: Drugs

—

Pure compound

1-1

30 x 6.3

Column (cm x mm)

25 x 0.25

Column Extraction (m x mm)

Extraction

Serum (0.5)

Specimen (m l)

|x-Bondapak-Clg (10)

Packing (pm)

Chirasil-Val

Packing (mesh) Heb

Gas (ml/min)

Flow (ml/min) 1.0

Elution E-l

Det. (nm)

14.8

RT (min)

Electrochemc

MS-EI

Det.

Liquid Chromatography

T.P.a

Oven temp (°C)

Gas Chromatography

N-ACETYLCYSTEINE

Internal standard (RT) —

10*

Methyl

Deriv.

RT (min)

N-Acetyl-Dcysteine (14.4)

Internal standard (RT)

Glutathione (4) L-Cysteine (8) Penicillamine (11.5)

Other compounds (RT)

—

Other compounds (RT)

2

Ref.

1

Ref.

Rumble, R. H., Roberts, M. S., and Wanwimolruk, S., Determination of aspirin and its major metabolites in plasma by high-performance liquid chromatography without solvent extraction, J. Chromatogr., 225, 252, 1981. Buskin, J. N., Upton, R. A., and Williams, R. L., Improved liquid-chromatography of aspirin, salicylate, and salicyluric acid in plasma, with a modification for determining aspirin metabolites in urine, Clin. Chem., 28, 1200, 1982. Bakar, S. K. and Niazi, S., High-performance liquid chromatographic determination of aspirin and its metabolites in plasma and urine, J. Pharm. Sci., 72, 1020, 1983. Reidl, U., Determination of acetylsalicylic and metabolites in biological fluids by high-performance liquid chromatography, J. Chromatogr., 272, 325, 1983. O’Kruk, R. J ., Adams, M. A., and Philip, R. B., Rapid and sensitive determination of acetylsalicylic acid and its metabolites using reversed-phase highperformance liquid chromatography, J. Chromatogr., 310, 343, 1984. Mays, D. C ., Sharp, D. E., Beach, C. A., Kershaw, R. A., Bianchine, J. R., and Gerber, N., Improved method for the determination of aspirin and its metabolites in biological fluids by high-performance liquid chromatography: applications to human and animal studies, J. Chromatogr., 311, 301, 1984. Fu, C. J ., Melethil, S., and Mason, W. D., Method for determination of aspirin and salicylic acid in rat whole blood by high pressure liquid chromatography, Anal. Lett., 18, 269, 1985. Klitgaard, N. A., Determination of acetylsalicylic acid in plasma by thin-layer chromatography, Arch. Pharm. Chem. Sci. Ed., 7, 128, 1979.

Specimen (ml)

8.

7.

6.

4. 5.

3.

2.

1.

REFERENCE

Volume III 21

20 x 5

Spherisorb-ODS (10)*

Nucleosil C18 (7)f UltrasphereODS (5)

Supelcosil LC-8 (5)e

Packing (μπι)

E-5

E-4

E-3

E-2

Elution

2.0

1.0

1.6

0.75

Flow (ml/min)

ABS (215)

FI (340, 418700) ABS (470) ABS (360)

Det. (nm)

2.7

7.8

8

18

RT (min)

Other compounds (RT)

A-iV-Diacetylcysteine (8.3) /V-S-Diacetylcysteine (7.6)

—

— N-Acetylpencillamine (13.3) L-Tyrosine (2.4)

_

_

Internal standard (RT)

5

4

1

3

Ref.

Extraction — 1-1. The sample (0.5 m l) is mixed with 20 μ ΐ of a solution of N-acetyl-D-cysteine (250 nmol/ml) in distilled water, one drop of ethanethiol and allowed to stand for 5 min. Acetone (1 m l) is added, centrifuged, supernatant collected, adjusted to pH 9 with a drop of aqueous ammonia (25%), and washed with hexane (1 ml). The aqueous phase is then concentrated to about half of its initial volume with a stream of nitrogen and under moderate heating, which is then applied to a 40 x 5 mm Dowex I-X8, OH column (50/100 mesh). After 10 min the column is washed with 4 m l of water, 5 m l of HC1 (0.1 N .) The last 1 m l containing the N-acetylcysteine is collected and dried in a vacuum centrifuge. Diazomethane in 0.9 m l of diethylether, 0.1 m l of methanol and a drop of ethanethiol are added to the residue, the mixture sonicated for 2 min and allowed to stand at room temp for 30 min. The solvent is removed with a stream of nitrogen and the residue dissolved in 10 μ ΐ of toluene, 0.1 to 1 μ ΐ is injected.

a Initial temp = 60°C, hold time = 0.7 min, rate = 40°C/min, to 145°C, 50°C/min to 175°C, 40°C/min to 200°C. Hold time = 6 min. b Inlet pressure = 100 k Pa. c Applied voltage = 1.0 V. Derivatives with N-(4-anilinophenyl) maleimide were prepared prior to chromatography. e The injector loop was replaced by 30 x 4.6 mm RP-8 column. After injection, the precolumn in load position was flushed backward with the mobile phase. f Column temp = 30°C. * Column temp = 60°C.

Dilution

25 x 4.6

1-4

Dosage

30 x 4.6

1-3

Serum, urine (0.25) Plasma (1.5)

25 x 4.6

1-2

Extraction

Column (cm x mm)

Plasma (1)

Specimen (m l)

Liquid Chromatography

N-ACETYLCYSTEINE (continued)

22 CRC Handbook of Chromatography: Drugs

1. Frank, H ., Thiel, D., and Langer, K., Determination of A-acetyl-L-cysteine in biological fluids, J. Chromatogr., 309, 261, 1984. 2. Shimada, K., Tanaka, M., and Nambara, T., Sensitive derivatization reagents for thiol compounds in high-performance liquid chromatography with electro­ chemical detection, Anal. Chim. Acta, 147, 375, 1983. 3. Kagedal, B., Kallberg, M., and Martensson, J ., Determination of non-protein-bound N-acetylcysteine in plasma by high-performance liquid chromatography, J. Chromatogr., 311, 170, 1984. 4. Lewis, P. A., Woodward, A. J ., and Maddock, J ., Improved method for the determination of N-acetylcysteine in human plasma by high-performance liquid chromatography, J. Chromatogr., 327, 261, 1985. 5. Farquhar, J ., Finlay, G., Ford, P. A., and Martin-Smith, M., A reversed-phase high-performance liquid chromatography assay for the determination of Nacetylcysteine in aqueous formulations, J. Pharm. Biomed. Anal., 3, 279, 1985.

REFERENCE

Elution — E-l. Acetonitrile-0.5% ammonium dihydrogen phosphate, pH 3.0 (4:7). E-2. Sodium phosphate buffer pH 7.4-methanol (70:30) containing tetramethylammonium ion (10 mmol/€). E-3. Aqueous disodium hydrogen phosphate (0.5%) acetonitrile (70:30). E-4. Methanol-0.067 M phosphate buffer, pH 8 containing 0.005 M tetrabutylammonium hydrogen sulphate. (50:50). E-5. 0.5% Aqueous sodium perchlorate (m/v), adjusted to pH 2 with phosphoric acid.

1-2. A l-m€ sample was treated with 0.2 m€ of 20% trichloroacetic acid. After vortex mixing and standing at room temp for 15 min, the samples were centrifuged, 0.6 m€ of supernatant was transferred to new tubes and mixed with 0.4 m€ of 20 mmol/€ Na2 EDTA and 1 m€ of sodium borate buffer, pH 10. Then 1 m€ of thiopropyl-sepharose 6 B suspension (20 μπιο1/πι€) was added and the tubes were placed in an end-over-end mixter for 30 min. Then, 1 m€ of 4 N acetic acid was added, the tubes mixed and centrifuged. The supernatant (3 m€) was transferred to a 1.3 x 0.7 cm column packed with p-acetoxymercurianiline-Sepharose 4B. The column was washed with 2 x 1 m€ water and eluted with 3 m€ of 10 mmol/€ cysteine solution. The eluate was transferred to a 2.5 x 0.5 cm column packed with AG 50W-X8 (H+) column. The effluent was collected in a tube containing 0.2 m€ of 0.18 mol/€ Na2EDTA together with a further l-m€ washing with 10 N HC1. From the combined effluent, 2 m€ was transferred to a new tube, 0.2 m€ of 0.1 N NaOH, 3 m€ of 50 mM carbonate buffer, pH 9 containing 10 mM ii Na2EDTA, and 0.5 m€ of 20 μπιο1/€ of N-(7-dimethylamino-4-methyl-3-coumarinyl) maleimide were added. The tubes were left overnight in a 37°C water bath. 1-3. To 250 μ€ of urine or deproteinized serum 10 μ€ of a solution of dithioerythritol (145 mg in 10 m€ water) and 10 μ€ of phosphate buffer pH 8 are added and allowed to stand at room temp for 30 min. An aliquot of 250 μ€ of this mixture is treated with 250 μ€ of EDTA-citrate buffer, pH 8.3 and 50 μ€ solution of NBDC1 (1 mg/m€ of methanol). The mixture is allowed to stand for 20 min and 20 μ€ are injected. 1-4. To the sample 100 μ€ of N-acetylpenicillamine (60 mg/100 m€ of 1% sodium bicarbonate), and 125 μ€ of 10 mg/m€ of dithiothreitol solution were added. The tubes were vortex mixed and placed in a water bath at 37°C for 30 min. Then to each tube 1 m€ of 1% sodium bicarbonate and 180 μ€ of 10% dinitrofluorobenzene in methanol were added. The tubes were vortex mixed and placed in a water bath at 60°C for 30 min. A 400 μ€ aliquot of the ultrafiltrate of the reaction mixture was acidified with 3 m€ of 0.2 M HC1 and extracted with diethyl ether (8 m€). The ether layer was collected and back extracted into 2 m€ of 1% sodium bicarbonate and ether layer discarded. The aqueous layer was made acidic and re-extracted with diethyl ether. The organic layer was collected and evaporated in a current of nitrogen at room temp. The residue was reconstituted in 100 μ€ of the mobile phase and 20 μ€ were injected.

Volume III 23

1-1

25 x 0.53

Column Extraction (m x mm) 5% Phenylmethyl silicone (5-μπι)*

Packing (mesh) 270

Oven temp (°C) Heb

Gas (ml/min) MS-CP

Det. 2.9

RT (min) Bromoacivicin (3.9)

Internal standard (RT) N- Isobutyloxycarbonyl, methyl ester

Deriv. —

Other compounds (RT)

1

Ref.

1. McGovren, J. P., Williams, M. G., Robins , R. H., and Roach, B. L., Quantitation of antitumor agent acivicin in plasma by gas chromatography/mass spectrometry, Anal. Chem., 57, 2046, 1985.

REFERENCE

Extraction — 1-1. The sample, 5 μg of the internal standard in 0.1 m l of water, and 0.05 m l of 85% phosphoric acid were mixed gently with 0.2 m l of 12% perchloric acid. After centrifugation, the supernatant was neutralized with 0.3 m l of 10% sodium carbonate, isobutyl chloroformate (0.5 m l) added and the mixture incubated at 50°C for 60 min. Excess isobutyl chloroformate was removed with two 1-ml ethyl ether washes. The aqueous phase was acidified with 0.3 m l of 10% phosphoric acid, saturated with sodium chloride and extracted twice with 2-ml portions of ethyl ether. The combined ether extracts were evaporated in a stream of nitrogen. The residue was treated with 0.5 to 1 m l quantity of freshly prepared diazomethane. When the yellow color faded, the mixture was evaporated, reconstituted in 0.1 m l of dichloromethane. An aliquot of 1 μ ΐ was injected.

a Film thickness. b Pressure drop across the column = 10 psi. c Reagent gas = friethane to yield a pressure of 0.8 torr.

Plasma (1)

Specimen (ml)

Gas Chromatography

ACIVICIN

24 CRC Handbook of Chromatography: Drugs

1-1

Extraction

30 x 4

Column (cm x mm) μ -Bondapak-Cu ( 10)

Packing (μιη) E-l

Elution 1.5

Flow (m€/min) ABS (254)

Det. (nm) 5.9

RT (min) Daunoru­ bicin (4)

Internal standard (RT) __

Other compounds (RT)

1

Ref.

Plasma* (0.5) Plasma (0.25) Plasma ( 1)

Specimen (m€)

25 x 4.6

15 x 4.2

30 x 3.8

1-1

1-2

1-3

Extraction

Column (cm x mm) Zorbax-ODSb (5) PRP-1C (10) μ-Bondapak-C l8 (10)

(μπι)

Packing

1.5 1.0

E-3

1.2

E-2

E-l

Elution

Flow (ml/min)

Liquid Chromatography

ACYCLOVIR

ABS (254) ABS (254) ABS (254)

Det. (nm)

5.5

3

6

RT (min)

Acetami­ nophen (6.5)

—

—

_

_ —

Other compounds (RT) Internal standard (RT)

1. Erttmann, R ., Determination of aclacinomycin A by reversed-phase high-performance liquid chromatography, J. Chromatogr., 277, 433, 1983.

REFERENCE

Elution — E-l. Acetonitrile-methanol-water (40:90:70) +1.5 m€/100 of 85% orthophosphoric acid.

3

2

1

Ref.

Extraction — 1[-1. The sample is spiked with 750 ng of daunorubicin and treated with an equal volume of 300 mM trichloroacetic acid. After centrifugation, the supernatant is applied to Sep-Pak C 18 cartridge which was then washed with 5 m€ water, 5 m€ 25% methanol, eluted with 0.7 m€ of acetonitrile-methanol (1:1), and 2 0 0 μ€ aliquots of the eluate were injected.

Plasma (10)

Specimen (m€)

Liquid Chromatography

ACLACINOMYCIN

Volume III 25

Plasma, urine (4)

Specimen (ml)

1-1

Extraction

20 x 4.6

Column (cm x mm) Silica-C2 (5)

Packing (μπι)

E-l

Elution

2.0

Flow (ml/min)

Liquid Chromatography

AD-022

ABS (220)

Det. (nm)

14.5

RT (min)

—

Internal standard (RT)

—

Other compounds (RT)

1

Ref.

1. Land, G. and Bye, A., Simple high-performance liquid chromatography method for the analysis of 9-(2-Hydroxyethoxymethyl)guanine (acyclovir) in human plasma and urine, J. Chromatogr., 224, 51, 1981. 2. Smith, R. L. and Walker, D. D., High-performance liquid chromatographic determination of acyclovir in serum, J. Chromatogr., 343, 203, 1985. 3. Bouquet, S., Regnier, B., Quehen, S., Brisson, A. M ., Courtois, Ph., and Fourtillan, J. B., Rapid determination of acyclovir in plasma by reversed-phase high-performance liquid chromatography, J. Liq. Chromatogr., 8 , 1663, 1985.

REFERENCE

Elution — E-l. 0.0025 M Heptane sulfonic acid in 0.005 M sodium acetate adjusted to pH 6.5. E-2. Methanol-0.1 M HC1-0.02 M sodium heptane sulfonate-0.25 M NaCl (10:10:20:60). E-3. Acetonitrile-0.01 M phosphate buffer, pH 2.3 (0.025:99.975) containing 90 mg heptane sulfonic acid.

Extraction — 1-1. The sample (0.5 ml) was treated with 200 μ ΐ of 5% W/V aluminum sulfate solution and vortex mixed; 400 μ ΐ of 0.15 M barium hydroxide was added and mixed. The tubes were allowed to stand overnight at 4°C to complete precipitation, were centrifuged (5000 g, 20 min, 10°C). The supernatant was collected and 60 μ ΐ were injected with an autosampler. 1-2. The sample (0.25 m l) was treated with 200 μ ΐ of mobile phase and 50 μ ΐ of trifluoroacetic acid, mixed and centrifuged. A 200-μ € aliquot of the supernatant was neutralized with an equal fraction of 0.1 M NaOH and 100 μ ΐ were injected. 1-3. To the sample (1 m l), 100 μ ΐ of an aqueous internal standard solution (200 μg/m€) and 100 μ ΐ 0.1 M HC1 saturated with NaCl were added. The mixture was mixed by slow rotation, 2 m l of acetonitrile was added, vortex mixed (1 min), and centrifuged at 2000 g for 10 min. The supernatant (2.5 m l) was washed with 6 m l of dichloromethane. An aliquot (30 μ ΐ) of the aqueous layer was injected (loop = 20 μ£).

* Modified conditions are described for the analysis of urine. b Column temp = 50°C. c Polystyrene divinylbenzene resin column from Hamilton Co.

ACYCLOVIR (continued)

26 CRC Handbook of Chromatography: Drugs

1-1

Extraction

20 x cm (Merck)

Plate (Manufacturer) Silica gel G (0.25)

Layer (mm) S-l

Solvent Remission (510)

Det. (nm)

0.6

Rf

—

Internal standard (Rf)

a Methanol (40 m€) is carefully added to a mixture of 30 m€ 65% nitric acid and 10 m€ 95 to 97% sulfuric acid. b To 20 m€ of methanol, 4 m i of 15% titanium (ΠΙ) chloride solution in 4% HC1 are added. Prepared just before use. c Sodium nitrite (400 mg) is dissolved in 20 m€ of 1 N HC1. Prepared just before use. d N-(1-Naphthyl)ethylenediamine dihydrochloride (1 g) is dissolved in 100 m€ of methanol. c Sodium nitrite (400 mg) is dissolved in 20 m€ of 1 N HC1. Prepared just before use. d N-(1-Naphthyl)ethylenediamine dihydrochloride (1 g) is dissolved in 100 m i of methanol.

Post-separation treatment Sp: (1) Sulfonitric acid* (2) Titanium chloride** (3) Sodium nitritec (4) N-(l-Naphthyl) ethvlene-diamined

Plasma (1)

Specimen (m€)

Thin-Layer Chromatography

ADAMEXINA

—

Other compounds (Rf)

1

Ref.

1. Rosenblum, M. G., Hersh, E. M., and Loo, T. L., Determination of the biological response modifier MVE-2 (AD-022) in biological fluids by high-performance liquid chromatography, J. Chromatogr., 272, 200, 1983.

REFERENCE

Elution — E -l. (A) 0.02 M Phosphate buffer, pH 4. (B) 0.02 M phosphate buffer in 80% methanol. Nonlinear gradient (curve 7) from 0% (B) to 100% (B) over 25 min.

Extraction — 1-1. The sample (4 m€) is rapidly mixed with a 400 μ€ of 0.2 N NaOH and 4 m€ of hot water (85°C), allowed to stand for 5 min and applied to a column made from 1 m€ pipette tips packed with macroporous anion exchange resin (AGMP-1), 100/120 mesh. The columns were washed with 3 m€ of water, adjusted to pH 11 with NaOH, then with 4 m€ of distilled water, 2 m€ of 1 M formic acid and finally with 3 m€ of 8.5 M formic acid. The column is then eluted with 2.5 m€ of 14 M formic acid and the eluate was applied to another column packed with Bio-Rex 70 cation exchange resin (bed size 2 x 0.5 cm, 100/200 mesh). The samples were washed through with 0.5 m€ of distilled water, 50 μ€ of 0.1 N NaOH added to the combined eluate and lypholized to dryness. The samples were reconstituted with 250 μ€ of mobile phase A.

Volume III 27

1-1

Extraction

30 x 4

Column (cm x mm) μ-Bondapak C 18 (10)

Packing (pm) E-l

Elution 2.0

Flow (ml/min) ABS (254)

Det. (nm)

8-Chloro-l-ethyl-4{(dimethylamino)methyl-6-phenyl-4H-s-triazolo [4,3-a][l,4]} benzodiazepine.

Plasma (1)

Specimen (ml)

Liquid Chromatography

ADINAZOLAM

5.6

RT (min)

a (8 )

Internal standard (RT)

N-Demethyladinazolam (4.4)

Other compounds (RT)

1

Ref.

1. Peng, G. W., Assay of adinazolam in plasma by liquid chromatography, J. Pharm. Sci., 73, 1173, 1984.

REFERENCE

Elution — E-l. Acetonitrile-methanol-phosphate buffer (0.006 M monobasic ammonium phosphate + 0.02% phosphoric acid) (30:20:85)

Extraction — 1-1. After the addition of internal standard, the sample (1 ml) was made alkaline with 1 m l of 0.1 M dibasic potassium phosphate and extracted with 5 m l of ethyl acetate. The aqueous phase was frozen in a dry-ice bath, the organic layer was transferred to new tubes and evaporated under a stream of nitrogen. The residue was dissolved in 2 m l aliquots of toleune which was washed with 2 m l of 2 M NaOH. The toluene layer was evaporated under a stream of nitrogen at a temperature 50°C. The residue was dissolved in 0.1 m l of acetonitrile-methanol-water (1:1:2), aliquots of which were injected.

a

REFERENCE

1. Albet, C., Sanchez, M. G ., and Colome, J., Determination of a new drug Adamexina in biological liquids by photodensitometry, J. Chromatogr., 181, 504, 1980.

Solvent — S-l. Ethyl acetate-acetic acid-water (60:15:15).

Extraction — 1-1. The sample was diluted with 4 m l of water and extracted with 10 m l of chloroform. The organic layer was dried over anhydrous sodium sulfate and evaporated. The residue was dissolved in 2 m l of 10% HC1, washed with 1 m l of diethyl ether, made basic with 1 N KOH (pH 9) and extracted with 5 m l of chlorofo m. The organic phase was dried over anhydrous sodium sulfate and evaporated. The residue was dissolved in 50 μ ΐ of ethanol and 25 μ ΐ were spotted.

ADAMEXINA (continued)

28 CRC Handbook of Chromatography: Drugs

a

Tissue

30 x 4.7

Column (cm x mm) LiChrosorbRP-18 (10)b

(pm)

Packing

E-l; grad­ ient

Elution 2.0

Flow (ml/min) ABS (254)

Det. (nm) 37

RT (min)

Internal standard (RT)

6

1

Ref.

min; 5 min linear; to 30%

Diethylamino­ ethanol (25.8) Diphenylacetic acid (12.5)

Other compounds (RT)

Plasma (0 . 1 )

Specimen (ml)

1-1

Extraction

12.5 x 4

Column (cm x mm) LiChrosorb RP-8 (5)

Packing (pm) E-l

Elution

1.5

Flow (ml/min)

Liquid Chromatography

ADRIAMYCIN»

FI (470;565)

Det. (nm)

3

RT (min)

Daunorubicin (7.2)

Internal standard (RT)

Adriamycinol ( 1 .8 )

Other compounds (RT)

2

Ref.

1. Michelot, J., Moreau, M. F., and Madelmont, J. C., Determination of adiphenine, diphenylacetic acid and diethylaminoethanol by high-performance liquid chromatography, J. Chromatogr., 257, 395, 1983.

REFERENCE

Elution — E-l. (A) 0.027 M aqueous ammonia; (B) 0.027 M ammonia in methanol. Nonlinear gradient from 100% (A) to 70% (A) in (A) in 15 min.

a Tissue homogenate chromatographed directly. b Separation on an alternative normal phase silica column is also described.

