Modern Analysis of Antibiotics (Drugs and the Pharmaceutical Sciences) 9780824773588

Table of contents :
Cover
Half Title
Series Page
Title Page
Dedication
Copyright Page
Foreword
Preface
Introduction
Contributors
Contents
1. Gas Chromatographic Analysis
2. Ultraviolet and Light Absorption Spectrometry
3. Infrared Spectroscopy
4. Mass Spectrometric Analysis
5. Electron Spin Resonance Spectroscopy
6. Thin-Layer Chromatographic Techniques and Systems
7. High-Performance Liquid Chromatography
8. Thermal Analysis
9. Microbiological Assay of Antibiotics in Body Fluids and Tissues
10. Assay of Antibiotics in Mammalian Cell Culture
11. Immunological Approaches
12. Determination of Antiviral Activity
13. Fertilized Sea Urchin Eggs as a Model for Detecting Cell Division Inhibitors
14. Critical Appraisal of Animal Models for Antibiotic Toxicity
Index

Citation preview

MODERN ANALYSIS of ANTIBIOTICS

DRUGS AND THE PHARMACEUTICAL SCIENCES A Series of Textbooks and Monographs Edited by James Swarbrick

School of Pharmacy University of North Carolina Chapel Hill, North Carolina

Volume 1.

PHARMACOKINETICS, Milo Gibaldi and Donald Perrier (out of print)

Volume 2.

GOOD MANUFACTURING PRACTICES FOR PHARMACEUTICALS: A PLAN FOR TOTAL QUALITY CONTROL, Sidney H. Willig, Murray M. Tuckerman, and William S. Hitchings IV (out of print)

Volume 3.

MICROENCAPSULATION, edited by J. R. Nixon

Volume 4.

DRUG METABOLISM: CHEMICAL AND BIOCHEMICAL ASPECTS, Bernard Testa and Peter Jenner

Volume 5.

NEW DRUGS: DISCOVERY AND DEVELOPMENT, edited by Alan A. Rubin

Volume 6.

SUSTAINED AND CONTROLLED RELEASE DRUG DELIVERY SYSTEMS, edited by Joseph R. Robinson

Volume 7.

MODERN PHARMACEUTICS, edited by Gilbert S. Banker and Christopher T. Rhodes

Volume 8.

PRESCRIPTION DRUGS IN SHORT SUPPLY: CASE HISTORIES, Michael A. Schwartz

Volume 9.

ACTIVATED CHARCOAL: ANTIDOTAL AND OTHER MEDICAL USES, David 0. Cooney

Volume 10. CONCEPTS IN DRUG METABOLISM (in two parts). edited bv Peter Jenner and Bernard Testa Volume 11. PHARMACEUTICAL ANALYSIS: MODERN METHODS (in two parts). edited by James W. Munson Volume 12. TECHNIQUES OF SOLUBILIZATION OF DRUGS, edited by Samuel H. Yalkowsky

Volume 13

ORPHAN DRUGS, edited by Fred E. Karch

Volume 14.

NOVEL DRUG DELIVERY SYSTEMS FUNDAMENTALS, DEVELOPMENTAL CONCEPTS, BIOMEDICAL ASSESSMENTS, edited hy Yie W Chien

Volume 15.

PHARMACOKINETICS, Second Ed1t1on, Revised and Expanded, Milo Gibaldi and Donald Perrier

Volume 16.

GOOD MANUFACTURING PRACTICES FOR PHARMACEUTICALS: A PLAN FOR TOTAL QUALITY CONTROL, Second Edition, Revised and Expanded, Sidney H. Willig, Murray M Tuckerman,

and William S. Hitchings IV Volume 17.

Volume 18.

FORMULATION OF VETERINARY DOSAGE FORMS, edited by Jack Blodinger DERMATOLOGICAL FORMULATIONS: PERCUTANEOUS ABSORPTION, Brian W. Barry

Volume 19. THE CLINICAL RESEARCH PROCESS IN THE PHARMACEUTICAL INDUSTRY, edited by Gary M Matoren Volume 20.

MICROENCAPSULATION AND RELATED DRUG PROCESSES, Patrick B. Deasy

Volume 21.

DRUGS AND NUTRIENTS: THE INTERACTIVE EFFECTS, edited by Daphne A. Roe and T. Colin Campbell

Volume 22.

BIOTECHNOLOGY OF INDUSTRIAL ANTIBIOTICS, Erick J Vandamme

Volume 23.

PHARMACEUTICAL PROCESS VALIDATION, edited by Bernard T. Loftus and Robert A. Nash

Volume 24.

ANTICANCER AND INTERFERON AGENTS SYNTHESIS AND PROPERTIES, edited by Raphael M Ottenhrite and George B. Butler

Volume 25.

PHARMACEUTICAL STATISTICS: PRACTICAL AND CLINICAL APPLICATIONS, Sanford Bolton

Volume 26.

DRUG DYNAMICS FOR ANALYTICAL, CLINICAL, AND BIOLOGICAL CHEMISTS, Benjamin J. Gudzinowicz,

Burrows T. Younkin, Jr., and Michael J. Gudzinowicz Volume 27.

MODERN ANALYSIS OF ANTIBIOTICS, edited by Adorjan Aszalos

Other Volumes in Preparation

MODERN ANALYSIS of ANTIBIOTICS edited by

Adorjan Aszalos

Department of Health and Human Services Food and Drug Administration Washington, D.C.

MARCEL DEKKER, INC.

New York and Basel

To my mother

Library of Congress Cataloging in Publication Data Main entry under title: Modern analysis of antibiotics. (Drugs and the pharmaceutical sciences ; v. 27) Includes index. 1. Antibiotics- -Analysis. I. Aszalos, A. A. ( Adorjan A.) II. Series. [DNLM: 1. Antibiotics- analysis. Wl DR 893B v. 2 I QV 350 M689] RS190.A5M63 1986 615'. 329 85-25432 ISBN 13: 978-0-8247-7358-8

COPYRIGHT© 1986 by MARCEL DEKKER, INC. ALL RIGHTS RESERVED Neither this book nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming, and recording, or by any information storage and retrieval system, without permission in writing from the publisher. MARCEL DEKKER, INC. 270 Madison Avenue, New York, New York 10016 Current printing (last digit) 10 9 8 7 6 5 4 3 2 1 PRINTED IN THE UNITED STATES OF AMERICA

Foreword

Why the need for a book devoted exclusively to instruction in methods of analysis for antibiotics? After all, antibiotics are not a chemically distinct or unique class of compounds, and methods for analysis of nonantibiotic compounds should serve equally well for antibiotics. These remarks deserve thoughtful consideration. Apart from being an investment by a publisher, a scientific book should have practical and intellectually defensible reasons for its existence. It should have some useful social purpose in meeting needs of practicing scientists and technicians. Such purposes for this book do exist. Analyses for antibiotics are employed in the search for new and improved antibiotics, in the discovery of mechanisms of biological activity, as a means for quality control of manufacture, as tools in following the stability of antibiotics exposed to different environments, and in support of government regulatory activities. These uses explain both the great demand for well-tested, reliable methods of analysis of antibiotics and the considerable volume of research performed on analytical methods historically , currently, and anticipated for the future. Antibiotics are defined by their biological origins and activities. Yet, chemically, they are not a distinct class of substances that are classifiably separate from all other kinds of compounds. In fact, a great variety of classes of compounds include antibiotics, and no doubt there remain classes of compounds within which antibiotics are still to be discovered. The very diversity in the chemical structure of antibiotics argues for the value to the laboratory scientific community in bringing together in one place, as a matter of convenience, methods of analysis developed for different cJasses of compounds and reported in widely scattered journals not only of chemistry, but of medicine too. The laboratory community served by a book on antibiotic analysis is itself diversified in the educational backgrounds and needs of its personnel and the social institutions it serves. This compendium of analytical methods for antibiotics provides a common meeting ground for help, advice, and exchange of information. In assays for antibiotics there is a choice to be made between biological and chemical methods. It is only the biological methods that directly detect biological activity and measure potencies. And it is the biological activities

iii

iv

Foreword

that necessitate analyses of antibiotics. A chemical method of analysis can only give presumptive evidence of biological activity. As a measurement for potency , the acceptance of chemical analysis for an antibiotic without a concurrent biological analysis is reasonable only after research has demon strated a reproducible correlation between the two kinds of measurement. When coupled to biological activity measurement by chemical analysis, it has its own unique value as a means for determining how molecular structural variations affect biological activities. Since an antibiotic can have more than one kind of biological activity, chemical analyses also permit judgments to made as to whether or not, and how, given molecular structural changes affect particular kinds of activity. For the analyst this book brings together an up-to-date account of instructions for biological and chemical assays. Given the option to choose among methods of analysis, chemical analyses are preferred. This is because chemical methods are generally less costly, take less time to perform , and are more precise than biological assays. The science of chemistry is in a continuing revolution of invention of new technologies and accompanying developments of new instruments with applications to chemical analyses. Gravimetric and volumetric analyses are increasingly abandoned. This reflects the fact that certain technologies and the instrumentation they have fathered have proven useful for application to analyses for particular classes of compounds. What method becomes a favored and then routine method of analysis results from experience in comparative evaluations of competing methods. One value of this book is that it makes the experience of its authors available to both the neophyte and the experienced antibiotic analyst. It provides sound guidance to making choices among tried and true methods and instruments. While great advances in chemical analytic technologies are being made, diversification and improvements have also proceeded at a rapid pace in bioassays for antibiotics. Thus, there are choices of methods to be made by bioanalysts, and this book should prove of great value to them. The developments of improved chemical and biological methods of analysis for antibiotics arise directly from both the application of well-tested scientific concepts and the design of new kinds of laboratory instruments. Yet the workaday analysis of antibiotics is not all science. As a human activity, laboratory analysis still has an element of art in it. Two analysts equally knowledgeable scientifically may still differ in the quality of the analyses they perform. This is because know-how is an important element in laboratory work and different persons possess know-how in different measure. Differences in informed intuition, patience, persistence, and intensity of experience are elements that affect individual quality performance. With experienced analysts as its authors, this book brings together the know- how so important to the actual performance of laboratory procedures. This also tells us that the step-by-step outlines of procedures recorded in this book are not simple cookbook recipes for analyses. Rather, they are distillations of the scientific competence, experiences, and sophisticated laboratory skills of capable scientists with exceptional know-how. It is fortunate that the book has as its editor (and contributor to individual chapters) Dr. Adorjan Aszalos. Dr. Aszalos brings to his task an exceptional variety of experiences as an organic chemist , biochemist, and chemical engineer. He knows the many theoretical chemical and practical technical problems faced by analysts of antibiotics. As he is himself a productive research worker, he understands the challenges faced by analysts

v

Foreword

in givmg support to efforts to discover antibiotics, and to mastering understanding of the mechanisms of biological activities of antibiotics. This understanding and experience of Dr. Aszalos assures the usefulness of this book to all scientific workers in the diversified community of industrial, medical, academic, and governmental antibiotic laboratories. Carl Lamanna, Ph.D. Associate Director

Pharmaceutical Research and Testing Department of Health and Human Services Food and Drug Administration Washington, D. C.

Preface

Antibiotics are defined as compounds that have inhibitory activity against microorganisms, eukaryotic cells, and viruses that are produced by secondary metabolism of living organisms, and that possess a variety of widely different chemical structures. Antibiotics have gained increasing importance in the last 40 years in a variety of scientific and commercial fields. Their successful application in human and veterinary therapy is well recognized. They have considerable utility as animal growth promoters and plant protection agents in agriculture, in the food industry as food preservatives, and as specific inhibitors in many different laboratory sciences. Because of their numerous applications, antibiotics have become the most important pharmaceutical preparations in recent years. The market value of manufactured antibiotics is probably larger than for any other type of drugs commercially available. The first practical antibiotic, penicillin, which was produced on a large scale about 40 years ago, instantly became a very desirable product. Since that time more than 10, 000 antibiotics have been isolated from fermentations, plant materials, animal tissues, and various marine sources. Probably no more than 50 of these antibiotics are manufactured on a large scale, in addition to the variety of the chemical analogs of about 10 commercially important ones. Because of strong industrial interest and wide medical application, the antibiotic field requires considerable support, especially in the analytical area. Naturally many scientific papers appeared on this subject but it has been many years since a book dedicated to the general analysis of antibiotics was published. The last such book was written by Grove and Randall in 1955. The many papers and chapters published since then deal with a specific analytical technique of antibiotics or with the different assay methods of a single antibiotic or class of antibiotics. A book in which up-to-date analytical techniques for the different types of antibiotics can be compared is missing from the libraries and laboratories. It was my pleasure, therefore, to accept the invitation of the publisher to edit such a book. My conceptions of the book are summarized as follows: A book dedicated to the modern analysis of antibiotics should contain chemical and biological methods that were developed in the last five to seven years and

vii

viii

Preface

have proved to be valuable tools in the laboratory. Special topics that are well detailed in the recent literature should not be treated again, even if that results in an incomplete anitbiotic treatment. An example of such a topic is the 13-lactams (Cephalosporins and Penicillins: Chemistry and Biology, E. H. Flynn, ed., Academic, N. Y., 1976 and Handbook of Experimental Pharmacology, Vol. 67, Antibiotics, Vol. I, Demain and Solomon, eds., Springer-Verlag, N.Y., 1983). Also, microbiological determination of antibiotics is well treated in the book by F. Kavanagh, Analytical Microbiology, Vols. I and II, Academic, N. Y., 1972. In some of the specialized areas like gas chromatography and infrared and ultraviolet spectrometry, we included some basic concepts for the sake of completeness in discussing these techniques. We also believed it is important that useful detailed procedures should be described so that one may get a practical impression about an analytical procedure without consulting other sources. Naturally, since not all published analytical procedures can be handled in one book , we must restrict ourselves to very good , selected procedures. In some areas, such as high-pressure liquid chromatography, infrared spectroscopy, or analytical techniques related to antibiotic determination in body fluids, in which a wealth of literature has appeared in recent years, we had to make some arbitrary selections in order to avoid unnecessarily large chapters. We would like to emphasize that only the qualitative and quantitative aspects of analysis are considered in this book. One of the most interesting chemical and biological aspects of the antibiotic field is the identification of supposedly new antibiotics and structure elucidation. This is the area where most intellectual excitement is created in dealing with antibiotic analysis. Unfortunately, the strategies and the chemical and biological means used to identify an antibiotic could not be included in this analytically oriented book. The method used to dereplicate chemical principles in crude antibiotic preparations alone would create one volume. Other volumes would come from structure elucidation with all the degradation chemistry and NMR and mass spectrometric and x-ray crystallographic studies. The modern aspects of these two spectroscopies are extremely interesting and greatly accelerate structure elucidation. Some quantitative antibiotic analyses are being done with NMR spectrometry; for example, gentamycin preparations are analyzed for their different gentamycin contents (Reuter et al., J. Assoc. Off. Anal. Chem., 65, 1413, 1982). However, NMR spectrometry is used mostly in structure elucidation studies and therefore we have not included a chapter dealing with this method. Readers interested in a descriptive treatment of the many aspects of antibiotic research, classification, characteri zation, modes of action, and microbiological quantitation are referred to the book by Vladimir Betina, The Chemistry and Biology of Antibiotics, published by Elsevier in 1983. I am pleased that all contributors accepted these principles and that, as a result, very useful chapters could be brought together without repeating what is in the recent literature. Indeed, I am grateful to all contributors for their understanding of the need for this book, for sharing their extremely valuable expertise, and for their diligence which made it possible to compile this information. I am also grateful to Carl Lamanna for his special encouragement. Adorjan Aszalos

Introduction

There are 14 chapters in this book, Half of them deal with chemical analysis and the remainder with biological analysis. One of the first instrumental analytical techniques used to analyze antibiotics is gas chromatography. This technique is still in use for certain antibiotics but in the past decade was mostly replaced by high-pressure liquid chromatography. Some antibiotic analysis is still being done by gas chromatography because this process is available in the laboratory and usually an analyst does not want to replace a well-functioning method. It is also important to realize that a gas chromatograph can be coupled with some mass spectrometers and therefore separation, quantitation, and identification can be done by one single analysis, namely GC /MS. Most gas chromatographic analyses of antibiotics used today are well detailed in the first chapter. This chapter also contains some basic chromatographic principles , like resolution and peak symmetry. We thought that a brief treatment of these principles would be of some value to the reader. But we have not repeated this treatment in relation to the other chromatographic methods, i.e., thin-layer chromatography and high-pressure liquid chromatography, These latter two methods are in a way competitors to the analysis of antibiotics. Thin-layer chromatography is the older method. It is still used when not very precise quantitation is required. For example, it is convenient to take a centrifuged experimental fermentation broth, spot it on a thin -layer plate along with a solution of known antibiotics and estimate the amount of produped antibiotic after several development trials. The required instrumentation and expense for this process are extremely minimal in comparison to those for other chromatographic methods. This chapter dealing with thin-layer chromatography lists recently developed systems used in identification and quantitation of antibiotics. Besides these data, a short treatment on the methodology and instrumentation is given. Finally, the brief discussion of antibiotic classification systems contains data that could be adapted by analysts to solve separation and semiquantitation of antibiotic combinations in biological fluids. The most frequently used chromatographic analysis of antibiotics is done by high-pressure liquid chromatography. The best features of this

ix

x

Introduction

method are sensitivity, accuracy, and adaptability to low and high molecular weight compounds and to serial analyses. The cost of the required instrumentation is moderate and all parts are commercially available. There are limitations in its detection method because up to now the only broadly accepted detection methods are based on the ultraviolet and fluorescence spectrometry. Antibiotics not detectable by these methods could be detected by electrochemical, refractive index, or infrared and mass spectrometric techniques; but at present none of these detection techniques is generally useful, sensitive , and inexpensive. Despite this difficulty, most of today's chemical quantitation of antibiotics is done by high-pressure liquid chromatography. One must emphasize that analysis by this method, or for that matter by all chemical methods , has to be correlated with the biological activity that is the principal mode of action of an antibiotic. A wealth of literature exists on the subject of high-pressure liquid chromatography. Because of space limitations, Kirschbaum and Aszalos had to restrict themselves to the most adopted methods in the most frequently analyzed antibiotics. Antibiotics are grouped in this chapter according to structural similarities, except that unclassified and antitumor agents are treated under such subheadings together. The most valuable features of this chapter may lie in the possibility to acquire immediate practical details for all the antibiotics quoted in it. Both spectroscopic chapters, ultraviolet and infrared, contain some introductory basic spectroscopic details. Such short treatments were thought to be necessary so that a practical analyst can understand explanations given later in the chapter about spectra of indivi,dual antibiotics. The chapter on infrared spectrometry gives excellent examples of how to use this method in the analysis of antibiotics. Besides general qualitative and quantitative analysis of bulk materials, infrared spectrometry is used to analyze stability, solvent effects, structure activity relations, metal bondings, and polymorphism of antibiotics. While infrared spectrometry is a relatively old method, some areas of its use, especially those based on computerized discriminant qualitative analysis of antibiotics, are quite recent and are well introduced here by Rose. Infrared spectra of antibiotics are quite adequately covered in the literature and therefore this chapter does not list these spectra. Contrary to the availability of infrared spectra of antibiotics, the ultraviolet spectra of antibiotics is only listed by absorption wavelength in the Antibiotic Handbook by Berdy et al., published by CRC Press and now available in 13 volumes. Therefore, it is a welcome achievement of Dinya and Sztaricskai that they give an analytical presentation of the ultraviolet spectra of antibiotics. Ultraviolet spectroscopy is still used frequently to qualitatively and quantitatively analyze antibiotics having chromophor groups. Before the advent of high-pressure liquid chromatography it was one of the most frequently used quantitative analytical tools for such antibiotics. Exciting new techniques were developed in mass spectrometry in the past five years. These developments make it possible to analyze practically all types of antibiotics by mass spectrometry today. Contrary to this, 10 years ago only limited types, mostly small molecular weight antibiotics, could be analyzed by this method. Therefore it was an excellent choice by Smith to deal with these new techniques in his chapter before describing the use of modern mass spectrometry with the individual types of antibiotics. Also,