Extraction

Specimen (ml)

Liquid Chromatography

ADIPHENINE

Volume III 29

1-2

—

1-3

Pure compounds

Plasma ( 1)

Extraction

Serum ( 1- 2 )

Specimen (m€) E-2

E-3C

E-4C

μ-BondapakC 18 ( 1 0 )

μ-BondapakC 18 ( 1 0 )

Cpspher C8 (8 )d

25 X 4.6

30 x 3.9

15 x 4.6

Elution

Packing (μη·)

Column (cm x mm)

1.0

1.0

2.5

Flow (ml/min)

Liquid Chromatography

FI (474;590)

ABS (NA)

FI (480;560)

Det. (nm)

ADRIAMYCIN" (continued)

6

5

7.3

RT (min) Daunorubicin (14.1)

Internal standard (RT) Adriamycinol (5) Adi^-aglycone (8.3) Adr-7-deoxyglycone (15.5) Adriamycinolaglycone (5.8) Adriamycinol (4.5) Daunomycinol (7) Daunomycin (8.5) N-Acetyldaunomycin (10) Adr-aglycone (14) 14-Bromodaunomycin (16) Daunomycinaglycone (2 1 ) Adriamy­ cinol (4)

Other compounds (RT)

6

5

3, 4

Ref.

30 CRC Handbook of Chromatography : Drugs

1-4

1-5

1-6

1-7

1-8

1-9

1-10

Plasma, pleural fluid (0.05—0.5)

Plasma ( 1)

Plasma ( 1)

Plasma ( 1)

Plasma (0.05—0.5)

Plasma, cell cultures ( 1 )

Cell suspension (0.25)

10 x 3

25 x 4.6

30 x 4

10 x 5

10 x 8

30 x 4

25 x 4.6

Silica gel SI-60 (7)

SpherisorbODS (5) 1.5

0.8

E-l 1

2.0

1.0

2.8

1.2

1.5

E-10

E-9

E-8

Hypersil-ODS (5)

μ -BondapakPhenyl (10)h

E-7

E- 6

E-5

Radial-PakPhenyl (10)

μ-BondapakPhenyl (10)f

Zorbax BP-SIL (5)

FI (488, 550)

2

9

3.9

Flg (480, 560)

FI (480, 580)

5

3.4

10.3

5

FI (450, 550)

FI (478)

Flg (480;560)

FI (480;590)

4-epiadriamycin (11) Daunomycin (1.4)

Daunorubicin (7.2)

Daunorubicin (20.1) 4-Demethoxyadriamycin (7.2) Daunorubicin (11)

Daunorubicin (3.5)

Adriamycinone (3) Adriamycinol (4) Adriamycinol (2.8) Adr-aglycone (5.5) Adr-7-deoxyaglycone (8.7) Adriamycinone (4.5) Adriamycinol (6.7) Adriamycinol (2.3) 4-epiadriamycin (1-5)' 4-Demethoxydaunomycin (1.4)

Adriamycinone (2) Adriamycinal (7) Adriamycinal (6 .6 )

14

12, 13

11

10

9

8

7

Volume III 31

H

3

P

O

4

.

Extraction — 1-1. the sample was mixed with 10 μ€ of an aqueous solution of the internal standard and 100 μ€ of borate buffer (pH 9) containing 10 mg/€ desipramine HC1. The mixture was extracted with 0.5 m€ of a chloroform- 1-heptanol (1:1). The organic layer was back extracted into 100 μ€ of 0.2 M phosphoric acid. After centrifugation 10 to 90 μ€ of the aqueous phase was injected. 1-2. The sample was spiked with 100 ng of the internal standard in 10 μ€ of methanol and extracted with 5 volumes of chloroform-propan-2-ol (2:1). The organic layer was evaporated at 40°C under reduced pressure. The residue was dissolved in 50 μ€ of methanol and 20 μ ί were injected. 1-3. The sample was mixed with 1 mf of borate buffer, pH 8.9 and extracted with 5 m€ of chloroform-isopropanol (4:1). The organic layer is back extracted into 1 After centrifugation 1 m€ of the aqueous layer was injected on the concentration column. A solution of desipramine HC1 (10 μg/m€) was used m€ of 0.1 Μ to transfer adriamycin from the concentration to the analytical column. 1-4. The sample; 40 to 100 μ€ of 0.1 M phosphate buffer, pH 7.8; and 20 μ€ of a 5 μg/m€ solution of the internal standard was poured onto the top of an Extrelut column. The column was eluted with a mixture of chloroform-methanol (10:1, 20 to 30 m€). The eluate was evaporated under vacuum below 35°C and the residue was dissolved in 100 to 200 μ€ of the mobile phase. Aliquots of 20 μ€ of this solution were injected. 1-5. The sample was spiked with 100 μ€ of normal plasma containing 5 ng of the internal standard and passed through a prewashed (2 m€ mobile phase, 4 m€ water) 200 μ€ cartridge packed with 50 mg of Bondapak-Phenyl. The draining was collected and repassed through the cartridge. The cartridge was washed with 4 m€ of water, dried, and eluted with 300 μ€ of the mobile phase containing 30% acetonitrile. The entire eluate was injected. 1-6. The sample was spiked with the internal standard colution and applied to a prewashed (10 m€ methanol, 10 m€ water) SepPak cartridge. After the sample had passed through, the cartridge was washed with 15 m€ of water, dried and eluted with 2 m€ of methanol. The eluate was dried under a gentle stream of nitrogen. The residue was dissolved in 100 μ€ of the mobile phase. Aliquots of 25 to 50 μ€ of this solution were injected with an autosampler. 1-7. To the sample was added 100 μ£ of the internal standard solution (200 ng in dichloromethane). The sample was then mixed with 1 m€ of 10 mM phosphatebuffered saline (pH 7.8) and extracted with 10 m€ of dichloromethane-isopropanol (4:1). The organic layer was evaporated at 50°C under a gentle stream of nitrogen. The residue was dissolved in 100 μ€ of the mobile phase and a 20 μ€ aliquot of this solution was injected. 1-8. The sample was mixed well with 20 μ€ (25 ng) of the internal standard solution and was loaded directly on the precolumn (2.3 x 0.39 cm packed with Corasil Phenyl 37 to 50 μπι) replacing the loop of the injector. With the injector still in the load position, the loop column was washed with 0.5 m€ of water and then the loop column was switched to the inject position. 1-9. The sample was spiked with the internal standard and treated with 2.5 m€ of ice cold isopropanol and 100 μ€ of buffer solution (1 M ammonium formate, pH 8.5 prepared just prior to use). After centrifugation the supernatant was extracted with a 5 m€ volume of ice cold chloroform. The organic layer was evaporated to dryness. The residue was dissolved in 100 μ€ of methanol and 50 μ€ were injected.

Assay of anticancer drugs has recently been reviewed. 1 Adr = adriamycin. Performances of different types of columns and of the same column with different mobile phases are compared. Injector loop is replaced with a 5 x 0.3 cm column filled with LiChrosorb RP-8 , 10 μπι. Different compositions are used for the assay of other anthracyclines. Protected by a 5 x 0.4 cm guard column packed with Corasil-Phenyl, 37 to 50 μπι. 8 An electrochemical detector (V = 0.7) was also used. h Protected by a 2.3 x 0.39 cm guard column packed with Corasil-Phenyl, 37 to 50 μπι. 1 Other anthracyclines have been assayed using similar extraction and chromatographic conditions but different internal standards.

a b c d e f

ADRIAMYCIN* (continued)

32 CRC Handbook of Chromatography: Drugs

1. Eksborg, S. and Ehrsson, H., Review: drug level monitoring: cytostatics, J. Chromatogr., 340, 31, 1985. 2. Bots, A. Μ. B., Van Oort, W. J ., Noordhoek, J., Van Dyk, A., Klein, S. W., and Van Hoesel, Q. G. C. M., Analysis of adriamycin and adriamycinol in micro volumes of rat plasma, J. Chromatogr., 272, 421, 1983. 3. Cummings, J ., Stuart, J. F. B., and Caiman, K. C., Determination of adriamycin, adriamycinol and their 7-deoxyaglycones in human serum by highperformance liquid chromatography, J. Chromatogr., 311, 125, 1984. 4. Cummings, J . and Willmott, N., Adriamycin-loaded albumin microspheres: qualitative assessment of drug incorporation and in vitro release by high-performance liquid chromatography and high-speed multidiode array spectrophotometric detection, J. Chromatogr., 343, 208, 1985. 5. Scourides, P. A., Brownlee, R. T. C., Phillips, D. R., and Reiss, J. A., Application of analytical and semi-preparative high-performance liquid chromatography to anthracyclines and fo's-anthracycline derivatives, J. Chromatogr., 288, 127, 1984. 6 . Oosterbaan, M. J. M., Dirks, R. J. M., Vree, T. B., and Van Der Kleijn, E., Rapid quantitative determination of seven anthracyclines and their hydroxy metabolites in body fluids, J. Chromatogr., 306, 323, 1984. 7. Ichiba, H., Morishita, M., Yajima, T., Kawatsu, Y., Kawamura, M., Komatsu, H., and Ikegami, T., Determination of adriamycin and its fluorescent metabolites in biological fluids of inpatients with lung cancer by high-performance liquid chromatography, Chem. Pharm. Bull., 33, 3868, 1985. 8 . Kotake, A. N., Vogelzang, N. J ., Larson, R. A., and Choporis, N., New high-performance liquid chromatographic assay for plasma doxorubicin, J. Chromatogr., 337, 194, 1985. 9. El-Yazigi, A. and Al-Saleh, I., Rapid analysis of doxorubicin in plasma by radial compression liquid chromatography, J. Pharm. Sci., 74, 1225, 1985. 10. Watson, I. D., Stewart, M. J ., and Farid, Y. Y. Z., The effect of surfactants on the high-performance liquid chromatography of anthracyclines, J. Pharm. Biomed. Anal., 3, 555, 1985. 11. Riley, C. A., Crom, W. R., and Evans, W. E., Loop-column extraction and liquid chromatographic analysis of doxorubicin and three metabolites in plasma, Ther. Drug Monit., 7, 455, 1985.

REFERENCE

Elution — E-l. Acetonitrile-water-0.1 M H3P 0 4 (31:61:8) containing 10 μg/m€ desipramine HC1 (pH 2.3). E-2. Acetonitrile-methanol-propan-2-ol-0.5 mM H3P 0 4 (12.5:12.5:12.5:62.5). E-3. Methanol-water (65:35) with 10 mM tetrabutylammonium phosphate. E-4. Acetonitrile-0.1 M citrate buffer, pH 2.2, water (30:10:60). E-5. Dichloromethane-methanol, acetic acid, 0.01 M magnesium chloride (200:50:7:5). E-6 . Acetonitrile-acetic acid-water (27:1:72), adjusted to pH 4.3 with a 20% (W/V) sodium acetate solution. E-7. Acetonitrile-methanol- 1 mM tetrabutylammonium phosphate (8.6:36.4:55). E-8 . Acetonitrile-10 mM phosphoric acid (35:65) containing 6 mM Brij. E-9. Acetonitrile-water-acetic acid (69:30:1) adjusted to pH 4 with 20% solution of sodium acetate. E-10. Acetonitrile-water (48:42) containing 20 mM phosphoric acid and 10 vaM sodium dodecyl sulfate. E -ll. Chloroform-methanol-acetic acid-water (72:21:4:3) containing 0.3 mM magnesium chloride and 10 μg/m€ desipramine.

I-10. The cell suspension was sonicated, supplemented with 100 μ€ of 1 M Tris buffer (pH 8 .8 ) containing the appropriate internal standard) and extracted twice with 3 m€ portions of chloroform-methanol (9:1). The combined organic extract was evaporated at 35°C in a current air. The residue was dissolved in 750 μ€ of chloroform-methanol (9:1). After addition of 50 μ€ of Tris buffer, mixing and centrifugation, 500 μ€ of the orgnic phase were injected.

Volume III 33

1-1

Extraction

25 x 4.6

Column (cm x mm) Zorbax-CN (5)

Packing (pm) E-l

Elution 2.5

Flow (ml/min) FI (295, 375)

Det. (nm) 5.2

RT (min)

Quinine ethylcarbonate (14.5)

Internal standard (RT)

Quinidine (16.8)

Other compounds (RT)

1

Ref.

REFERENCE

1. Hori, R., Okumura, K., Inui, K. I., Yasuhara, M., Yamada, K., Sakurai, T., and Kawai, C., Quinidine-induced rise in aimaline plasma concentration, J. Pharm. Pharmacol, 36, 202, 1984.

Elution — E-l. Acetonitrile-2.5% acetic acid (44:56).

Extraction — To the sample (0.5 ml), 1 m l of 0.1 M glycine buffer, pH 10, and 5 m l of diethylether containing 0.3 μg/ml of quinine ethylcarbonate were added. The tubes were vortex mixed for 2 min and centrifuged (10 min) and the upper organic phase was transferred to tapered tubes. After the addition of 0.1 m l of 2 propanol, the organic phase was evaporated under a stream of nitrogen. The residue was reconstituted in 0.5 m l of 2% acetic acid and a 50-μ € aliquot was injected.

Plasma (0.5)

Specimen (ml)

Liquid Chromatography

AJMALINE

12. Van Lancker, M. A., Nelis, H. J. C. F., and De Leenheer, A. P., Reversed-phase ion-pair chromatography of anthracyclines, J. Chromatogr., 254, 45, 1983. 13. Van Lancker, M. A., Bellemans, L. A., and De Leenheer, A. P., Quantitative determination of low concentrations of adriamycin in plasma and cell cultures, using a volatile extraction buffer, J. Chromatogr., 374, 415, 1986. 14. Speth, P. A. J., Linssen, P. C. M., Boezeman, J. B. M., Wessels, J. M. C., and Haanen, C., Rapid quantitative determination of four anthracyclines and their main metabolites in human nucleated haematopoietic cells, J. Chromatogr., 377, 415, 1986.

ADRIAMYCIN* (continued)

34 CRC Handbook of Chromatography: Drugs

25 X 4

30 X 4

1-2

1-3

1-4

Plasma ( 1)

Serum, bile* ( 1)

Blood, plasma (0 . 1 )

μ-Bondapak C 18 (10) Partisil (5)

RP-8 (Merck) ( 1 0 )b

ODS-HYPERSIL (10)

(μπι)

Packing

ABS (225)

1.5

E-4

4.4

6 .6

8 .2

ABS (290)

ABS (254)

7.2

RT (min)

ABS (292)

1.5

2 .0

1.5

Det. (nm)

E-3

E-2; gradient

E-l

Elution

Flow (ml/min)

Meben­ dazole (7)

—

—

_

Internal standard (RT)

Sulfone meta­ bolite ( 1 1 ) Sulfoxide metabolite (15)

Oxfendazole (3) Thiabendazole (3.5) Cambendozole (4) Mbendazole (4.4) Oxibendazole (5) Fenbendozole (10) Parbendazole (11) Sulfoxidemetabolite (2 .6 ) Sulfone meta­ bolite (3.8) c

Other compounds (RT)

4

3

2

1

Ref.

Extraction — 1-1. To the sample (4 m€), 4 m€ of phosphate buffer, pH 7.4 and 20 m£ of ether were added. The tubes were mixed on a rotary mixer for 10 min and 16 m€ of ether layer was collected. The aqueous layer was extracted with another 20-m€ portion of ether, and 20 m€ or organic layer was collected, combined with the first extract and evaporated to dryness in a current of nitrogen. The residue was dissolved in 50 μ€ of methanol and 5 μ€ were injected. 1-2. The sample (1 m€) is diluted with 1 m€ of phosphate buffer, pH 7.4 and extracted with 20 m€ of deethyl ether. The organic layer is collected and the aqueous

* Ruminal fluid and abomassal fluid are also extracted in a similar manner. b Column temp = 30°C. c Sulfoxide metabolite of albendazole was analyzed separately on a normal phase column using the same serum extract.

25 x 4.6

10 x 8

1-1

Extraction

Column (cm x mm)

Plasma* (4)

Specimen (m€)

Liquid Chromatography

ALBENDAZOLE

Volume III 35

Plasma (2)

Specimen (ml)

1-1

1.8 x 2

Column Extraction (m x mm) 3%-OV-l Supelcoport (80/100)

Packing (mesh) 210

Oven temp (°C)

He (2 0 )

Gas (ml/min)

MS-CIa

Det.

Gas Chromatography

ALBUTEROL

1.8

RT (min)

[2H3]-Albuterol ( 1 .8 )

Internal standard (RT)

Trimethylsilyl

Deriv.

—

Other compounds (RT)

1

Ref.

1. Bogan, J. A. and Marriner, S., Analysis of benzimidazoles in body fluids by high-performance liquid chromatography, J. Pharm. Sci., 69, 422, 1980. 2. Delatour, P., Gamier, F., Benoit, E., and Longin, Ch., A correlation of toxicity of albendazole and oxfendazole with their free metabolites and bound residues, 7. Vet. Pharmacol. Ther., 7, 139, 1984. 3. Meulemans, A., Giovanangeli, M. D., Mohler, J., Vulpillat, M., Hay, J. M., and Saimot, A. G., High performance liquid chromatography of albendazole and its sulfoxide metabolite in human organs and fluids during hydatatidosis, J. Liq. Chromatogr., 7, 569, 1984. 4. Alvinerie, M. and Galtier, P., Simultaneous determination of albendazole and its principal metabolites in plasma by normal phase high-performance liquid chromatography, J. Pharm. Biomed. Anal., 2, 73, 1984.

REFERENCES

Elution — E-l. Methanol-0.05 M ammonium carbonate (65:35). E-2. (A) Acetonitrile; (B) 1% phosphoric acid. Gradient: From 80 to 40% (B) in 10 min. E-3. Methanol-water (65:35). E-4. Hexane-ethanol (445:55).

layer re-extracted with another 2 0 -m l aliquot of diethyl ether. The combined ether layer was evaporated at 45°C under a current of nitrogen, the residue dissolved in 0 .2 m l of dimethyl-formamide and 2 0 μ ΐ injected. 1-3. The sample (1 m l) was treated with 1 m l of phosphate buffer, pH 7.4 and extracted with 1 m l of ethyl acetate by mixing for 15 min on a rotary mixer. The organic layer (700 μ ΐ) was collected, evaporated, the residue dissolved in 100 μ ΐ of the mobile phase and 35 μ ΐ injected with an auto-injector. 1-4. To 100 μ ΐ of the sample, 10 μ ΐ of the working internal standard solution (0.2 μg of mebendazole) and 1 m l of ethyl acetate were added. After mixing for 15 min on a table-top shaker and centrifugation (2 min), the organic layer was collected and evaporated under a strem of nitrogen. The residue was dissolved in 50 μ ΐ of the mobile phase and all of which was injected.

ALBENDAZOLE (continued)

36 CRC Handbook of Chromatography: Drugs

25 x 4.6

1-5

1-6

1-7

Plasma (2 ) Urine® ( 1)

Extraction

7.5 x 4.6

1-4

x 2 0 cm (Merck)

10

x 1 0 cm (Merck)

10

Silica gel 60®

Kieselgel 60F 254c

Layer (mm)

25 X 4.6

1-3

Plate (Manu­ facturer)

LiChrosorb RP-2 (10) Zorbax-ODS (5) UltrasphereODS (3) Zorbax-Cg ( 1 0 )b

25 X 4.6

1-2

Extraction

Packing (μπι)

Column (cm x mm)

Urine (10)

Specimen (mi)

Plasma ( 1) Plasma ( 1) Serum (0.5) Plasma (0.5)

Specimen (m i)

0.5 1.0

E-3 E-4

S-2

S -ld

Solvent

—

Sp:2, 6 Dichloroquinonechlorimide (5 g/€ in ethanol); heat at 60°C for 5 min; E: ammonia vapors

Post-sepa­ ration treatment

Det. (nm)

f (650)

Visual

Det. (nm)

Electrochem FI (230, 309) Electrochem FI (273, 310)

Thin-Layer Chromatography

1.7

2 .0

E-2

E-l

Elution

Flow (ml/min)

Liquid Chromatography

0.3

0.67

Rf

—

—

Internal standard (Rf)

Fenoterol (13.5) Ethenzamide (19)

7 15

—

Bamethan (10)

Internal standard (RT)

6

5

RT (min)

—

Terbutaline (0.60)

Other compounds (Rf)

—

—

7

6

Ref.

5

4

3

2

__ —

Ref.

Other compounds (RT)

Volume III 37

Extraction — 1-1. To the sample (2 m€), 50 μ€ of ethanolic solution of the internal standard (50 ng/m€ of [2H3] albuterol) and 6 m€ of diethyl ether were added. After mixing and centrifugation, the organic layer was discarded, the aqueous phase diluted with 2 m€ of 0.05 M phosphate buffer, pH 7.6 and the mixture adjusted to pH 10.6 by the addition of 200 μ€ of cone, ammonium hydroxide. Sodium chloride (1 g) was added and the samples extracted twice with 5-m€ aliquots of ethyl acetate. The combined ethyl acetate layers were evaporated to dryness under a stream of nitrogen. The residue was treated with 50 μ€ of pyridine and 50 μ€ of Ν,Οfcw-(trimethylsilyl) acetamide. The mixture was heated at 80°C for 30 min and aliquots of 1 to 10 μ€ were injected. 1-2. The sample (1 m€) was spiked with 20 μ€ of the internal standard solution (banethan, 1 μg/m€ of water), was applied to washed (5 m€ methanol, 5 m€ water) Sep-Pak columns. The tubes containing sample were rinsed with 1 m€ water and the washings were also applied to the column. The drainings were discarded and the column eluted with 5 m€ of methanol-diethyl ether (25:75). The eluates were evaporated under nitrogen at 60°C. The residue was dissolved in 500 μ€ of 0.015 M phosphate buffer, pH 7.5 and 250 μ€ of this solution was applied to 50 x 4.6 mm column packed with Partisil SCX (10 \jM ). The column was washed with the above mentioned buffer for 5 min at 3 m€/min with an auxiliary pump. This column was then switched on line to the analytical column with a switching valve. 1-3. Plasma (1 m€) was buffered to pH 1.2 with 0.2 m€ of 0.42 M phosphate buffer and extracted with a solution of 0.1 M di(2-ethylhexyl) phosphate in chloroform (6 m€) by vortexing for 2 min. Chloroform layer was collected and back extracted with 500 μ^ of 0.5 M HC1. A 200-μ€ aliquot of the aqueous phase was injected. 1-4. The sample (0.5 m€) was diluted with 1 m€ of water, 15 μ€ of the internal standard solution (100 ng/m€ of fenoterol bromide in water) added and was applied to Sep-Pak C ,8 cartridge which was previously washed with 10 m€ of methanol and 10 m€ of water. The cartridge was washed with 2 m€ of water and eluted with 2 m€ of methanol — the first 2 drops of eluate being discarded. The rest of the eluate was evaporated to dryness at 40°C under a stream of nitrogen. The residue was treated with 70 μ€ of phosphate buffer containing Cl and heptane sulfonic acid (aqueous component of the mobile phase) and extracted with 300 μ€ of 0.05% solution of di(2-ethylhexyl phosphate in ethyl acetate). The organic layer was collected and washed with 40 μ€ of above mentioned phosphate buffer. The organic layer was collected and back extracted into 70 μ€ of 10 mAf HC1. The organic layer was discarded and the aqueous layer was washed with 150 μ€ of chloroform. A 40- to 60-μ€ aliquot of the aqueous phase was injected. 1-5. To the sample (0.5 m€) were added 2 m€ of 2.5 mM solium heptane-sulfonate and 200 μ€ of an aqueous solution of ethenzamide (2 mg/m€). The mixture was applied to prewashed (20 m€ of methanol-diethyl ether (1:1) and 20 m€ of water) Sep-Pak C 18 cartridge. The cartridge was washed twice with 2 m€ of water, eluted with 7 m€ methanol-diethyl ether (1:1) and the eluate was evaporated to dryness under vacuum at 20°C. The residue was dissolved in 200 μ€ of the mobile phase and a 60-μΙ aliquot of this solution was injected. 1-6. The sample (10 m€) was adjusted to pH 5.5 and applied to 1 x 10 cm plastic column packed with Amberlite CG-50 Type II ion exchange resin (acetate form). The column was washed with 5 m€ of water and 10 m€ of methanol. The columns were eluted with 1 mol/€ ammonia. First 3 m€ of the eluate was discarded and the next 3 m€ were collected and evaporated. The residue was dissolved in 25 μ€ of methanol and 5 μ€ of this solution was applied to the plate. 1-7. The sample (2 m€ of plasma) was applied to washed Sep-Pak C 18 cartridge (5 m€ methanol, 5 m€ water). The cartridge was washed with water (2 m€) and acetonitrile (2 m€) and was eluted with 1.5 m€ of methanol. The eluate was evaporated at 45°C under vacuum. The residue was dissolved in 0.2 m€ of water, 60

a Ammonia was used as the Cl reagent gas. b Column temp = 60°C. c High performance TLC plates. d Radiad development at a flow rate of 4 sec/μ ί. e Urine is extracted with Prep-1 automated sample processor with Type W cartridge. f Zeiss KM-3 densitometer with micro-optics using reflectance/20% transmission mode.

ALBUTEROL (continued)

38 CRC Handbook of Chromatography: Drugs

1. Weisberger, M ., Patrick, J. E ., and Powell, M. L., Quantitative analysis of albuterol in human plasma by combined gas chromatography chemical ionization mass spectometry, Biomed. Mass Spectrom., 10, 556, 1983. 2. Oosterhuis, B. and Van Boxtel, C. J., Determination of salbutamol in human plasma with bimodal high-performance liquid chromatography and a rotated disc amperometric detector, J. Chromatogr., 232, 327, 1982. 3. Hutchings, M. J., Determination of salbutamol in plasma by high-performance liquid chromatography with fluorescence detection, J. Chromatogr., 277, 423, 1983. 4. Tan, Y. K. and Soldin, S. J., Determination of salbutamol in human serum by reversed-phase high-performance liquid chromatography with amperometric detection, J. Chromatogr., 311, 311, 1984. 5. Kurosawa, N., Morishima, S., Owada, E., and Ito, K., Reversed-phase high-performance liquid chromatographic determination of salbutamol in rabbit plasma, J. Chromatogr., 305, 485, 1984. 6 . Plavsic, F ., High-performance radial chromatography for semi-quantitative salbutamol and terbutaline determination in urine, Clin. Chem., 27, 771, 1981. 7. Colthup, P. V., Dallas, F. A. A., Saynor, D. A., Carey, P. F., Skidmore, L. F., and Martin, L. E., Determination of salbutamol in human plasma and urine by high-performance thin-layer chromatography, J. Chromatogr., 345, 111, 1985.

REFERENCES

Solvent — S-l. Ethanol-25% ammonium hydroxide (10:1). S-2. Ethylacetate-chloroform-methanol (60:40:1).

Elution — E -l. Sodium perchlorate (40 g/€)-2-propanol (45 m£/£) in phosphate buffer (pH 7) containing 14 g of Na2HP04, 2H20 and 7.5 g of KH2P 0 4 per liter. E-2. Acetonitrile-0.15% phosphoric acid (8:92). E-3. Methanol-70 mAf phosphate buffer, pH 6 . 8 , containing 1 mM chloride ions and 2 mM sodium 1-heptanesulfonic acid (25:75). E-4. Methanol-5.8 mAf phosphate buffer, pH 6.0 (31:69), containing 5 mAf sodium heptanesulfonate.

μ€ of aqueous sodium bicarbonate (5%) and 60 μ€ of aqueous A,./V-dimethyl-p-phenylenediamine (0.1%) were added. The mixture was vortex mixed 60 μ€ of 4(2-dimethylaminoethyl) phenol sulfate (0.2%) and 60 μ€ of aqueous potassium ferricyamide (8 %) were added. After vortex mixing, the reaction mixture was allowed to stand in the dark for 5 min at room temp and then extracted for 20 sec with 0.10 m€ of chloroform. The aqueous layer was discarded and 6 μ€ of the chloroform extract was applied to the plate.

Volume III 39

Column at 40°C. Oxidation potential = 1.0 V.

1-2

Plasma (0.25)

25 x 4.6

30 x 6.4

Column (cm x mm)

Brownlee RP-8 (10)

μ-Bondapak C 18 (10Y

Packing (pm)

E-2

E-l

Elution

1.5

1.5

Flow (ml/min)

Electrochemb

ABS (292)

Det. (nm)

12

10

RT (min)

1

2

—

—

Tubocurarine (7) —

Ref.

Other compounds (RT)

Internal standard (RT)

1. 2.

Parkin, J. E., Determination of alcuronium chloride in biological fluids by high-performance liquid chromatography, J. Chromatogr., 225, 240, 1981. Tovey, C., Bourne, D. W. A., and Schneider, J., Determination of alcuronium dichloride in plasma by high-performance liquid chromatography without solvent extraction, J. Chromatogr., 278, 216, 1983.

REFERENCE

Elution — E -l. Methanol-water (80:20) containing 0.25% acetic acid and 0.005 M sodium lauryl sulfate. E-2. Acetonitrile-0.001 M potassium dehydrogen orthophosphate-methanol (10:22:68).

Extraction — 1-1. To the sample (1 ml), 1 m l of 0.25 M disodium hydrogen phosphate, 2.0 m l of picrate reagent, 1.0 m l of tubocurarine internal standard (7.5 pg/€ for plasma and 50 pg/m€ for urine), and 4 m l of 15% isopropyl alcohol in dichloromethane were added. The tubes were mixed at 13 rpm for 5 hr. A 2-ml aliquot of organic phase was collected and evaporated in a stream of nitrogen. The residue was dissolved in 50 μ ΐ and the solution injected. The picrate reagent was prepared by dissolving 2.5 g of picric acid in 5.8 m l of (10%) of anhydrous sodium carbonate and the solution made up to 100 m l with water. The solution was washed with three equivalent volumes of 15% isopropyl alcohol in dichloromethane and the organic layers discarded. 1-2. To 250 μ ΐ of plasma were added 500 μ ΐ of acetonitrile. The solution was vortexed for 1 min, then centrifuged for 5 min at 2000 x g, and an aliquot of 40 μ ΐ of clear supernatant was injected.

a b

1-1

Extraction

Plasma, urine (1)

Specimen (ml)

Liquid Chromatography

ALCURONIUM CHLORIDE

40 CRC Handbook of Chromatography: Drugs

1-1

1x3

Column Extraction (m x mm) 3%-OV-17 Supelcorport (80/100)

Packing (mesh) 290

Oven temp (°C) N2 (35)

Gas (ml/min) NPD

Det. 2.7

RT (min) R 38 527 (3.6)

Internal standard (RT) —

Deriv.

Sufentanil ( 1 .6 )

Other compounds (RT)

1

Ref.

1-1

Extraction

30 x 3.9

Column (cm x mm) μ-Bondapak C 18 (10)

Packing (pm) E-l

Elution

Flow (ml/min)

ABS (2 1 0 )

Det. (nm)

4.3

RT (min)

STH 2199 (5.9)

Internal standard (RT)

—

Other compounds (RT)

1

Ref.

Extraction — I-1. To the sample (1 ml) were added, 5 μ ΐ of the internal standard (300 ng), 50 μ ΐ of 5 N NaOH, and 5 m l of diethyl ether. The samples were mixed for 25 min on a rotary mixer at 25 rpm and centrifuged at 100 x g for 10 min. The organic layer was collected and back extracted twice with 2 m l portions

Plasma ( 1)

Specimen (ml)

Liquid Chromatography

ALINIDINE

1. Woestenborghs, R., Mechielsen, L., and Heykants, J., Rapid and sensitive gas chromatographic method for the determination of alfentanil and sufentanil in biological samples, J. Chromatogr., 224, 122, 1981.

REFERENCE

Extraction — 1-1. To 1-ml aliquots of plasma, were added 0.5 μ§ of the internal standard (1 μ§/ιηΙ in methanol), 1 m l of 1 M sodium hydroxide, and 4 m l of nheptane-isoamyl alcohol (98.5:1.5). The tubes were rotated for 10 min at 25 rpm and centrifuged for 5 min at 1000 x g. The organic phase was collected and the aqueous phase was re-extracted with 4-ml aliquot of the extraction solvent. The combined organic layers were back extracted with 3 m l of 0.05 M sulfuric acid and the organic phase was discarded. The aqueous phase was made alkaline by the addition of 0.1 m l of concentrated ammonia and extracted twice with 2-ml aliquots of heptane-isomyl alcohol mixture. The combined organic layer was evaporated to dryness under nitrogen in a water bath at 60°C. The residue was reconstituted with 50 μ ΐ of methanol and 5-μΙ aliquots were injected.

Plasma ( 1)

Specimen (ml)

Gas Chromatography

ALFENTANIL

Volume III 41

1-1

Extraction

25 X 4

Column (cm x mm) LiChrosorbRP-18 (10)

Packing (pm) E-l

Elution 1.5

Flow (ml/min) FI (323, 370)

Det. (nm) 3.0

RT (nm)

Griseofulvin (3.5)

Internal standard (RT)

—

Other compounds (RT)

1

Ref.

1. Coulais, Y., Campistron, G., Caillard, C., and Houin, G., Quantitative determination of alizapride in human plasma by high-performance liquid chromatorgraphy, J. Chromatogr., 374, 425, 1986.

REFERENCE

Elution — E-l. Methanol-0.05 M monobasic potassium phosphate-triethylamine (77:20:1) adjusted to pH 7.6 with 2.6 M HC1.

Extraction — 1-1. To the sample (1 m l) were added, 50 μ ΐ of the internal standard, 1 m l of 0.2 M Tris buffer (pH 8.1), and 5 m l of chloroform. After vortex mixing and centrifugation, the organic layer was collected and evaporated under a stream of nitrogen. The residue was dissolved in 100 μ ΐ of the mobile phase and a 2 0 -μ Ι aliquot of the solution was injected.

Plasma (1)

Specimen (ml)

Liquid Chromatography

ALIZAPRIDE

1. Weigand, U. W., Meinertz, T., Kasper, W., and Jahnchen, E., Determination of alinidine in human plasma by high-performance liquid chromatography, J. Chromatogr., 223, 238, 1981.

REFERENCE

Elution — E-l. 50 mM sodium phosphate in methanol-water (50:50), adjusted to pH 5.0 with HC1.

of 0.01 N HC1. The combined aqueous phase was made alkaline with 50 μ€ of 5 A NaOH and extracted with chloroform. The aqueous layer was discarded and the organic layer evaporated in a current of nitrogen. The residue was dissolved in 25 μ€ of the mobile phase and a 20 μ ΐ of this solution was injected.

ALINIDINE (continued)

42 CRC Handbook of Chromatography: Drugs

1-1

15 x 4.6

Zorbax-ODS (5)b

E-l gradient

Elution 1.0

Flow (ml/min)

RT (min)

Det. (nm)

Internal standard (RT)

Other compounds (RT)

Ref.

1. Hirota, K., Kawase, M ., and Ohmori, S., High-performance liquid chromatographic method for the determination of plasma allantoin, J. Chromatogr., 277, 165, 1983.

REFERENCE

Elution — (A) 27% Acetonitrile; (B) 70% methanol. Mobile phase A is pumped for 12 min after injection and then mobile phase B for 18 min when the mobile phase was again changed to A. Next injection was made after equilibration with solvent A for 27 min.

Extraction — 1-1. The sample (1 m l) was mixed with methanol (1.5 m l) and was allowed to stand overnight in the refrigerator. After centrifugation the supernatant was collected. The precipitate was homogenized twice with 1 m l of methanol and the supernatant collected after centrifugation each time. The pooled three supernatants were evaporated to dryness. The residue was homogenized with methanol and the supernatant obtained after centrifugation was again evaporated to dryness. The residue was dissolved in 1 m l of water and washed with three 1-ml portions of ether-ethyl acetate (1:1). The aqueous solution was then passed through a Sep-Pak C 18 cartridge and the cartridge washed with water (0.5 ml) and eluted with 3 m l of methanol. The combined solution of the eluate and washings was evaporated to dryness. The residue was dissolved in 0.7 m l of 50% acetic acid and the solution cooled to 0°C in an ice bath. A solution (0.2 m l) of zanthydrol (25 mg/ml in dimethylformamide) were added and allowed to stand at 0°C for 1 hr and then evaporated to dryness. The residue was suspended in 100 μ ΐ of 50% dimethylformamide in water, diluted with 1 m l of mobile phase A, and allowed to stand in the refrigerator. After centrifugation, 50 μ ΐ of the supernatant was injected with an autosampler.

a Allantoin is the hydrolysis product of anti-ulcer drug aluminium dihydroxyallantoinate. b Column at 43°C. c One sample is analyzed per hour because of extraneous peaks.

Plasma (1)

Extraction

Packing (μπι) £ on

0d

Column (cm x mm) S?

m

Specimen (ml) 1

Liquid Chromatography

1

ALLANTOIN*

Volume III 43

Plasma (0.5)

Plasma (0.5) Plasma ( 1) Plasma (0.5)

Plasma (0.5) Plasma (0 .0 1 ) Plasma (0.5)

Specimen (m€)

Serum, urine (0.5, 0.1)

Specimen (m€)

22 x 4.6

15 x 4.6

1-7

1-8

25 x 4.6

1-6

30 x 3.9

1-4

30 x 3.9

30 x 4.1

1-3

1-5

25 x 3.2

Column (cm x mm)

15 x 2

1-2

Extraction

1-1

Column Extraction (m x mm)

Brownlee RP-18 (5)

μ-Βοη(&ρ3Κ-0,8 GO) Perkin Elmer RP-8 (10) Hypersil-ODS (3)

LiChrosorb-RP-18 GO) SAS-Hypersil (5) μ-Βοη(&ρ3ΐ^18 ( 1 0 )b

(μπι)

Packing

3% OV-17 GasChrom Q ( 100/ 1 20)

Packing (mesh)

Gas (ml/min) MS-EI

Det.

ABS (254)

1.5 1.5

1.3

E-5 E-6

E-7

ABS (254)

ABS (254) c

1.0

E-4

ABS (254) ABS (252) ABS (254)

2.5

2 .0

2 .0

Det. (nm)

3.2

RT (min)

E-3

E-2

E-l

Elution

Flow (ml/min)

Liquid Chromatography

T.P.a He (2 0 )

Oven temp (°C)

Gas Chromatography

ALLOPURINOL

13

8

7

18.8

7.2

10

8

RT (min)

[7,9-15N] xanthine (5.5)

Internal standard (RT)

—

9-Methylxanthine (6.5)

—

—

Acetami­ nophen (18)

—

_

Internal standard (RT)

Butyl

Deriv.

Oxipurinol (9)

Oxipurinol (16) Oxipurinol (5.5) Oxipurinol (7.1)

Oxipurinol (6) Oxipurinol (7.8) Oxipurinol (6.7)

Other compounds (RT)

Oxipurinol (6) Hypoxanthine (5.2)

Other compounds (RT)

8

7

6

5

4

3

2

Ref.

1

Ref.

44 CRC Handbook of Chromatography: Drugs

45

REFERENCES

1. Lartigue-Mattei, C., Chabard, J. L., Bargnoux, H ., Petit, J., and Berger, J. A., Simultaneous determination of allopurinol and oxipurinol in biological fluids by mass fragmentography, J . Chromatogr., 229, 211, 1982. 2. Sved, S. and Wilson, D. L., Analysis of allopurinol and oxypurinol in plasma and its application to metabolic studies, Biopharm. Drug Dispos., 1, 111, 1980.

Elution — E-l. 0.01 M sodium heptanesulfonate, pH 5.9. E-2. 0.1 M citric acid-0.2 M disodium phosphate-water (19:81:200). E-3. 0.05 M phosphate buffer, pH 6.0. E-4. 4 mM sodium phosphate buffer, pH 6.0. E-5. 0.025 M phosphate buffer, pH 6.1 with 6 to 8 % methanol. E-6 . 0.02 M Potassium dihydrogen phosphate, pH 3.65 (adjusted with phosphoric acid). E-7. 0.02 M sodium acetate, pH 7.2.

Extraction — 1-1. Serum after the addition of internal standard ([7,9-15N]xanthine) was deproteinized by ultrafiltration in Amicon CF 25 centriflo cones and was then assayed as the urine samples (0.1 m€). The samples were extracted by shaking with 2 m€ of n-butanol at pH 4.2. The organic layer was evaporated at 50°C under a current of nitrogen. The residue was dissolved in 120 μ€ of 7V,7V-dimethylacetamide and 50 μ€ of tetrabutylammonium hydroxide. After vortex mixing for 10 sec, a 20-μ€ aliquot of butyl iodide was then added, vortexed for 1 min and allowed to stand for 15 min at room temp. After evaporation at 50°C under a stream of nitrogen the residue was dissolved in 2 0 μ€ of methanol and 2 -μ€ aliquots were injected. 1-2. The sample (0.5 m€) was treated with 0.2 m€ of 20% trichloroacetic acid, vortexed, and centrifuged for 10 min at 2000 rpm. A 50-μ€ aliquot was injected into a 2 -m€ loop. 1-3. Plasma is diluted 1:2 with the mobile phase and 20-μ€ aliquots are injected. 1-3. Plasma is diluted 1:2 with the mobile phase and 20-μ€ aliquots are injected. 1-4. A 75-μ€ volume of internal standard solution (acetaminophen in distilled water, 10 μg/m€) was added to 0.5 m€ of plasma and mixed. A 20-μ€ aliquot of this sample was injected directly onto the column. 1-5. Plasma (0.5 m€) was mixed with 0.4 m€ of water and then with 0.1 m€ of 20% perchloric acid in an ice bath. The mixture was centrifuged at 1300 x g at 4°C for 10 min. An aliquot of 0.5 m€ of the supernatant was combined with 0.5 m€ of 0.2 M disodium phosphate and 50 μ€ of the mixture was injected. 1-6. To 1 m€ of the sample were added in sequence: 0.1 m€ of 15% (W/W) zinc sulfate, 0.2 m€ of saturated barium hydroxide, 0.1 m€ of 0.5 mol/€ phosphate buffer pH 6.1, and 1 m€ of methanol. After each addition the sample was vortex-mixed and finally centrifuged at 1200 x g for 5 min. An aliquot of the supernatant was injected. 1-7. A 500-μ€ aliquot of plasma was spiked with the internal standard solution (12.5 μιηο1/€) and was deproteinized with 150 μ€ of 12% trichloroacetic acid. 1-8. An aliquot of 0.5 m€ of plasma with 100 μ€ of 20% trichloroacetic acid. The supernatant was neutralized with 50 μ€ of 1.22 M sodium hydroxide and 50 μ€ of 1 M pH 4.5 acetate buffer.

a From 190 to 260°C at 10°C/min. b The column is protected by a 23 x 3.9 mm precolumn packed with Bondapak-C18 (37 to 50 μπι). c A PAR model 310 detector was used as polarographic detector.

Volume III

Extraction

1-2

Plasma (1)

1-1

30 x 4.6

Column (cm x mm)

2x3

Column Extraction (m x mm)

Specimen (m€)

Plasma ( 1)

Specimen (m€)

p-Bondapak-C18 GO)

Packing (pm)

3% OV-1 Gas Chrom Q (80/100)

Packing (mesh) N2 (30)

Gas (m€/min) FID

Det.

E-l

Elution

0.85

Flow (ml/min)

Liquid Chromatography

200

Oven temp (°C)

Gas Chromatography

ALMINOPROFEN

ABS (235)

Det. (nm)

5.5

RT (min)

Deriv.

8

RT (min)

Glafenic acid (5.6)

Internal Standard (RT)

2-(p-AllylTrimethylaminophenyl) silyl propionic acid (4)

Internal Standard (RT)

—

Other compounds (RT)

Other compounds (RT)

2

Ref.

1

Ref.

3. Breithaupt, H. and Goebel, G., Determination of allopurinol and oxipurinol in biological fluids by high-performance liquid chromatography, J. Chromatogr., 226, 237, 1981. 4. Nissen, P., Simultaneous determination of allopurinol, oxipurinol and uric acid in human plasma by high-performance liquid chromatograph, J. Chromatogr., 228, 382, 1982. 5. Miyazaki, H., Matsunaga, Y., Yoshida, K., Arakawa, S., and Hashimoto, M., Simultaneous determination of plasma and urinary uric acid, xanthine, hypoxantine, allopurinol, oxipurinol, orotic acid, orotidine and creatinine by high-performance liquid chromatography, J. Chromatogr., 274, 75, 1983. 6 . Palmisano, F., Desimoni, E., and Zambonin, P. G., High-performance liquid chromatography with polarographic and voltammetric anodic detection: simultaneous determination of allopurinol, oxipurinol and uirc acid in body fluids, J. Chromatogr., 306, 205, 1984. 7. Boulieu, R., Bory, C., and Baltassat, P., Simultaneous determination of allopurinol, oxipurinol, hypoxanthine and xanthine in biological fluids by highperformance liquid chromatography, J. Chromatogr., 307, 469, 1984. 8 . Failler, J. M., Farinotti, R., and Dauphin, A., Quantitative liquid chromatography of allopurinol and oxypurinol in human plasma and urine, Ther. Drug Monit., 7, 324, 1985.

ALLOPURINOL (continued)

46 CRC Handbook of Chromatography: Drugs

a

1-2

Plasma (1)

a

Column (cm x mm)

0.5 x 2

Waters radial compression module.

Extraction

1-1

Column Extraction (m x mm)

Specimen (ml)

Plasma, blood ( 1)

Specimen (ml)

RP-C18 (10)

Packing (pm)

5% OV-1 Chromosorb W (80/100)

Packing (mesh) N2 (40)

Gas (ml/min) NPD

Det.