Introduction

xi

the description of the combination of mass spectrometry with other methods, such as liquid chromatography or second mass spectrometry (MS /MS), broadens the horizon of possible choices of antibiotic analyses for the future. Two other physicochemical analytical methods, electron spin resonance and thermal analysis, are special techniques and not so widely used. However, the different techniques of thermal analysis as introduced by Jacobson (differential thermal analysis, differential scanning calorimetry, and thermogravimetric analysis) make the reader aware of possible areas where these techniques can be used in the analyses of antibiotics. Electron spin resonance is mostly used to analyze for the presence of antibiotic radical metabolites, which are thought to be important in the mode of action of the particular antibiotics in consideration. The method is not used frequently but in the case of free radical forming antibiotics and metal complex forming antibiotics it has proved itself indispensable. We believe that the use of electron spin resonance spectrometry will spread in the future. Chemical assays are usually less sensitive than biological assays. However, immunological assays that are basically biochemical assays are far more sensitive than regular chemical assays. It was natural, therefore, to consider determination of antibiotics by immunological approaches. These approaches are especially important when low concentrations of antibiotics have to be assayed in fluids or tissues, when separation techniques fail or are cumbersome, and when many assays have to be carried out in a short period of time. A very recent summary of these immunological approaches was put together by Dixon, Steiner, and Katz. We believe that this chapter will serve as a seed for further development work of this sensitive method. Antibiotics are used to combat microorganisms, viruses, and sometimes specific mammalian cells. It follows that the most accurate assay for an antibiotic is related to its activity against these targets. Such assays are biological assays. The usual microbiological assays were not considered in this book simply because they are adequately treated in recent literature. However, special microbiological assays, assays dealing with viruses and mammalian cells , are treated in this book. One of the most important special microbiological assays deals with quantitation of antibiotics in body fluids and tissues. It is realized now that activity of an antibiotic is related to its distribution in the living organism and to its half-life in the different organs. Residues of antibiotics in edible animal tissues are of concern because of the possible developments of antibiotic-resistant microbes. All the ramifications of this important area of microbiological assay are well summarized by Platt. Very useful, practical assay procedures are likewise detailed in his chapter. As in Platt's chapter, the chapter "Assay of Antibiotics in Mammalian Cell Culture" by Garretson gives very practical details of the use of this important biological assay. This type of assay was developed mostly for the sake of antitumor antibiotics where the target of an antibiotic is a special mammalian cell. More than a thousand antibiotics were discovered and assayed for purity in cell cultures in the past three decades. We believe that in the future more cell culture assays will be done , but mostly in a new direction. Some toxicity assays will be run in cell cultures instead of in whole animals. It is important, then, that examples of cell culture assays as described by Garretson can be compared in the same book

xii

Introduction

with the animal models for antibiotic toxicity discussed by Williams and Hottendorf. Needless to say, whole animal toxicity studies cannot be replaced entirely (mostly for pharmacological reasons): They are the ultimate safety assay to perform before putting an antibiotic on the market. Two special biological assays are included in this book. One is the determination of antiviral activity of antibiotics, well presented by Sidwell. This assay method will be used quite frequently in the future because more and more antiviral agents are marketed in recent times. Vidarabin, acyclovir, and cytarabin are commercially used antiviral agents, and others are waiting for approval for clinical use. The other special biological assay utilizes the sea urchin egg for detecting cell division inhibitors. The assay has been used mostly to detect and quantitate unusual, biologically active agents obtained from marine animals and plants. The assay is of importance since , in the last two decades, a great deal of effort went into exploring the sea for unusual antibiotics. It is conceivable that this effort will continue and even increase as isolation of a novel antibiotic from microbiological sources becomes less and less likely. One thing should be mentioned here: Because of the unique cell cycle system of the sea urchin egg it can be utilized to assay antibiotics with different modes of action, side by side.

Contributors

Thomas G. Alexander Adorjan Aszalos James A. Chan Pennsylvania

Food and Drug Administration, Washington, D.C.

Food and Drug Administration , Washington , D. C. Smith Kline

&

French Laboratories, Philadelphia,

Zoltan M. Dinya Research Group for Antibiotics of the Hungarian Academy of Sciences, L. Kossuth University of Debrecen, Hungary Deborah E. Dixon* Cook College, New Jersey Agricultural Experiment Station, Rutgers University, New Brunswick, New Jersey Aline L. Garretson

Gaithersburg, Maryland

Girard H. Hottendorf Robert S. Jacobs Harold Jacobson Jersey

Bristol-Myers Company, Syracuse, New York University of California, Santa Barbara, California

E. R. Squibb

&

Sons, Inc. , New Brunswick, New

Stanley E. Katz Cook College, New Jersey Agricultural Experiment Station, Rutgers University, New Brunswick, New Jersey Joel J. Kirschbaum Squibb Institute for Medical Research, New Brunswick, New Jersey Thomas B. Platt New Jersey

Squibb Institute for Medical Research, New Brunswick,

*Present affiliation: Michigan State University, East Lansing, Michigan

xiii

Contributors

xiv

John J. Rose

E. R. Squibb

Robert W. Sidwell

&

Sons, Inc., New Brunswick, New Jersey

Utah State University, Logan, Utah

Ronald G. Smith* The University of Texas M.D. Anderson Hospital and Tumor Institute, Houston, Texas Susan J. Steiner Cook College, New Jersey Agricultural Experiment Station, Rutgers University, New Brunswick, New Jersey Ferenc J. Sztaricskai Research Group for Antibiotics of the Hungarian Academy of Sciences, L. Kossuth University of Debrecen, Hungary Leslie Wilson

University of California, Santa Barbara, California

Patricia D. Williams George C. Yang

*Present affiliation:

Bristol-Myers Company, Syracuse, New York Food and Drug Administration, Washington, D.C.

Monsanto Agricultural Products, Chesterfield, Missouri

Contents

Foreword Preface Introduction Contributors 1. 2. 3. 4. 5. 6. 7. 8. 9, 10. 11.

Gas Chromatographic Analysis Thomas G. Alexander

iii vii ix xiii

1

Ultraviolet and Light Absorption Spectrometry

19

Infrared Spectroscopy John J. Rose

97

Zoltan M. Dinya and Ferenc J, Sztaricskai

Mass Spectrometric Analysis

141

Electron Spin Resonance Spectroscopy

183

Thin-Layer Chromatographic Techniques and Systems

197

High- Performance Liquid Chromatography

239

Thermal Analysis

323

Microbiological Assay of Antibiotics in Body Fluids and Tissues

341

Assay of Antibiotics in Mammalian Cell Culture

387

Immunological Approaches

415

Ronald G. Smith

George C. Yang and Adorjan Aszalos James A. Chan and Adorjan Aszalos

Joel J. Kirschbaum and Adorjan Aszalos Harold Jacobson

Thomas B. Platt

Aline L. Garretson

Deborah E. Dixon, Susan J. Steiner, and Stanley E. Katz

xv

xvi 12.

Contents

Determination of Antiviral Activity

433

13.

Fertilized Sea Urchin Eggs as a Model for Detecting Cell Division Inhibitors Robert s. Jacobs and Leslie Wilson

481

14.

Critical Appraisal of Animal Models for Antibiotic Toxicity Patricia D. Williams and Girard H. Hottendorf

Robert W. Sidwell

Index

495 525

MODERN ANALYSIS of ANTIBIOTICS

1 Gas Chromatographic Analysis THOMAS G. ALEXANDER

Food and Drug Administration, Washington, D. C.

Gas Chromatography Principles Instrumentation Columns Detectors Performance Criteria Special Techniques Derivatization Temperature Programming Pyrolysis Applications Simpler Molecules Aminoglycosides Lincomycin Antibiotics Penicillin and Cephalosporin Derivatives Antitumor Drugs Other Antibiotics Impurities in Antibiotics Summary and Conclusion References

2 2 3 3

4 4

6 6

7

7 8 8

10 12 12

14 14 16 17 17

Although antibiotics and related compounds have been known for only the past four decades, there has been tremendous activity in the area of the analysis of their preparations, residues, and metabolites, presumably due to the medical and economic significance of these materials. Consequently, as gas chromatography (GC) came on the scene as a practical tool of analytical chemistry, soon after the advent of the penicillins, its capabilities to impart qualitative and quantitative insight into the composition of antibiotic preparations and related materials were immediately utilized. Antibiotics, per se, include a wide variety of types of organic chemical structures. Only a few of them are sufficiently volatile to be gas 1

Alexander

2

chromatographed without preliminary treatment (griseofulvin). Consequently, it was necessary to devise means to obtain chromatographable forms of the samples, as through derivatization or pyrolysis. The analysis of antibiotics is not only of importance in pharmaceutical preparations, but also in foods and biological fluids. Consequently, there is discussion in this chapter of sample preparation to eliminate interference caused by excipients, meat tissues, and other substances. Also discussed are several examples in which the analysis involves the chromatographic separation of impurity or precursor of antibiotics, such as N ,N-dimethylaniline and 6-aminopenicillanic acid. The structural formulas of antibiotics are readily available and thus are not included in this chapter, except for the depicting of the thermolizing of lasalocid in the last section. The reader is referred to the ninth edition of The Merck Index (published by Merck & Co., Inc., Rahway, NJ, 1976) or USAN 1984 (published by The United States Pharmacopeial Convention, Inc., 12601 Twinbrook Parkway, Rockville, MD 20852, 1984). GAS CHROMATOGRAPHY

Principles

All chromatographic separations involve the selective partitioning of analytes between a carrier gas or mobile liquid phase (mobile phase) and a stationary liquid or solid phase. Separations are effected by means of differences in partition coefficients (K) of the various analytes in the sample. The term K is the ratio, at equilibrium, of the concentrations of a particular analyte in the stationary phase, Cs, and the mobile phase, Cm. That is,

K =

cs cm

For chromatography to serve as an useful analytical tool, the K values of closely related analytes must be enough different from each other to effect separation, yet the analytes must be sufficiently mobile (small K) that the analysis is accomplished in a reasonable length of time. Further, the K terms must be large enough so that all the analytes do not appear at the solvent front. Gas chromatography, by definition, includes those separations that involve gas as the mobile phase. Two primary categories are gas-solid chromatography and gas-liquid chromatography (GLC), which involve solids or liquids, respectively, as stationary phases packed in columns. In the former case, the analyte is adsorbed by the solid, and in the latter the analyte dissolves in the stationary liquid. The volatility of the analyte effects partitioning as more gas is passed along the column. The preponderance of GC methods involving antibiotics are by GLC. At some point in the past, the term "vapor phase chromatography" was used to describe what is now generally referred to as GC. Presumably, this was because the analytes were in the form of vapors when in motion. The usual carrier gases are indeed gases since all their critical temperatures are well below normal operating temperatures. Although the term "vapor phase chromatography" is still used in an official document [ 1] , it should vanish in the next few years.

3

Gas Chromatographic Analysis

The essential components to gas chromatographic systems are 1.

2. 3. 4.

The carrier gas, usually delivered from a cylinder and controlled by a two-stage regulator. An injection device, often by means of syringe needle through a septum, for introducing the sample into the gas stream [ 2] . The column that effects the separation. A detection device for monitoring the effluent from the column. It is necessary that this device be sensitive and produce a signal that is linear with respect to concentration over a workable range. Coupled to the detector is a means of recording the quantitative signal, as well as certain qualitative features, such as peak shape. Quantitation is usually accomplished by electronic integration of area under the analyte peak.

Much of the utility value of GC lies in the versatility afforded by varying some of the parameters, specifically (in order of their probable importance) 1. 2. 3. 4.

Temperature, usually from about 40 to 400°C Column material Diameter and length of column Flow rate of the carrier gas

Each of these parameters is easily varied, and the variation of each has significant effect on resultant chromatograms. Instrumentation

Generally, for the sake of careful control, chromatographic systems consist of rather compact cabinets housing the injection device, column oven, and detector. It is best that there be separate thermostatic controls and monitors of the temperatures in each of the three areas. The materials (including valves) for supplying the gases and electronics for the detector may either be contained in the same cabinet as the oven, or the arrangement may be modular, making for greater versatility.

Because of the necessity for strict tempera-

ture control from injection to detection, modular arrangement and rearrangement of the apparatus is more difficult with GC than with high-performance liquid chromatography (HPLC). Columns

For the most part, the methods used in the analysis of antibiotics involve GLC; that is, the column materials are inert solids coated with liquid film and packed into tubing, usually glass. The solid support material may be diatomaceous earths (Chromosorb Wand Supasorb, for example) or polymers (such as Chromosorb T or Columnpak) . The particle sizes are specified as part of the procedure in terms of mesh. Throughout this chapter meshs are reported by two numbers separated by a slash, such as 80/100. This means that all the material will go through an 80-mesh screen, but none through 100. Liquid phases are hydrocarbons, polyethylene glycols (Carbowax), silicone oil (DC- 550, the OV), esters (LAC 296), and silicone elastomers (SEs).

4

Alexander

Detectors A variety of detectors is used in GC, but there appear to be only two types used in analyzing antibiotic materials, flame ionization and electron capture. Some of the characteristics sought in detectors are fast response, linear response, sensitivity, precision, and, if possible, selectivity. Also, it is important that the detector response be readily recorded and calculable. The flame ionization detector (FID) is certainly the "workhorse" in the gas chromatographic analysis of pharmaceuticals. It responds to organic compounds, approximately on the basis of the number of carbon atoms. The range of linear response is quite wide, 106 or 107 [ 3] , making the simultaneous analysis of main component and impurities quite feasible. A drawback to the FID is that it is quite non selective. The electron capture detector (ECD) is somewhat selective in that it responds to halogens, conjugated carbonyls, and some nitrogen and sulfur compounds. In the detector chamber, the effluent gas is flowed between charged electrodes and a source of radioactivity ( 3tt or 63Ni). Nitrogen carrier gas tends to form electrons and positive ions, making a steady flow of current. When a halogen atom, or other forms of negative ions, appears and attracts the positive ions, there is a decrease in current between the electrodes, resulting in detector response. The ECD have a moderate range of linearity [ 4] , 102-103. They require more frequent cleaning than FID and a special license is needed because of the radioactivity. Performance Criteria A most important aspect of analytical methodology is that the analyst is assured that the system is properly performing. Although there is not yet complete uniformity of agreement among all analytical scientists about the most proper and best criteria, the United States Pharmacopeia [ 5] establishes five criteria for the analyst to use in deciding whether the system is satisfactorily performing. All these factors can be hand measured without elaborate electronic gear, and the first four often are. A number of the recent, sophisticated data processing systems provide programs or can be programmed to calculate automatically all five criteria. The analyst does have to be aware of "pig in a poke" pitfalls in which the data reported are not what are expected. This problem can arise from the assumption of baseline corrections and just when the integrator is activated. Plate Count

Plate count is a measure of the efficiency of the column, that is, how many

apparent transitions of the analyte there are between the mobile and stationary

phases. The concept is derived from the physical-chemical treatment of reflex distillation columns. Plate counts n are based on the baseline width of the analyte peak w and retention time of the analyte t. That is,

Gas Chromatographic Analysis

5

Peak width is at times measured at the base, at one-tenth peak height, and at half peak height. It is the author's belief that half peak height is more straightforward and objective. It is advantageous to run the chart paper rapidly through the peak area so that measurements are more precise.

Peak Symmetry This is a good diagnostic tool for determining the overall condition of the system. Dead volume and chemical reactions of analyte with stationary materials are depicted in peak symmetry measurements. For this, a perpendicular is drawn from the tip of the peak to the baseline and then the portions on either side are measured. Peak symmetry is expressed in terms of tailing factor T,

where peak width at 1/ 20 the distance from base to peak distance from the peak perpendicular to the leading edge of the peak

Resolution In all analytical chemistry determinations, it is essential that the analyte is separated from whatever impurities about which there may be concern. To be most rigorous, standards of both the analyte and the possible impurities are mixed and the results observed. Of course, in the development of the method, the investigator will do just that. However, it is very difficult to maintain an adequate supply of standards of the analytes, let alone impurities. In GC, an internal, neutral, weighable standard is often used. This is for both qualitative and quantitative purposes. The prescribed resolution factor R will be based on the widths of the peaks and the difference in the retention times. R

where ti and t2 are the retention times of the two substances being separated and w1 and w 2 are the corresponding widths of the peaks at baseline. Usually, readily purchased, high-purity materials are chosen as internal standards.

Capacity Factor k' This is a measure of the retention time. As mentioned elsewhere in this chapter, it is necessary that the retention time be such that the analyte is not at the solvent front, yet that the analysis is accomplished in a reasonable length of time. k' = amount of analyte in stationary phase amount of analyte in mobile phase

Alexander

6

time spent by analyte in stationary phase time spent by analyte in mobile phase

k'

t

ta

- 1

where t

a

retention time of nonretarded component (air). If necessary, this is measured with a thermoconductivity detector.

t

retention time of analyte.

Precision

The analyst is expected to make repeated injections and calculate the coefficient of variation (CV). Assuming that the column is properly functioning, the CV serves as a measure of the adequacy of the detection and quantitation equipment. In the absence of an internal standard, it also serves as a measure of the reproducibility of the injection process. SPECIAL TECHNIQUES Derivatization

Since very few of the antibiotics are sufficiently volatile and stable at elevated temperatures to chromatograph, means other than direct injection must be used. The aminoglycosides and some others have amino or alcohols functional groups that are readily esterified. Silyl acids render stable volatile esters, which chromatograph easily using the FID. A typical reaction is Me I R -OH + R -Si-Me 1

2

I

Me I

+

Me Antibiotic

Silylating agent

R H + R -0-Si-Me 2

1

I

Me Volatile, stable

It must be noted that several of the silylating agents are extremely hydrophilic, which requires that much caution be applied in working with these compounds. It should also be pointed out that, where there are several derivatizable groups, mixtures of esters are often formed making for many an analyst's dilemma. Investigators have varied the formulas for the silylating reactions until only one peak was obtained. They then checked with a gas chromatograph-mass spectrometer interfaced combination to ascertain the identity. Several of the more common derivatizing agents are usually referred to by acronyms, which will be used throughout this chapter. Some of these are

BSA BSTFA HFBA

N, 0-bis(trimethylsilyl)acetamide N, 0-bis( trimethylsilyl)trifluoroacetamide heptafluorobutyric anhydride

Gas Chromatographic Analysis

HFBI

HMDS TMCS TMS TMSDEA TMSI

7

N- heptafluorobutyrylimidazole hexamethyldisilazane trimethylchlorosilane trimethylsilyl N -trimethylsilyldiethylamine N -trimethylsilylimidazole

Temperature Programming As mentioned, two of the parameters of GC that can be varied to develop useful chromatographs are temperature and column material. When a specific pharmaceutical ingredient, or a simple mixture of similar materials, is being analyzed, a method involving a column and a specific temperature (isothermal) often works quite well. However, the need sometimes arises to analyze complex mixtures. It is impractical to change the column material during the course of analysis; however, the column temperature can be changed readily. Many of the commercial instruments are intended to be used with temperature programming. The usual approach is to first chromatograph at a low temperature so that the more volatile analytes are discernible and analyzable without being part of the solvent front. Then the column temperature is raised according to a preset program to the point at which the least volatile analytes of interest chromatograph within a reasonable time. Often, at the end of the analysis, the instrument is programmed to return to the initial temperature automatically. Obviously, by temperature programming, many analyses are accomplished that would otherwise be impossible. A difficulty that arises with temperature programming is that of baseline drift since the background current of the detectors changes as the chromatographic conditions change. In many models, this problem is largely compensated for by the use of dual detectors. Two gas streams are piped to two identical detectors and the difference in their signals recorded. The sample stream comes from the analytical column, and the reference stream is carrier gas. It should be noted that temperature programming only involves changing the column oven temperature. The injector temperature is kept high enough to assure that the sample is completely vaporized, but not so high as to decompose the sample. Likewise, the detector area heater is kept high enough to prevent condensation of analytes. Pyrolysis This technique is another approach used to obtain analyses of nonvolatile substances. The sample is flash heated at a temperature rise rate of 20 or 30°C per microsecond, to as high as 1200°C and for as long as 30 sec. The resultant gases are then passed onto the gas chromatograph column. Usually, the flash heating is performed in a precolumn space immediately at the head of the column. With proper design and execution, band spreading is kept at a minimum. The chromatographic peaks obtained usually do not represent known substances. Instead, the analyst expects "fingerprint" patterns that are often reproducible and from which both qualitative and quantitative information can be obtained.