E-l

Elution

1.6

Flow (ml/min)

Liquid Chromatography

270

Oven temp (°C)

Gas Chromatography

ALMITRINE

ABS (225)

Det. (nm)

2.5

RT (min)

4

RT (min)

S-2082 (4.5)

Internal standard (RT)

S-2082 (6 )

Internal standard (RT)

—

Deriv.

—

Other compounds (RT)

—

Other compounds (RT)

2

Ref.

1

Ref.

1. Premel-Cabic, A., Allain, P., Pidhorz, L., and Streichenberger, G., Pharmacokinetic of 2 -(p-methylallylaminophenyl) propionic acid, alminoprofene, in man after single and multiple oral doses, Eur. J. Clin. Pharmacol., 18, 419, 1980. 2. Paillet, M., Merdjan, Brouard, A., Doucet, D., Barreteau, H., and Fredj, G., Rapid determination of alminoprofen in plasma by high-performance liquid chromatography, J. Chromatogr., 343, 455, 1985.

REFERENCES

Elution — E-l. Methanol-water (50:50) containing 1% glacial acetic acid.

Extraction — 1-1. The sample ws mixed with 50 μ ΐ of an ethanolic solution of the internal standard (1 mg/ml) and 50 μ ΐ of 6 N HC1. The mixture was extracted with 10 m l of diethyl ether. The ether layer was dried over anhydrous sodium sulfate and evaporated to dryness at 50°C under a stream of nitrogen. The residue was treated with 25 μ ΐ of pyridine and 75 μ ΐ of N,-6 /s-(trimethylsilyl)-trifluoroacetamide at 70°C for 10 min. Aliquots of 1 μ ΐ of the reaction mixture were injected. I2. The sample was mixed with 50 μ ΐ of the internal standard solution (200 μg/ml in the mobile phase) and 100 μ ΐ of 0.017 M glacial acetic acid. The mixture was extracted with 7 m l of diethyl ether. The organic layer was evaporated under reduced pressure. The residue was dissolved in 200 μ ΐ of the mobile phase and a 50μ ΐ aliquot was injected.

Volume III 47

1-1

2.1x4

3% OV-17 Celite 545 (80/100)

Packing (mesh)

Initial temp = 235°C; rate = l°C/min; final temp = 280°C.

Plasma (0.5)

Column Extraction (m x mm) T.P.a

Oven temp (°C) N2 (40)

Gas (ml/min)

NPD

Det.

10.8

RT (min)

Pregnenolone (7.8)

Internal standard (RT)

O-Methyloxime-3acetate

Deriv.

—

Other compounds (RT)

1

Ref.

Extraction — 1-1. Plasma (0.5 m€), to which 200 μ€ of 1 Λ/ NaOH and 50 μ€ of a pyridine solution of the internal standard pregenenolone (1 μg) had been added was extracted with 4 m i of light petroleum by shaking for 4 min. The organic layer was removed and evaporated to dryness at 40°C. o-Methyl oximes were prepared

a

1

Specimen (m€)

Gas Chromatography

ALPHAXALONE

1. Baune, A., Bromet, N., and Courte, S., Trace determination of almitrine in plasma by gas-liquid chromatography using a nitrogen-phosphorus detector, J. Chromatogr., 223, 219, 1981. 2. Parkhurst, G. W., Bromet, N., MacLeod, C., Bachand, R. T., Jr., and Carson, P. E., Quantitative determination of almitrine in plasma by high-performance liquid chromatography, J. Chromatogr., 278, 209, 1983.

REFERENCES

Elution — E-l. Acetonitrile-2-propranol-0.006 M K2HP0 4 buffer, pH 7.8 (66.7:8.3:25).

Extraction — 1-1. The sample (1 m i) was extracted 3 times with 7 m i portions of cyclohexane. The combined organic layer was concentrated to about 5.7 m i in a current of nitrogen at 50°C; and was washed with 2 m i of 1 N NaOH. The organic layer was collected in another tube, 100 μ€ of the internal standard (2 μg/m€) in acetone was added, mixed, and evaporated at 50°C in a current of nitrogen. The residue was dissolved in 100 μ€ of acetone and 1 to 2 μ€ were injected. Cells were hemolyzed by diluting 1:1 with water prior to extraction of whole blood. 1-2. To the sample (1 m i) were added a 20-μ€ volume of internal standard (145 ng/m€ in acetonitrile) and 1 m i of acetonitrile. The tubes were vortex mixed, allowed to stand at room temperature for 25 to 30 min, and centrifuged at 4100 x g for 10 min at 4°C. The supernatant was collected in alcohol washed tubes containing 20 μ€ of 5 M NaOH. The protein precipitate was discarded. The alkaline supernatant was then extracted twice with 1 m i of cyclohexane by vortex mixing for 30 sec and centrifugation for 10 min at 4100 x g for 10 min. The combined organic layer was evaporated at 40°C under a gentle stream of nitrogen. The residue was dissolved in 100 μ€ of the mobile phase and an aliquot of 35 μ€ injected with the auto injector.

ALMITRINE (continued)

48 CRC Handbook of Chromatography: Drugs

1-3

Serum

(1)

1-2

Extraction

1-1

25 X 4.6

25 X 4

Column (cm x mm)

1.8 X 2

Column Extraction (m x mm)

Plasma (2 )

Specimen (m€)

Plasma (0.5—2)

Specimen (m€)

Zorbax SIL (6 )d

Bio-Sil ODS (I 0 )b

(μπι)

Packing

1% OV-17 Chromosorb W (80/100)

Packing (mesh) He (50)“

Gas (ml/min) ECD

Det.

E-2

E-l

Elution

1.5

2.5

Flow (ml/min)

Liquid Chromatography

290

Oven temp (°C)

Gas Chromatography

ALPRAZOLAM

ABS (214)

ABS (2 0 2 )

Det. (nm)

3.3

RT (min)

9

8.4

RT (min)

U-31485 (2 .2 )

Internal standard (RT)

Triazolam (6.5)

U-31485 (6.9)

Internal standard (RT)

_

Deriv.

—

a-Hydroxyal-prazolamc (6.5) 4-Hydroxyal-prazolamc (8 )

Other compounds (RT)

Triazolam (4.1)

Other compounds (RT)

3

2

Ref.

1

Ref.

1. Sear, J. W., Holly, J. Μ. P., Trafford, D. J. H., and Makin,, H. L. J., Plasma concentrations of alqhaxalone by gas chromatography: comparison with other gas chromatographic methods and gas chromatograph-mass spectometry, J. Pharm. Pharmacol., 32, 349, 1980.

REFERENCE

by the addition of 50 μ€ of pyridine saturated with methoxyamine hydrochloride, incubating the mixture for 5 min at 50°C, addition of 1 m€ of 5% HC1 saturated with ammonium sulfate and extraction with 2 m€ of light petroleum. The organic layer was evaporated and the residue treated with acetic anhydride-pyridine (1:1) at 50°C for 1 hr. Reagents were then removed under vacuum and the residue dissolved in 20 μ€ of light petroleum and 10-μ€ aliquot was injected.

Volume III 49

1-4

Extraction

12 x 4.6

Column (cm x mm) Perkin-Elmer C 18 (5)

Packing (μπι) E-3

Elution 1.0

Flow (ml/min) ABS (254)

Det. (nm) 5.5

RT (min) Nitrazepam (4)

Internal standard (RT) Temazepam (6.2) Chlordiaze-poxide (6 .8 ) Nordiazepam (7.6) Diazepam (9.1) Prazepam (14.7)

Other compounds (RT) 4

Ref.

Elution — E-l. Acetonitrile-50 mAf phosphate buffer, pH 4.5 (300:700). E-2. Acetonitrile-water (96:4). E-3. Methanol-water (60:40).

Extraction — 1-1. The sample was mixed with the residue after evaporation of 100 μ ΐ of the internal standard solution (0.1 μg/ml in benzene) and extracted with 3 m l of benzene containing 1.5% isoamyl alcohol. The organic layer was evaporated at 40 to 50°C under mildly reduced pressure. The residue was dissolved in 25 μ ΐ of toluene containing 15% isoamyl alcohol, of which 3 to 6 μ ΐ were injected. 1-2. The sample was mixed with 100 μ ΐ of the internal standard solution (1 μg/ml in methanol) and 0.5 m l of water. The mixture was extracted with 10 m l of toluene containing 1% isoamyl alcohol. The organic layer was evaporated at 37°C under a stream of nitrogen. The residue was reconstituted in 150 μ ΐ of the mobile phase. This solution was washed with 0.5 m l of hexane and an aliquot of 100 μ ΐ of the aqueous phase was injected. 1-3. The sample was buffered with 4 volumes of 4 M sodium hydroxide and extracted with 10 m l of toluene containing 10 ng/ml of the internal standard. The organic phase was evaporated at 50°C under a stream of nitrogen. The residue was dissolved in 50 μ ΐ of acetone and diluted to 300 μ ΐ with acetonitrile for injection with an autosampler. 1-4. The sample was mixed with 100 ng of the internal standard as a methanolic solution and 0.5 m l of pH 9 borate buffer. The mixture was extracted with 5 m l of toluene. A 4-ml aliquot of the toluene phase was passed through an Acrodisc-CR 0.45-μπι pore size filter assembly. The filtrate was evaporated at 60°C under a stream of nitrogen. The residue was reconstituted in 50 μ ΐ of methanol and a 10-μΙ aliquot was injected.

a Argon-methane (95:5) at a flow rate of 80 ml/min was used as the detector purge gas. b Column temp = 45°C. c A different mobile phase is described for the separation of hydroxy metabolites. d Protected by a 30 x 4.6 mm precolumn packed with SPHER-5 silica.

Blood 0)

Specimen (ml)

Liquid Chromatography

ALPRAZOLAM (continued)

50 CRC Handbook of Chromatography: Drugs

Extraction

1-4

Pure compounds

22 X 0.32

1-3

3x4

Column (cm x mm)

20 X 0.25

1-2

Plasma ( 1) Urine (0.5)

1.5 x 2

1-1

Column Extraction (m x mm)

Plasma (2 )

Specimen (ml)

Specimen (ml)

1.

8

α -AGP silicaf (10)

Packing (pm)

CP Sil

CP Sil 5a

3% OV-17 GasChrom Q (100/ 120)

Packing (mesh)

He (3.5) N2d

(40)

n2

Gas (ml/min)

FID; NPD

FID

ECD

Det.

E-l

Elution

0.5

Flow (ml/min)

Liquid Chromatography

T.P.C

T.P.b

235

Oven temp (°C)

Gas Chromatography

ALPRENOLOL

ABS (215)

Det. (nm)

NAe

7

5

RT (min)

g

RT (min)

Oxprenolol (8 ) H 56/60 + H 155/48

p- Analog (7)

Internal standard (RT)

—

Internal standard (RT)

2,4-Dichloro-benzene-boronate Trimethylsilyl Oxazolidine

Deriv.

g

Other compounds (RT)

4-Hydroxyalprenolol

—

—

Other compounds (RT)

4

Ref.

3

2

1

Ref.

Greenblatt, D. J., Divoll, M., Moschitto, L. J., and Shader, R. I., Electron-capture gas chromatographic analysis of the triazolobenzodiazepines alprazolam and triazolam, J . Chromatogr., 225, 202, 1981. 2 . McCormick, S. R., Nielsen, J., and Jatlow, P., Quantification of alprazolam in serum or plasma by liquid chromatogrpahy, Clin. Chem., 30, 1652, 1984. 3. Adams, W. J., Bombardt, P. A., and Brewer, J. E ., Normal-phase liquid chromatographic determination of alprazolam in human serum, Anal. Chem., 56, 1590, 1984. 4. Edinboro, L. E. and Backer, R. C., Preliminary report on the application of a high performance liquid chromatographic method for alprazolam in postmortem blood specimens, J. Anal. Toxicol., 9, 207, 1985.

REFERENCES

Volume III 51

1-5

Pure compounds

20 x 20 (Merck)

Plate (Manufacturer) Silica 60 (0.25)

Layer (mm) S-l

Solvent —

Postseparation treatment Visual

Det. (nm) 0.84

Rf —

Internal standard (Rf) h

Other compounds (Rf)

5

Ref.

Extraction — 1-1. The sample was mixed with 25 μ ΐ of the internal standard solution (5.6 mg/ml in 0.1 M HC1) and 0.3 m l of 0.1 M sodium hydroxide. The mixture was extracted with 5 m l of hexane-dichloromethane (4:1). A 4-ml aliquot of the organic phase was evaporated at 40°C under a stream of nitrogen. The residue was dissolved in 25 μ ΐ of transboronation reagent prepared by mixing 500 μ ΐ of 5.5 μΛί solution of 2,4-dichlorobenzene-boronic acid in acetonitrile, 50 μ ΐ of 55 μΜ solution of 1 ,3-propanediamine in acetonitrile and 450 μ ΐ of acetonitrile. After vigorous mixing a l -μ ΐ aliquot of the solution was injected. 1-2. The sample was mixed with an aqueous solution of the internal standard and 0.3 m l of 1 N sodium hydroxide solution. The mixture was extracted with 5 m l of dichloromethane-diethyl ether (1:4). The organic phase was back extracted into 2 m l of 0.1 N HC1. The aqueous phase was made alkaline with 0.3 m l of 1 N sodium hydroxide and re-extracted with 5 m l of the extraction solvent. The organic phase was washed with 2 m l of 5% sodium acetate solution and evaporated at 45°C under a stream of nitrogen. The residue was dissolved in an aliquot of ethylacetate. A l-μ ΐ aliquot of this solution and 2 μ ΐ of N,0-/?/s(trimethylsilyl)trifluoroacetamide reagent were injected. The precolumn was back flushed at 85 sec. 1-3. The sample was mixed with 1 m l of buffer (pH 12.3), 50 μ ΐ of methanol, 150 μ ΐ of a solution of the mixed internal standards and water to make a total volume of 2 m l. Phosgene (2 M in toluene) was added with vigorous shaking in aliquots of 20 μ ΐ at 0, 2, and 4 min. After 6 min, 2 m l of dichloromethane containing the marker (trichloro-ethylcarbamate) were added and the mixing continued for another 2 min. The organic layer was evaporated. The residue was dissolved in 150 μ ΐ of ethyl acetate and a 3-μΙ aliquot was injected. 1-4. About 1 mg of the racemic β-blocker was dissolved in 0.5 m l of diethyl ether and 50 μ ΐ of 0.5 M sodium hydroxide. After mixing, the solution was treated with 50 μ ΐ of phorgene (20% in toluene). After mixing for 1 hr at room temperature, the organic phase was evaporated and the residue dissolved in the mobile phase for injection.

The column was connected to a 15 cm x 3 mm precolumn packed with 3% SE-30 on Supelcoport (80/100 mesh) to a length of 7 cm. Initial temp = 70°C; rate = 20°/min to 160°C; 10°/min to final temp (240°C). Initial temp = 100°C, rate = 207min, final temp = 280°C. Linear velocity = 60 cm/sec. The last peak of the internal standard (H 155/48) elutes at 10.9 min. Packing material contains 147 mg of aj-acid glycoprotein per gram of silica. 8 Capacity and resolution factors of enantiomers of a number oxazolidones of β-blockers are given. h Separation of dansyl derivatives by two dimensional chromatography of a number of β-blockers is shown.

a b c d e f

Extraction

Specimen (ml)

Thin-Layer Chromatography

ALPRENOLOL (continued)

52 CRC Handbook of Chromatography: Drugs

REFERENCES

1-1

1-2

1-3

Plasma, urine (1)

Urine

1.8x4

2 x 3

1.8x3

Column Extraction (m x mm)

Plasma (2)

Specimen (ml)

200

180

Oven temp (°C)

10% APiez145 on —L + 2% KOH Chromosorb W (80/100)

3% OV-225 GasChromQ (80/100) 5% SE-30

Packing (mesh)

—

FID

Phenylethylamine (3.5)

9

ECD

Ar:90Methane 10 (75) N2 (50)

Deriv. p-MethoxyphenIsothioylethylamine cyanate8 (4.9) NA Amphetamine (NA) Trichloroacetyl

2.3

Internal standard (RT)

MS-EI

Det.

RT (min)

He (30)

Gas ml/min

Gas Chromatography

AMANTADINE

—

—

—

Other compounds (RT)

3

2

1

Ref.

1. Poole, C. F., Johansson, L ., and Vessman, J., Formation of electron-capturing derivatives of alprenolol by transboro-nation. Application to the determination of alprenolol in plasma, J. Chromatogr., 194, 365, 1980. 2. Christophersen, A. S. and Rasmussen, K. E., On-column silylation of β-blocking agents applied to a back-flushable precolumn capillary system, J. Chromatogr., 246, 57, 1982. 3. Gyllenhaal, O., Direct derivatization of alprenolol and its 4-hydroxy metabolite in urine with phosgene and methanol prior to analysis by capillary column gas chromatography, J. Chromatogr., 349, 447, 1985. 4. Hermansson, J., Resolution of racemic aminoalcohols (β-blockers), amines and acids as enantiomeric derivatives using a chiral a r acid glycoprotein column, J. Chromatogr., 325, 379, 1985. 5. Schulz, H. G. and Zapka, R., Dansylation and thin-layer chromatography of beta blockers, Fresenius Z. Anal. Chem., 323, 162, 1986.

Solvent — S-l. Benzene-cyclohexane-methanol (75:10:15).

Elution E-l. 2-Propanol-0.02 M phosphate buffer, pH 7 (10:90).

1-5. A 2-ml aliquot of an aqueous solution of the drug is mixed with 16 m l of a solution of 20 mg of sodium bicarbonate in acetone-water (1:1) and 4 m l of dansyl chloride reagent (3 mg/ml in acetone). The mixture was heated under reflux at 55°C for 20 min. After cooling, the reaction mixture was extracted with 8 m l of ether. The organic layer was concentrated to about 1 m l for application on TLC plate.

Volume III 53

0.6 x 1

1.2 x 4

Oven temp (°C)

5% Apiezon L 150 GasChrom Q (100/ 120) 5% OV-101 Chro- T.P.b mosorb G HP (100/ 120)

Packing (mesh)

He (7)

N2 (60)

Gas ml/min 4.2

RT (min)

MS-EIC d

FID

Det. Chlorphentermine (8 )

Internal standard (RT)

Acetyl

—

Deriv.

—e

—

Other compounds (RT)

4

Ref.

Extraction — 1-1. To 2 m l of plasma were added 2 m l of borate buffer (0.5 M, pH 10.5), and 1 g of NaCl. The mixture was shaken with 10 m l of ethyl acetate for 10 min and centrifuged for 5 min. The organic layer was collected and the aqueous layer re-extracted with another 5 m l of ethyl acetate. An aliquot of 14 m l of combined extract was back extracted into 1 m l of 0.5 N HC1. The acid extract was made alkaline with 0.5 m l of 2 N sodium hydroxide and extracted into 5 m l of ethyl acetate. An aliquot of 4.5 m l of the ethyl acetate extract was treated with 0.5 m l of carbon disulphide, mixed and allowed to stand for 4 hr. Then the solution was washed with 0.5 m l of 0.5 N HC1 and the organic layer evaporated to dryness. The residue was then dissolved in 20 μ ΐ of ethyl acetate solution containing 10 ng/ml of p-methoxyphenylethylamine isothiocyanate. 1-2. To the sample are added 1 m l of internal standard solution (200 ng/ml of water), 1 m l of 1 M NaOH, and 2 m l of toluene. The tubes are mixed on a rotary mixer at 300 rpm for 15 min and centrifuged 480 x g for 10 min. An aliquot of the toluene phase is transferred to another tube, 10 μ ΐ of 2% trichloroacetyl chloride solution in toluene (prepared immediately before use) are added, and the stoppered tube is heated at 70°C for 30 min. After cooling, 1 m l of 1 M NaOH is added. The tubes are mixed for 5 min, centrifuged, and a 3-μΙ portion of the organic layer is injected into the gas chromatograph by solvent flush technique using 1 μ ΐ of the toluene as the guard. 1-3. To the sample are added 20 μ ΐ of the internal standard (95.8 μg), 0.5 m l of the 6 M NaOH and 300 μ ΐ of chloroform. The tubes are vortex mixed for 1 min and centrifuged at 800 x g for 10 min. Upper layer is discarded and 1 to 3 μ ΐ of the chloroform layer is injected. 1-4. To the sample (1 to 3 ml) were added, 1 m l of chlorphentermine solution (10 μg/ml) and 0.5 m l of 2 N NaOH. The mixture was extracted three times with freshly distilled diethyl ether. Isopropanol (0.1 m l) was added to the combined ethereal extracts, concentrated to 20 to 40 μ ΐ at 44°C and aliquots of the concentrated extract were injected.

Isothiocyanate derivative of p-methoxyphenylethyl amine is added just prior to injection. Initial temp = 100°C, rate = 20°C/min; final temp = 310°C; final time = 3 min. FID and NDP were also used after 1:1 split of the column effluent. Retention index = 1640. Retention indices of a number of other antiparkinsonian drugs are given.

1-5

Urine (10)

a b c d e

1-4

Column Extraction (m x mm)

Plasma, urine (1— 3)

Specimen (ml)

Gas Chromatography

AMANTADINE (continued)

54 CRC Handbook of Chromatography: Drugs

a

1-1

Extraction

30x4

Column (cm x mm) μ-Bondapak-CN ( 1 0 )·

Packing (pm) E-l

Elution 1.5

Flow (ml/min) ABS (2 2 0 )

Det. (nm)

6

RT (min)

Azidocillin (8 )

Internal standard (RT)

—

Other compounds (RT)

1

Ref.

1. Lee, T. L. and Brooks, M. A., Determination of amdinocillin in plasma and urine by high-performance liquid chromatography, J. Chromatogr., 227, 137, 1982.

REFERENCE

Elution — E -l. Water-methanol-1 M phosphate buffer, pH 7 (70:30:0.5)

A guard column packed with Bondapak phenyl-corasil was used. Extraction — 1-1. To the sample (0.2 m l) was added, 25 μ ΐ of internal standard (2.5 pg/m l of water), and 0.4 m l of acetonitrile. The tubes were vortex mixed for 10 to 20 sec and centrifuged at 1400 x g for 5 min at 0 to 5°C. The supernatant was collected and washed with 5 m l of diethyl ether. The tubes were kept in an ice bath until 20-μΙ aliquots were injected. Urine samples (0.1 ml) after the addition of internal standard (200 μ ΐ) were diluted to 1 m l, mixed and kept in an ice bath until 10 μ ΐ were injected.

Plasma, urine (0 .2 )

Specimen (m l)

Liquid Chromatography

AMDINOCILLIN

Sioufi, A. and Pommier, F., Gas chromatographic determination of amantadine hydrochloride (Symmetrel) in human plasma and urine, J. Chromatogr., 183, 33, 1980. 3. Stumph, M. J., Noall, M. W., and Knight, V., Gas-chromatographic determination of amantadine in human urine, Clin. Chem., 26, 295, 1980. 4. Belanger, P. M. and Grech-Belanger, O., Gas-liquid chromatographic determination of plasma and urinary levels of amantadine in man, J. Chromatogr., 228, 327, 1982. 5. Maurer, H. and Pfleger, K., Screening procedure for the detection of antiparkinsonian drugs and their metabolites in urine using a computerized gas chromat­ ographic-mass spectrometric technique, Fresenius Angew. Anal. Chem., 321, 363, 1985.

2.

1. Narasimhachari, N., Helgeson, E., and Prakash, U., GC-MS-SIM Quantitation of amantadine in biological samples, Chromatographia, 12, 523, 1979.

REFERENCES

1-5. Ten milliliters of urine were refluxed with 3 m l of HC1 (37%) for 15 min, made basic with 3 g of KOH pellets and mixed with 10 m l of 30% aqueous ammonium sulfate to obtain a pH between 8 and 9. The samples were extracted twice with 10 m l each of a mixture of 2 parts of dichloromethane, 2 parts of isopropanol, and 6 parts of ethyl acetate. The combined organic extracts were evaporated to dryness under vacuum and the residue dissolved in 0.1 m l of methanol.

V o lu m e III

55

Column (cm x mm)

25 x 4

Extraction

1-1

LiChrosorbRP-8 (7)

Packing (μπι)

Flow (ml/min) 2

Elution E-l

ABS (280)

Det. (nm) 3.5

RT (min)

Other compounds (RT) —

Internal standard (RT) p-Methylamezinium (7)

1

Ref.

REFERENCE

a

1-1

Extraction

30 x 3.9

Column (cm x mm) μ-Bondapak phenyl ( 1 0 )a

(μπι)

Packing

E-l

Elution

1.6

Flow (ml/min)

The column is protected by a 23 x 3.9 mm guard column packed with Corasil phenyl.

Plasma, urine ( 1)

Specimen (ml)

Det. (nm) ABS (280)

Liquid Chromatography

AMIFLOXACIN

1 1 .8

RT (min)

Rosoxacin (14.1)

Internal standard (RT)

Piperazinyl-N-desmethyl metabolite (9.7)

Other compounds (RT)

1. Hotz, D. and Brode, E., High-performance liquid chromatographic determination of amezinium in human plasma, J. Chromatogr., 227, 217, 1983.

Elution — E-l. Acetonitrile-water (25:75).

1

Ref.

Extraction — 1-1. Disposable syringe columns (2 ml) packed with cellulose acetate (bed volume 0.9 to 1.1 m€) are washed twice with 2 m l of water. After addition of the internal standard (45 ng), the plasma sample (2 ml) is applied to the column. The columns are centrifuged for about 10 min at 1000 x g, washed with two consecutive 2 m l portions of water, and eluted with 1 m l of 1 A# NaCl. To this eluate 0.1 m l of 10 M NaOH is added, kept at room temperature for 30 min, and extracted with 4 m l of dichloromethane. The organic layer is evaporated to dryness in a stream of nitrogen (temp. 30°C), the residue dissolved in 100 μ ΐ of mobile phase and the maximum volume possible injected into the HPLC system.

Plasma (2 )

Specimen (ml)

Liquid Chromatography

AMEZINIUM

56 C R C H a n d b o o k o f C h r o m a to g r a p h y : D r u g s

30 x 3.9

12.5 X 4.6

1-1

1-2

1-3

Ultrasphere Octyl (5)e

Ultrasphere-ODS (5)a,b LiChrosorb RP-18 (1 0 ) Nucleosil-C18 (5)

Packing (pm)

E-4

2.0

1.0

2.5

E-2 E-3

NA

E-l

Elution

Flow (m€/min)

7.5

Kanamycin (12.5)

—

d

—

Kanamycin (17) Neamine (2 0 . 1 ) 7.5 15.7

—

— 5.1

Other compounds (RT)

ABS (360) ABS (365) FL (UGI, Kv418)c ABS (340)

Internal standard (RT)

RT (min)

Det. (nm)

The column is protected by a Brownlee RP-18 column (30 x 4.6 mm, particle size 5 μΛΓ) Conditions for phase chromatography are also described. Excitation and emission filters, respectively. Conditions for the analysis of dibekacin, gentamicin, sisomicin, tobramycin, and netilmicin are also described. The column at 50°C.

25 X 4.6

25 X 4.6

Extraction

1-4

Column (cm x mm)

4

3

2

1

Ref.

Extraction — 1-1. To 25 μ€ of sample were added 10 μ€ of 0.1 M borate buffer (pH 9.3) and 100 μ€ of methanol. The vials were vortexed and centrifuged at 2000 x g for 5 min. A 7 5 -μ€ aliquot of the supernatant was incubated with 10 μ€ of l-fluro-2, 4-dinitrobenzene (180 mg/m€ in methanol) for 30 min at 80°C, then

a b c d e

Serum (0.05)

Plasma (0.025) Serum (0 .2 ) Serum (0 .0 2 )

Specimen (m€)

Liquid Chromatography

AMIKACIN

1. McCoy, L. F., Crawmer, B. P., and Benziger, D. P., Analysis of amifloxacin in plasma and urine by high-pressure liquid chromatography and intravenous pharmacokinetics in rhesus monkeys, Antimicrob. Agents Chemother., 27, 769, 1985.

REFERENCE

Elution — E-l . 0.05 M Sodium chloride + 3 mAf tetrabutyl ammonium phosphate adjusted to pH 2.3 with phosphoric acid - acetonitrile (92:8).

Extraction — 1-1. To each 1 m€ of plasma sample was added 50 μ€ of the internal standard solution (20 μg/m€ in 0.01 M NaOH) and 0.3 m€ of 1 M sodium phosphate buffer (pH 6 .6 ). Chloroform (7 m€) was added, mixed for 10 min, and centrifuged. The chloroform layer was back extracted with 0.6 m€ of 0.1 M NaOH. After centrifugation, to 0.3 m€ of the aqueous layer was added 30 μ€ of 1 N sulfuric acid and 1 0 0 μ€ of the mobile phase, aliquots of this mixture were injected.

Volume III 57

REFERENCES

1. Wong, L. T., Beaubien, A. R., and Pakuts, A. P., Determination of amikacin in microlitre quantities of biological fluids by high-performance liquid chromatography using l-fluoro-2, 4-dinitrobenzene derivatization, J. Chromatogr., 231, 145, 1982. 2. Barends, D. M., Blauw, J. S., Smits, Μ. H., and Hulshoff, A., Determination of amidacin in serum by high-performance liquid chromatography with ultraviolet detection, J. Chromatogr., 276, 385, 1983. 3. Essers, L., An automated high-performance liquid chromatographic method for the determination of aminoglycosides in serum using pre-column sample clean­ up and derivatization, J . Chromatogr., 305, 345, 1984. 4. Kabra, P. M., Bhatnager, P. K., and Nelson, M. A., Liquid chromatographic determination of amikacin in serum with spectrophotometric detection, J. Chromatogr., 307, 224, 1984.

Elution — E-l. Acetonitrile-water (68:32). E-2. Acetonitrile-water-acetic acid (470:530:1). E-3. Methanol-0.2 Af sodium acetate buffer, pH 7.4 (74:26). E-4. Acetonitrile-20 mAf phosphate buffer, pH 3 (520:480).

evaporated in a current of nitrogen and 6 . 8 mg of binder-free silica gel was added to the residue. After adding 600 μ ΐ of diethyl ether, the vials were shaken for 5 min and centrifuged at 2000 x g for 5 min. The ether was discarded by decanting and the washing procedure repeated once more with 600 \it of ether, then with 400 μ€ of 0.2 Af acetate buffer (pH 4.0). The silica gel pellet was then extracted by vortexing with 300 μ£ of the mobile phase. After centrifugation for 5 min, 50μ ΐ aliquots of the supernatant were injected on to the column. 1-2. Pasteur pipettes are filled with CM-Sephadex C25 soaked overnight in 0.2 Af Na2S0 4 to a height of 1.5 cm. Sample (200 μ€) is spiked with 40 μ€ of aqueous solution of kanamycin (250 ng/m€) and is applied to the column. The column is washed with 2 mf of a solution containing 0.001 M HC1 and 0.2 Af Na2S 0 4 and with 250 μ€ of 0.05 Af NaOH. The column is then eluted with 1 m€ of 0.05 Af NaOH. The eluate is treated with 2.5 m€ solution of 1-fluorodinitrobenzene in methanol (30 mg/m€), and the mixture heated in a boiling water bath for 5 min. After cooling 150 μ€ of the mixture is injected. 1-3. An automated system of sample clean up by adsorption of aminoglycoides on a precolumn, subsequent derivatization with o-phthalaldehyde and on-line separation of derivatives by column switching is described. 1-4. The sample (50 μ€) was spiked with 100 μ€ of the internal standard (16 mg/€ in acetonitrile) and 25 μ€ of 2 M Tris buffer were added. The tubes were vortex mixed, centrifuged, and decanted into new tubes. The supernatant was treated with 30 μ€ of 2, 4, 6 -trinitrobenzene-l-sulfonic acid (250 g/€ in acetonitrile) and incubated at 70°C for 30 min. The reaction mixture was applied to a prewashed (2 m€ methanol, 2 m€ water) Bond-Elut C ,8 extraction columns along with 0.7 m€ of wash phosphate buffer (10 mAf, pH 8 .6 ). The columns were drained and washed three times with wash phosphate buffer and eluted with 300 μ€ of acetonitrile. An aliquot of 50 μ€ of the eluate was injected.

AMIKACIN (continued)

58 C R C H a n d b o o k o f C h r o m a to g r a p h y : D r u g s

x 10 cm (Merck)

10

1.0

1.0

2.0

S-l

Solvent —

Post­ separation treatment

Det. (nm)

FI (Reflec­ tance) (365, 435)

Det. (nm)

FI (355, 410)

FI (286, 395) FI (368, 417)

Thin-Layer Chromatography

E-3

E-2

E-l

Elution

Flow (ml/min)

0.34

Rf

7.5

5.8

2.3

RT (min)

(Rf)

_

_

Other compounds

—

—

—

Other compounds (RT)

(Rf)

Internal standard

Triametrene (3.8) 6 -Fluoroanalog (4.8) Quinidine (10.5)

Internal standard (RT)

4

Ref.

3

2

1

Ref.

Extraction — 1-1. The sample was mixed with 1 m l of ethyl acetate and 10 μ ΐ of the internal standard solution (7 μg/ml in 1 % lactic acid). While vortexing, 50 μ ΐ of 5 M sodium hydroxide were added. An aliquot of 0.8 m l of the organic layer was back extracted into 0.5 m l of 0.1 M HC1. The aqueous layer was concentrated at 60°C under a stream of nitrogen and aliquots of 10 to 50 μ ΐ of the residual aqueous solution were injected. 1-2. The sample was mixed with 50 μ ΐ of the internal standard solution (0.2 mg/ml in methanol-0.01 N HC1, 1:1) and 20 μ ΐ of methanol-0.01 N HC1 (1:1). The mixture was applied to a prewashed (1 m l acetonitrile, 1 m l water) Baker 3-ml silica column. The column was washed with 2 m l water, 1 m l acetonitrile, 1 m l water, and 0.25 m l 2 M sodium perchlorate. Finally, the column was eluted with 0.5 m l of acetonitrile. The eluate was concentrated to 50 μ ΐ at 40°C under a stream of nitrogen. The residual solution was diluted with 0.2 m l of 2 M sodium perchlorate solution for injection. 1-3. The sample was mixed with 0.5 m l of acetonitrile containing the internal standard. The supernatant was concentrated to about 200 μ ΐ and aliquots of 75 μ ΐ were injected.

a

1-4

Plasma (1)

Layer (mm)

Ultrasphere Si (5)

Spherisorb-ODS (10) μ-Bondapak-Qg ( 1 0 )“

(μπι)

Packing

Silica gel 60 (HPTLC) (0.25) Protected by a 3 cm Bio-Rad ODS (10 μιη) guard column.

Extraction

Plate (Manu­ facturer)

25 x 4.6

30 x 3.9

1-2

1-3

25 x 4.1

Column (cm x mm)

1-1

Extraction

Specimen (ml)

Plasma (0 .2 )

Plasma, urine (0 . 1) Serum, urine ( 1)

Specimen (ml)

Liquid Chromatography

A M IL O R ID E

Volume III 59

a b

1-1

Extraction

15 x 4.6

Column (cm x mm) Nucleosil C,8 (5)

Packing (pm) E-l

Elution

(Dihydro-10,1 l-dibenzo-[a,d]cycloheptenyl-5-amino)-7-octanoic acid. (Dihydro-10,1 l-dibenzo-[a,d]cycloheptenyl-5-amino)-7-pentanoic acid.

Plasma (2)

Specimen (ml)

NA

Flow (ml/min)

Liquid Chromatography

AMINEPTINE

ABS (220)

Det. (nm)

6.5

RT (min)

a (8.5)

Internal standard (RT)

Metabolite1* (4)

Other compounds (RT)

1

Ref.

1. Yip, M. S., Coates, P. E., and Thiessen, J. J., High-performance liquid chromatographic analysis of amiloride in plasma and urine, J. Chromatogr., 307, 343, 1984. 2. Vincek, W. C., Hessey, G. A., II, Constanzer, M. L., and Bayne, W. F., Amiloride: biological fluid analysis by reverse-phase HPLC, Pharm. Res., 3, 143, 1985. 3. Shi, R. J. Y., Benet, L. Z., and Lin, E. T., High-performance liquid chromatographic assay of basic amine drugs in plasma and urine using a silica gel column and an aqueous mobile phase, J. Chromatogr., 377, 399, 1986. 4. Reuter, K., Knauf, H., and Mutschler, E., Fluorimetrische Bestimmung von Amilorid in Humanplasma mittels Dunnschichtchromatographie, J. Chromatogr., 233, 432, 1982.

REFERENCES

Solvent — S-l. (A) Ethyl acetate. (B) 2-Propanol-diisopropyl ether-25% ammonia (70:30:10).

Elution — E -l. Acetonitrile-0.15 M perchloric acid, pH 2.2 (32:68). E-2. Methanol-0.1 M sodium perchlorate, pH 4 (40:60). E-3. Acetonitrile + water (600:400) containing 0.99 g dibasic ammonium phospahte, pH 7.

1-4. The sample was mixed with 0.2 m l of 1 N NaOH and extracted with 5 m l of w-butanol-diisopropyl ether (1:1). An aliquot of 2 m l of the organic phase was evaporated at 80°C under nitrogen. The residue was dissolved in 0.2 m l of methanol-diisopropyl ether (3:2) and an aliquot of 50 μ ΐ was spotted. The plate was developed in solvent A to a height of 8 cm, dried, developed to a height of 8 cm in solvent B, dried, dipped in paraffin-cyclohexane mixture (1:2), and dried for scanning.

AMILORIDE (continued)

60 CRC Handbook of Chromatography: Drugs

1.

1-1

1-2

1-3

Plasma ( 1)

Urine

Extraction

Urine ( 1)

Specimen (m l)

30 x 4

25 x 4

25 x 4

Column (cm x mm) E-l

E-2

E-3

Spherisorb ODS (5)

p-Bondapak-C18 ( 1 0 )d

Elution

Yanapak ODS-Tb GO)

Packing (pm)

Det. (nm) ABS (254)c

ABS (254)

ABS (254)

Flow (ml/min) 0.7

1.5

1.4

Liquid Chromatography

p-AMINOBENZOIC ACID*

5

6.5

7.5

RT (min)

m-Hydroxybenzoic acid (9.5) m-Hydroxybenzoic acid (7.5) —

Internal standard (RT)

p-Aminohippuric acid (6.5) p-Acetamido-benzoic acid (8 ) p-Acetamido-hippuric acid (14)

—

—

Other compounds (RT)

4

2,3

1

Ref.

Nicot, G. and Lachatre, G., High-performance liquid chromatographic method for the determination of amineptine and its main metabolite in human plasma, J. Chromatogr., 306, 279, 1984.

REFERENCE

Elution — E -l. Acetonitrile-water containing 1.2 g/l heptanesulfonate adjusted to pH 3 with phosphoric acid (38:62).

Extraction — 1-1. The sample (2 m l) was added to 1 m l of 0.05 M phosphate buffer (pH 7.0), 100 μ ΐ of 10 μg/m l solution in water of the internal standard and 10 m£ of heptane-octanol-tetr^heptylammonium bromide (98:2:0.5). The tubes were shaken for 10 min centrifuged at 900 x g for 10 min. An 8 -m l aliquot of the organic phase was collected and 200 μ£ of 0.1 M acetic acid-methanol (90:10) were added. After mixing (5 min) and centrifugation (10 min), the organic phase was discarded and 50 μ£ of the lower aqueous phase was injected.

V o lu m e III

61

REFERENCES

1. Ito, S., Manila, K., Imai, Y., Kato, T., Ito, M., Nakajima, S., Fujita, K., and Kurahashi, T., Urinary p-aminobenzoic acid determined in the pancreatic function test by liquid chromatography, with electrochemical detection, Clin. Chem., 28, 323, 1982. 2. Lawson, N., Berg, J. D., and Chesner, I., Liquid-chromatographic determination of p-aminobenzoic acid in plasma to evaluate exocrine pancreatic function, Clin. Chem., 31, 1073, 1985. 3. Berg, J. D., Chesner, I., and Lawson, N., Practical assessment of the NBT-PABA pancreatic function test using high performance liquid chromatography determination of p-aminobenzoic acid in urine, Ann. Clin. Biochem., 22, 586, 1985. 4. Karnes, Η. T., Riley, C. M., Curry, S. H., and Schulman, S. G., Analysis of N-benzoyl-L-tryosyl-p-aminobenzoic acid (bentiromide) metabolites in urine by ion-pair high-performance liquid chromatography, J. Chromatogr., 338, 377, 1985.

Elution — E-l. Acetonitrile-0.2 M phosphate buffer, pH 3.5 (50:350). E-2. Acetonitrile-0.2 M phosphate buffer, pH 4 (1:7). E-3. Methanol-0.01 M tetrabutylammonium chloride (1:9).

Extraction — 1-1. To the sample (1 ml) is added 1 m l of 8 M NaOH containing 1 g/l of m-hydroxybenzoic acid. The tubes are heated at 120°C for 60 min. After cooling 10 μ ΐ of the hydrolysate is diluted to 1 m l with 50 mM phosphoric acid, centrifuged, and 10 μ ΐ of the supernatant injected. 1-2. The sample was deproteinized with 1 m l of 2 M perchloric acid containing 0.65 mAf/l of m-hydroxybenzoic acid as the internal standard. After centrifugation at 6000 x g for 20 min, 0.5 m l of the clear supernatant was added to 0.5 m l of 8 M NaOH. The tubes were heated at 120°C for 1 hr. After cooling 0.1 m l of the hydrolysate was diluted to 1 m l with 0.5 M phosphoric acid and 200 μ ΐ of this acidified hydrolysate was injected. 1-3. Urine was diluted. The extent of dilution was based on the results of preliminary experiments. The diluted samples were injected directly.

* Hydrolysis product of N-benzoyl-L-tyrosyl-p-aminobenzoic acid by the action of pancreatic chymotrypsin during pancreatic function test. b Column temp = 55°C. c An electrochemical detector at the applied potential of +1100 mV is also used. d Column temp = 40°C.

p-AMINOBENZOIC ACID* (continued)

62 CRC Handbook of Chromatography: Drugs

1-1

Serum (0.02)

LiChrosorb RP-18 (10)

iranj-4-Aminomethylcyclohexane carboxylic acid.

25 x 4.6

Extraction

Packing (pm) E-l

Elution 2

Flow (m€/min) FI (390, 475)

Det. (nm) 6

RT (min) a (9)

Internal standard (RT)

Ref. 1

Other compounds (RT) __

1-1

1-2

(2

g) Plasma, blood8 (0.5— 1)

Extraction

Fed

Specimen (m€)

30 X 4

30 X 3.9

Column (cm x mm) p-Bondapak-C1;8 (10) μ -Porasil (10)

Packing (pm)

E-2

E-l

Elution

2.2

1.5

Flow (m€/min)

Liquid Chromatography

ABS (254) ABS (254)

Det. (nm)

—

—

—

— 6.5 5

Other compounds (RT)

Internal standard (RT) RT (min)

l-(2-AMINOETHYL)-3-(2,6-DICHLOROPHENYL)THIOUREA

2

1

Ref.

1. Sacroix, C., Levert, P., Laine, G., and Goulle, J. P., Microdosage de deux antifibrinolytiques (acid e-aminocaproique et acide tranexamique) par chromatographie liquide et detection fluorim&rique, J. Chromatogr., 309, 183, 1984.

REFERENCE

Elution — E-l. Acetonitrile-water-acetic acid-tetrahydrofuran (300:690:5:5) containing 40 mM /i sodium acetate.

Extraction — 1-1. The sample (20 μ€) after the addition of 2 μ€ of the internal standard (1 g/€) was deproteinized with 20 μ€ of acetonitrile. After centrifugation, 5 μ€ of the supernatant was diluted with 100 μ€ of 0.025 M phosphate buffer, pH 8 ; and 100 μ€ of fluorescamine solution (300 mg/€ of acetone) was added. After vigorous vortex mixing, 2 0 μ€ of this solution was injected.

a

Column (cm x mm)

Specimen (m€)

Liquid Chromatography

e-AMINOCAPROIC ACID

Volume III 63

2.

1.

Plasma, urine (1)

Specimen (ml)

1-1

Extraction

30 x 4

Column (cm x mm) μ-Porasil (10)

Packing (μπι) E-l

Elution

1.0

Flow (ml/min)

Liquid Chromatography

ABS (254)

Det. (nm)

4.2

RT (min)

5-Methyl analog

Internal standard (RT)

4-AMINO-5-ETHYL-3-THIOPENECARBOXYLIC ACID METHYL ESTER

4-Amino-5ethyl 3-thiophenecarboxylic acid"

Other compounds (RT)

1

Ref.

Quadrel, R. R. and Bogdansky, F. M., A rapid chromatographic procedure for the isolation and quantitation of l-(2-aminoethyl)-3-(2.6-dichlorophenyl)thiourea from dietary admix followed by HPLC, J. Chromatogr. Sci., 18, 622, 1980. Lee, T. L., Brooks, M. A., and de Silva, J. A. F., Determination of the antihypertensive agent l-(2-aminoethyl)-3-(2,6-dichlorophenyl)thiourea in plasma and urine by high-performance liquid chromatography, J. Chromatogr., 224, 275, 1981.

REFERENCES

Elution — E-l. Methanol-0.01 M dibasic ammonium phosphate, adjusted to pH 7.4 with 1 N phosphoric acid (43:57). E-2. Dichloromethane-methanol-heptane-ammonium hydroxide (85:10:5:0.1).