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APPLICATIONS Simpler Molecules Chloramphenicol

The antibiotic most often made reference to in gas chromatographic literature is chloramphenicol. This is probably because of its widespread use in both human and veterinary medicine as well as on plant crops. There is much concern regarding overuse and abuse, which results in increasing calls for analytical results. Margosis [ 6] developed a method for pharmaceuticals that involved silylation with BSA in acetonitrile, m-phenylene dibenzoate as internal standard, and columns of low-polarity methyl silicone gum or fluid coatings (OV-1, OV101, SE-30, and others) on 80/100 or 100/120 silanized, acid-washed, fluxcalcined diatomite (Gas Chrom Q). Detection was by FID maintained at 250°C. The injection port was also at 250°C and the column at 240°C. A rather largescale interlaboratory collaborative study was undertaken with bulk samples. The overall coefficient of variation for three samples and 14 participants was 2. 97. Mean recovery was 100. 01%. This is probably as good an agreement as one can reasonably expect under these circumstances. Dosage forms of chloramphenicol were extracted with ethyl acetate, which was then washed with water and filtered if necessary. Aliquots in conical centrifuge tubes were evaporated to dryness under N 2 and then dissolved in derivatizing reagent. Ointments were cleaned with gravity-flow columns of diatomaceous earth ( Celite) treated with pH 5. 80 phosphate buffer. Excipient material was extracted with cyclohexane and the chloramphenicol with ethyl acetate. Subsequently, Irwin and coworkers [ 7] reported derivatizing with phenylboronic acid with triphenylbenzene for internal standard, all dissolved in ethyl acetate. The structure of the derivative was verified by spectroscopic studies. The column was 1.5% OV-17 coating on Supasorb M, AW-DMCS, 100/ 120, and was maintained at 260°C. The flash-heater-injection port was kept at 300°C. Capsules, eye drops, and ear drops were dissolved and diluted with ethanol and water then extracted into ethyl acetate. Eye ointments were diluted with petroleum ether (octane) and extracted into acetonitrile. Pickering et al. [8] derivatized with TriSil (HMDS and TMCS) in pyridine and then diluted with cyclohexane. The injection port was at 300°C. The column of 3% OV-17 on Gas Chrom Q was at 240°C, and ECD c63Ni) was at 300°C. The internal standard was D(-)threo-N-(13-hydroxy-a.-[hydroxymethyl] -p-nitrophenethyl)acetamide. The authors noted that this GLC method was particularly useful for detecting 6-desmethylgriseofulvin. Pickering also reported coupling the GC to a mass spectrometer. Chloramphenicol was extracted from phosphate buffer with ethyl acetate and then derivatized. The injection port was at 250°C, and the column, using OV-1, was at 240°C . The following illustrate additional methods used to extract chloramphenicol from various media. Holtmannspoetter and Thier [ 9] analyzed meat, milk, and eggs for chloramphenicol along with six sulfonamides. The samples extracted with acetonitrile, which was then shaken with NaCl and methylene chloride. The organic layers were combined and evaporated. After further cleanup, chloramphenicol was derivatized with diazomethane and heptafluorobutyric acid. The column was a capillary coated with SE-30-SE-52 (1: 1). The detection limit is less than 0.01 mg/kg (10 ppb).

Gas Chromatographic Analysis

9

Three other teams of investigators all used ECD in their ~nalysis of nonpharmaceutical materials. Wal and colleagues [ 10) were also looking at chloramphenicol levels in milk. Their detection limit was 50 ppb using 63Ni at 290°C. The injection port temperature was 220°C and the column ( 3% Dexsil 300 on Supelcoport AW DWCS) at 195°C. The column is maintained by injecting HFBA and then heating 24 hr at 250°C. Milk samples were extracted with acetonitrile, which was then evaporated to dryness. The residue was dissolved in H20 and mixed with pH 6 phosphate and iso-octane. The aqueous layer was then adjusted to pH 6. 5 and extracted with ethyl acetate. The excipients caused no interferences. Residues were reacted with HFBA and injected promptly. At 25 ppb the error is ±9 ppb. It was thought that 50 ppb was the level at which a high degree of confidence can be assumed. · Sasaki and coworkers [ 11) were assaying vegetable crops for chloramphenicol. They used columns of 0.1% diethylene glycol succinate and 0. 03% H3Po 4 on Gas Chrom Q 60/80 held at 200°C. Inlet and ECD (63Ni) temperature were held at 230°C. The sample preparation involved three successive evaporations. The sample was extracted with ethyl acetate, which was filtered and evaporated to dryness using an air jet. The residue was taken up in n-hexane, which was then extracted with acetonitrile and that evaporated to dryness. This residue was dissolved in benzene and passed through a gravity-flow column of silicic acid and sodium sulfate. Benzene and ethyl acetate eluted the chloramphenicol, and again, there was evaporation to dryness. The residue was acetylated with acetic anhydride and injected. The recovery was 61-86%. In an environmental study, LeCloirec et al. [ 12) also used electron capture detector in their study of chloramphenicol in water. They extracted with methylene chloride. Cycloheximide

Brown [ 13) reported on the analyses of formulations of this fungicide. The flask-heater-injection port and column (1% QF-1 on Gas Chrom Q 80/100) were both at 200°C and the FID at 225°C. The internal standard was cholesteryl acetate and silylating agent was a combination of TMCS and BSTFA. Initially, two peaks of derivatized cycloheximide were observed, the mono- and disubstituted derivatives. By adding isopropanol, quantitative yields of only the monosubstitution derivative were obtained. Mass spectrometry was used to identify the observed peaks. The sample preparation is rather straightforward. Where the excipients are soluble in organic solvents, the sample is leached in water, which dissolves the cycloheximide. It is then extracted into chloroform and evaporated to dryness. Where the excipients are insoluble in organic solvents, the sample is leached with benzene. In the analysis of 15 different weighings, the coefficient of variation was about 2. O. In comparing microbiological and GLC assays of nine samples of bulk,

Average CV

GLC

Microbiological

100.3 2.0

99.6 5.8

Cycloserine

Sondack et al. [14) chromatographed cycloserine with the injection and FID areas at 150°C. The columns of 3. 8% UC W98 on Diatoport S 80/ 100 was

10

Alexander

at 115°C. The internal standard was hexamethylbenzene and the silylating agent a combination of BSA and TMCS. A coefficient of variation of 0.5 was obtained.

Griseofulvin Shah et al. [15] were able to chromatograph underivatized griseofulvin extracted from skin, using ECD, which was very helpful because of its selectivity. Margosis [ 16] also chromatographed griseofulvin without derivatization. He was particularly looking for dechlorogriseofulvin since this substance resulted in erroneous assays by the ultraviolet absorption technique. Up to 8. 5% of the dechloro compound was found in some preparations. By adding together the GLC readings for both griseofulvin and the dechloro, good agreement with the spectrophotometric method was obtained. In an interlaboratory collaborative study of the GLC among 19 participants, an overall coefficient of variation of 1. 5 was obtained. In later work, Kamimura and colleqgues [ 17] found that 6-desmethylgriseoful vin, a metabolite of griseofulvin, would not chromatograph well without derivatization. They found it necessary, for the analysis of this metabolite in plasma, to form the methylated derivative, griseofulvin, with diazomethane. Two assays were made, with and without the diazomethane reaction. Griseofulvin was measured both times, and the difference was the desmethyl compound. The injection port and ECD ( 63 Ni) were at 300°C, and the column, 1. 5% OV-225 on Gas Chrom Q 100/200, was at 275°C. Blood samples were centrifuged immediately after withdrawing and the plasma frozen until analysis. In a second method, the plasma extract was treated with diazobutane, resulting in the separation of griseofulvin and its metabolites.

Phosphonomycin Shafer et al. [18] analyzed for this antibiotic in human urine. They obtained very complicated chromatograms with numerous peaks. With preparative paper chromatography, the gas chromatograms were rendered very much simpler and the recovery of phosphonomycin was quantitative. After BSA silylation, samples were gas chromatographed with the column ( 4% F-60 DC560 coated over 1.5% SE-30 on acid-washed, silanized Gas Chrom P 100/120) at 65°C initially and programmed to 220°C. The detector was FID. Identification of the TMS derivative was done by mass spectrometry. Ami nogl ycosides The aminoglycosides present the chemical analyst with one of the greatest of challenges in analytical medicinal chemistry. These compounds have little or no ultraviolet-absorbing chromophores, and their infrared and nuclear magnetic resonance spectra are ill defined. Thus, even though it was necessary to derivatize and perhaps to use ECD, there still is much interest in the GLC analysis of aminoglycosides. All but one (griseofulvin) of the official methods in the Code of Federal Regulations ( CFR) [ 1] using GC pertain to aminoglycosides.

Kanamycin and Paromomycin Tsuji and Robertson [ 19] formed TMS derivatives of kanamycin, neomycin, and paromomycin with TMSDEA. Chromatographic separation was accomplished

Gas Chromatographic Analysis

11

with OV-1 on Gas Chrom Q 100/120 and FID. Baseline or better separations were obtained of kanamycins A and B , neomycins B and C , and paromomycin I and II. Murata et al. [ 20] formed derivatives of the kanamycins and neomycins with bis(trifluorosilyl)acetamide and chromatographed them directly into a mass spectrometer. Neomycin

Margosis and Tsuji [ 2, 21] collaborated on this difficult analysis. Since the stereoisomers, neomycins B and C, have different therapeutic effects, it is important that the analysis include separate assays of the two. In reviewing earlier work, Margosis found poor correlation of values reported for neomycin C and excessive solvent tailing. After close and critical examination of the procedure, the authors arrived at several improvements, the most spectacular a redesign of the injection port. The original design of the instrument involved an inlet sleeve. Between it and the column was a joint with Teflon and brass exposure. The Margosis-Tsuji redesigned port eliminated the junction. The brass barrier was drilled out and a modified column placed in the instrument so that the injection area walls were all glass. Following that, good correlation was obtained for assays of neomycin

c.

The evolved procedure involves 3% OV-1 on Gas Chrom Q 100/120 with trilaurin as internal standard and derivatization with TMSDEA. The temperatures are injection port, 300°C, and oven, 290°C. To condition the column, Silyl- 8 was injected and then the column heated to 330°C with no flow. Spectinomycin

Brown and Bowman [ 22] described a procedure that is nearly identical to the current one in the CFR [ 1] . The conditions were injection port, 265°C; column (5% SE-52 on Diatoport 5 {80/100}), 215°C; FID, 270°C; internal standard, triphenylantimony; and derivatizer, HMDS. Considerable research had to be done to establish the best conditions for obtaining a single derivative peak. There are six possible positions for silyl substitution, but the one arrived at had four silyl groups. By obtaining and studying the mass spectral patterns of several related compounds, it was concluded that the substitutions were occurring at the four hydroxyl and enol oxygens. Gentamicin, Tobramycin, Netilmicin, and Amikacin and Their Combinations

Mayhew and Gorbach [ 23] found it necessaJ.>y to measure serum levels of several antibiotics simultaneously, rapidly, and at reasonable costs. The targeted antibiotics were gentamicin, tobramycin, netilmicin, and amikacin with kanamycin A or paromomycin B as internal standards. The sample preparations included precipitation of interfering blood substances, centrifugation, evaporation and the supernatant layer, and then derivatization. The chromatographic conditions are listed in Table 1. Reasonably good agreements with microbiological assays were reported for gentamicin and amikacin. It is interesting to note that the three prominent isomers of gentamicin (C1, ClA• and C2) are separated into two bands, C1 and C1A-C2. New columns were flow-conditioned before the first use and attachment to the ECD. During this time, derivatized aminoglycoside was injected. With care, a year of use was obtained from each column.

12

Alexander

Table 1 Chromatographic Conditions for Aminoglycoside Combination Gentamicin, tobramycin , netilmicin

Conditions

Amikacin

Injection port temperature (°C)

287

277

Column temperature (°C) ECD ( 63 Ni) temperature ( °C)

272

262

287

277

Internal standard

Kanamycin A

Paromomycin B

Derivatizing agent

TMSI + HFBl

TMSI + HFBI

Immobile liquid phase

3%0V-101

1% QV-,17

Column support

Chromosorb W AW DMCS 80/100

Same

Source:

From Ref. 23.

Lincomycin Antibiotics Clindamycin

Brown [ 24] reported the analysis of the hydrochloride, 2-phosphate, and 2-palmitate hydrochloride salts of clindamycin as well as several dosage forms. Nearly the same methods appear in the CFR, which is reasonable to expect since the Upjohn Company proposed the CFR methods and since Brown was from the Upjohn Laboratories. The chromatographic conditions are given in Table 2. Brown's sample preparation for the hydrochloride essentially consists of extracting clindamycin into chloroform from aqueous sodium carbonate solution. In the case of the phosphate, the chloroform layer contained free clindamycin, which was assayed. The phosphate-containing aqueous borate layer was treated with phosphatase and then extraction and assay done for the liberated clindamycin. The palmitate was directly leached into chloroform. The CFR preparations are essentially the same except that no separate assay for free clindamycin is performed. Linocomycin

Margosis [ 25] described the procedure that is essentially the one currently in the CFR [ 1]. It involves column of 5% SE-30 on Gas Chrom Q 80/ 100 maintained at 257°C. The injection port and FID were at 280°C. To alleviate a problem with deposits forming on an electrode of the FID, methanol and ethanol were used as solvents. Good agreement with microbiological assays was obtained. Penicillin and Cephalosporin Derivatives Penicillin G has been important and widely used since the 1940s. Thus it is to be expected that one of the first GC references [ 26] involves that drug. In spite of the difficulty the analyst experiences in gas chromatographing these nonvolatile compounds, references still appeared in the late 1970s on

13

Gas Chromatographic Analysis

Table 2 Chromatographic Conditions for Clindamycin Salts Conditions Clindamycin HCL Injection port temperature (oC) Column temperature ( °C) FID temperature (°C) Internal standard Derivatizing agent Immobile liquid phase Column support Clindamycin 2-phosphate Injection port temperature ( °C) Column temperature ( 0 c) FID temperature ( °C) Internal standard Derivatizing agent Immobile liquid phase Column support Clindamycin 2-palmitate HCl Injection port temperature (OC) Column temperature ( °C) FID temperature (°C) Internal standard Derivatizing agent Immobile liquid phase Column support

CFR [ 1]

Brown [24] 170 170 200 Hexacosane (CF3C0)20 3%0V-17 60/80 Oas Chrom Q

Ambient 200 215 Cholestane (CH3C0)20 1% SE-30 60/80 Diatoport S

170 170 200 Hexacosane (CF3C0)20 3%0V-17 60/80 Gas Chrom Q

Ambient 180 215 Same Same 1% SE-30 80/ 100 Diatoport S

260 260 285 Cholesteryl benzoate (CH3C0)20 1% SE-54 60/80 Diatoport S

280 275 290 Same

the GC of ampicillin [ 27] and cephalexin [ 28] . Roy and Szinal [ 29] pyrolyzed the sample.

Same 1% UC-W98 80/ 100 Chromosorb WHP

To circumvent the non volatility,

Derivatization Evrard et al. [ 26] prepared methyl esters of pencillin G and a-phenoxyethylpenicillin with diazomethane dissolved in ether and chromatographed on silanized Gas Chrom P 100/ 120 coated with SE-30, SE- 52, or QF-1. They used an ECD with strontium foil. Hisnta et al. [ 30] chromatographed derivatives made with HMDS of cloxacillin, dicloxacillin, methicillin, penicillin G, penicillin V, and phenethicillin plus isomers. They used 2% OV-17 on Supelcoport 80/ 100 with FID. Injection port, column, and detector were maintained at 275°C. (For methicillin, the column temperature was 240°C.) Wu et al. [ 27] gas chromatographed ampicillin after derivatization with a complex mixture of pyridine, HMDS, TMCS, TMSI, and BSA (0.3:0.3:0.3: 0.1: 0. 5 by volume). Mas spectrometric data ind.icate that the one peak, mono-TMS-ampicillin, was obtained with both ampicillin and sodium ampicillin.

Alexander

14

Also, a-aminobenzylpenicilloic acid, a degradation product, was found to give a separate peak. The exerpimental conditions were injection port and FID at 270°C, 5a-cholestane as internal standard, and the column ( 1. 5% OV-17 on Chromosorb WAW DMCS 60/80) temperature programmed from 230 to 270°C. In analyzing cephalexin, Nakagawa et al. [ 28] also used a mixture of silylating agents, acetonitrile, BSA, and TMCS (2:5:5 by volume). The mass spectra indicated the derivative to be substituted at the amide nitrogen, carboxylic acid, and amine positions. The injection port was at 280°C, the column (1. 5% OV-101 on chromosorb W 60/80, DMCS treated) at 175°C, and the FID at 280°C. The authors concluded that this method had good potential as a sensitive assay method for cephalexin in aqueous solutions and biological fluids. Pyrolysis

Roy and Szinal [ 29] pyrolyzed with a platinium ribbon in a modified injection port. The sample (up to 0.1 mg) placed on the ribbon was heated at the rate of 20°C per microsecond to 875°C for a duration of 1 sec. The gases were then passed onto a column of 3% XE-60 on Gas Chrom Q 80/ 100 at 100°C and then to the FID. Individually characteristic chromatograms, suitable for identification, were obtained with 10 penicillins and four cephalosporins. The pyrolysis-chromatography was repi oducible. Concentration -response curves were prepared for cephalexin, methicillin, oxacillin, and penicillin G and found to be linear over a range of 10- 2 to nearly 102 µg. Several of the peaks were identified by mass spectrometry. The usual excipients did not interfere. Antitumor Drugs Watson and Chan [ 31] developed a method for testing carminomycin, chromomycin A3, daunorubicin, doxorubicin, and mithramycin as well as their metabolites. These compounds could not be silylated directly, but their aglycones could be silylated. The antibiotics were heated in 0. 1 N HCl and extracted with ethyl acetate, which was then evaporated off. The aglycones were then derivatized with methoxylamine and a mixture of BS TF A, TMC S, and TMSI (3: 3: 2 by volume). The instrumental settings were 260°C for the flash heater injection port and 260°C for the column, 3% OV-101 on Gas Chrom Q 100/ 120. From the column, the sample was passed into a molecular separator and restrictor at 210°C for mass spectrometry. Excess reagent was vented off. Andrews et al. [ 32] also formed aglycones by HCL hydrolysis, Daunorubicin and other danosamine aglycones were extracted with chloroform, evaporated to dryness, and then silylated with a pyridine-TMSI reagent. The column of 3% OV-17 on Gas Chrom Q 100/200 was programmed from 120 to 250°C. The transfer lines to the mass spectrometer were kept at 2002500C. Other Antibiotics Erythromycin

Tsuji and Robertson [ 33] demonstrated very good accuracy, recovery, and precision with the analysis of erythromycin preparations. They used 3% OV-225 on Gas Chrom Q 100/120 at 275°C and FID. It was necessary to "condition" the column at 330°C, no flow, for 45 min after injections of TMS donors and silylated erythromycin. Ground tablet mix was extracted with

15

Gas Chromatographic Analysis

methylene chloride, evaporated to dryness, and then silylated. A 100. 6% recovery from simulated mixes was obtained with a CV of 2. 3. In the analysis of 13 commercial tablets, microbiological assay reported 254 mg per tablet and GC, 250 mg per tablet. Lasalocid

Westley et al. [ 34] found that this complex antibiotic could not be gas chromatographed directly, and attempts to form a consistent derivative were unsuccessful. However, they obtained chromatographable degradation products by flash heating ("thermolizing") in the injection port. Solutions of lasalocid in tetrahydrofuran formed the retroaldol ketone with one- half of the molecule and the remainder formed dihydro-1-naphthol and dihydro- 2naphthol (see Figure 1). Aliquots of whole broth were mixed with tetrahydrofuran containing methylbehenate (internal standard). The supernatant portions were injected into the injection port at 310°C. The column (10% OV-17 on Gas Chrom Q 100/120) was maintained at 250°C and the FID at 290°C. The coefficients of variation obtained were 0. 9 for the drug substance and 3. 7 for the broth. Tetracyclines

Despite the very wide use of tetracyclines for years, there are very few reports on their analysis by GC. This clearly indicates the difficulty involved. Tsuji and Robertson [35] did report a method for chlorotetracycline,

COOH OH

OH 0

l

LASALOCID

H

RETROALDOL KETONE HO and

CH 3 DIHYDR0-1-NAPHTHOL

CH 3 DIHYDR0-2-NAPHTHOL

Figure 1 Degradation of lasalocid by "thermolizing."

(From Ref. 34.)