Extraction — 1-1. The sample was agitated with 25 m l of methanol in an ultrasonic bath for 5 min, then shaken vigorously for 15 min, and centrifuged for 5 min at 2200 rpm. A glass column (15 cm x 6 mm) was packed with preswollen CM52 cation exchanger to a height of 12 mm. The column washed with methanol (2 ml) and a 2-ml aliquot of the feed extract was applied to the column. The column was allowed to drain, washed with 2 x 1-ml aliquots of 0.01 M dibasic ammonium phosphate, adjusted to pH 7.3 with 1 N phosphoric acid, and finally the column was eluted with 2 m l of 0.1 N NaOH. The eluate was adjusted to pH 7 with a few drops of 1 N phospharic acid. Aliquots of this solution were injected. 1-2. The sample was treated with 1 M phosphate buffer (pH 10) to make up to a volume of 2.5 m l. The mixture was extracted with 6 m l of methylene chloride by slowly shaking for 10 min on a reciprocating shaker. The tubes were then centrifuged in a refrigerated centrifuged for 10 min at approximately 1200 x g. The organic layer was evaporated under a current of nitrogen at 35 to 40°C. The residue was dissolved in 200 μ ΐ of dichloromethane-methol-heptane (85:10:5) and the tubes were kept in an ice bath until 2 0 μ ΐ aliquots were injected.

* Conditions for the analysis of urine ate also described.

l-(2-AMINOETHYL)-3-(2,6-DICHLOROPHENYL)THIOUREA (continued)

64 CRC Handbook of Chromatography: Drugs

Separate conditions for the analysis of this metabolite as a free acid by reversed phase chromatography are described.

REFEREN CE

1-1

1-2

Plasma (0 . 1 )

Extraction

1-1

10 x 8

15 x 4.6

Column (cm x mm)

1.5 x 4

Column Extraction (m x mm)

Plasma, saliva (0.5)

Specimen (ml)

Plasma, saliva (0.5)

Specimen (ml) N2 (100)

Gas (ml/min) NPD

Det.

E-l

E-2

Magnusphere C 18 (7) Radial Pak C18

2.0

1.2

Elution

(μπι)

Liquid Chromatography

240

Oven temp (°C)

Flow (ml/min)

Packing

2% CDMSa Chromosorb W (80/100)

Packing (mesh)

Gas Chromatography

ABS (254)c

ABS (254)

Det. (nm)

2 .8

RT (min)

A M IN O G L U T E T H IM ID E

2.5

—

RT (min)

Primidone (3.5)

Internal standard (RT)

_

—

Heptabarbitone (5.6)

—

Acetamidoglutethimide (3.5)

Other compounds (RT)

b

Deriv.

Internal standard (RT)

Other compounds (RT)

2

1

Ref.

1

Ref.

1. Brooks, M. A. and Lee, T. L., Determination of 4-amino-5-ethyl-3-thiopenecarboxylic acid methyl ester and its acid metabolite in plasma and urine by highperformance liquid chromatography, J. Chromatogr., 345, 333, 1985.

Elution — E-l. Hexane-ethanol (95:5).

Extraction — 1-1. To the sample are added, 100 μ ΐ of the internal standard (2.5 μ§/ιη€ of hexane), 1 m l of 1 M phosphate buffer (pH 11) and 1 m l of hexane. The mixture is mixed on a resiprocating shaker for 15 min and then centrifuged at 0 to 5°C for 10 min at approximately 1500 x g. Aliquots of 100 μ ΐ of the organic layer are injected.

a

Volume III 65

1-5

Plasma (0.05)

10 x NA

10 x 4.8

E-3

E-4

E-5

Nucleosil C 18 (5)d Hypersil ODS (3)

Elution

Spherisorb ODS (5)

Packing (μιη)

2.0

1.0

1.5

Flow (ml/min)

ABS (242)

ABS (235)

ABS (234)

Det. (nm)

4.5

8.9

12.6

RT (min)

Cyclohexanedimethanol succinate. The metabolite acetamidoglutethimide was analyzed by liquid chromatography. Absorbance is also monitored at 240 nm to check the purity of the peak. Column temp = 30°C. 2-(p-N-acetylaminophenyl)-2-methylglutamide. Conditions for the determination of minor metabolites N-formyl glutethimide and nitroglutethimide are also described.

1-4

Plasma (0.5 g)

30 x 4

Column (cm x mm)

—

e (6 .2 )

Aniline (8 .6 )

Internal standard (RT) Acetamidoglutethimide (14.8) Acetamidoglutethimide 0 2 .2 ) Acetamidoglutethimide 0 7 )f

Other compounds (RT)

5

4

3

Ref.

Extraction — 1-1. To the sample were added, 15 μ ΐ of an aqueous solution of primidone (100 μg/ml) followed by 1 m l of 0.1 M citrate buffer, pH 4 and 5 m l of dichloromethane. The tubes were mixed for 10 min, centrifuged at 800 x g for 15 min, and the organic layer evaporated in new clean dry tubes at 35°C in a stream of air. The residue was dissolved in 20 μ ΐ of methanol and 1 to 2 μ ΐ of this solution was injected. For the extraction of acetamidoglutethimide, the internal standard was 1 0 0 μ ΐ of 1 0 0 μg/ml of heptabarbitone in water, otherwise the extraction procedure was the same as for the parent drug. 1-2. The sample is treated with 0.2 m l of acetonitrile, vortexed for 15 sec, and then centrifuged at 12,500 x g for 1 min. The supernatant is decanted, recentrifuged and 2 0 μ ΐ of the clear supernatant is injected. 1-3. The sample was treated with 5 m l of 0.1 M phosphate buffer, pH 7 and 1 m l of aqueous aniline HC1 (0.1 mg/ml) and 10 m l of dichloromethane. The mixture was vortexed for 2 min, centrifuged, and the aqueous layer re-extracted with another 10-ml aliquot of dichloromethane. The combined organic phase was dried with anhydrous sodium sulfate and evaporated at reduced pressure at 60°C. The residue was disolved in methanol (0.5 m l) and 10-μΙ aliquots were injected. 1-4. The sample was treated with 1 g of water, 0.5 m l of working internal standard (1 μg/ml in 10% aqueous ethanol), and 7 m l of extraction solvent (diethyl etherdichloromethane 2:1). The mixture was shaken on a horizontal shaker for 12 min at 200 rpm and centrifuged for 5 min at 940 x g. The aqueous phase was frozen,

a b c d e f

1-3

Extraction

Urine (5)

Specimen (m l)

Liquid Chromatography

AMINOGLUTETHIMIDE (continued)

66 CRC Handbook of Chromatography: Drugs

5.

4.

2. 3.

1.

Plasma, urine ( 1)

Specimen (m l)

1-1

NA

Column Extraction (m x mm) 10% Carbowax 20 M + 2% KOH Chromosorb WAW (80/100)

Packing (mesh) 200

Oven temp (°C) N2 (12)

Gas (m€/min)

NPD

Det.

Gas Chromatography

4-A M IN O PY R ID IN E

7.5

RT (min)

—

Internal standard (RT)

—

Deriv.

—

Other compounds (RT)

1

Ref.

Adam, A. M. and Rogers, H. J ., Gas-liquid chromatographic assay of aminoglutethimide and a high-performance liquid chromatographic assay for its acetyl metabolite in biological fluids, J. Chromatogr., 307, 129, 1984. Robinson, B. A. and Cornell, F. N., Liquid chromatographic determination of aminoglutethimide in plasma, Clin. Chem., 29, 1104, 1983. Kamblawi, M. W., Stevens, R. G., and Nicholls, P. J ., High-performance liquid chromatographic assay for aminoglutethimide and its acetylated metabolite in urine, J. Chromatogr., 309, 431, 1984. Menge, G. and Dubois, J . P., Determination of aminoglutethimide and N-acetylaminoglutethimide in human plasma by high-performance liquid chromatography, J. Chromatogr., 310, 431, 1984. Schanche, J. S., Lonning, P. E ., Ueland, P. M., and Kvinnsland, S., Determination of aminoglutethimide and N-acetylaminoglutethimide in human plasma by reversed-phase liquid chromatography, Ther. Drug Monit., 6 , 221, 1984.

REFERENCES

Elution — E -l. Methanol-0.1 M citrate buffer, pH 3.4 (280:500). E-2. Acetonitrile-water (100:100) + 3 m€ Pic A (tetrabutyl ammonium phosphate reagent form waters) adjusted to pH 6.3 with phosphoric acid. E-3. Acetonitrile-0.01 M phosphate buffer. E-4. Acetonitrile-methanol-water (5:20:75). E-5. Acetonitrile-100 mM ammonium formate buffer, pH 3.5 (11:89).

the organic layer transferred to disposable tubes and evaporated to dryness in a stream of nitrogen at 40°C. The residue was dissolved in 0.2 m€ of the mobile phase and aliquots of this solution were injected with an auto injector. 1-5. Plasma was mixed with an equal volume of a solution containing acetonitrile (50% and perchloric acid (0.8 N). After centrifugation, the supernatant was collected and 25-μΙ aliquots were injected.

Volume III 67

1-4

1-5

Serum (1)

Serum (0.1)

30 x 4

25 x 4.6

15 x 3

Column (cm x mm)

1.8 x 2 N2 (20)

Gas (m€/min) ECDa

Det.

E-2

E-3

Micropak C 18 GO)

E-l

Elution

2.0

1.0

0.8

Flow (m€/min)

Liquid Chromatography

170

Oven temp (°C)

Ultrasphere C ,8 (5)b

Nucleosil C 18 (5)

Packing (pm)

3% Poly(A)-103 GasChromoQ (100/120)

Packing (mesh)

ABS (260)

ABS (263)

ABS (245)

Det. (nm)

3.3

RT (min)

3,4-Diaminopyridine (6.4) 2-Aminopyridine (4.7) —

Internal standard (RT)

Pentafluoropropionyl

Deriv.

3,4-Diaminopyridine (2.9)c

5

4

3

_

—

Ref.

2

_

Other compounds (RT)

Ref.

Other compounds (RT)

Extraction — 1-1. To the sample were added 1.3 g of anhydrous potassium carbonate, 2 m€ of saturated solution of potassium carbonate, and 4 m€ of diethyl ether. The mixture was shaken on a vortex for 3 min, centrifuged for 10 min at 4000 rpm, and 3 m€ of the upper organic layer was collected. Acid activated alumina (10 mg) was added to the 3 m€ organic layer and the mixture shaken on a rotary mixer for 10 min and centrifuged for 5 min at 4000 rpm. The solvent was discarded and the alumina pellet eluted with 100 μ€ ethanol containing (0.2 g/100 m€) NaOH. Aliquots of 2μ€ of this eluate were injected.

3.6

4

5.8

RT (min)

3-Methyl-4-amino pyridine (6 )

Internal standard (RT)

a Conditions for the use of NPD and the preparation of monochlorodifluoroacetyl derivative are also described. b Protected by a guard column (50 x 3.2 mm) packed with Vydac RP (30 to 44 μιη). c This compound is monitored separately at 290 nm.

1-3

Extraction

1-2

Column Extraction (m x mm)

Serum, saliva, urine (0.25)

Specimen (m€)

Plasma (0.2)

Specimen (m€)

Gas Chromatography

4-AMINOPYRIDINE (continued)

68 CRC Handbook of Chromatography: Drugs

Urine (10)

Specimen (m€)

1-1

0.6 x 1 (Ni)

Column Extraction (m x mm) 5% OV-101 Chromosorb G (100/ 120)

Packing (mesh) T.P.a

Oven temp (°C)

He (7)

Gas (ml/min)

MS-EI

Det.

Gas Chromatography

AMINOPYRINE

b

RT (min)

—

Internal standard (RT)

Acetyl

Deriv.

b

Other compounds (RT)

1

Ref.

1. Evenhuis, J., Agoston, S., Salt, P. J., De Lange, A. R., Wouthuyzen, W., and Erdmann, W., Pharmacokinetics of 4-amino-pyridine in human volunteers, Br. J. Anaesth., 53, 567, 1981. 2. Watson, E., Determination of 4-aminopyridine in plasma, Anal. Biochem., 113, 139, 1981. 3. Uges, D. R. A. and Bouma, P., Liquid-chromatographic determination of 4-aminopyridine in serum, saliva, and urine, Clin. Chem., 27, 437, 1981.

REFERENCES

Elution — E-l. Acetonitrile-methanol-aqueous ammonium carbonate (10 g/i) (61:35:4). E-2. 0.015 M Sodium heptanesulfonate, 0.002 M tetrabutylammonium iodide, and 0.01 M phosphate buffer, pH 3 in acetonitrile-water (7.5:92.5). E-3. Aceonitrile-0.05 M phosphate buffer containing 0.02 M tetramethylammonium chloride, pH 7.4 (60:40).

1-2. To 200 μ i of plasma were added 120 ng of the internal standard, 0.3 m i of 0.5N NaOH, and 2.5 m i of dichloromethane-isopropanol (90:10). The tubes were vortexed for 2 min, centrifuged, and approximately 2 m i of the organic layer was collected in tubes containing 1 0 0 μ€ of ascorbic acid solution (0 .0 2 % in methanol) and evaporated to dryness under nitrogen at 45°C. The residue was dissolved in 50 μ ΐ of ethyl acetate and 20 μ€ of pentafluoropropionic anhydride were added. The tubes were allowed to stand at room temperature for 30 min and the solvents removed with nitrogen. The residue was dissolved in 300 μ i of ethyl acetate and 1 to 2 μ€ were injected. 1-3. Vortex-mix 0.25 m i of the sample and about 300 mg of potassium carbonate for about 10 sec, then add 0.1 m i of internal standard (40 mg/100 m i of dichloromethane). Vortex-mix the tubes for 1 min and centrifuge at 3000 rpm for 5 min. Collect the organic layer, add 50 \Li of 1-pentanol and evaporate at reduced pressure at 25°C. Reconstitute the oily residue in 50 μ ΐ of the mobile phase and inject 50 μ ί onto the column. 1-4. The sample after the addition of 0.1 m i of the internal standard (25 μg/m€ in water) was made alkaline (pH 12) with 20 μ i o i l N NaOH and extracted with 10 m i of dichloromethane on a rotary mixer for 30 min. After centrifugation for 5 min at 3000 x g, the organic layer was collected, 0.1 m i of 2 M trifluoroactic acid in methanol added, and after vortex mixing the solvent was evaporated under a stream of nitrogen at 40°C. The residue was dissolved in 0.1 m i of 10% acetonitrile by ultrasonification for 5 min and centrifuged. A 50-μ€ aliquot of the solution was then injected. 1-5. To 100 μ i of serum were added 200 μ ΐ of methanol. The mixture was vortex mixed for 30 sec, centrifuged for 5 min at 2600 x g and a 10-μ€ portion of the supernatant was injected.

V o lu m e III 69

* b c d e

ABS (260)

ABS (257)

ABS (254)

Det. (nm)

37.1

24.9

19.8

RT (min)

Initial = 100°C; rate = 20°C/min; final = 310°C. Retention indices of aminopyrine, its metabolites and a number of other analgesics and their metabolites are given. Polystyrenedivinylbenzene. Column temp = 30°C. A precolumn (50 x 4.6 mm) packed with LiChroprep RP-8 (25 to 40 μιη) replaces the injector loop.

0.7

E-3

ODS-120A (5)

25 X 4.6

1-4

Plasma (0 . 1 )

NA

E-2; grad

LiChrosorb RP-18 (5)

25 X 4

I-3ef

Plasma (0.05— 1)

1.2

Elution

Flow (mf/min)

E-l

Hitachigel 30 lO0 (2 0 )d

50 x 2.1

1-2

Extraction

Packing (pm)

Column (cm x mm)

Urine (10)

Specimen (m€)

Liquid Chromatography

AMINOPYRINE (continued)

Other compounds (RT)

Pyrrole-2Acetylaminopyrine carboxylic (3.4) acid (2.5) 4-Aminoantipyrine (7.3) 4-Monomethyl aminoantipyrine ( 1 2 . 1) FormylaminoantiIsopropylam pyrine (12.4) inoanti4-Acetylaminoantipyrine pyrine (13.8) (28)f 4-Aminoantipyrine (19.8) 4-Methylaminoantipyrine (22.7) Pheno4-Aminoantipyrine bar(26) bital 4-Formylaminanti(42.1) pyrine (12.3) 4-Acetylaminoantipyrine (13.8) 4-Methylaminantipyrine (23.7)g

Internal standard (RT)

4

3

2

Ref.

70 CRC Handbook of Chromatography: Drugs

On column sample preparation was compared to solvent extraction using isopropylan tipyrine as the internal standard. A number of additional metabolites have also been detected in plasma.

4.

3.

2.

1.

8

REFEREN CES

min isocratic at 13% (B), 8

to 30 min from 13 to 43%(B) (linear).

Maurer, H. and Pfleter, K., Screening procedure for detecting anti-inflammatory analgesics and their metabolites in urine using a computerized gas-chromat­ ographic-mass spectrometric technique, Fresenius Z. Anal. Chem., 314, 586, 1983. Shimada, K. and Nagase, Y., Quantitative high-performance liquid chromatographic determinations of aminopyrine and its metabolites in man, J. Chromatogr., 181, 51, 1980. Voelter, W., Kronbach, T., Zech, K., and Huber, R., A simple high-performance liquid chromatographic precolumn technique for investigation of drug metabolism in biological fluids, J. Chromatogr., 239, 475, 1982. Miyagi, N., Hikichi, N., and Niwa, H., Simultaneous determination of aminopyrine and its metabolites in rat plasma by high-performance liquid chromatography, J. Chromatogr., 375, 91, 1986.

Elution — E-1. Methanol-water (1:1). E-2. (A) 20 mM NaH2P 04, pH 8 , (B) acetonitrile: gradient: from 0 to E-3. Methanol-0.05 M phosphate buffer, pH 3.9 (30:70).

Extraction — 1-1. Ten milliters of urine were refluxed with 3 m l of 37% HC1 for 15 min, then made basic with about 3 g of KOH and mixed with 10 m l of 30% aqueous ammonium sulfate to obtain a pH between 8 and 9. The samples were extracted twice with 10 m l each of a mixture of 2 parts of dichloromethane, 2 parts of isopropanol, and 6 parts of ethyl acetate. After centrifugation, the combined residue was treated with 40 μ ΐ of a mixture of 3 parts of acetic anhydride and 2 parts of pyridine and incubated for 30 to 60 min at 60°C. An aliquot of 1.4 μ ΐ of this mixture was injected. 1-2. Ten milliters of urine was brought to pH 9 with 1 N NaOH which was then concentrated to a small volume and extracted successively with 30 m l each of ethyl acetate, chloroform, and ethyl ether. The combined extracts were dehydrated over anhydrous sodium sulfate. Pyrrole-2-carboxylic acid (250 μg as an ethanolic solution) was added to the dried extract which was then evaporated in vacuo. The residue was dissolved in 50 μ ΐ of methanol and 1.2 μ ΐ aliquot of this solution was injected into the chromatograph. 1-3. The precolumn was purged in the load position with 1 m l of water, 50 to 1000 μ ΐ of the plasma sample was loaded on the precolumn and washed with 1 m l of water. The sample was injected by changing precolumn to inject position and precolumn remained in this position until analysis was completed. 1-4. Plasma (0.1 m l) was extracted with 5 m l of chloroform by shaking for 30 min and centrifugation for 10 min. A 4-m l aliquot of the organic layer was collected. The pH of the remaining mixture was adjusted to 3.5 with 0.1 M HC1 and the extraction was repeated. A 4-ml aliquot of chloroform was collected and the combined extracts were evaporated to dryness. The residue was reconstituted in 0.1 m l of the mobile phase containing the internal standard (300 μg/ml) and 20-μΙ aliquots were injected.

8

f

Volume III 71

25 x 3.2

25 x 4.6

1-1

1-2

Extraction

Column (cm x mm) LiChrosorb C 18 (10) Datasorb ODS (NA)

Packing (pm)

E-2

E-l

Elution

1.5

1.0

Flow (ml/min) FI (270, 385) ABS (254)

Det. (nm)

7

7

RT (min) Anthranilic acid (15) Aceta­ minophen (6 ) Sulfani­ lamide (3.5)

Internal standard (RT)

Ref. 1

2

Other compounds (RT) — Metaminophenol (4.8)

1. Honigberg, I. L., Stewart, J. T., Clark, T. C., and Davis, D. Y., Non-extractive fluorometric measurement of p-aminosalicylic acid in plasma by ion-pairing techniques and high-performance liquid chromatography, J. Chromatogr., 181, 226, 1980. 2. Melvin, J. L., Bailey, L. C., and Medwick, T., Simultaneous dosage-form analysis of p-aminosalicylic acid and its degradation product, m-aminophenol, by high-performance liquid chromatography, J. Chromatogr. Sci., 19, 146, 1981.

REFERENCES

Elution — E-l. Methanol-water (20:80) containing 0.005 M tetrabutylammonium hydroxide and 0.01 M disodium acid phosphate, pH 5.5. E-2. Methanol-tetrabutylammonium hydroxide (1 M in methanol)-phosphate buffer (15:150:850). Final pH = 7.2.

Extraction — 1-1. The sample (100 μ ΐ) was treated with 100 μ ΐ of methanol. The mixture was vortexed for 1 min and centrifuged at 3000 rpm for 15 min. An aliquot of 50 μ ΐ of the supernatant was mixed with 100 μ ΐ of the mobile phase containing 25 μg/ml of anthranilic acid as the internal standard. After mixing 50 μ ΐ of the solution was injected. 1-2. An accurately weighed portion of the powdered tablets is placed in a 100 m l volumetric flask. Methanol (15 ml) and 1 m l of sulfanilamide solution (0.5 mg/ m l of methanol) are added. The mixture was shaken thoroughly for 5 min and the volume was made up to the mark. After mixing, the solution was filtered and 10 m l of the filtrate was transferred to a second 100 m l volumetric flask, to which 5 m l of acetaminophen (1 g/100 m l of methanol) was added and the volume made up to mark with the mobile phase.