Alexander

16

doxycycline , oxytetracycline , tetracycline, 4-epi-tetracycline, and 4-epianhydrotetracycline. The silylation, with freshly prepared BSA plus TMCS dissolved with internal standard (trioctanoin) in pyridine, took 24 hr at room temperature. Higher temperatures caused degradation. Mass spectrometric data indicated that there were five silyl substitutions per tetracycline molecule. The flash-heater-injection was at 260°C, the column (3% JXR [methylsilicone] on Gas Chrom Q 100/ 120) at 260°C, and the FID at 290°C. The CV was about 2. 3 by peak height ratio. Excellent agreements with microbiological and ultraviolet assays were obtained. Impurities in Antibiotics. N, N-Dimethylaniline

This substance is often used in the preparation of the semisynthetic penicillin derivatives, yet it is purported to be carcinogenic. Consequently, there has been much concern and call for analysis of this possible contaminant. Because of the volatility of N, N -dimethylaniline, GLC is well suited to this analysis. It was necessary to extract the amine from the antibiotic prior to chromatography. Margosis [36) extracted with cyclohexane from 5% NaOH. Naphthalene was used as internal standard. The experimental conditions were injection port at 60°C, column ( 3% OV-17 on Gas Chrom Q 100/ 120) at 60°C, and FID at 150°C. The column had to be silylated by injection of a silyl reagent with the temperature at 275°C prior to the analysis. At the level of 1. 7 ppm of N, N dimethylaniline in antibiotic, the CV was 7. 9. The limit of detection was about 0. 5 ppm. Amounts found in commercial samples ranged up to 1500 ppm. Quercia et al. [ 37) had considerably higher temperatures, with injection port and detector at 220°C and column ( 3. 3%of a silicone on Chromsorb WHP 80/ 100) at 120°C. They found N, N-dimethylaniline only in penicillins, not cephalosporins. Of 124 samples, 18. 5% contained "higher than permissible" quantities of the contaminant. Choi and Park [ 38) used selective ion focusing generated by electron impact ionization as detector. Their extraction and column composition were the same as those of Margosis. Their temperatures were injection port at 250°C, column at 150°C, molecular separator at 250°C, and current monitor at 200°C. They recovered about 97% from spiked samples. The detection limit was about 50 ppb. Nachtmann and Gstrein [39) were looking for both N ,N-dimethylaniline and formaldehyde used in sterilization. For the former, they extracted with n-heptane and used either a regular FID or a "N-P FID," a nitrogen- and phosphorus-sensitive FID. Their temperatures were injection port at 200°C, column (3% OV-17 on Gas Chrom Q 60/120) at 105°C, and detector at 250°C. The turnaround time was only 4 min , and the detection limit was less than 1 ppm. Nachtmann and Gstrein had to make a separate chromatographic analysis to analyze for formaldehyde, which was converted to formaldorime with aqueous hydroxylamine hydrochloride. The settings were injection port at 150°C, column (10% Carbowax on Chrom V- HP 801100) at 100°C, and detector (N-P FID) at 250°C. The detection limit was about 1 ppm.

Gas Chromatographic Analysis

17

6-Aminopenicillanic Acid

Silingardi et al. [ 40] were concerned that reaction intermediates remained in the finished antibiotic. They reported a method for 6-aminopenicillanic acid and 7-amino-3-methyl-fi3-cephem-4-carboxylic acid using BSA in pyridine for the former and TMCS and HMDS for the latter. The instrument settings were injection port at 220°C, column ( 4% OV-17 on HP Chromosorb 80/ 100) at 180°C, and FID at 200°C. The column was conditioned by repeated injections of Silyl S and overnight at 300°C with gas flow. Comparisons with assay from ultraviolet absorption and iodometric methods indicated the GLC method to be quite satisfactory. Water

Casana-Barber and Gassiot-Matas [41] investigated methods for the analysis of water in ampicillin trihydrate. They found GLC with a thermoconductivity detector to give about the same values as Karl Fischer, but with less precision. The thermoconductivity detector, the original detector used in GC, is sensitive to the ability of the effluent to conduct heat away from a heated element. It is probably the only detector sensitive to water vapor. SUMMARY AND CONCLUSION

Gas chromatography has played an active role in the analysis of antibiotics over the last two decades, particularly with the simpler molecules. However, with the advent of HPLC, investigative work in method development has turned away from GC. Future developments in GC will involve two aspects. First, "direct into the mass spectrometer" studies of metabolites is more readily accomplished by GC than LC since there is so much less carrier material with GC. Second, the analysis of such impurities as N ,N-dimethylaniline is often quite readily accomplished by GC, and I expect to see more developments in this area.

REFERENCES 1.

2. 3. 4. 5. 6. 7. 8. 9. 10.

U.S. Government Printing Office, Code of Federal Regulations, Title 21, Part 436, April 1, 1983. M. Margosis and K. Tsuji, J. Pharm. Sci., 62:1836 (1973). L. Szepesy, Gas Chromatography, CRC Press, Cleveland, 1970, p. 149. H. W. McNair, J. Chromatogr. Sci., 16:578 (1978). The United States Pharmacopeia, 20th revised ed., Mack Publishing Co., Easton, PA, 1980, p. 945. M. Margosis, J. Chromatogr., 47:341 (1970); J. Pharm Sci., 63:435 ( 1974). W. J. Irwin, L. W. A. Po, and R. R. Wadwani, J. Clin. Hosp. Pharm., 5: 55 ( 1980). L. K. Pickering, J. L. Hoecker, W. G. Kramer, J. G. Liehr, and R. M. Caprioli, Clin. Chem., 25:300 (1979). H. Holtmannspoetter and H. P. Thier, Dtsch. Lebensm-Rundsch., 78: 347 ( 1982) (through CA 98: 015557j). J. M. Wal, J. C. Peleran, and G. Bories, J. Chromatogr., 168:179 ( 1979).

18

11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41.

Alexander

K. Sasaki, M. Takeda, and M. Uchiyama, J. Ass. Offic. Anal. Chem. 59: 1118 ( 1976). C. LeCloirec, P. LeCloirec, M. Elmghari, J. Morvan, and G. Martin, Intern. J. Environ. Anal. Chem., 14:127 (1983). L. W. Brown, J. Agr. Food. Chem., 21:83 (1973). D. L. Sondack, F. E. Gainer, and H. J. Wesselman, J. Pharm. Sci., 62:1344 (1973). V. P. Shah, S. Riegelman, and W. L. Epstein, J. Pharm. Sci., 61:634 ( 1972). M. Margosis, J. Chromatogr., 70: 73 (1972); J. Pharm. Sci., 64: 1020 ( 1975). H. Kamimura, Y. Omi, and Y. Shiobarba, J. Chromatogr., 163:271 ( 1979). H. Shafer, W. J. A. VandenHeuvel, R. Ormand, F. A. Kuehl, Jr., and F. J. Wolf, J. Chromatogr., 52: 111 (1970). K. Tsuji and J. H. Robertson, Anal. Chem., 42: 1661 (1970). T. Murta, S. Takahashi, and T. Takeda, Bunseki Kagaku, 22:405 ( 1973) (through CA 79: 83498y). K. Tsuji and J. H. Robertson, Anal. Chem., 41:1332 (1969). L. W. Brown and P. B. Bowman, J. Chromatogr. Sci., 12:373 (1974). J. W. Mayhew and S. L. Gorbach, J. Chroma to gr. , 151: 133 ( 1978) . L. W. Brown, J. Pharm. Sci., 63:1597 (1974). M. Margosis, J. Chromatogr., 37:46 (1968). E. Evrard, M. Clasen, and H. Vanderhaeghe, Nature, 201: 1124 ( 1964). H.-L. Wu, M. Masada, and T. Uno, J. Chromatogr., 137:127 (1977). T. Nakagawa, J. Haginaka, M. Masada, and T. Uno, J. Chromatogr., 154:264 (1978). T. Roy and S.S. Szinal, J. Chromatogr. Sci., 14:580 (1976). C. Hisnta, D. L. Mays, and M. Garofalo, Anal. Chem., 43: 1530 ( 1971). E. Watson and K. K. Chan, J. Pharm. Sci., 67: 1243 (1978); K. K. Chan and E. Watson, J. Pharm. Sci., 67:1748 (1978). P. A. Andrews, P. S. Callery, F. E. Chon, M. E. May, and N. R. Bachur, Anal. Biochem., 126: 258 (1982). K. Tsuji and J. H. Robertson, Anal. Chem., 43:818 (1971); J. H. Robertson and K. Tsuji, J. Pharm. Sci., 61:1633 (1972). J. W. Westley, R. H. Evans, and A. Stempel, Anal. Biochem. , 59: 57 4 (1974). K. Tsuji and J. H. Robertson, Anal. Chem., 45:2136 (1973). M. Margosis, J. Pharm. Sci., 66:1634 (1977). V. Quercia, C. Desena, G. Iela, G. Paghozzi, and N. Pierini, Boll. Chim. Farm., 119: 619 (1980). J. K. Choi and M. K. Park, Arch. Pharmacol. Res., 4:85 (1981). F. Nachtmann and K. Gstrein, Mikrochim. Acta, 2:83 (1981). S. Silingardi, M. diBitetto, and A. Mangia, J. Pharm. Sci., 66: 1769 ( 1977). A. Casana-Barber and M. Gassiot-Matas, Afinidad, 298:635 (1972).

2 Ultraviolet and Light Absorption Spectrometry ZOLTAN M. DINYA and FERENC J. SZTARICSKAI Research Group for Antibiotics of the Hungarian Academy of Sciences, L. Kossuth University of Debrecen, Hungary

Ultraviolet and Visible Spectrometry as an Analytical Tool Amino Acid Derivatives, Oligo- and Polypeptide Antibiotics Amino Acid Derivatives S-Lactam Antibiotics Polypeptide and Cyclic Polypeptide Antibiotics Depsipeptide Antibiotics Glycopeptide Antibiotics Carbohydrate-Glycoside Antibiotics Aminoglycosides Anthrone and Anthracycline Glycoside Antibiotics Nucleoside Antibiotics Antibiotics Containing Condensed Aromatic Systems Antibiotics with a Naphthacene Skeleton (Tetracyclines) Antibiotics with a Phenanthrene Skeleton Polyene and Polyyne Antibiotics Macrolide Antibiotics (Large Lactones) Nonpolyene Macrolide Antibiotics Macrocyclic Di- and Tetralactones Polyene Macrolides Ansa Macrolides (Macrocyclic Lactams) Polyether Antibiotics Heterocyclic Antibiotics Nitrogen-Containing Heterocyclic Oxygen -Containing Heterocylic Additional, Unclassified Antibiotics Conclusions References

20 22 22 22

32 34

35 39 39

42

44

53 53

59 59 61 61 67 67 76 79 79 79

81 81 82 83

19

20

Dinya and Sztaricskai

The research of antibiotics, growing to huge dimensions throughout the world, has enriched the treasury of medications for humankind to a great extent. It is quite natural that the extensive development of modern isolation techniques and physicochemical structural investigation methods has gone a long way to make this research more successful. By application of various fields of spectroscopy, the structure elucidations, previously taking years of laborious work, can be now significantly shortened. Due to their speed and accuracy, these methods have become indispensible in pharmacological studies and in the qualification of the intermediates and formulated products of medicinal industrial technology [ 1-10] . Therefore, it is not surprising that these procedures have been included in official pharmacopeia [ 11-16] , allowing their practical application. One of the earliest methods of physicochemical analysis, ultraviolet and visible absorption spectrometry, has a huge literature, as demonstrated by a large number of monographs [ 17-28] as well as by the excellent review materials dealing with the ultraviolet [ 29-36] and visible [ 34-38] absorption spectrometry of organic compounds. In the pharmaceutical industry, ultraviolet and visible spectrometry has been used as a routine analytical assay method [ 1-7, 9-16], reviewed by earlier volumes of Analytical Chemistry [39-44]. References to the most important ultraviolet and visible spectral data of antibiotics are given in Volumes 1-X of the CRC Handbook of Antibiotic Compounds, edited by Berdy et al. [ 45] , as well as in Volumes I and II of the Index of Antibiotics from Actinomycetes [46,47] compiled by Umezawa. These excellent monographs, however, do not serve analytical or spectroscopic purposes, but are prepared, first, with the goal of providing help in the identification of classification of newly isolated antibiotics. Although spectral data of a few marketed antibiotics are included in collections of spectra and handbooks [ 4, 5, 7, 16], these do not satisfy the growing demands. The review materials give information primarily about the ultraviolet and visible spectral properties and analysis of (3-lactam [ 48- 52] , polyene macrolide [ 5355] , and aminoglycoside groups of antibiotics [ 56] and also tetracyclines [ 57] . Therefore, the authors believe that summarization of topics in ultraviolet and visible spectral properties of the individual groups of antibiotics-with respect to their structural feature-is extremely reasonable, together with the discussion of the possible analytical applications. In addition to the term, "ultraviolet and visible spectrometry," in the treatment of this subject the internationally accepted abbreviation UV-VIS will be also used. This review is based primarily on the references of Chemical Abstracts and CA Selects and extends the series of reviews sponsored by Analytical Chemistry. The literature on the ultraviolet and light absorption spectrometry of antibiotics continues to be extensive and the citations in this chapter represent a huge effort on the authors' part to select those results of most probable interest to (analytical) chemists working in the field of antibiotics. The authors apologize, in advance, for any error of judgment in omitting certain references. ULTRAVIOLET AND VISIBLE SPECTROMETRY AS AN ANALYTICAL TOOL

Of the physical methods of structure elucidation, ultraviolet and visible spectrometry (UV-VIS) is the oldest and most commonly used, considered

UV and Light Absorption Spectrometry

21

nowadays as a routine analytical tool. It is suitable for both qualitative and quantitative purposes. A spectrum, applicable for structure analysis, is obtainable with compounds containing chromophores. The technique UV-VIS is not qualified, in general, for detection of the presence or absence of function al groups, but it is especially servicable for the examination of conjugated (unsaturated) compounds. If the degree of conjugation changes as a result of an effect, such as steric or electron-shift effects, substitution, formation of salt, solvent effect, or tautomerization, this change is characteristically expressed in the ultraviolet and visible absorption spectra. The character of the spectra (shape, number, position, and intensity) provides a possibility for the settlement of the identity or dissimilarity of the chromophoric systems in a given molecule. Consequently, UV-VIS offers a method for classification of organic compounds, and this is particularly of great importance in the field of antibiotics. By using UV-VIS, a small sample of an antibiotic with unknown structure can be quickly, conveniently, and cheaply classified into the respective group. As compared to other physical methods, UV-VIS has several advantages and disadvantages. The advantages are High sensitivity (-1 ng [ 57a]) in the case of suitable chromophores. Requires small amount of sample (10-200 µg). A quick and cheap method for structure elucidation. Applicable as routine analytical method. No special qualification is required for handling of the instrument. It is not a destructive method of structure investigation; the samples can be recovered unchanged after the measurement. (This is particularly important upon examination of antibiotics isolated, at first, in small quantities. ) It can be combined with suitable separation techniques (column, layer, and high-performance liquid chromatography). It is applicable for both qualitative and quantitative analyses, such as for checking quality (detection of contamination containing chromophores), for the determination of reaction rate constant, and for studying equilibrium systems. It is useful for the quantitative examination of multicomponent mixtures. Its disadvantages are The range of application is limited due to structural requirements, and it is useful primarily for compounds with chromophoric systems. It is suitable only for the examination of solutions or liquid samples. There is no possibility for the determination of the presence or absence of functional groups. The information obtained by means of UV-VIS in itself, is not sufficient for the identification of a compound with unknown structure. The numerical values of the UV-VIS measurements are more dependent upon the conditions applied (solvent effect, pH-dependence, and others) than that of the other physical methods of structure investigation. It cannot be generally applied for trace analysis [ 58] . Special attention is directed to one of the analytical applications of UV-VIS, namely, for homogeneous antibiotic samples, allowing the determination of the molecular weight by means of a spectrophotometric method [17,25,26,59].

22

Dinya and Sztaricskai

This method is based on the Lambert-Beer law and involves the derivatization of the unknown substance with a model compound exhibiting known, characteristic, and stable absorption properties. In new derivatives obtained this way, the model compounds approximately maintain their own absorption behavior. Thus, in the knowledge of the average value of molar absorptivity (E:) of the applied model compound the unknown molecular weight can be calculated from an equation by measuring the A value of a solution with given concentration. The model compound should be suitable for the chemical reaction demanded and should not exhibit absorption at the range where the unknown material has absorbance. One of the most commonly used derivatizing agent is picric acid, showing intense absorption at about 378 nm. It forms picrate with amine readily, with an average molar absorption of (s) = 13,400. Using this value the molecular weight can be determined with an accuracy of 1- 5%. In the case of new antibiotics this approximate molecular weight may offer a useful basis for further structure examination. In the following, recent developments in the ultraviolet and visible absorption spectrometry of individual groups (types) of antibiotics are surveyed, emphasizing primarily analytical applications. AMINO ACID DERIVATIVES, OLIGO- AND POLYPEPTIDE ANTIBIOTICS Amino Acid Derivatives Absorption of the representatives of the amino acid type of antibiotics is usually insufficient for analytical purposes. The color reactions (for example, with ninhydrin) are, however, quite suitable for their determination with colorimetric methods. For such colorimetric determination of cycloserine, its colored complex with sodium pentacyanonitrosoferrate (exhibiting absorption at 625 nm) is utilized [ 60] . The diazo group of aza-amino acids shows characteristic ultraviolet absorption. These antibiotics are amphoteric, unstable, and highly toxic. The spectra of these compounds show two absorption maxima at 220- 250 and 272- 278 nm, with the one at higher wavelength the more intense. These bands are due to the TI-+ TI* transition of the diazo group. Some examples are duazomycin (diazomycin A) [61], /.. (E 1 % ) = 245 (313) and 275 (530); max 1cm DON ( 6-diazo- 5-oxo-L-norleucin) [ 62] , /.. (E 1 % ) = 224 ( 376) and 274 ( 683) max 1cm in pH 7. 0 buffer. 1% = At the same time, azaserine [63] has only one band at 250 nm (E1 cm 1140 in pH 7. 0 buffer). [3-Lactam Antibiotics The ultraviolet and visible spectrophotometry of 13-lactam antibiotics is very well documented, and most of the UV-VIS analyses have been achieved with [3-lactam molecules. The uniform discussion of the absorption of these molecules is extremely difficult, as the UV-VIS properties of the natural and semisynthetic substances are more or less different, depending on the character of the 6acylamido side chain ( l,R) of penicillins and of the 7-acylamido (2, R), 7o:-R 1 , and c 3-R2 substituents of cephalosporins.

23

UV and Light Absorption Spectrometry

R-HN~~¥

Ci-~

R-HN}

-~~~COOH 0

O

[1]

COOH [~]

Esterification of the carboxyl groups generally does not influence the original absorption properties. However, exceptions have been found with talampicillin (the phthalidyl ester of ampicillin) and carindacillin (carbenicillin indanyl ester). Natural and Semisynthetic Penicillins

In contrast to cephalosporins, penicillins usually do not exhibit characteristic UV absorption, and end-absorption is observed in their spectra. When the acylamido side chain contains an aromatic ring (as in penicillin G and penicillin V), the absorption band of low intensity is seen at about 255 nm ( E = 25-55 m2/mol; B band, TI+ TI*) that is characteristic of benzene derivatives and possesses fine structure. In some cases an extremely low intensity band can also be observed that shows a bathochromic shift of 2-4 nm upon H20+EtOH solvent exchange. Based on this observation, this band is ascribed to n+TI* transition (R band). The UV spectral data of natural penicillins are summarized in Table 1. Table 1 UV Absorption Characteristics of Naturally Occurring Penicillins TI+ TI* Transition (B band) Compounda

Emax

6-APA

MeOH

Penicillin G

252 264 275.5

Penicillin V

268 274

29.5 17.8 24.2

x

Water

278

1.700

Penicillin K Dihydropenicillin F

Water Water

Penicillin F Penicillin

Solvent

EtOH/water Water

265

34

Water

Penicillin N

Water

Penicillin 0

Water

Penicillin T

MeOH

aPotassium or sodium salts.