Plasma (0 . 1 ) Dosage

Specimen (ml)

Liquid Chromatography

p-AMINOSALICYLIC ACID

72 CRC Handbook of Chromatography: Drugs

Serum ( 1) Serum (0.5) Plasma (0.5) Serum, red cells (0.25) Plasma (0.5) Plasma, urine, bile (0 . 1 ) Serum ( 1)

(1)

Serum ( 1) Serum (0.5) Serum ( 1) Plasma, urine, saliva ( 1) Plasma, tissue ( 1) Plasma, urine, tissues

Specimen (m€)

30 x 4

25 X 4.6

12.5 x 5 x

1-3

1-4

1-5

1-6

30 x 3.9

30 x 4

25 x 4

15 x 4.6

15 x 4.6

30 x 3.9

15 x 4.6

1-7

1-8

1-9

1 -1 0

1-11

1-1 2

1-13

8

30 x 4

1-2

10

30 x 3.9

1-1

Extraction

Column (cm x mm)

Ultrasphere-octyl (5)

μ-Bondapak-Qg (10) Micropak-NH2 ( 1 0 )e,f LiChrosorb SI-100 (5) Supelcosil LC-18 DB (5) Supelcosil LC-18 DB (5)h μ -Bondapak-Cjg ( 1 0 )*

Spherisorb Si (5) Radial-Pak-C18 ( 1 0 )d

Micropack-C18 (10) LiChrosorb RP8 (10)

μ-Bondapak-Qg ( 10) μ-Porasil (10)

Packing (pm)

1.5

2 .0

E-13

0 .8

E-l 1 E-12

NA

E-10

1.5

E-8 2 .0

1.3

E-7

E-9

2 .0

2 .0

1.5

a

0 .8

2 .0

E- 6

E-5

E-4

E-3

E-2

E-l

Elution

Flow (ml/min)

Liquid Chromatography

AMIODARONE

ABS (240)

ABS (254) ABS (248) ABS (242) ABS (240) ABS (254) ABS (244)

ABS (240) ABS (242)

ABS (254) ABS (254) ABS (242)b ABS (243)

Det. (nm)

L 8040 (3) L 8040 (9.4)

4 6.3

L 8040 (6 .8 ) Trifluo-perazine (5.4)

7.2

L 8040 (6.5) L 8040 (6 )

L 8040 (4.6) g (2.9)

5.5

5.6

4.5

5.8

6 .2

10.9

—

Imipramine (4.4)

9.2

8 .1

L 8040 (10) c (10.3)

—

Internal standard (RT)

8 .2

2 .2

RT (min)

13

12

11

10

9

8

Desethyl-amiodrone 14 (6.4)

Desethyl-amiodarone (3.5) Desethyl-amiodarone (5.2) Desethyl-amiodarone (4.6)

Desethyl-amiodarone (9.2) Desethyl-amiodarone —

7

5,

4

Desethyl-amiodarone (6 .8 ) Desethyl-amiodarone (2.5) Desethyl-amiodarone (5.3)

3

2

1

6

Ref.

—

—

—

Other compounds (RT)

Volume III 73

15 x 3.2

15 x 4.6

1-14

1-15

Extraction

Column (cm x mm) Spherisorb-octyl (5) Ultrasphere-octyl (5)

Packing (pm)

E-15

E-14

Elution

2.0

0.9

Flow (ml/min) ABS (254) ABS (240)

Det. (nm)

11.8

9.7

RT (min)

4

L 8040 (11.9) g (6.5)

Internal standard (RT) Desethyl-amiodarone (6.9) Desethyl-amiodarone (8 .2 )

Other compounds (RT)

16

15

Ref.

Extraction — 1-1. The sample was mixed with 2 vols of 96% ethanol for 20 sec on a whirl mixer and allowed to stand for 15 min. After centrifugation, 20 p i of the supernatant was injected. 1-2. The sample was mixed with 1 m l of 0.5 pg/m l of the internal standard solution in 0.2 M acetate buffer, pH 3.8 and extracted with 5 m l of π-hexane. The organic extract was evaporated at 60°C under a gentle stream of nitrogen. The residue was dissolved in 50 p i of the mobile phase and 20 p i was injected. 1-3. The sample was spiked with an appropriate volume of the methanolic solution of the internal standard (20 pg/m l), and 5 m l of 0.2 M acetate buffer, pH 3.6 and was extracted twice with 5-ml aliquots of diethyl ether. The combined ether layers were evaporated at 56°C under nitrogen. The residue was dissolved in 160 p i of methanol and aliquots of this solution were injected. 1-4. The sample was spiked with 10 p i of a 0.1 mg/ml aqueous solution of imipramine hydrochloride, the pH of the mixture adjusted to 6 by adding 1 m l of phosphate buffer and extracted with 8 m l of isopropyl ether. The organic layer was evaporated at 50°C under a stream of nitrogen. The residue was reconstituted in 60 p i of acetonitrile and 2 0 p i were injected. 1-5. The sample was spiked with 100 p i of a 0.2 mg/l solution of the internal standard in 2 M aqueous sodium dihydrogen ortho-phosphate containing 2 g /l human serum albumin, pH 4.5 and extracted with 1 m l of disisopropyl ether. The organic phase was evaporated with a stream of compressed air. The residue was dissolved in 1 2 0 p i of methanol and 1 1 0 p i of the solution was injected to fill a loop of 1 0 0 p i. 1-6. Plasma sample (1ml) was treated with 2 m l of the solution of the internal standard (5 pg/ml) in acetonitrile. After mixing and centrifugation 50 p i of the supernatant were injected. (Different procedures are described for the extraction of urine and tissue samples.)

Linear gradient 0.1 ml/min; initial = 1 ml/min; final 2 ml/min. b Conditions for normal phase chromatography are also described. In this mode absorbance is monitored at 255 nm. c 3-(3,5-Dibromo-4-p-dibutylaminopropoxy benzoyl)benzothiophene. d Protected by a guard column packed with Corasil-C18 (37-50 pAf). e Protected by a 4 x 0.4 cm precolumn packed with 40 pm silica. f Column temp = 30°C. g 2-Ethyl-3-benzofuranyl-[4-(2-(dimethylamino)-propoxy)3,5-diiodophenyl]methanone. h Column temp = 50°C. * Protected by a 5 x 0.32 cm precolumn packed with CO:Pell ODS (30 to 38 pm).

Serum (0.1) Plasma (0.5)

Specimen (ml)

Liquid Chromatography

AMIODARONE (continued)

74 CRC Handbook of Chromatography: Drugs

Elution — E -l. Methanol-0.01 Af phosphoric acid-acetic acid (84:14:2). E-2. Chloroform-methanol-ammonium hydroxide (98.95:1.00:0.05). E-3. Methanol-1 % ammonium hydroxide (99:1). E-4. Acetonitrile-1% acetic acid, pH 4.7 with NH4OH (85:15). E-5.Methanol-diethyl ether (85:15) containing 0.03% perchloric acid. E-6 . Methanol-25% ammonia (999.3:0.7). E-7. Methanol-water-ammonium hydroxide (89.7:10:0.3). E-8 . Dichloromethane-hexane-methanol-acetic acid-0.117 N perchloric acid in methanol (50:39:6:1:1). E-9. Methanol-diethyl ether (70:30) containing 0.02% perchloric acid. E-10. Acetonitrile-ammonium acetate, pH 6 . 8 (2:8). E -ll. Acetonitrile-10 mM KH2P0 4 containing 20 mM tetramethylammonium chloride - 85% phosphoric acid (90:9:1). E -l2. Methanol-water-58% ammonium hydroxide (94:4:2). E-13. Acetonitrile-methanol-0.01 M ammonium acetate (45:45:10).

1-7. The sample was treated with 30 μ£ of 1.2 M HC1 and 2 m£ of absolute ethanol. After vortex mixing and centrifugation, 50 μ£ of the supernatant were injected. 1-8. The sample was spiked with 10 μ£ of the internal standard solution (100 μg/m€) in methanol, 0.2 m£ of 3 M sodium acetate buffer, pH 4.8 added and extracted with 5 m£ of hexane. The organic extract was evaporated and the residue dissolved in 65 μ£ of the mobile phase. From this solution 35 to 40 μ£ was used to load a 25-μ£ loop. 1-9. To the sample were added 2 m£ of 0.2 M acetate buffer (pH 3.8) and 20 μ£ of the internal standard solution (50 μg/m€) in methanol and the mixture was extracted twice with 4 m£ portions of diethyl ether. The combined ether extract was evaporated under vacuum at room temp. The residue was dissolved in 500 μ£ of the mobile phase and 50 μ£ were injected with an autosampler. 1-10. The sample was mixed with an equal volume of phosphate buffer (pH 5.4) and extracted with 2 m£ of isooctane-isopropyl alcohol (85:15) containing 500 ng/ m£ of the internal standard. The organic extract was evaporated at 60°C under a nitrogen stream. The residue was dissolved in 200 μ£ of the mobile phase and 30 μ£ of this solution were injected. 1-11. The sample was mixed with 100 μ£ of 2 M phosphate buffer (pH 6 ) and extracted with 3 m£ of methyl-f-butyl ether containing 330 μg/€ of the internal standard. The organic layer was evaporated at 30°C under nitrogen. The residue was dissolved in 1 m£ of acetonitrile and 200-μ€ aliquots of this solution were injected. 1-12. The sample was treated with 200 μ£ of the internal standard solution (1 μg/m€) in acetonitrile. After mixing and centrifugation 200 μ£ of the supernatant was injected. (A separate extraction procedure for urine and bile is described.) 1-13. The sample was mixed with 20 μ£ of the internal standard solution (100 mg/£) in methanol, 0.5 m£ of 1 M sodium acetate buffer, pH 5.5, and was extracred with 5 mL of hexane. The organic phase was evaporated under nitrogen. The residue was dissolved in 250 μ£ of methanol and 25 μ£ of this solution were injected. 1-14. The sample and 100 μ£ of the internal standard solution (2 mg/€) in methanol-water (2:8) and 0.5 m£ of water were applied to a prewashed (lm < methanol, 3 m£ water) l-m£ Baker-CN column. The sample mixture was allowed to pass through the column by gravity. The column was then washed three times in water; one time in methanol-water (1:1), and finally eluted with 0.5 m£ of methanol containing 10 m£/£ of triethylamine. The eluate was evaporated at 37°C under nitrogen and the residue was reconstituted with 100 μ£ of acetonitrile-buffer (40:60). Aliquots of 20 μ£ of this solution were injected in duplicate. 1-15. The sample was mixed with 1 m£ of 0.25 M phosphate buffer, pH 6.5, 100 μ£ of the internal standard solution (20 mg/€) in methanol, and extracted with 7 m£ of pentane. The organic layer was evaporated at 40 to 45°C. The residue was dissolved in 100 μ£ of the mobile phase and 10 to 20 μ£ of this solution were injected.

Volume III 75

16.

15.

14.

13.

12.

11.

10.

9.

8.

7.

6.

5.

4.

3.

2.

1.

Andreasen, F., Agerbaek, H., Bjerregaard, P., and Gotzsche, H., Pharmacokinetics of amiodarone after intravenous and oral administration, Eur. J. Clin. Pharmacol., 19, 293, 1981. Lesko, L. J ., Marion, A., Canada, A. T., and Haffajee, C., High-pressure liquid chromatography of amiodarone in biological fluids, J. Pharm. Sci., 70, 1366, 1981. Cervelli, J. A., Kerkay, J ., and Pearson, K. H., Quantative serum amiodarone determination by high performance liquid chromatography, Anal. Lett., 14, 137, 1981. Gobbato, S., Padrini, R., Candelpergher, G., Bettero, A., Cargnelli, G., and Ferrari, M., Monitoring of amiodarone levels in various body fluids, Int. J. Clin. Pharm. Res., 2, 279, 1982. Storey, G. C. A. and Holt, D. W., High-performance liquid chromatographic measurement of amiodarone and desethylamiodarone in plasma or serum at the concentrations attained following a single 400-mg dose, J. Chromatogr., 245, 377, 1982. Storey, G. C. A., Adams, P. C., Campbell, R. W. F., and Holt, D. W., High-performance liquid chromatographic measurement of amiodarone and desethylamiodarone in small tissue samples after enzymatic digestion, J. Clin. Pathol., 36, 785, 1983. Plomp, T. A., Engels, M., Robles De Medina, E. O., and Maes, R. A. A., Simultaneous determination of amiodarone and its major metabolite desethyla­ miodarone in plasma, urine and tissues by high-performance liquid chromatography, J. Chromatogr., 273, 379, 1983. Brien, J. F., Jimmo, S., and Armstrong, P. W., Rapid high-performance liquid chromatographic analysis of amiodarone and N-desethyl-amiodarone in serum, Can. J. Physiol. Pharmacol., 61, 245, 1983. Mostow, N. D., Noon, D. L., Myers, C. M., Rakita, L., and Blumer, J. L., Determination of amiodarone and its N-deethylated metabolite in serum by high-performance liquid chromatography, J. Chromatogr., 277, 229, 1983. Duranti, L., Caracciolo, M., and Oriani, G., New, accurate semiautomatic high-performance liquid chroamtographic method for routine monitoring of amiodarone plasma levels, J. Chromatogr., 277, 401, 1983. Heger, J. J ., Solow, E. B., Prystowsky, E. N., and Zipes, D. P., Plasma and red blood cell concentrations of amiodarone during chronic therapy, Am. J. Cardiol., 53, 912, 1984. Shipe, J. R., Liquid-chromatographic determination of amiodarone and its desethyl metabolite in plasma, Clin. Chem., 30, 1259, 1984. Weir, S. J. and Ueda, C. T ., Rapid liquid chromatographic assay for the determination of amiodarone and its N-deethyl metabolite in plasma, urine, and bile, J. Pharm. Sci., 74, 460, 1985. Muir, K. T. and Kook, K. A., Analysis of amiodarone and desethyl-amiodarone in serum and tears by reversed-phase high-performance liquid chromatography, J. Chromatogr., 374, 394, 1986. Poliak, P. T ., Carruthers, S. G., and Freeman, D. J., Simplified liquid-chromatographic assay of amiodarone and desethylamiodarone after solid-phase extraction, Clin. Chem., 32, 890, 1986. Gupta, R. N., Lew, M ., and Stefanec, M ., unpublished observations.

REFERENCES

E-14. Acetonitrile-10 mM phosphate buffer, pH 3.5 (62:38) containing 0.5 m€ of triethylamine/€. E-15. Acetonitrile-water-70% perchloric acid-20% tetramethylammonium hydroxide in methanol (550:450:0.5:0.5).

AMIODARONE (continued)

76 CRC Handbook of Chromatography: Drugs

1-6

1-7

Plasma ( 1— 8 )

Brain tissue (2 0 0 mg)

1 X 2

1 X4

1 X 2

15 X 0.32

1-4

1-5

1 X 2

1-3

Serum ( 1)

25 X 0.31

1-2

Plasma (2 ) Plasma ( 1) Plasma ( 1)

1.2 X 1.8

1-1

Column Extraction (m x mm)

Plasma ( 1)

Specimen (m i)

2% OV-17 ChromosorbW (80/100) 10% OV-17 GasChromQ (80/100) 3% OV-17 Chromosorb W ( 10 0 / 2 0 0 )

Supelcoport (100/ 120) DB-5e

3% OV-17 Gas ChromQ (100/ 120) OV-101

Packing (mesh)

Gas (ml/min) Det.

n2

230

230

235

NA

(75)

n2

Heh

(17) T.P.f He*

240

NPD

FID

MS-EI

NPD

NPD

ECD Ar:95Methane:5 (30) T.P.b Hec MS-EI 310

Oven temp (°C)

Gas Chromatography

AMITRIPTYLINE

4.2

6

2

5.1

2 .6

6

3

RT (min)

Maprotiline 00.3)

N-7084* (5)

N-7084 (8.5) Clomipramine (5) Protriptyline (5.4) Chlorprothixene (7.7)

Clomipramine (5.5)

Internal standard (RT)

—

—

Nortriptyline (5)

Nortriptyline (6.9)k

Nortriptyline (5.2) lO-Hydroxy-(E) amitriptyline (6 .2 ) lO-Hydroxy-(E) nortriptyline (6.4) 10-Hydroxy (Z) nortriptyline (6.5) Trifluoro- 10-Hydroxy-amiacetyl· triptyline» (2.5)

—

2,4-Dich- Nortriptyline3 (NA) lorophenyl Trifluoro- Nortriptyline*1 (7.5) acetyl — Nortriptyline (3)

Deriv.

Other compound (RT)

7

6

5

4

3

2

1

Ref.

Volume III 77

30 X 3.9

1-10

1-11

Plasma, serum (2 )

Plasma ( 1)

Supelco LC-1 (5)

μ-Bondapak-Cu (10)

25 X 2

1-9

Blood ( 1)

25 X 4.6

μ-Bondapak-Cjg (10) MicroPak Silica (5)

30 X 3.9

1-8

Extraction

Packing (urn)

Column cm x mm

Brain tissue

Specimen (m€)

0.9

1.8

E-4

1.5

1.0

E-3

E-2

E-l

Elution

Flow m€/min

Liquid Chromatography

ABS (254)

ABS (254)

ABS (254) ABS (2 2 0 )

DET (nm)

AMITRIPTYLINE (continued)

19

6 .1

3

NA

RT min

Loxapine (13)

Diazepam (7.6)m

(NA) Propanolol (5)

1

Internal standard (RT)

Nortriptyline(9) 10-Hydroxyamitriptyline (4) 10-Hydroxy-c/snortriptyline (12) 10-Hydroxy-irawsnortriptyline (14) Nortriptyline (5.6) Amitriptyline oxide (5.2)m Trans- 10hydroxy nortriptyline (5) Trans- 10hydroxy amitriptyline (5.8) Cis-10-hydroxynortriptyline (6.4) Cis- 10-hydroxy amitriptyline (7)

—

Other compounds (RT)

11

10

9

8

Ref.

78 CRC Handbook of Chromatography: Drugs

1-12

10x3

LiChrosorbSI60 (5)

E-5

1.2

ABS (240)

1.5

Perazine (2.8)

Nortriptyline (16.5) E/Z-10-Hydroxy amitriptyline (1.7) Desmethyl-nortriptyline (2.3) Nortriptyline (3.9) E-1O-Hydroxy-nortriptyline (5.3) Amitriptyline-N-oxide (5.4) Z-1O-Hydroxy-nortriptyline (5.9) 12

Extraction — 1-1. Plasma (1 m l) in a small centrifuge tube is mixed with 1 m l of a phosphate buffer solution, pH 6.0 containing 35 ng/ml of chloroimipramine and 1 M sodium iodide, and 2 m l of 3 mM solution of 2,4-dichlorophenyl chloroformate. The mixture is shaken for 10 min, centrifuged for 2 min and the organic phase is transferred to another tube containing 0.5 m l of toluene. The organic phase is reduced to about 0.5 m l by evaporation. Alcoholic alkali (0.5 m l containing 2.8 g of KOH/100 m l of methanol-water 75:25) is added and after 5 min, 2 m l of 2 M sodium hydroxide is added and the tubes vigorously shaken for 30 sec. A few microliters of the organic phase is injected. 1-2. Plasma (2 m l) was dialysed for 3 hr against 0.15 M phosphate buffer, pH 7.4 at 37°C. To 1 m l of dialysate were added 50 ng each of N-7084 and maprotiline as internal standards, 2 m l of carbonate buffer, and finally 100 μ ΐ of toluene/isoamyl alcohol 85:15). The mixture was agitated for 15 min, centrifuged and the organic phase evaporated in a current of nitrogen. The residue was dissolved in 50 μ ΐ of toluene, 10 μ ΐ trifluoroacetic anhydride was added and the mixture incubated

a Nortriptyline is removed by prior extraction in the presence of trichloroethylchloroformate and analyzed separately. b Initial temp = 160°C; rate = 30°C/min; final temp = 270°C. c Column pressure = 0.75 kg/cm2. d Maprotiline was used as the internal standard for quantitation of nortriptyline. e Equivalent to SE-54. f Initial temp = 120°C; rate = 32°C/min; final temp = 250°C. 8 Column pressure = 69 kPa (linear velocity = 48 cm/sec). h Column pressure = 0.9 kg/cm2. 4 During trifluoroacetylation 10-hydroxy amitriptyline is dehydrated. j Conditions for the analysis of nortriptyline and 10-hydroxynor-triptyline are also described. k Conditions for the determination of hydroxy metabolites as dehydro-compounds are described. 1 2-(Dibenz-[b3]-azepin-5-yl)-N-methylamine. m Different extraction and chromatographic conditions for the analysis of amitriptyline-N-oxide and the internal standard diazepam. No internal standard is used for the analysis of amitriptyline.