24

Dinya and Sztaricskai

Although penicillins do not generally have characteristic absorption, their UV-VIS spectrometry has emerged as a uniform analytical method for their determination. The British Pharmacopoeia 1980 recommends [ 13) UV analysis of oral penicillins. The United States Pharmacopeia [14) and the Code of Federal Regulations [16) permit the application of UV-VIS [50) for several penicillin derivatives. Methycillin, na:ficillin , penicillin V, penicillin G , cloxacillin, and oxacillin are all controlled in the Code of Federal Regulations [ 16) by UV-VIS measurements at particular wavelengths between 230 and 280 nm. Hetacillin content is controlled by colorimetric estimation of a condensation product formed between liberated ampicillin and p-dimethylaminocinnamaldehyde. The phenethicillin content of phenethicillin potassium is determined in the Code of Federal Regulations [ 16) by an UV absorption measurement. UVVfS has been found valuable in elucidation of the structure of some degradation products of penicillin, and in connection with this several excellent review articles are available [ 47, 48, 51). Most applications of UV-VIS in the field of penicillins are based on degradation methods performed according to various procedures (Figure 1). For estimation of penicillin G and ampicillin in the presence of their degradation products, Wahbi et al. [ 64) have introduced a mathematical method, according to absorbance measurement in the ranges 274-269 and 249-271 nm, respectively, for penicillin G and ampicillin. Smith et al. [ 65) elaborated a process for the determination of ampicillin at 320 nm in the presence of CuS04 •5H20, and later this procedure became the fundamental principle of the official method given by the British Pharmacopoeia 1973. Angelucci and Baldieri [ 66) used this process for the determination of ampicillin in body fluids and for the analysis of methycillin [ 67) , pivampicillin [ 68, 69) , carbenicillin -sodium [ 70) , cloxacillin, dicloxacillin, propicillin, and hetacillin [ 71) , flucloxacillin, cloxacillin, and dicloxacillin [ 72) . Penicillin G was measured by Yusim et al. [ 73) in fermentation broths by means of this method. Benzylpenicillin and 6-APA were simultaneously determined by Saccani and Pitrolo [74,75) in fermentation broth by means of degradation catalyzed by copper ions, and penicillins were also identified in various pharmaceutical preparations (Fig. 2) . Saha [ 76) measured the penicillin G content of injections and tablets through its green complex, formed with copper acetate at 750 nm. In 1972, Bungaard and Ilver [ 77) introduced a quick and convenient method for the determination of the nine most commonly used penicillins (Figure 2; 3->-14->-16), which serves as the official prescription in the British Pharmacopoeia 1980 for ampicillin, amoxicillin, cloxacillin, and phenethicillin. The performance of the reaction is very simple and proceeds quantitatively even at room temperature at pH 6. 8. However, a higher reaction temperature (50-60°C) is most advantageous as the process is quicker, and it is accompanied by fewer side reactions. It was aiso Bungaard [77a-80] who recognized that in the case of ampicillin this reaction is not identical with those observed for other penicillins. Namely, with ampicillin the product is not the mercuric mercaptide derivative ( 16) exhibiting absorption near 325 nm, but an unstable compound absorbing at 311 nm. Although this latter band is suitable for the spectrometric determination of ampicillin, a preceding N-acetylation or N-benzuylation is strongly recommended. For the determination of dicloxacillin, Cervera et al. [69) adapted the imidazole-HgCl2 method. Besides the determination through penicillenic acid, the other most commonly used method for the spectrophotometric analysis of penicillins is based

?i

j

H'

s

Figure 1

[ru

enzyme

OH-,~O

pH

1-1+

~

[fil

s'l
y~"

OJ5~

~"~~ R

I

R [58]

[57]

(56]

OOH H

(61]

~'lNH

l.,)o N H

(63]

[64]

[62]

0 OH

[65]

In the case of 4-hydroxypyrimidine ( 65), two amide forms ( 66) and ( 67) may be present, and 2- or 4-hydroxy- and 2,4-dihydroxypyrimidines exist predominatly as amides in solution. The tautomeric properties, dependent upon pH, play a decisive but hitherto not entirely known role in the explanation of the UV spectra. The absorption band of pyrimidine derivatives in the range 240- 290 nm can be regarded as formally correspondent to the lLb band ( n + n *) of benzene. In spectra of the purine derivatives, two broad absorption bands are detected at about 220 and 260 nm, the actual position of which is dependent on pH. By substitution of the purine skeleton the band near 260 nm shows a batochromic shift. (The lLb band for the 2-substituted analogs can generally be detected above 300 nm.

Pyrimidine Nucleosides The antibiotics belonging to this group are structurally citozin and uracil derivatives. Typical representatives of this family are amicetin [allomycin (68); blasticidin S (69), Ri = H) and polioxins (69), Ri = CH3, CH20, and

[68]

[69]

50

Dinya and Sztaricskai

Table 5 UV Data of Pyrimidine Nucleosides

Name

Neutral

Acidic

c

305

316

c

232

u

270-290

Amicetin Blasticidin

s

Polyoxins ac

UV data, Amax (nm) ( E, m2/mol)

Parent a compound

Basic 322

References 160

274 ( 1340)

266 ( 885)

161

275-290

262-271

162

= cytosine' u = uracyl.

so on] , the UV spectral data of which are summarized in Table 5. No selective assay methods have been elaborated for their spectrophotometric determination. Adenine and Purine Nucleosides

The UV spectral properties of some typical representatives of this group are shown in Table 6. Up to this time no spectrophotometric methods have been described for their analysis. The pyrrolopyrimidine nucleosides ( 70) are currently in the limelight due to their antitumor effects. The UV spectral parameters of some antibiotics of this type are given in Table 7.

R = B-D-ribofuranosyl

IZ.Ql

C-Nucleosides Among the representatives of this group the antiviral pyrazomycin, as well as showdomycin, which possessed a broad spectrum of antimicrobial and antitumor activity, have found practical application. Pyrazomycin contains a substituted pyrazole chromophore [(58), B-D-ribofuranosyl] and exhibits absorption at 263 nm ( E = 620 m2Jmol) in neutral medium and at 307 nm ( E = 810 m2/mol) in alkaline solution [ 171]. The bathochromic shift in alkaline medium is compatible with the 4-hydroxyl group. Showdomycin ( 71) has a maleinimide chromophoric system displaying absorption at 220 nm ( E = 1012 m2/mol). The antibiotic is unstable in alkaline medium: the absorption band at 220 nm disappears within 5 min in dilute aqueous ammonium hydroxide [ 172] . There is no spectrophotometric assay method for the analysis of this group of antibiotics.

( 55)

Spongosin

260 ( 1710) 259 ( 1700)

-

-

( 54)

Angustmycin C ( psicofuranine)

274 ( 1250)

249 ( 835)

259 (1310)

267 ( 1950)

( 54)

267 ( 1250)

(nm), (

Acid

max

Angustmycin A ( decoyinine)

R 2 = OCH 3

R 1 = NH 2

212 (2060)

( 54)

Aristeromycin 258 ( 1450)

258 (1310)

( 54)

266 ( 1900)

Cordycepin

R2 = H

R 1 = N(CH 3 ) 2

( 55)

Neutral

UV data, A.

UV Characteristics of Purine and Adenine Nucleosides

Puromycin

Name

Table 6 E,

261 (1740)

260 (1710)

268 ( 1270)

-

260 (1370)

275 ( 2030)

Basic

m 2 /mol)

c::

168

167

166

165

164

s·

163

CJ1 .....

'


,

tr

):..

;:J' .....

cq·

I:'"'

~

;:!

i:i

'!!9e

Ect..atilloa

......

"'I

......

"'
-CH-

< > ...I ·10 < 2 0

o Pen G Pot \

Amp1c1lhn

...I

al

Pot Pen V-1 CID Pot Pen V-2

Amoxic1llin

-15




'


DMSO,

1188

1201. 5

v

)

v

CH3CN \)

N-Ring methyl-2-sulfanilamido I. Pyridine II.

1212.5 1205

Pyrimidine

1209

1225

III.

3 (or 5)-Methylpyrimidine

1202

1216

IV.

3, 5- Dimethylpyrimidine

1201

1214.5

Thiazole

1207.5

1230

v.

N 1-Methyl-2-sulfanilamido VI.

Pyridine

1247 1239

1253.5

1257

1255.5

1257.5

1259

3-Methylpyrimidine

1235

1251. 5

3, 5-Dimethyl pyrimidine

1237

1353

Thiazole

1258.5

1260

VII. Pyrimidine VIII. IX.

x.

1260.5

Source: Data from Reference 77.

environment. That stretching bands are lower for the imide form than for the amide form supports earlier theoretical treatments and may serve as a means of identifying the two tautomeric forms of the drugs. It is difficult to determine whether frequency differences within each group of compounds is due to electronic differences or merely intermolecular or solvation differences. Murakami et al. [ 78] extended this approach in their study of 1-oxacephalosporins. A number of 1-oxa congeners of cephalosporins were prepared and compared to their 1-sulfur analogs. Thus, for each Ri. R2 combination in Figure 10, two compounds, one in which t:,, = 0 and one in which t:,, = S, were prepared. This approach neutralized the effect of side chains by comparing molecules in which they were identical. The f3-lactam infrared absorption

Figure 10 Cephalosporin analogs: t:,, = S for 1-sulfur cephalosporins. for 1-oxa congeners. (Adapted from Reference 78.)

t:,,

=0

128

o~ high frequency

Rose

~

-o

low frequency

Figure 11 Effect of resonance delocalization on S-lactam absorption frequency.

frequencies of six of these pairs were measured. In all six pairs the 1-oxa compound showed a higher frequency than its 1-sulfur congener, indicating that oxygen replacement of sulfur decreases resonance in the S-lactam ring (Figure 11). A correlation was found between !ability to S-lactamases and infrared frequency. It was theorized that, when the antibiotic is bound to transpeptidase, the enzyme that catalyzes the cross-linkage of peptidoglycan (the terminal stage of cell wall biosynthesis), the S-lactam amide bond is cleaved with concomitant acylation and loss of enzyme activity. A similar mechanism is suggested for the S-lactamase hydrolysis of S-lactam compounds. The 1-oxa analogs had higher frequency (more strained) S-lactam carbonyl stretching vibrations and, in general, showed lower or equal minimum inhibitory concentrations (MIC). Blanpain et al. [ 79] studied the effect of side-chain conformation on Slactamase resistance of several penicillins using infrared spectra among other tools; O. 01 M solutions in CHCl3 and CCl4 as well as solid-phase spectra using KBr disks were obtained. The carbonyl stretching frequency of the S-lactam ring was found to be the same for all penicillins studied for a given solvent. On the other hand, the exocyclic amidic CO stretching frequency is very sensitive to the nature of the side chain as well as the solvent. Better hydrogen donor ability is associated with lowering the vibration frequency, but this vibration does not correlate with the resistance of the penicillin. Both IR and 13c-NMR support the conclusion that the side chain has no effect on the electronic properties of the nucleus. The relative resistance, then, was attributed to the structure and nature of the side chain, the resistant penicillins appearing to have a rigid side chain. Morin and coworkers [ 80] studied the synthesis of cephalosporin compounds via transformations of penicillin sulfoxide. Infrared spectra were used to elucidate the structure of intermediates. Thus, penicillin V sulfoxide methyl ester, when refluxed with acetic anhydride, provided a material in 60% yield that contained a strained lactam, as indicated by a bond at 1790 cm-1, in contrast to the characteristic location at 1770 cm-1. An interesting result of this work is that the correlation found between the infrared frequency of the S-lactam frequency is an indicator of acylating power-the higher the frequency, the better the acylating agent (Table 9).

Infrared Spectroscopy

129

Table 9 Comparison of S-Lactam Absorption Frequency and Biological Activity Frequency (cm -l)a

Bioassayb

1790

1800

1795

High

1792

300

1785

25

1776

4

1784

6

1780

15

1780

Low

aDetermined in CHCl3 solution on the methyl esters. b Assay on the salts in Oxford units against a penicillin a-sensitive Staphylococcus aureus strain. Source: Data from Reference 80.

POLYMORPHISM

Polymorphism is of importance because of the impact of this physical property on the bioavailability of solid dosage forms. Infrared spectroscopy, along with other tools, such as differential thermal analysis and x-ray diffraction, has been used to shed light on this property of some drug substances. Pelizza and coworkers [81] studied the polymorphism of rifampicin (Figure 12).

Two crystalline modifications, one amorphous form,

32

CH3

37

CH30

29

Figure 12 Hydrogen bonding in rifampicin. (Adapted from Reference 81.)

130

Rose

and four solvates were studied.. The infrared data on the major bands of these various forms are summarized in Table 10. That the functions that can be involved in the H bonding do not change in CDCl3 solution indicate that such H bonding is intramolecular. The C-21 hydroxyl is H bonded to the C-23 hydroxyl, which in turn is H bonded to the C-25 acetyl. The C-8 hydroxyl is H bonded to the C-1 hydroxyl, which in turn is bonded to the amide carbonyl. The C-4 hydroxyl is bonded to the furanone carbonyl and the amide NH to the imine nitrogen of the substituent at C-3. These interactions are indicated in Figure 9. The spectrum of form I indicates that, in this crystal, the C-23 hydroxyl is not H bonded to the acetyl group, as indicated by the acetyl frequency at 1725 cm-1 and the OH at 3550, although H bonding is seen at other possible sites. In form II, on the other hand, C-4 OH is not H bonded to the furanone carbonyl and the C-1 OH-amide carbonyl interaction is lacking. The IR spectrum of the amorphous form in dicates that it is a mixture of that of form I and that found in CDC1 3 solution. This implies two conformations of the acetyl group, which prevents formation of an ordered crystalline state. The pentahydrates SI and Sil demonstrate differing IR spectra. The band at 1725 cm-1 indicates no H bonding between CO 23 OH and the acetyl group, but the appearance of this bond at 1750 cm-1 in Sil supports the existence of such a bond. Chapman and coworkers [82] reported that cephaloridine can exist in at least six polymorphic forms depending on the conditions of recrystallization. The infrared spectra of the various forms in Nujol mulls are quite different. The a. and S forms show no OH absorption, confirming their anhydrous nature. The µ form contains 1 mol of methanol, and its spectrum contains a broad OH bond at 3350 cm-1. The OH and CO bands at 3270 and 1028 cm-1 disappear when methanol is removed and the sample is converted to the e: form. The o form, obtained when the sample is recrystallized from water, and the µ form (recrystallized from methanol) are quite hydroscopic, as reflected by weak peaks at 3575 cm-1, attributed to water incorporated in the mulling process.

Evidence for a hydrate 3/4 mol of water comes principally from the infrared spectrum. Upon hydration with up to 3/4 mol of H20, characteristic OH stretching and OH bending bands for water of crystallization appear at 3757, 3366, and 1665 cm-1, The intensity of these bands is directly related to the Karl Fischer water content. This water of crystallization hydrogen bonds to the S-lactam carbonyl, and decrease in the carbonyl bond at 1762 cm-1 is accompanied by a corresponding increase in the new carbonyl bond at 1745 cm-1, The relative intensities of these two bonds is a measure of free and H-bonded S-lactam carbonyl. If one increases the water content up to 4 mol, no significant change is seen in the water bands at 3575 and 3366 cm-1 or in the carbonyl bands at 1762 and 1745 cm-1. The additional water gives rise to broad bands centered at 3380 and 1665 cm-1 and does not H bond to the S-lactam carbonyl. This additional water is more readily lost than the first 3/ 4 mol. Kuhnert-Brandstaetter and colleagues [ 83, 84] , in a series of papers, report the use of infrared spectroscopy with thermal analysis to study enantiomorphic polymorphs of drugs. Although the drugs under study are not antibiotics, the techniques are useful to all interested in structure-related properties of drugs.

1665d 1610 1660

1725c 1750c 1725c 1725c

1630 1750c 1640 1645 1640

OBW OBW 3460 3480 3400b

SI

SU

SU after 130°C

SUI

SIV

OBW = overlapped by water band. bH bonded. CNo H bonding. dcl+C8 OH. einternal salt. Source: Data from Reference 81.

a

Absente

1660d

1715

1645

3480

CDC1 3 solution

1735

2800 1625

1725, 1710

1645

3460

1625

1625

Absent

Absent

Absent

Absent

2800

2780

Amorphous form

1680

1715

1740

3500-3150

2800

Form II

1625

1725

N-CH 3

1645

Amide C-0 (amide I)

3550, 3480

Acetyl C=O

Form I

ansa OH

Furanonic C=O

Table 10 Important Rifampicin Absorption Bandsa

.....

c:..o .....

~

't:l

0 0

6en

0 .....

(l)

tfJ 't:l

s:l.

(l)

i:i

;:I

.,.,.....

132

Rose

METAL BONDING

Preti and Tosi [85] have observed that there is considerable interest in the effect of metal ions on antibacterial activity and on the rate of hydrolysis of the S-lactam ring in penicillins, and that group SB complexes, especially those of radium, iridium, and platinum, have been reported to have considerable antibacterial power and induce lysis in lysogenic bacteria. Additionally, there is interest in metal complexes as dosage forms, as part of a sustained release formulation or as a form that would be less toxic. The authors studied the complexes of Cr(III), Mn(II), Rh(III), and lv(Ill) with the antibiotic cycloserine. This compound exists as a zwitterion. Assignments were made using a model compound in which the NH2 function was missing, thus permitting no zwitterionic equilibrium. With Cr(Ill), 1: 3 and 1: 6 complexes were formed. With Mn(ll), 1: 4 complexes resulted, but Rh(Ill) and lr(Ill) yielded 1:3 complexes. In Cr(lll) 1:6 complexes and Mn(ll) complexes, vibrational modes due to NH2, NH, and NH3+ are still present. The C=O bands are intense and shifted to lower frequencies by about 70-90 cm-1, This implies that the C=O oxygen is the donor center of the ligand and that the zwitterionic form is still present even in the ligand. The 1: 3 Cr( III) complexes are significantly different from the 1: 6 complexes All NH3+ bands disappear. The NH 2 stretch and C=O stretch are shifted to higher energies. This implies coordination through the N of NH. The absence of NH 3+ implies the ligand is stabilized by coordination in the amino keto form. Rh(lll) and Ir(llI) are different. The NHt disappears. The C=O stretch is shifted to lower energies by -60 cm-1. This implies that the donor site of the ligand is the 0 of C=O. The ligand is coordinated without zwitterionic equilibrium. Far infrared spectra of Cr(Ill) 1:6 derivatives reveal no Crhalogen vibrations. A single bond in the 415-409 cm-1 range indicates a Cr-ligand vibration involving the C=O. The Cr(llI) 1: 3 complexes have three bands in the far infrared, which are different with Br- than with C, implying a Cr- X vibration within the range of octahedral symmetry. This implies Cr-X covalent bonding. Three halogen-independent bands in the 238-189 cm-1 range imply Cr-N stretch modes. With Mn(II) there are some bands whose frequency becomes lower as the mass of halogen increases, implying Mn-X bands. Four halogen-independent bands in the 379-272 cm-1 range imply Mn-0 vibrations. The data suggest octahydral symmetry. Three M-0 and three M-X stretch vibrations are present in all Rh(Ill) and Ir(III) spectra, indicating octahedral complexes of the type [M(cycloserine)3X3]. Fonina et al. [86) used infrared spectroscopy in conjuction with circular dichroism and NMR spectroscopy to study the conformation of valinomycin analogs in homogeneous solution as well as in a two-phase membrane model system. A correlation was found between the ability of a compound to complex metal ions in solution and its capacity to transport them across membranes. The analogs studied were cyclo-(D-isoleucyl-lactyl-isoleucyl-D- a.-hydroxyvaleryl)n in which n = 2(1), 3(11), or 4(111). In nonpolar medium, II.and Ill had substantially the same conformation as valinomycin; that of I was very different. The infrared spectra of Ill in alcohol showed no changes upon addition of K+. Large excesses of Na+ did induce change. Wong et al. [87] examined the binding of methylmercury by the sulfur amino acid DL-penicillamine as a model for the methylmercury poisoning of

Infrared Spectroscopy

133

proteins. Mid- and far infrared as well as Raman spectra were used in conjunction with NMR and x-ray crystallographic measurements, The infrared spectra are that of strongly hydrogen-bonded zwitterionic amino acids rather than free amine and carboxyl functions. The y(S-H) bond is weak in the infrared but is the strongest band in the Raman spectrum of amino acids that contain it. Its disappearance in the Raman spectra of these compounds is strong evidence of deprotonated sulfydryl-metal coordination. When deuterated, the o(+NH3) bands shift from 1508-1659 cm-1 to 11321183 cm-1, With [Hg2(CH3)2(pen)] these bands were not found, indicating attachment of HgCH3+ to the amine function. The oasym(NH2+) are obscured by the strong o(C02-) bands and osym(NH2+) bands that occur around 1350 cm-1 in [HgCH3(pen)] •D20. There is little change in the carboxyl stretching modes upon complexation by methylmercury, implying that little complexation of mercury with these functions takes place. In the far infrared a medium-intensity band in the infrared spectra and an intense bond in the Raman spectra represent Hg-C stretching modes. This band is so close to p(Co 2-) bonds in the [Hg 2(CH3)2(pen)] that one sees only a single band at 546 cm-1 in the Raman and at 547 cm-1 in the infrared. Upon deuterium exchange with D20, the peaks of p(Co 2-) (543 cm- 1) and y(Hg-C) (540 cm-1) become identifiable. Medium-intensity Raman bonds at 322 cm-1 for [HgCH 3(pen)] •H 20 and 334 cm-1 in [ Hg2( CH 3) 2(pen)] are not observed in the free ligand or in HgCH3Cl and are assigned to y(Hg-S) stretching. A medium-intensity Raman bond at 432 cm-1 for [Hg2(CH3) 2(pen)] have been tentatively assigned to y(Hg-N), The infrared counterpart was a broad medium-strong bond at 441 cm-1, The authors conclude that, at neutral pH, attachment of HgCH3 + to sulfyhydryl will result in disappearance of the strong y(S-H) bond at -2500 cm-1 and the appearance of a medium-strong bond at 300 cm-1 in the Raman spectrum due to y(Hg-S). If the HgCH 32 is bound to the amine group, bands at -1625 and 1520 cm-1 will disappear in the infrared and a medium-intensity peak will appear at 400-500 cm-1 due to y(Hg-N). Tsatsas et al. (88] determined the frequencies of quantized motion of cations encaged in cyclic polyether systems-the so-called crown-ethersusing far infrared spectroscopy. Such structures occur as antibiotics, such as eniatin B , noactin, or valinomycin , and are known to selectively enchange ion binding in solution, as well as ion transport across membranes. The work was undertaken to study the effect of encagement on cation motion frequencies in solution and to determine the ion-cage interaction force information that these frequencies reflect. Generally speaking, frequencies due to the motion of a cation are a function of the size, shape, and charge distribu tion of its counterion, as well as the nature of the solvent. It follows that cations in solution should exhibit frequencies different from those of encaged cations. In this work, the macrocyclic polyether dibenzo-18-crown-6 was chosen as a model ion-encaging compound. This compound, among others, is known to enhance the solubility of inorganic salts in organic solvents and to increase the permeability of biological membranes to alkali metals-behavior similar to that of the antibiotics already mentioned. The crystalline structure of 1: 1 complexes of this crown with MSCN (M = Na+, K+, Rb+) has been determined to be that illustrated in Figure 13. The metal ion is centrally located within the polyether ring and the counterion above or below the ring. Tsatsas et al. have postulated the structure illustrated in Figure 14. for the complex in solution, although a structure in which one solvent molecule is replaced by the counterion is also held to be feasible.

134

Rose

•c

00 OM+ Figure 13 Crystalline structure of 1: 1 complexes of crown: MSCN (M =Na+, K+, Rb+). (Reprinted with permission from Reference 88. Copyright 1972 American Chemical Society. )

The far infrared spectra of crown-MX complexes were obtained from 100650 cm-1 in THF, DMSO, or pyridine. The M was Na+ or K+; X was PF5-, SCN-, or B04-. In each spectrum one band was found that was strongly cation dependent. These were in the range 180-205 cm-1 for Na+ and 135150 cm-1 for K+. The location of these bands is illustrated in Figure 15. In DMSO, the frequencies occur 15-20 cm-1 higher than the analogous frequency in pyridine solution. No counterion dependence was found with these solvents. However, the Na+ motion bands were found to be anion dependent in THF, indicating significant anion participation in the near-neighbor environment of the cation. The motion frequency of crown-encaged Na+ is 213 ± 4 cm-1, is independent of solvent and anion, and is higher than that for simple salts in the same solvent. Likewise, the vibrational motion of K+, which is crownencaged, appears at 167 ± 3 cm-1 higher than that found for salts in the same solvent. That the ion motion frequency is shifted by encagement, that it is independent of solvent and anion variation, and that the shift is a strong function of cation mass lead to the conclusion that the bands at 213 cm-1 for Na+ and 167 cm-1 for K+ are due to an infrared active vibration of M+ in the plane defined by M+ and the six ethereal oxygen atoms. The authors present a detailed vibrational analysis of the spectra. The far infrared method is a powerful tool for studying metal-antibiotic interactions and transport properties. A wealth of literature exists on materials that serve as models for antibiotic compounds.

s I

S Solvent 0 Oxygen

M M+

,

7

o---1----o s,..... \

,,, 's

'

/

\

0-4-M-1-0 \ 3// '2 /' ./ ' ' 0------0 8 I

s

Figure 14 Postulated structure for 1: 1 crown: MSCN complex in solution. (Reprinted with permission from Reference 88. Copyright 1972 American Chemical Society. )

Infrared Spectroscopy o Na-DMSO • Na-PY • K-DMSO 6. K-PY

cm-1

250

200

202 "R 181

150

100

135

2:

• •

150 I I 136 :zi: A I

l'

168

c

.._

t.>

• • z l..r s::. I u a.. LL D > C > B > A). (From Reference 30.) reduced daunorubicin (Figure 5(b)). In a recent study, Kalyanaraman et al. [ 30] showed that superoxide, formed by the reduction of oxygen with daunorubicin semiquinone, can be trapped by DMPO or BPN spin traps, proving the biological events responsible for the lethal action of these antibiotics (see Figure 4). It was also observed that much less superoxide is formed when antibiotics were intercalated into DNA during the microsomal reduction process. Experimentally, 1 mM daunorubicin or adriamycin, 0. 08 M DMPO, and the NADPH generating system are mixed. The NADPH generating system contained 0.39 mM NADP+, 5.5 mM glucose-6-phosphate, and 0.67 units/ml of glucose6-phosphate dehydrogenase. During their study, Kalyanaraman et al. [ 30] observed that in time the ESR spectra line shape of the microsomal reduced danuorubicin changes and this change indicates the immobilization of the daunorubicin free radical. These spectral changes are illustrated in Figure 6. It is conceivable that the distribution of the semiquinone or its metabolite changes between the aqueous and the lipid compartment of the microsome. Similar conclusions were down in the rubiflavin free radical [ 23] immobilization studies. Mitomycin is also a quinone type of antibiotic, and its mode of action was thought to be· similar to that of adriamycin, daunorubicin, and streptonigrin. Lown et al. [31] and Kalyanaraman [30] both applied spin trapping reagents to detect the existence of the superoxide and hydroxyl radicals and the formation of the mitomycin free radical. Lown et al. [31] produced the mitomycin

Figure 5 ESR spectra of reduced daunorubicin. (a) Chemically reduced with the assigned unpaired electron distribution in the molecule, and (b) biologically reduced. (From Reference 29.)

190

Yang and Aszalos

2G ~

Figure 7 ESR spectrum of chemically reduced mitomycin C with the assigned unpair electron distribution in the molecule. (From Reference 30.)

free radical by the reduction of 20 x 10- 3 M mitocyin in 0. 5 ml of DMSO-H20 (1:9) solution with 8 x 10-3 M NaBH4 in 60 mM oxygenated, potassium phosphate buffer, pH 7. 0. Computer simulation revealed a spectrum with lorentzian line shapes and linear widths of 0.150, confirming the hyperfine splitting values assigned to the mitomycin C free radical. The experimentally obtained spectrum for mitomycin is shown in Figure 7. With the addition of 0.06 M benzyl tert-butylnitroxide (PBN) in 60 mM potassium phosphate buffer, pH 7.0, a new spectrum was obtained characteristic of the PBN-OH radical adduct characterized by Harbour et al. [32]. This spectrum consists of a triplet of doublets with AN= 16.0 0, A~= 3.4 O, and g = 20061. When the enzymes catalase (0.04 mg/mL) or superoxide dimutase (0.8 mg/ml) were added to the 0.06 M PBN solution, no PBN-OH radical could be detected. This indicates that the formation of the hydroxy radical requires the previous formation of H 2o 2 and/or superoxide species. Kalayanaraman et al. [30] used microsomal reduction of mitomycin C and obtained an ESR spectrum whose lifetime is shorter than 4 min. In addition the g value of the mitomycin C free radicals could not be accurately determined due to the overlapping spectrum of cytochrome C reductase flavin semiquinone, present in the microsomal preparation. In this redox system, superoxide was trapped by the addition of a spin trap (DMPO). The experimental procedure is similar to that used with daunorubicin [30]. The observed spectrum shows hyperfine couplings of AN = 14. 30, A~ = 11. 70, and AH = 1. 250, in agreement with the reported values for the DMPO-superoxide adduct [ 32]. One could conclude from these experiments, as well as from those with adriamycin, daunorubicin, and rubiflavin, that the antibiotic free radicals are formed first, followed by formation of superoxide, H 2o 2 , and hydroxyl radicals, as shown in Figure 4.

191

Electron Spin Resonance Spectroscopy

MEMBRANE FLUIDITY

Drugs may change the physical state of the biological membranes by specific or nonspecific interactions. The specific interactions involve site binding (receptor or membrane protein with specific functions); nonspecific interactions involve insertion into the lipid domain of the membrane. These druginduced changes in the physical state, as well as the dynamic properties of cellular membrane components, result in the alteration of cellular functions, such as metabolism, differentiation, hormonal regulation, cell adhesion, and transport [34-3B]. Some drugs may interact with cellular membranes in such a way that it causes membrane "fluidity" changes that can be measured by ESR. When spin probes, such as the fatty acid analogs with the nitroxide ring located at the hydrophilic end (5-nitroxystearate, Figure BA), the hydrophobic end of the molecule ( 12-nitroxystearate, Figure BB) , or a spin label phospholipids (Figure BC), are inserted into the membrane, the probe undergoes an isotropic rotation; a parameter known as the rotational correlation time (Tc) can be calculated from the following equation:

(1)

OH

(a}

0

N-0

'-,(

(b}

r lN-0

0

OH

II 0

(c}

Figure B Spin labels of (a) 5-nitroxystearic acid, (b) 12-nitroxystearic acid, and (c) phospholipids.

192

Yang and Aszalos

1-- - -

--2Tu----

ho

h -1

Figure 9 Spectral parameters used in the determination of molecular motion of spin-labeled marcomolecules.

where W1 and hl are the width and height, respectively, of the low field line of the first derivative spectrum and h_ 1 is the height of the high field line, as shown in Figure 9. The rotational correlation time is an approximate measure of the time required for the nitroxide to tumble through an arc of 1 rad. It can be related to the viscosity of lipids by the Stokes relation. Since viscosity and fluidity are universely related, 1/'Ti can be defined as fluidity. The order parameter S in Equation ( 1) can be calculated with the following equation

s =

A

zz

-1/2(A

xx

-A

yy

)

where All

Al Amax' Amin Axx, Ayy

=

= = =

1/2 A max 1/ 2 A . + 0. 86

mm

hyperfine splitting, shown in Figure 9 principle elements of the A tensor.

A listing of A tensors for the various spin labels has been compiled by Berliner [38]. Experimentally, a 3.3 to 6.6 x 10-8 M solution of a spin label, such as 5-nitroxylstearic acid in 10- 20 µl EtOH in a glass tube is evaporated to dryness in vacuum; this residue can be stored at 5°C for many weeks. A cell suspension containing 106 cell in 0.1-0.2 ml phosphate buffered saline solution (pH = 7. 2) is pipetted into this glass tube. Capillary tubes or flat cells are used for ESR measurement. In studying the effect of drugs on

Electron Spin Resonance Spectroscopy

193

Untreated cells

( V."

0

x

5

~5. Figure 10 ESR spectra of spin-labeled myocardial cells, untreated and treated with 10 µ g of nystatin.

membrane fluidity, a particular drug will be added to the cell suspension buffer prior to ESR measurement. As a typical example, myocardial cells (106) treated with 10 µg of the polyene antibiotic nystatin introduces a 1.6 G increase in the 2T II value (Figure 10), indicating that intercalation of nystatin into the membrane results in a less fluid environment for the spin label [39]. Using the same technique, with the addition of 1.5 µg of adriamycin, results in a more rigid myocardial membrane, as indicated in an increase of about 2G in 2T II splitting. Order parameter calculations indicate membrane fluidity changes, reflecting the increase in the 2T11 values when myocardial cells are treated by these two antibiotics (Figure 9) • These spectra were obtained using 5-nitroxylstearic acid spin label. The interaction of antibiotics plymixin B, gramicidin S, and chlorothricin was initially studied by Packe et al. [ 40, 41] using artificial and bacterial membranes. In the ESR experiments, the fluidity of egg yolk lecithin and the phospholipids of Bacillus subtilis was investigated in the presence and absence of chlorothricin with two spin probes, 12-nitroxylmethylstearate and distearoylglycerophosphate, derivatized on the phosphate with tetramethyl-3hydroxymethylpyrrolidin-1-oxyl. The rotational correlation times (Tc) obtained with different mole percent chlorothricin and the 12-nitroxylmethylstearate probe were calculated. Results indicated that increasing the concentration of the antibiotic increases the rigidity (immobilization) of inner areas of the

Yang and Aszalos

194

membrane. However, using the distearoylglycerophosphate spin label, very few changes in the rotational freedom and correlation time were observed with increasing amounts of antibiotic added to the membrane preparation. The 12-nitroxylmethylstearate probe inserts deep into the membrane and senses fluidity changes; the phospholipid spin probe resides near the membrane surface and senses no fluidity change. It could be concluded that chlorothricin also penetrates deep inside the membrane near the position where the 12-nitroxylmethylstearate probe resides. In contrast to chlorothricin, polymixin B and gramicidin S remain at the outside of membranes. Experimentally, the addition of these two antibiotics to egg yolk lecithin does not alter position of the 12-nitroxylmethylstearate. The mode of action of various antibiotics may depend on their depth of membrane penetration. This can be determined by ESR spectroscopy with different spin probes. In addition to the application of ESR to membrane fluidity studies, Peterson et al. [42] used ESR to measure the competitive bindings between dodecyldimethylammonium-1-oxyl- 2, 2, 6, 6-tetramethylpiperidine spin label and aminoglycoside antibiotics to gram-negative bacterial membrane lipopolysacharides. They observed that the toxicity of the different aminoglocoside antibiotics correlate to their binding ability.

SUMMARY Selected applications of ESR spectroscopy have been briefly reviewed. The formation of transition metal-antibiotic coordination complexes and the detection and measurement of g factors and hyperfine splittings, together with other spectroscopic techniques, aided in the structure elucidation for these complexes. The bifunctionality of metal binding and DNA intercalation contributed to elucidation of the biological activities of these antibiotics. The detection of reactive oxygen radicals by the spin trapping technique considerably strengthened the belief that mechanism of oxygen activation with metal-antibiotic complexes is essential for DNA breakage. The isolation and identification of enzymatically reduced antibiotic radical intermediates lead to the suggestion of lipid peroxidation [ 43] . The antibiotic-induced fluidity changes in the lipid domain of membrane, as deduced from the ESR spectra of nitroxyl probes, presented models of antibiotic-membrane interaction.

REFERENCES 1. 2. 3. 4. 5. 6.

E. A. Sausville, R. W. Stein, J. Peisach, and S. B. Horwitz, Biochemistry 17, 2746, 1979. T. Takita, Y. Muraoka, T. Nakatani, A. Fujii, Y. litaka, and H. Umezawa, J. Antibiot. (Tokyo) 31, 1073, 1978. K. Negal, H. Yamaki, H. Suzuki, N. Tanaka, and H. Umezawa, Biochim. Biophys. Acta 179, 165, 1969. W. E. B. Muller, Z. Yamazaki, H. J. breter, and R. K. Zahn, Eur. J. Biochem. 31, 518, 1972. M. Chien, A. P. Grollman, and S. B. Horwitz, Biochemistry, 16, 3640, 1977. H. Kasai, H. Naganawa, T. Takita, and H. Umerzawa, J. Antibiot. (Tokyo), 31, 1316, 1978.

Electron Spin Resonance Spectroscopy

195

Y. Sugiura, K. Ishizu, and K. Miyoshi, J. Antibiot. (Tokyo), 32, 453, 1979. 8. J. L. Lown and S. Sims, Biochem. Biophys. Res. Commun. 77, 1150, 1977. 9. Y. Sugiura, Biochem. Biophys. Res. Commun. 87, 2, 649, 1979. 10. E. G. Jansen, Acc. Chem. Res. 4, 31, 1971. 11. L. W. Oberley, and G. R. Buettner, FEBS Lett. 97, 47, 1979. 12. Y. Sugiura and T. Kikuchi, J. Antibiot. (Tokyo) 31, 1210, 1978. 13. Y. Sugiura, J. Amer. Chem. Soc. 102(16), 5208, 1980. 14. Y. Sugiura, J, Antibiot. (Tokyo) 31, 1206, 1978. 15. H. Umezawa, Y. Suhara, T. Takita, and K. Maeda, J. Antibiot. (Tokyo) 19, 210, 1966. 16. J. C. Darbowiak, F. T. Greenway, W. E. Ionzo, M. Van Husen, and S. T. Crooke, Biochim. Biophys. Acta 517, 1978. 17. Y. Sugiura, Biochem. Biophys. Res. Commun. 90(1), 375, 1979. 18. I. Yamazaki, Free radicals in enzyme-substrate reactions, in Free Radicals in Biology, Vol. III, edited by W. A. Pryor, Academic Press, New York, 1977. 19. R. P. Mason, Free radicals intermediate in the metabolism of toxic chemicals, in Free Radicals in Biology, Vol. 5, edited by W. A. Pryor, Academic Press, New York, 1982, p. 161. 20. H. P. Misra and I. Fridovich, J. Biol. Chem. 247, 188, 1972. 21. K. V. Rao, K. Bienann, and R. B. Woodward, J. Amer. Chem. Soc. 85, 2532, 1963. 22. A. Aszalos, M. Jelinke, and B. Berk, Antimicrob. Agents Chemother. 68, 1964. 23. J. R. White and H. H. Dearman, Proc. Nat. Acad. Sci. U.S. 54, 887, 1965. 24. H. H. Hasson and I. Fridovich, J. Bacteriol. 129, 6885, 1977. 25. S. Sato, M. Iwaizumi, K. Banda, and Y. Tamura, GANN 67, 523, 1976. 26. N. R. Bachur, S. L. Gordon, and M. V. Gee, Cancer Res. 38, 1475, 1978. 27. T. Oki, T. Komiyama, H. Tone, T. Tnui, T. Takeuchi, and H. Umezawa, J. Antibiot. (Tokyo) 30, 613, 1977. 28. N. R. Bachur, S. L. Gordon, and M. V. Gee, Mol. Pharmacol. 13, 901, 1977. 29. J. W. Lown and H. H. Chen, Can. J. Chem. 59, 3212, 1981. 30. B. Kalyanaraman, E. Perez-Reyes, and R. P. Mason, Biochim. Biophys. Acta 830, 119, 1980. 31. J, W. Lown, S. K. Sim, and H. H. Chen, Can. J. Biochem. 56, 1042, 1978. 32. J. R. Harbour, V. Chow, and J. R. Bolton, Can. J. Chem. 52, 3549, 1974. 33. J. R. Harbour and J. R. Bolton, Biochem. Biophys. Res. Commun. 64' 803' 1975. 34. M. Edidin, Annu. Rev. Biophys. Bioeng, 3, 179, 1974. 35. G. M. Edelman, Science, 192, 218, 1976. 36. G. I. Bell, Science, 200, 618, 1978. 37. K. P. Pauls, J. Thompson, and J. R. Lepack, Arch. Biochem. Biophys. 220, 122, 1980. 7.

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L. J. Berliner, Spin Labeling: Theory and Applications, Vol. I, Academic Press, New York, 1976, p. 565. A. Aszalos, J. A. Bradlow, G. C. Yang, E. R. Reynoldo, and A. N. El Hage, Biochem. Pharmacol., 33, 3779, 1984. W. Pache, D. Chapman, and R. Hillaby, Biochim. Biophys. Acta 255, 358, 1972. W. Pache and D. Chapman, Biochim. Biophys, Acta 255, 348, 1972. A. A. Peterson, R. E. W. Hancock, and E. J. McGroarty, 28th Annual Meeting of the Biophysical Society, San Antonio, Texas, Abs. W-POS 80, Febuary 19-23, 1984. J. Goldman and P. Hochstein, Biochem. Biophys. Res. Commun. 77, 797, 1977.