Plasma (1)

Volume III 79

for 1 hr at 62°C without allowing the solvent and reagent to evaporate. After cooling 1 m l of carbonate buffer (pH 9.4) was added and the solution extracted into 50 μ ΐ of toluene/isoamyl alcohol (85:15). 1-3. To 1 m l of plasma were added, 500 ng of clomipramine in aqueous solution, and 0.4 m l of 5% NaOH. The tubes were shaken for 45 min with 10 m l of heptane containing 1.5% isoamyl alcohol. After centrifugation for 5 min, the organic phase was transferred to another tube, shaken for 15 min with 1.5 m l of 0.1 N H2S 04, and centrifuged for 2 min. The organic layer was discarded, the aqueous phase made alkaline with 0.1 m l of 6 N NaOH and extracted for 15 min with 0.5 m l of toluene containing 15% isoamyl alcohol. The organic phase was collected and evaporated under a stream of nitrogen. The residue was dissolved in 20 μ ΐ of heptane containing 20% isoamyl alcohol. Aliquots of 2 μ ΐ were injected. 1-4. To 1 m l of plasma were added, 1 0 0 -μ Ι aliquot of internal standard solution of chloroprothexene and protripyline (0.5 μg/ml in methanol of each), 1 m l of 4 Af NaOH and 8 m l of hexane-2-butanol (98:2). This mixture was mixed (2 min), centrifuged (2 min), and the organic layer transferred to another tube. One milliliter of 0.001 Af HC1 was added, the mixture mixed (2 min) and centrifuged (2 min). The organic layer was discarded, the aqueous layer transferred to a 15-ml conical tipped tube containing 0.5 m l of 4 AfNaOH and the solution was mixed for 10 sec. A 100-μΙ aliquot of n-butyl acetate was added and the mixture mixed (2 min), centrifuged (2 min) and 0.5 to 0.8 μ ΐ of the organic layer was injected. 1-5. To 1-ml aliquot of the sample were added 200 μ ΐ of aqueous solution containing the internal standard-1 (1.5 μg/ml), 200 μ ΐ of internal standard-2 in n-hexane (0.4 μg/ml) and 0.1 m l of 5 Af NaOH. The serum was then extracted with 6 m l of n-hexane by shaking for 10 min. After centrifugation at 1300 x g for 10 min, 5 m l of the hexane layer was evaporated to dryness. To the resdue were added 100 μ ΐ of 0.1% triethylamine in n-hexane and 50 μ ΐ of trifluoroacetic anhydride. The mixtrue was incubated at 50°C for 10 min, the solvent was evaporated and the residue vortexed with 0.1 m l of benzene and 0.1 m l of distilled water and centrifuged. An aliquot of the resulting benzene layer was injected into the GC-MS system. 1-6. A 1-ml volume of a solution prepared by diluting 125 m l of 4 Af NaOH and 10 m l of diethylamine to 200 m l was pipetted into a 35-ml tube. To the sample (1.8 ml) in a polystyrene tube was added, 1 m l of 0.01 Af HC1 containing 1 μg of maprotiline as internal standard and was transferred quantitatively to a 35-ml tube. Then 10 m l toluene, 0.1 m l amyl alcohol, and water added so as to nearly fill the 35-ml tube. After mixing for 30 min on a rotary tumbler at 30 rpm, the tube was centrifuged for 15 min at 2000 x g. The toluene layer was transferred to a 15-ml tube. After adding 2 m l of 0.5 Af sulfuric acid, the tube was tumbled at 30 rpm for 15 min and centrifuged. Organic layer was discarded, the aqueous layer was washed with pentane. Then 0.1 m l of 1 Af HC1 was added to the aqueous layer and heated at 1 bar excess pressure for 70 min. After cooling to room temperature, 1 μg of desmethylclomipramine in 0.1 m l of 0.01 Af HC1 was added. The solution was made alkaline with 0.3 m l of a solution prepared by diluting 100 m l of 4 Af NaOH and 45 m l of diethylamine to 300 m l of water and extracted with 50 μ ΐ of heptane containing diethylamine and 250 ng of tetracosane. 1-7. Brain tissue (200 μg) was homogenized in about 5 volumes of 0.1 Af HC1. The homogenates were centrifuged at 50,000 x g for 15 min and to the supernatant 20 μ ΐ of 5 Af NaOH was added to adjust the pH to 11.5. The mixture was extracted with 5 m l of heptane-isoamyl alcohol (9:1). The organic layer (4 ml) was transferred to another tube and extracted with 0.2 m l of methanol-0.1 Af HC1 (1:1). The organic layer was discarded and the aqueous layer washed with heptaneisoamyl alcohol, made alkalline with 20 μ ΐ of 4 Af NaOH and extracted into 0.8 m l of heptane-isoamyl alcohol. The organic layer was collected and evaporated under a stream of nitrogen. The residue was dissolved in 50 μ ΐ of heptane/isoamyl alcohol and 2 μ ΐ were injected. 1-8. The harvested brains were weighed and homogenized in 4 m l of water. A 2-ml sample of the homogenate was spiked with 100 μ ΐ of 1 μg/m l internal standard solution, 200 μ ΐ of 1.5 N NaOH, and extracted into 4 m l of hexane-isoamyl alcohol (99:1) by gentle shaking for 60 min. The organic layer was collected and back extracted into 200 μ ΐ of 0.1 Af perchloric acid by shaking for 30 min. The organic layer was discarded and an aliquot for 25 μ ΐ of aqueous layer was injected. 1-9. To 1-ml of hemolyzed blood were added 10 μ ΐ solution of the internal standard (10 μg/ml of methanol), 1 m l of water, 100 μ ΐ of 5 Af NaOH, and 6 m l of hexane-n-butanol (98:2). The mixture was shaken for 10 min followed by centrifugation at 2800 rpm for 5 min. The aqueous phase was frozen in dry ice-acetone

AMITRIPTYLINE (continued) 80 CRC Handbook of Chromatography: Drugs

1. Karlsson, K. E ., Two-phase derivatization of amitriptyline and structurally related tertiary amines for gas chromatography with electron-capture detection, J. Chromatogr., 219, 373, 1981. 2. Baumann, P., Koeb, L ., Tinguely, D., and Rivier, L., A method for the analysis of free, total plasma and saliva amitriptyline and nortriptyline by dialysis and gc/ms, Eur. J. Mass Spectrom. Biochem. Med. Environ., 2, 19, 1982. 3. Hals, P. A., Lundgren, T. I., and Aarbakke, J ., A sensitive gas chromatographic assay for amitriptyline and nortriptyline in plasma, Ther. Drug Monitor., 4, 365, 1982. 4. Jones, D. R ., Lukey, B. J., and Hurst, Η. E., Quantification of amitriptyline, nortriptyline, and 10-hydroxy metabolite isomers in plasma by capillary gas chromatography with nitrogen-sensitive detection, J. Chromatogr., 278, 291, 1983. 5. Ishida, R ., Ozaki, T ., Uchida, H., and Irikura, T., Gas chromatographic-mass spectometric determination of amitriptyline and its major metabolites in human serum, J. Chromatogr., 305, 73, 1984. 6 . Burch, J. E ., Roberts, S. G ., and Raddats, M. A., Amitriptyline and its basic metabolites determined in plasma by gas chromatography, J. Chromatogr., 308, 165, 1984. 7. Hsu, L. L., Regional distribution of amitriptyline in rat brain: determination by gas chromatography, IRCS Med. Sci., 13, 252, 1985. 8 . Preskorn, S. H. and Glotzbach, R. K., A liquid chromatographic method for quantitating amitriptyline in brain tissue, Psychopharmacology, 78, 23, 1982.

REFERENCES

Elution — E-l. Acetonitrile-perchlorate solution (44:56). Perchlorate solution was prepared by mixing 0.005 M perchloric acid with 0.045 M sodium perchlorate (9:1). E-2. Acetonitrile-methanol-aqueous ammonium hydroxide (93:7:0.4). E-3. Acetonitrile-0.6 % potassium dihydrogen phosphate (50:50). E-4. Acetonitrile-0.1 M acetate buffer (30:70) with 0.005 M heptanesulfonate and 0.01 M triethylamine added. E-5. Acetonitrile-methanol-ammonium hydroxide (1 M) (250:55:13).

bath, the organic layer was poured into new tubes with elongated cones at its base, and evaporated to dryness at 40°C under vacuum on a vortex evaporator. The residue was dissolved in 1 0 0 μ€ of the mobile phase, vortexed, centrifuged, and all of the solution was injected. 1-10. The sample (2 mf) was made alkaline with 0.2 m€ of 0.5 N NaOH and extracted with 10 m€ of hexane by mixing for 15 min. After centrifugation the organic layer was collected in separate rubes and the aqueous phase saved for the extraction of amitriptyline N-oxide. An 8 -m€ portion of the hexane extract was evaporated at 20°C with a current of nitrogen. The residue was dissolved in 200 μ€ of the mobile phase and 5 to 100 μ€ of this solution were injected. 1-11. To the sample (1 mf) were added, 100 μ€ (1 μg/m€ in 0.01 N HC1) of internal standard, 0.5 m€ of 0.5 N NaOH and 8 mf of 1.5% isoamyl alcohol in nheptane. The mixture was shaken for 15 min and centrifuged at 1500 x g for 10 min. The organic layer was collected and back extracted into 1.2 m€ of 0.1 N HC1. The aqueous layer was made alkaline with 0.5 m€ of 0 .1 A NaOH, extracted with 0.5 m€ of 0.5% isoamyl alcohol in n-heptane by shaking for 10 min and centrifugation at 1500 x g for 5 min. The organic layer was collected and evaporated at 45°C under vacuo. The residue was reconstituted with 100 μ€ of the mobile phase. 1-12. To 1 mf of the sample are added, 100 μ£ of 1.5 M NaOH, 20 μ€ of internal standard (1 mg/€ in ethanol) and 6 m f of hexane-isoamyl alcohol (99:1). The tubes are rolled at 60 rpm for 30 min and centrifuged for 4 min at 3000 x g. The organic layer is transferred to a conical tube, 20 μ€ of maprotiline (2.5 g/€ in methanol) is added, and the mixture evaporated at 40°C in a current of nitrogen. The residue is dissolved in 50 μ€ of the mobile phase and 20 μ€ of this solution are injected.

Volume III 81

1-1

1.5x4

Column Extraction (m x mm) 10% SE-30 ChromosorbW AW DMCS (80/100)

Packing (mesh) 180

Oven temp (°C) N2 (45)

Gas (ml/min) FID

Det. 3.5

RT (min) Octadecane (7)

Internal standard (RT)

Methyl

Deriv.

—

Other compounds (RT)

1

Ref.

1.

Dilli, S. and Weekley, A., Direct derivatization and gas chromatographic determination of barbiturates in autopsy liver tissues, J. Chromatogr. ,195, 349, 1980.

REFERENCE

Extraction — 1-1. A sample (5 g) of the tissue was extracted three times by boiling in 50-ml portions of methanol for 30 min. The combined extracts were concentrated in a rotary evaporator to —20 m l, 50 m l of 0.2 M HC1 added and extracted three times by shaking with 20 ml-portions of chloroform. The combined extracts were evaporated to dryness at 35°C under vacuum and the residue treated with methanol-HCl reagent (30 m l) at 85°C for 30 min. The cooled solution was reduced to about 10 m l, diluted with water (20 ml), and extracted with chloroform (2 x 10 ml). The combined extracts were back extracted into 2 x 10 m l of 0.1 N NaOH. The combined aqueous layer was made acidic with 10 M HC1 and extracted with 2 x 10 m l chloroform. The residue was methylated by treating it with 0.07 g dimethyl sulfate, potassium carbonate solution (0.3 m l), and methanol (0.3 ml) and heating the mixture at 85°C for 5 min. After cooling, the reaction mixture was diluted with water (2 ml) and extracted with benzene ( 2 x 2 ml). The organic phase was evaporated and dissolved in an accurately measured volume of the internal standard solution. Aliquots of 1 to 2 μ ΐ were injected.

Tissue (5 g)

Specimen (ml)

Gas Chromatography

AMOBARBITAL

9. Smith, G. A., Schulz, P., Giacomini, K. M ., and Blaschke, T. F., High-pressure liquid chromatographic determination of amitriptyline and its major metabolites in human whole blood, J. Pharm. Sci., 71, 581, 1982. 10. Jensen, K. M ., Determination of amitriptyline-N-oxide, amitriptyline and nortriptyline in serum and plasma by high-performance liquid chromatography, J. Chromatogr., 183, 321, 1980. 11. Suckow, R. F. and Cooper, T. B., Simultaneous determination of amitriptyline, nortriptyline and their respective isomeric 10-hydroxy metabolites in plasma by liquid chromatography, J. Chromatogr., 230, 391, 1982. 12. Edelbroek, P. M ., de Haas, E. J. M ., and de Wolff, F. A., Liquid-chromatographic determination of amitriptyline and its metabolites in serum, with adsorption onto glass minimized, Clin. Chem., 28, 2143, 1982.

AMITRIPTYLINE

82 CRC Handbook of Chromatography: Drugs

1-1

Extraction

10

x 8

Column (cm x mm) μ-BondapakRad-Pak Phenyl (10)

Packing (pm)

Flow (ml/min) 3

Elution E-l

6 -Methoxy-

3.2

ABS (340) quinoline (4.4)

8 -amino-

Internal standard (RT)

RT (min)

Det. (iun) —

Other compounds (RT)

1

Ref.

1-2

Plasma, serum ( 1)

Extraction

1-1

Specimen (ml)

30 x 3.9

30 x 3.9

Column (cm x mm)

μ-Porasil silica ( 1 0 )b

p-Bondapak-Cig ( 1 0 )*

Packing (pm)

2.0

2.0

E-l

E-2

Elution

Flow (ml/min)

Liquid Chromatography

AMOXAPINE

ABS (254)

ABS (254)

Det (nm)

3

6 .0

RT (min)

Promazine (5)

loxapine (3.5)

8 -Methoxy-

Internal standard (RT)

amoxapine (1.7) 7-Hydroxyamoxapine (2.7) 8 -Hydroxyamoxapine (3.5)c

8 -Hydroxy-

Other compounds (RT)

2

1

Ref.

Mihaly, G. W. and NichoU, D. D., High-performance liquid chromatographic analysis of amodiaquine in human plasma, J. Chromatogr., 337, 166, 1985.

Serum ( 1)

1.

REFERENCE

Elution — E-l. Methanol-water (27:73) containing triethylamine (1%) adjusted to pH 2.8 with orthophosphoric acid.

Extraction — 1- 1 . To sample (1 ml) containing the internal standard (200 ng) was added ammonia solution (2 ml). This mixture was extracted twice by mechanical tumbling for 15 min with diethylether (5 ml) and centrifugation. The combined organic phases were evaporated to dryness under a steady stream of nitrogen at 25°C. The residue was reconstituted in the mobile phase (55 μ ΐ) and 50 μ ΐ of this was injected on the column.

Plasma ( 1)

Specimen (ml)

Liquid Chromatography

AMODIAQUINE

Volume III 83

1-4

1-5

1-6

Plasma ( 1)

Plasma ( 1)

Serum ( 1)

15 x 4.6

25 x 4.6

25 x 4.6

15 x 4.6

Column (cm x mm)

Comosil C ,8 (5)

E-6

E-5

E-4

Zorbax-CN (NA)d

Supelco LC-1 (5)

E-3

Elution

Supeliosil-CN (5 )

Packing (μπι)

1.0

1.8

3.0

1.2

Flow (ml/min)

ABS (254)

Electrochem

ABS (250)

ABS (2 1 1 )

Det. (nm)

8 .8

12.5

6 .6

10.4

RT (min) 8 -Hydroxy-

Other compounds (RT)

Ref.

3 amoxapine (6.4) 7-Hydroxyamoxapine (7.6) Protriptyline (22.4)c Trimi­ 7-Hydroxy4 amoxapine (5.6) pramine (8.4) 8 -Hydroxyamoxapine (4.7) 8 -Hydroxy- 7-Hydroxy5 loxapine amoxapine (5) 8 -Hydroxy(7) amoxapine (6 ) Carbam7-Hydroxy6 azepine amoxapine (3.7) (7.2) 8 -Hydroxyamoxapine (4.3)

Protrip­ tyline

Internal standard (RT)

Extraction — 1-1. To a 1-ml Clin-Elut extraction column 0.2 m l of saturated sodium carbonate buffer was added and allowed to absorb onto the column for 2 min, then 25 μ ΐ of the internal standard solution (10 μ§/πιΙ) was added. After 2 min, 1 m l of serum sample was introduced. The column was eluted with 4 m l of 1 butanol-hexane (1:5). After 5 min, the column was again eluted with 4 m l of the eluting solvent. The combined eluate was back extracted into 1 m l of 0.1 M HC1. The organic layer was discarded, the aqueous layer buffered with 0.6 m l saturated sodium carbonate solution and extracted with 2-ml aliquot of extraction solvent. The organic layer was evaporated at 50°C under reduced pressure. The residue was dissolved in 150 μ ΐ of the mobile phase and 30 to 70 μ ΐ were injected. 1-2. Bond-Elut C ,8 columns were washed with 2 column volumes of methanol followed by 2 column volumes of water. The sample (1 ml) was applied and then 2 0 0 μ ΐ of working internal standard mixture (trimipramine -I- promazine 20 mg/100 μ ΐ of each in methanol/0.1 N HC1). After aspiration of the sample through, the

a Column temp = 60°C. b Alternative column (25 cm x 4.6 m) packed with 5 μΛί silica from Supelco was also used. c Conditions for separate determination of maprotiline are also described. d Column = 45°C.

1-3

Extraction

Serum (2 )

Specimen (m l)

Liquid Chromatography

AMOXAPINE (continued)

84 CRC Handbook of Chromatography: Drugs

1. 2.

Tasset, J. J. and Hassan, F. M., Liquid-chromatographic determination of amoxapine and 8 -hydroxyamoxapine in human serum, Clin. Chem., 28, 2154, 1982. Beierle, F. A. and Hubbard, R. W., Liquid chromatographic separation of antidepressant drugs. II. Amoxapine and maprotiline, Ther. Drug Monit., 5, 293, 1983. 3. Ketchum, C ., Robinson, C. A., and Scott, J. W ., Analysis of amoxapine, 8 -hydroxyamoxapine, and maprotiline by high-pressure liquid chromatography, Ther. Drug. Monit., 5, 309, 1983. 4. Johnson, S. M., Nygard, G ., and Khalil, S. K. W ., Isocratic liquid chromatographic method for the determination of amoxapine and its metabolites, J. Pharm. Sci., 73, 696, 1984. 5. Suckow, R. F. and Cooper, T. B., Determination of amoxapine and metabolites in plasma by liquid chromatography with electrochemical detection, J. Chromatogr., 338, 225, 1985. 6 . Kobayashi, A., Sugita, S. and Nakazawa, K., Determination of amoxapine and its metabolites in human serum by high-performance liquid chromatography, Neuropharmacology, 24, 1253, 1985.

REFERENCES

Elution — E-l. Acetonitrile-water (74:26) plus 26 μ ΐ of n-butylamine. E-2. Acetonitrile (77 m l) + terf-butyl amine (1.9 m l) + ethanol (1 gal). E-3. Acetonitrile-0.5 M phosphate buffer, pH 7.0 (60:40). E-4. Acetonitrile-0.03 M acetate buffer, pH 6.0 (65:35). E-5. Acetonitrile-0.05 M potassium dehydrogen phosphate (75:25) with 1.2 m l/l n butylamine, 1 m l/l orthophosphoric acid, and 0.005 N heptanesulfonic acid added. E-6 . Methanol-tetrahydrofuran-1% triethylamine (adjusted to pH 3 with phosphoric acid (20:10:70).

column was washed with 2 volumes of each water and 0.1 N acetic acid followed with one volume of methanol-water (15:85, in 5 mM HC1). The columns were eluted three times with 200 μ ΐ of methanolic ammonium acetate (10 mM). The combined eluate was evaporated at 56°C under compressed air. The residue was reconstituted with the mobile phase (200 μ ΐ) and aliquots of 75 μ ΐ were injected. 1-3. To 2 m l of the sample were added to 30 μ ΐ of working protriptyline solution (10 μg/ml in methanol/water), 100 μ ΐ of 1.5 N NaOH, and were extracted with 5 m l hexane-ethyl acetate (1:1) by shaking with 5 m l hexane-ethyl acetate (1:1) by shaking for 5 min. After centrifugation, 4 m l of the organic layer was evaporated under nitrogen. The residue was reconstituted with 100 μ ΐ of the mobile phase and 30 μ ΐ were injected. 1-4. To the sample (1 m l) was added, 0.5 m l of 0.1 M NaOH and 10 m l of extraction solvent (ethyl acetate containing 20 ng/ml of the internal standard). The tubes were vortexed for 10 sec and centrifuged for 5 min at 900 x g. A 9-ml portion of the organic phase was evaporated at 50°C under a stream of nitrogen. The residue was dissolved in 100 μ ΐ of methanol and 30 μ ΐ were injected. 1-5. To 1 m l of the sample, 50 μ ΐ of the internal standard (1 μg/ml in 0.1 N HC1), 1 m l of 0.6 M carbonate buffer (pH 9.8), and 7 m l of tert butyl ether were added. The mixture was shaken for 15 min and centrifuged at 1500 x g for 15 min. The organic layer was collected and back extracted into 250 μ ΐ of 0.1 M phospahte buffer (pH 2). Aliquots of the aqueous layer were injected with an auto sampler. 1-6. The sample (1 m l), after the addition of 0.1 m l of 3 M acetate buffer (pH 4.5) was incubated with 1 μ ΐ β-glucuconidate/arysulfatase for 20 hr at 30°C. Carbamazepine (200 ng) and 0.5 m l of methanol were added. After mixing, 1.5 m l of 6% perchloric acid was added, mixed, centrifuged for 3 min at 11,000 x g, and to the supernatant 0.5 m l of 4 N KOH was added. The resulting solution was shaken with 5 m l of ethyl ether-ethylacetate (85:15) for 15 min. The organic phase was evaporated under a stream of nitrogen gas and the residue dissolved in 200 μ ΐ of 1% triethylamine phosphate (pH 2)-methanol (9:1) and an aliquot of 100 μ ΐ was injected.

Volume III 85

25 x 4.6

10 x 4.6

15 x 4.6

1-2

1-3

1-4

Microspher C18c (3) Nucleosil C 18 (5)

μ-Bondapak-C1g (10)“ Zorbax-C8 (6 )

Packing (μπι)

E-4

E-3

E-2

E-l

Elution

1.0

1.0“

2.0

2.5

Flow (ml/min) ABS (227) ABS (225) FI (372, 470)* ABS (229)

Det (nm)

2.8

10.5

7

1.5

RT (min)

Benzoic acid (5.9)

—

—

—

Internal standard (RT)

—

—

—

Clavulanic acidb

Other compounds (RT)

The column is protected by a guard column packed with CO:Pell ODS. Conditions for separate analysis of clavulanic acid are described. Two similar analytical columns were used in series separated by a switching valve. The first analytical column is protected by a guard column. Flow rate for the second analytical column. Flow rate and the mobile phase for the Fust analytical column are different. The eluate of the column is treated with fluorescamine prior to detection.

25 x 4.6

1-1

Extraction

Column (cm x mm)

4

3

2

1

Ref.

Extraction — 1-1. The serum sample is diluted with an equal volume of 0.1 M sodium phosphate buffer (pH 7) and was filtered through prewashed Amican YMB membrane filters by centrifugation at 5°C in a 35°C angle-head rotor at 1000 x g for 15 min. The ultrafiltrate was stored at 5°C until aliquots were injected. 1-2. The sample (1 m€) was diluted with 1 m€ of 0.02 M KH2PO and passed through Sep-Pak C 18 cartridges prewashed with 4 m€ of methanol followed by 1 m€ of 0.02 M KH2P 0 4. The cartridges were washed with 1 m€ of 0.02 M KH2P 0 4 and 0.5 m€ of water; and then eluted with 2-m€ aliquot of water-methanol (85:15). The eluate was placed in the vials of autosampler and 80-μ€ aliquots were injected. 1-3. The sample 0.5 m€ was treated with 250 μ€ of perchloric acid-citrate/phosphate buffer (pH 5.4) (10:90). After mixing and centrifugation 450 μ€ of the supernatant was treated with citrate/phosphate buffer (pH 5.4) — 1 M NaOH (110:40). The solution was placed in the sample vial of the autoinjector and 25 to 100 μ€ were injected. The desired compound is switched from the first analytical column to the second analytical column at the prescribed time automatically. 1-4. The sample was mixed with 0.25 m f of the internal standard solution (0.25 mM with 0.01 M tetrabutylammonium hydrogen sulfate in 0.1 M phosphate buffer, pH (7). An aliquot of 0.25 m€ of this mixture was applied to a l-m€ Baker-Clg extraction column. The column was washed three times with 500-μ€ aliquots of 0.01 M tetrabutylammonium hydrogen sulfate in 0.1 M phosphate buffer (pH 7) and eluted three times with 50 μ€ of methanol and once with 350 μ€ of 0.005 M tetrabutylammonium hydrogen sulfate in 0.05 M sodium dihydrogen phosphate (pH 6 ). The eluate was stored at 5°C until an aliquot of 10 μ€ was injected.

a b c d e

Serum (0.5— 1) Plasma ( 1) Plasma (0.5) Plasma (0.25)

Specimen (m€)

Liquid Chromatography

AMOXICILLIN

86 CRC Handbook of Chromatography: Drugs

25 x 0.2

0.9 x 2

12 x NA

1-2

1-3

1-4

Pure compound, street drug samples Serum, tissue ( 1) Brain extract (4 ml)

1.8 x NA

Column Extraction (m x mm)

1-1

Specimen (m l)

Carbowax

3% OV-17

SP-2100

3% OV-225 Chromosorb W (NA)

Packing (mesh)

T.P.e

T.P.a

190

Oven temp