6 Thin-Layer Chromatographic Techniques and Systems JAMES A. CHAN Smith Kline & French Laboratories, Philadelphia, Pennsylvania ADORJAN ASZALOS

Food and Drug Administration, Washington, D. C.

Principles and Techniques Adsorption and Partition Chromatography Apparatus and Techniques Quantitative TLC Reversed- Phase Thin- Layer Chromatography Identification of Antibiotics Quantitative Analysis of Antibiotics TLC Classification in the Discovery of Novel Antibiotics References

198 198 198 200 201 203 223 227 237

Thin-layer chromatography (TLC) is a frequently used laboratory technique in the field of antibiotic separation, purification, and quantitation. The technique is inherently simple and relatively inexpensive and requires little time. It was initially used in the separation of natural products, like lipids, steroids, and nucleic acids, but has been subsequently well adapted by chemists in laboratories dealing with antibiotic fermentations, purifications, or identifications. Since numerous reviews on TLC techniques are thoroughly covered in the literature [ 1- 5] and TLC systems for most antibiotics are also well reported [ 6-8] , this chapter only briefly describes general TLC techniques and selected antibiotic chromatographic systems. However, since high-performance liquid chromatography (HPLC) seems to have taken the place of TLC especially in quantitation, we will also briefly discuss the transferability of TLC data to HPLC systems. We have divided this chapter into four sections. The first section deals with the principles, techniques, and adsorbent materials related to TLC. The second part describes TLC systems use in the identification of some of the most important or recently discovered antibiotics. The third part gives examples and techniques on the application of TLC for the quantitative analysis of antibiotics. The last section deals with the use of TLC classification in the discovery of novel antibiotics.

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PRINCIPLES AND TECHNIQUES Adsorption and Partition Chromatography The physicochemical phenomenon, adsorption, is used to separate mixtures of compounds. The separation occurs because of the differences in the reversible physicochemical forces, that is, ionic, van der Waals, and London dispersing forces, exerted by the active centers of a solid stationary phase, for which starch, cellulose, silica gel, aluminum oxide, calcium phosphate-activated carbon, among others can be used. For elution, one can use apolar solvents or mixtures of polar and apolar solvents or small amounts of polar materials dissolved in an organic solvent. The relationship between the mechanism of adsorption and the chromatographic mobility of the different compounds is described in the literature [ 9, 10] • Partition chromatography utilizes the differences in solubility in two solvent systems of the different constituents of a mixture. In this case, members of the mixture are separated according to the influences on solubility exerted by their functional groups, by conformation of the molecules, and by solvation properties of the molecules. Substances used for the stationary phase are chiefly water, glycols, formamides, dimethylsulfoxide, and fatty acids or their derivatives alone or in combination with another. A variety of solvent or solvent mixtures can be used for the mobile phase. In partition TLC, the plates are covered with a solid support that carries the stationary phase. Silica gel, cellulose, or alumina is commonly used as the solid support. In partition TLC, the chamber is usually saturated with both phases. Theories of partition chromatography, which are based on either equilibrium or kinetic approaches, are adequately treated in the literature [ 11, 12] • Apparatus and Techniques The general technique and apparatus of TLC is simple and for most purposes quite similar. These topics are outlined here so that descriptions of the methods are not dealt with during the discussions of the individual antibiotics. However, for more complete descriptions of TLC techniques and apparatus, the excellent textbook of Stahl [ 13] is strongly recommended. In TLC, the stationary phase of a chromatoplate is either prepared in the laboratory by spreading a suspension of the desired adsorbent onto a glass plate or obtaining a commercially glass plate or plastic sheet already precoated with the desired solid support. The sample to be examined is applied in a solution to the chromatoplate, at about 1 cm distance from the side of the plate. The applied sample should occupy the smallest area possible on the plate and should be in a quantity that allows separation of the components without interference from each other. Naturally, it should be in such quantity that each component can easily be detected. The desired quantity is usually experimentally determined by multiple spotting at different concentrations. The type of adsorbent or stationary phase is important and is specified for each antibiotic. However, it is important that the moisture content of the plates be regulated and kept in line with the given experimental conditions. After sample application and drying of the spots, the chromatoplate is placed into a chamber that contains the mobile phase. It is important that the level of the mobile phase is about O. 5 cm below the spots of applied samples. Most often, TLC employs an ascending technique, that is, the mobile phase travels upward. It is also important that the chromatographic chamber be equilibrated with vapors of the mobile phase. For this purpose,

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the chambers are usually lined with an adsorbing paper saturated with the mobile phase. This condition results in the so-called frontal elution chromatography. A chromatogram can be developed in one, two, or even three dimensions. The chromatoplate is placed either into the same or a different mobile phase at a 90° angle to the first direction of development. However, the initial mobile phase must be evaporated before the next development. Occasionally multiple development is used, with the same or a different solvent, in the same direction, to achieve a better separation. Chromatographic chambers range from simple coffee jars to commercially available ground glass chambers , to chambers for horizontal development (Brenner and Niederwieser) and to controlled-moisture GS (Geiss and Schlitt) chambers [ 14-16]. Chambers are set up in a controlled temperature room and are not disturbed during the development of a chromatogram. The last step in TLC is evaporation of the mobile phase and detection of the spots. Evaporation techniques depend on the nature of the solvent used. Spots can be detected by visual color; ultraviolet (UV) light; fluorescence quenching, iodine vapor, spraying with specific reagent, spraying with dilute sulfuric acid, followed by heating, or biological means. Both short- and longwave ultraviolet detection boxes are available commercially. The number of specific spraying reagents is quite extensive, and details can be found in the literature [ 17] • Biologically active materials can be selectively detected by bioautographic method, a technique in which chromatoplates that have been developed are placed on agar seeded with a sensitive microorganism and clear inhibition zones appear in the agar after incubation. In some cases TLC requires special techniques. A good review of most special techniques can be found in the textbook Practice of Thin Layer Chromatography by Touchstone and Dobbins [ 18]. We shall mention some special techniques applicable to antibiotics. Chromatoplates can be sprayed or immersed into buffers for a short time before they are used [ 19, 20] . Such a technique permits the separation of differently charged antibiotics. Gradient TLC is a technique that utilized a layer with a progressive change or gradient in separating ability, derived, for example, from a gradient in buffer concentration or in pH [ 21] . In gradient-elution TLC, solvents are mixed together continuously for changing concentration to change the eluting powers of the mobile phase [ 22, 23). Programmed multiple development (PMD) is a technique in which the distance traveled by the mobile phase is controlled by intermittent application of infrared radiation to evaporate the solvent between consecutive developments [ 24). In circular chromatography the mobile phase is introduced at the center of a circular chromatoplate and the materials are spotted at a different part of the circle, close to the center. The technique was used to separate quinomycin and actinomycin antibiotics [ 25) • Reversed-phase partition TLC is based on liquid-liquid distribution, when the less polar phase of a pair of immiscible solvents is used as the stationary phase. A more detailed description of this technique can be found latter in this chapter. The system was used to separate penicillins [ 26] and cepholosporins [ 27] • Ion-exchange TLC is similar to TLC as far as technique is concerned. It was used successfully to separate amino acids and water-soluble basic antibiotics [ 28, 29). Reflectance spectrometry is applied to TLC to detect and quantitate spots on the TLC plate. The method involves scanning the chromatogram and recording the reflectance spectrum [ 30] • Materials that absorb in the visible, ultraviolet, or fluorescence are scanned directly. Other materials are made

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visible by spraying them with chromagenic reagents [31]. This method was used successfully to separate and quantitate components of the polyene antibiotics candihexin and candidin [32]. Quantitative TLC Quantitative analysis of antibiotics can be carried out by TLC. Data obtained by evaluation of chromatograms are frequently based on relative, rather than absolute measurements, because of inaccuracies inherent in the technique. Two common types of quantitation are possible with TLC: ( 1) comparing intensities or sizes of spots on the chromatoplate; and ( 2) determining concentration in solution of materials that have been eluted from the stationary phase. In the first case, care must be taken to obtain spots of regular shape and to compare only the spots that have migrated the same distance on the same chromatoplate. The comparison can be made by direct measurement of spot sizes, by photodensitometry, by measurement of clear zones after bioautography, or by measurement of colored zones on bioautography as a result of a special enzymatic reaction. Densitometric measurements are discussed in general and for many types of compounds in Densitometry in Thin Layer Chromatography, edited by Touchstone and Sherma [ 33] • Photodensitometry was used in the quantitative estimation of such antibiotics as sicanin [34], tetracycline [35], and different penicillins [36]. Basically, such measurement can be made with single-beam densitometry and double-beam densitometry. The first type of instrument is simple and inexpensive but of low precision. The double-beam instruments are more expensive but much more precise because the system cancels baseline optical noise. Fluorescence measuring instruments are almost as precise as double-beam densitometers, and their accuracy is almost independent of the concentration of the spots. Quantitation of materials that have been eluted from the stationary phase are of several types: weight determination, spectrophotometry, fluorometry, bioassay, gas chromatography, and high-performance liquid chromatography. The most crucial point of this method is the selective and quantitative elution of the separated materials from the mobile phase. Elution can be done manually or by specially designed instruments. The method of quantitation after elution from the chromatoplate has been used to quantitate tetracyclines, chroamiphenicol, and sulfadimethoxine individually [ 37] and simultaneously [ 38] . Biologically active materials, such as antibiotics, can be estimated by bioautography. This technique is based on the measurement of the zone of inhibition on agar seeded with sensitive microorganisms due to the presence of biologically active material. The developed and dried chromatoplate is placed directly on a seeded agar preparation or more frequently with a sterile filter paper between them. The chromatoplate with the filter paper is left on the seeded agar for an optimal time of diffusion, then removed and incubated. The technique was used, for example, to quantitate oleandomycin [ 39] . This short account of TLC techniques may be helpful when examples of TLC systems used for identification and quantitative analysis of individual antibiotics are described in a later section. More detailed accounts can be found in the excellent textbooks of Stahl [ 13] and Helftmann [ 40] • Reproducibility in TLC is detailed in a book by Lederer et al. [41]. Preparation of antibiotic samples from fermentation broth for TLC application is described by Aszalos et al. [ 19].

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Reversed-Phase Thin-Layer Chromatography

Reversed-phase TLC is a technique in which a nonpolar stationary phase is fixed to the thin-layer support and a polar mobile phase is used for development. This technique deserves special attention because data can be obtained from it that can be useful in developing a system for HPLC. There are several types of reversed-phase TLC plates. The first is manufactured by Whatman Co., in which octadecyl monochlorosilane is chemically bonded to silica gel. The second type, Analtech RPTLC plates, is prepared by coating silica gel with C-18 hydrocarbons, without chemical bonding. The third type is similar to the second except that very small particle size silica gel is used in a very thin layer, which permits compact spot formations and therefore precise quantitation (high-efficiency plates, Analtech). Also, there are plates coated with C-2, C-8 chain length hydrocarbons, and they are available from Merck. The mobile phases used with these plates are mostly twocomponent mixtures of water and an organic solvent, sometimes called organic modifiers. Organic modifiers commonly used are methanol, acetonitrile, tetrahydrofuran, and dioxane. Water can be substituted by buffer. In general, as the polarity of the compounds to be chromatographed increases, the water or buffer content of the mobile phase also increases. With the Analtech plates, the mobile phase must at least contain 20% water to prevent elution of the hydrocarbon coating. If 40-50% aqueous systems are used, the salt concentration must be raised to 0.1-0.5 M to maintain stability of the stationary layer. As mentioned before, the possibility of developing HPLC systems based on thin-layer chromatographic results was explored many times, and this is especially true for reversed-phase systems. For example, Golkiewicz [42] has found that the relation between TLC and HPLC values can be described by the equation RF= 1 (1 + k'), where k' is the capacity factor of HPLC. Gilpin and Sisco [ 43] found that TLC data can be used to predict HPLC data more accurately if dodecyl hydrocarbon chains are used to derivatize the support. This type of derivatized support was used by Von Arx and Faupel [ 44] to study the relation of TLC and HPLC with a few classes of compounds, such as steroids, penicillins, and calcitonin. They found that, within each class of compounds-as represented by three or four members of the class-a good correlation can be found in terms of chromatographic mobility. Recently, Aszalos and Aquilar [ 45] investigated the transferability of reversed-phase TLC systems to reversed-phase HPLC for peptide antibiotics. A group of 19 antibiotics, with molecular weights between 102 and 25,000 and of different chemical character, were chosen for this study. Analtech C-18 plates and a mobile phase consisting of either 0. 01 M or 0. 05 M phosphate buffer or 0. 01 M heptanesulfonic acid at a pH of 2. 0, 3. 4, or 6. 6 and organic modifiers (methanol, acetonitrile, and tetrahydrofuran) were used. All antibiotics moved with reasonable RF value ( 0 < RF < 1. 0) with all buffers, if either 80% methanol, 60% acetonitrile, or 40% tetrahydrofuran were used. All these TLC systems could be used with little or no modification in HPLC for all antibiotics, if a Vydac C-18 column was employed. Usefulness was defined at V0 < tR < 50 min for HPLC. This usefulness was achieved if 72% methanol was used instead of 80% methanol, or 52% acetonitrile was used instead of 60% acetonitrile, with all aqueous mobile-phase systems. It was concluded then that if a peptide antibiotic moves with a reasonable RF value in the TLC systems described, it would also yield a useful tR in the corresponding HPLC system. However, these studies did not show any correlations among RF, tR, molecular weight of the antibiotic, and chemical nature of the antibiotic. A list and properties of the investigated antibiotics are shown in Table 1.

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Table 1 Peptide Antibiotics Used in the Study of Correlation Between TLC and HPLC Antibiotic

General character

Molecular weight

Chemical nature

Use

Cycloserine

Amino acid

102

Amphoteric

Antibacterial

Hadacidin

Amino acid

119

Acidic

Antitumor

Azaserine

Amino acid

173

Amphoteric

Antitumor

Viomycin

Cyclopeptide

686

Basic

Antibacterial

Echinomycin

Cyclopeptide, aromatic

1,050

Basic

Antimicrobial

Polymyxin Bl

Lipopeptide

1,220

Basic

Antibacterial

Colistin S

Lipopeptide

1,250

Basic

Antibacterial

Actinomycin C2

Cyclopeptide, aromatic

1,296

~eutral

Antitumor

Bacitracin A

Cyclopeptide

1,470

Basic

Antibacterial

Phleomycin

Glycopeptide aromatic

1,500

Basic

Anti tumor

Bleomycin

Glycopeptide aromatic

1,550

Basic

Antitumor

Thiostrepton

Peptide, aromatic

1,650

Amphoteric

Antimicrobial

Saramycetin

Peptide, aromatic

2,200

Acidic

Antibacterial

Gramacidin A

Cyclopeptide

3,100

Neutral

Antibacterial

Cinnamycin

Polypeptide

5,000

Amphoteric

Duramycin

Polypeptide

5,000

Amphoteric

Neocarzinostatin

Polypeptide

12,500

Acidic

Antitumor

Restrictocin

Polypeptide

15,000

Amphoteric

Antitumor

Largomycin F-11

Glycoprotein

25,000

Acidic

Antitumor

Source: By permission of Elsevier Scientific Publishing Co., Amsterdam.

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203

IDENTIFICATION OF ANTIBIOTICS

This section gives examples of TLC system use in the identification of some of the most important and recently discovered antibiotics from fermentation sources. The antibiotics are arranged in alphabetical order, and the TLC systems described include the type of adsorbent, solvent system use, spotting solution, method of detection, RF value, and literature reference.

Antibiotic : Adsorbent: Solvent: Spotting solution: Detection: RF: Reference:

Aculeximycin Silica gel 60 F254 (Merck) n-Butanol-acetic acid-water (3: 1: 1) Pure compound isolated from fermentation broth Iodine vapor 0.29 T. Ikemoto, T. Katayama, A. Shiraishi, and T. Haneishi, J. Antibiot. (Tokyo), 36, 1097 ( 1983).

Antibiotic : Adsorbent: Solvent: Spotting solution: Detection: RF: Reference:

Althiomycin Silica gel F254 (Merck) Chloroform-acetone ( 1: 1) Pure compound isolated from fermentation broth

Antibiotic: Adsorbent: Solvent: Spotting solution: Detection: RF: Reference:

AM 2604A Precoated silica gel 60 F254 (Merck) Chloroform-methanol (9: 1) Pure compound isolated from fermentation broth

Antibiotic: Adsorbent:

Aminoglycosides Precoated Kieselgel 60 plates (Merck) or silica gel H plates containing 1% carbomer Al, Chloroform-methanol-25% NH40H-water ( 1:4: 2: 1) A2, Chloroform-methanol-25% NH40H (2:3:2) B 1, 15% aqueous solution of potassium dihydrogen phosphate (pH 4. 4) B2, 10% aqueous solution of potassium dihydrogen phosphate (pH 4. 5)

Solvent:

UV

0.4 B. Kunze, H. Reichenback, H. Augustiniak, and G. Holfe, J. Antibiot. (Tokyo), 35, 635 ( 1982).

UV

o. 54 S. Omura, H. Shimizu, Y. Iwai, K. Hinotozawa, K. Otoguro, H. Hashimoto, and A. Nagawa, J. Antibiot. (Tokyo), 35, 1632 ( 1982).

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Spotting solution: Detection:

Chan and Aszalos

10 µl or 20 µl aliquot of different concentration 1. Ninhydrin 2. Napthoresorcinol (2%) in ethanol, followed by 9N H2S04 and heated for 5-10 min at 120°C

RF:

TLC systema Aminoglycoside antibiotic Streptomycin Dihydrostreptomycin (DHSM) Neomycin B (N-B) Neamine C (N-C) Neamine (NEA) Paromomycin I(P-1) Paromomycin II (P-11) Paromamine (PAM) Kanamycin A (K-A) Kanamycin B (K-B) Kanamycin C (K-C) Tobramycin ( TOB) Dibekacin (DBK) Amikacin (AM) Gentamicin C1 (G-C1) Gentamicin C2 (G-C2) Sisomicin (SIS) Netilmicin (NET) Apramycin (APR) Spectinomycin (SPEC)

Alb

A2b

Blb

B2c

0.00

0.00 0.00 0.16 0.16 0.32 0.22 0.22 0.39 0.25 0.27 0.38 0.37 0.45 0.08 0.75 o. 72 0.67 0.84 0.33 0.50

0.66 0.64 0.27 0.27 0.42 0.38 0.38 0.61 0.47 0.34 0.47 0.36 0.31 0.57 0.21 0.28 0.29 0.25 0.37 0.48

0.56 0.54 0.17 0.17 0.37 0.33 0.33 0.57 0.42 0.27 0.42 0.39 0.27 0.51 0.20 0.24 0.26 0.23 0.32 0.54

o.oo

0.20 0.22 0.38 0.30 0.31 0.49 0.35 0.35 0.46 0.42 0.47 0.10 0.65 0.70 0.65 o. 79 0.38 0.49

aMeans of two values bobtained by chromatography on E. Merck precoated silica gel plates cobtained by chromatography on Carbomer-silica gel H plates Source: By permission of Elsevier Scientific Publishing Co., Amsterdam. Reference:

P. J. Claes and H. Vanderhaeghe, J. Chromatogr. , 248, 483 (1982).

Antibiotic: Adsorbent: Solvent: Spotting solution: Detection:

Ampicillin and its degradation products Precoated silica gel 60 (Merck) n-Butanol-formic acid-water (80: 3: 1) 10-30 mg/ml in 0.05 M citrate buffer (pH 6.5) 1% starch solution-acetic acid-0.1 N iodine solution (100:8:1), dried at 100°c for 15 min. Ampicillin, 0. 28; dimer, 0. 34; tetramer, 0. 42; hexamer, O. 47; oxtamer, O. 55. C. Larsen and M. Johansen, J. Chromatogr., 246, 360 ( 1982).

Reference:

TLC Techniques and Systems

Antibiotic : Adsorbent: Solvent: Spotting solution: Detection: Rp: Reference:

205

Ancovenin Cellulose (Merck) n-Butanol-methanol-water (4: 1: 2) Pure compound isolated from fermentation broth Ninhydrin 0.73 Y. Kido, T. Hamakado, T. Yoshida, M. Anno, and Y. Motoki, J. Antibiot. (Tokyo), 36, 1295 (1983).

Asparenomycins A , B , and C Precoated cellulose plates (Eastman) pretreated with 10 mM phosphate buffer, pH 7.0, containing 10 µg/ml EDTA. Solvent: 80% aqueous acetonitrile Spotting solution: Pure compounds isolated from fermentation broth Detection: Ehrlich reagent Asparenomycin A, 0.49; asparenomycin B, 0.39; Rp: asparenomycin C, O. 55 J, Shoji, H. Hinoo, R. Sakazaki, N. Tsuji, K. Nagashima, Reference: K. Matsumoto, Y. Takahashi, S. Kozuki, T. Hattori, E. Kondo, and K. Tanaka, J. Antibiot. (Tokyo), 35, 15 ( 1982).

Antibiotic: Adsorbent:

AT-265 Silica gel 60 F254 or silanized silica gel 60 F254 (Merck) 1. Methanol-1 N acetic acid ( 20: 1) for silica gel plate 2. Methanol-acetonitrile-1 N acetic acid ( 5: 5: 1) for silanized silica gel Spotting solution: Pure compound isolated from fermentation broth. Detection: UV Solvent 1, 0.69; solvent 2, 0.83 Rp E. Takahashi and T. Beppu, J, Antibiot. (Tokyo), 35, Reference: 939 ( 1982).

Antibiotic : Adsorbent: Solvent

Antibiotic: Adsorbent: Solvent: Spotting solution: Detection: Rp: Reference:

Awamycin Silica gel Chloroform-methanol ( 96: 4) Pure compounds isolated from fermentation broth

Antibiotic : Adsorbent:

Bu-2743E Silica gel

UV

0.36 I. Umezawa, H, Oka, K. Komiyama, K. Hagiwara, S. Tomisaki, and T. Miyano, J. Antibiot. (Tokyo), 36, 1144 (1983).

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Ethyl acetate-methanol-water ( 10: 3: 1) Solvent: Spotting solution: Pure compound isolated from fermentation broth Detection: UV 0.23 RF: S. Kobaru , M. Tsunaka wa, 1\1. Hanada, M. Konishi, Reference: K. Tomita, and H. Kawaguchi, J. Antibiot. (Tokyo), 36, 1396 (1983).

Antibiotic: Adsorbent: Solvent: Spotting solution: Detection: RF: Reference:

Antibiotic: Adsorbent: Solvent: Spotting solution: Detection: RF: Reference:

C-19393 Es Cellulose (Tokyo Kasei) CH3CN-H20 (4: 1) Culture filtrate ( 10 µl) after treating with methylene chloride containing tri-n-octylmethyl ammonium chloride Bioautography versus E. coli PG 8 0.56 S. Harada, Y. Nozaki, S. Shinajawa, and K. Kitano, J. Antibiot. (Tokyo), 35, 957 (1982).

Candiplanecin Precoated silica gel plate F254 Chloroform-methanol ( 4: 1) Pure compound isolated from fermentation broth Iodine vapor 0.4 Y. Itoh, A. Torikata, C. Katayama, T. Haneishi, and M. Arai, J. Antibiot. (Tokyo), 34, 934 (1981).

Carbapenems Antibiotic: Precoated silica gel plate (Merck) Adsorbent: Acetonitrile-0. 75% acetic acid (9: 2) at 5-8°C Solvent: Spotting solution: Authentic samples of Carbapenems PS-5, PS-6 and PS-7 and epithienamycins A, B, C, and D dissolved in 0.01 M phosphate buffer pH 8.0 Dipped in Ehrlich reagent, heated at 100°C for 5 min Detection: PS-5, 0.44; PS-6, 0.50; PS-7, 0.53; epithienamycin C, RF: 0.025; epithienamycin D, 0.42; epithienamycin A, 0.26; epithienamycin B, 0. 42. M. Ika, K. Kiyoshima, I. Kojima, R. Okamoto, Reference: Y. Fukagawa, and T. Ishikura, J. Chromatogr., 256, 47 ( 1983).

Antibiotic: Adsorbent: Solvent:

Cervinomycins Silica gel G 60 F254 (Merck) Chloroform-methanol ( 40: 1)

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Spotting solution: Pure compounds isolated from fermentation broth Detection: UV Cervinomycin A, 0.39; cervinomycin A2, 0.32 RF: Reference: A. Hirano, H. Shimizu, and K. Haneda, J. Antibiot. (Tokyo), 35, 646 (1982).

Antibiotic: Cinodine Adsorbent: Silica gel plates F254 (Brinkmann) Solvent: Methanol-1 M sodium acetate ( 30: 70) Spotting solution: Pure compound isolated from fermentation broth Detection: UV 0.48 RF: Reference: W. McGahren, F. Barbatshi, N. Kuck, G. Morton, B. Hardy, and G. Ellested, J. Antibiot. (Tokyo), 35, 794 (1982).

Cyanocycline A Antibiotic : Adsorbent: Precoated silica gel 60 F254 (Merck) Chloroform-methanol ( 9: 1) Solvent: Spotting solution: Pure compound isolated from fermentation broth Detection: Ninhydrin or UV 0.83 RF: Reference: T. Hayashi, T. Noto, Y. Nawata, H. Okazaki, M. Sawada, and K. Ando, J. Antibiot. (Tokyo), 35, 771 ( 1982).

Antibiotic : Adsorbent: Solvent:

Daunorubicin and related antibiotics Precoated silica gel 60 (Merck) 1. Chloroform-methanol-H 20 ( 120: 20: 1) 2. Chloroform-methanol-H20 (80: 30: 3) Spotting solution: Fermentation broth adjusted to pH 10. 0 and extracted with half its volume using chloroform. Detection: UV Solvent 1, daunorubicin, 0.45; N-acetyldaunorubicinol, RF: 0.25 Solvent 2, daunorubicin, 0.10; N-acetyldaunorubicinol, 0.04 A. A. Aszalos, N. R. Bachur, A. F. Langlykke, P. P. Reference: Roller, M. Y. Sheikh, M. S. Sutphin, M. C. Thomas, D. A. Wareheim, and L. H. Wright, J. Antibiot. (Tokyo), 30, 50 (1977).

Antibiotic: Adsorbent:

Daunorubicin and related antibiotics Precoated silica gel 60 F254 (Merck)

Chan and Aszalos

208

Solvent:

Chloroform-methanol-formic acid (80: 20: 2) 2. Chloroform-heptane-methanol (5: 5: 1) Spotting solution: n-Butanol extracts of whole broth or standards with various concentration Detection: UV 1.

An thracycline

e:- Rhodomycinone Daunorubicinone 7-Deoxydihydrodaunorubicinone Glycoside I (baumycin A2) Descarbomethoxybisanhydro- e:rhodomycinone Daunorubicin

Solvent 1

Solvent 2

0.79 0.74 0.63 0.21 0.83

0.48 0.25 0.16 0.00 0.74

0.15

0.03

Source:

By permission of Elsevier Scientific Publishing Co., Amsterdam.

Reference:

R. C. Pandey and M. W. Toussaint, J. Chromatogr. , 198, 407 (1980).

Antibiotic: Adsorbent: Solvent:

Desertomycin Polygram Cel 400 sheets (Brinkmann) Ethanol (95%)-pyridine-acetic acid-water ( 56: 56: 14: 100, v/v) Fermentation broth Bioautography against Micrococcus luteus UC 130

Spotting solution: Detection: RF: Reference:

Antibiotic: Adsorbent: Solvent: Spotting solution: Detection: RF: Reference:

-0.9

L. Dolak, F. Reusser, L. Bae zynskyj, S. Mizsak, B . Hannon, and T. Castle, J. An tibiot. (Tokyo) , 36, 13 (1983).

N-(2,6-Diamino-6-hydroxymethylpimelyl)-L-alanine Precoated silica gel 60 F (Merck) Isopropanol-14% NH40H (2: 1) Pure material isolated from fermentation broth Ninhydrin 0.28 J. Shoji, H. Hinoo, T. Kato, K. Nakauchi, S. Matsuura, M. Mayama, Y. Yasuda, and Y. Kawamura, J. Antibiot. (Tokyo), 34, 374 ( 1981).

TLC Techniques and Systems

Antibiotic : Adsorbent: Solvent: Spotting solution: Detection: RF: Reference:

3, 6-Dihydroxyindoxazene Silica gel G (Merck) Chloroform-methanol ( 4: 1) Pure compound isolated from fermentation broth

Antibiotic : Adsorbent: Solvent: Spotting solution: Detection: RF:

Ditrisarubicins A, B, and C Silica gel 60 F254 (Merck) Ethyl acetate-methanol (10: 1) Pure compounds isolated from fermentation broth

Reference:

Antibiotic: Adsorbent: Solvent: Spotting solution: Detection: RF: Reference:

Antibiotic : Adsorbent: Solvent:

Spotting solution: Detection: RF value:

209

UV

0.4 T. Ikeda, D. Ikeda, K. Iinuma, S. Gomi, Y. Ikeda, T. Takeuchi, and H. Umezawa, J. Antibiot. (Tokyo), 36, 445 ( 1983).

UV

Ditrisarubicin A, 0.65; ditrisarubicin B, 0.47; ditrisaurubicin D , 0. 11 T • Uchida, M. Imoto, T. Masuda, K. Imamura, Y. Hatori, T. Sawa, H. Naganawa, N. Hamada, T. Takeuchi, and H. Umezawa, J. Antibiot. (Tokyo), 36, 1080 (1983).

EM 5519 Silica gel 60 F254 (Merck) 1. n-Butanol-acetic acid-water (3: 1: 1) 2. Acetone-methanol ( 5: 1) Pure compound isolated from fermentation broth Bioautography against E. coli SC 2927 Solvent 1, 0.35; Solvent 2, 0.52 E. Meyers, R. Cooper, W. Trejo, N. Georgopapadakou, and R. Sykes, J. Antibiot. (Tokyo), 36, 191 (1983).

Erythromycins Prepared from standard equipment using a slurry of silica gel G (Merck) in phosphate buffer (pH 8) 1. CH2Cl2-CHCla-CH30H (80: 20: 20) 2. CH2ClrCH30H-HCONH2 (100:20:2) 3. CH2Cl2-CH30H (100: 20) 4. CHCl3-CH30H (20: 100) 1 µl of 10 mg/ml in methanol Phenol-sulfuric acid reagent Mono- and dipropionates tested, (E, erythromycin; EO, erythromycin oxime; EA, erthromycylamine; EPr , E propionate; EOMPr, EO monopropionate; EMA Pr, EA monopropionyl derivative; EODPr, EO dipropionate; EASPr, EA dipropionyl derivative)

210

Chan and Aszalos

Compound E

EO EA EPr EOMPr EAMPr EODPr EADPr

1

2

0.26 0.23 0.05 0.76 0.22 0.11 0.81 0.60

0.48 0.48 0.07 0.93 0.51 0.23 0.94 0.85

3

0.25 0.28 0.06 0.81 0.28 0.12 0.81 0.70

4

0.28 0.27 0.07 0.80 0.27 0.25 0.77 0.78

Mono- and Dimethylsuccinates tested (E, erythromycin; EO, erythromycin oxime; EA, erythomyclamine: EES, E ethylsuccinate; EOMMS, EO monomethylsuccinate; EAMMS , EA menomethylsuccinyl derivative; EODMS , EO dimethylsuccinate; EADMS, EA dimethylsuccinyl derivative; for solvent systems 1-4, see Table 1) Compound E EO EA EES EOMMS EAMMS EODMS EADMS

1

2

3

4

0.18 0.16 0.04 0.51 0.21 0.07 0.51 0.04

0.33 0.32 0.06 0.81 0.39 0.18 0.90 0.72

0.14 0.13 0.03 0.66 0.17 0.07 0.70 0.55

0.22 0.21 0.05 0.69 0.22 0.19 o. 70 0.68

Source: By permission of Elsevier Scientific Publishing Co., Amsterdam. Reference:

G • Kobrehel, Z. Tamburasu, and S. Djokic , J. Chroma togr., 133, 415 (1977).

Antibiotic: Adsorbent: Solvent:

Feresimycins A and B Silica gel 1. n-Hexane-ethyl acetate ( 1: 2) 2. Benzene-ethyl acetate (1: 1) Pure compounds isolated from fermentation broth 2, 4-Dinitrophenylhydrazine, Dragendorff, I 2 vapor 1. Ferensimycin A, 0. 56; ferensimycin B, 0. 62; 2. Ferensimycin A , 0. 28; ferensimycin B , 0. 3 7 Y. Kusakabe, T. Mizuno, S. Kawabata, S. Tanji, and A. Seino, J. Antibiot. (Tokyo), 35, 1119 (1982).

Spotting solution: Detection: RF: Reference:

An tibiotic: Adsorbent:

FR-900109 Silica gel sheet (Eastman)

TLC Techniques and Systems

211

Solvent: Spotting solution: Detection: Rp: Reference:

Chloroform-methanol ( 20: 1) Pure compound isolated from fermentation broth Iodine vapor 0.3 M. Yamashita, M. Iwami, K. Ikushima, T. Komari, H. Aoki, and H. Imanaka, J. Antibiot. (Tokyo), 36, 1123 (1983).

Antibiotic: Adsorbent: Solvent:

Fredricamycin A Silica gel or alumina 1. Chloroform-methanol-acetic acid (87:3:3) for silica gel 2. Chloroform-methanol-acetic acid (78:20:2) for alumina Pure compound isolated from fermentation broth UV Solvent 1, 0.76; solvent 2, 0.69 R. Pandey, M. Toussaint, R. Stroshane, C. Kalita, A. Aszalos, A. Garretson, T. Wei, K. Byrne, R. Geoghegan, and R. White, J. An tibiot. (Tokyo) , 34, 1389 (1981).

Spotting solution: Detection: Rp: Reference:

Antibiotic : Adsorbent: Solvent: Spotting solution: Detection: Rp:

Reference:

Fungichromin, lagosin, cogomycin, and filipin III polyene macrolide antibiotics Precoated TLC plates (Analtech) Solvent 1, chloroform-methanol (85: 15); Solvent 2, n-butanol-acetic acid-H20 ( 4: 1: 5) Pure standards UV, iodine, or sulfuric acid Solvent 1, Fungichromin, lagosin, and cogomycin are indistinguishable with Rp 0. 28; filipin III, O. 45 Solvent 2, Fungichromin, lagosin, and cogomycin are indistinguishable with Rp 0.68; filipin III, 0.81 R. Pandey, E. Guenther, and A. As zalos, J. Antibiot. (Tokyo), 35, 988 (1982).

Antibiotic: Adsorbent: Solvent:

G-3678 and related aminoglycoside antibiotics Precoated silica gel 60 F254 plate (Merck) 1. Chloroform-methanol-28% ammonium hydroxide (1: 1: 1) lower layer 2. Chloroform-methanol-14% ammonium hydroxide

Spotting solution:

Standards and pure compound isolated from fermentation broth Ninhydrin Solvent 1: G-367S, 0.36; sisom1cm, 0.44; verdamicin, 0.53; gentamicin C1a. 0.36; gentamicin C2, 0.48; gentamicin C1, 0. 54

Detection: Rp:

(1: 2: 1)

212

Reference:

Antibiotic : Adsorbent: Solvent: Spotting solution: Detection: Rp: Reference:

Antibiotic: Adsorbent: Solvent: Spotting solution: Detection: Rp: Reference:

Chan and Aszalos

Solvent 2: G-367S, O. 56; sisom1cm, 0. 50; verdamicin, 0.53; gentamicin c19' 0.42; gentamicin c2' 0.52; gentamicin C1, O. 57 s. Satoi, M. Awata, N. Awata, N. Muto, M. Hayashi, H. Sagai, and M. Otari, J. Antibiot. (Tokyo), 36, 1, 1983.

Gilvocarcins Silica gel Ethyl acetate-acetic acid (9: 1) Ethyl acetate-acetone ( 1: 1) extract of fermentation broth UV at 395 nm Gilvocarcin V, 0.45; gilvocarcin M, 0.32 H. Nakano, Y. Matsuda, K. Ito, S. Ohkubo, M. Morimoto, and F. Tomita, J. Antibiot. (Tokyo), 34, 266 (1981).

Glysperins A, B , and C Precoated silica gel 60 F254 (Merck) Chloroform-methanol-28% ammonium hydroxide Pure compound isolated from fermentation broth UV or ninhydrin Glysperin A, 0.36; glysperin B, 0.50; glysperin C, 0.30 H. Kawaguchi, M. Konishi, T. Tsuno, T. Miyaki, K. Tomita, K. Matsumoto, K. Fujisawa, and H. Tsukiura, J. Antibiot. (Tokyo), 34, 381 (1981).

Antibiotic: Adsorbent: Solvent: Spotting solution: Detection: Rp: Reference:

Haloquinone Silica gel plate F254 (Merck) Chloroform Pure compound isolated from fermentation broth

Antibiotic : Adsorbent: Solvent: Spotting solution: Detection: Rp:

Isohematinic acid Silica gel plate F254F (Merck) Benzene-ethyl acetate-acetic acid ( 10: 10: 1) Pure compound isolated from fermentation broth Iodine and bromocresol green 0.5

UV

0.32 B. Ewersmeyer-Wenk, H. Zahner, B. Krone, and A. Zeeck, J, Antibiot. (Tokyo), 34, 1531 (1981).

TLC Techniques and Systems

213

Reference:

Y. Itoh, M. Takeuchi, K. Shimizu, S. Takahashi, A. Terahara, and T. Haneishi, J. Antibiot. (Tokyo), 36' 497 ( 1983).

Antibiotic: Adsorbent: Solvent: Spotting solution: Detection: Rp: Reference:

Karabemycin Avicel cellulose plate n-Propanol-pyridine-acetic anhydride-water ( 15: 10: 3: 6) Pure compound isolated from fermentation broth Ninhydrin 0.47 S. Omura, A. Nakagawa, H. Aoyama, Y. Iwai, M. Kuwahara, and T. Furusato, J. Antibiot. (Tokyo), 36, 1129 (1983).

Antibiotic: Adsorbent: Solvent: Spotting solution: Detection: Rp: Reference:

Kijanimicin Silica gel GF plate (Analtech) Methanol Pure compound isolated from fermentation broth UV 0.74 J. Waitz, A. Horan, M. Kalyanpur, B. Lee, D. Loebenberg, J. Marquez, G. Miller, and M. Patel, J. Antibiot. (Tokyo), 34, 1101 (1981).

Antibiotic: Adsorbent: Solvent: Spotting solution:

Lavendamycin Silica gel GHLF Uniplates (Anal tech, Inc.) Toluene-acetonitrile-trifluoroacetic acid ( 70: 20: 10) Silica gel column fractions of crude lavendamycin from fermentation broth Visible color, bright red spot 0.3 D. M. Balitz, J. A. Bush, W. T. Bradner, T. W. Doyle, and D. E. Nettleton, J. Antibiot. (Tokyo), 35, 259 ( 1982).

Detection: Rp: Reference:

Antibiotic : Adsorbent: Solvent:

Macrolides Pre-coated Kieselgel 60 F254 1. Ethyl acetate-methanol-25% ammonia (85: 10: 5) 2. Diethyl ether-methanol-25% ammonia (90:9:2) 3. Dichloromethane-methanol-25% ammonia (90: 9: 1. 5) 4. Ethyl acetate-ethanol-15% ammonium acetate buffer pH 9.6 (9:4:8), upper phase 5. Di-isopropyl ether-methanol-25% ammonia (75:35:2) 6. Chloroform-ethanol-15% ammonium acetate pH 7.0 ( 85: 15: 1) 7. Chloroform-ethanol-3. 5% ammonia ( 85: 15: 1)

Chan and Aszalos

214

Spotting solution: Detection:

1 or 10 mg/ml in CH2Cl2 Anisaldehyde-sulfuric acid-ethanol ( 1: 1: 9), heated for 1 min at 110°

RF: Mobile phase Compound Midecamycin ethylcarbonate Midecamycin Josamycin Tylosin Troleandomycin Oleandomycin Leucomycin Erythromycin ethylcarbonate Erythromycin estolate Erythromycin ethylsuccinate Spiramycin I Spiramycin II Spiramycin III Rosamicin Megalomycin

Color of spot

1

2

3

4

5

6

7

o. 74

0.76

0.76

0.80

0.79

0.69

o. 71

Blue

0.64 0.63 0.31 0.65 0.36 a 7 sp. 0.58

0.57 0.55 0.13 0.53 0.21 9 sp~ 0.45

0.53 0.53 0.35 0.67 0.32 a 6 sp. 0.48

o. 76 0.78 0.58 0.60 0.24 7 sp~ 0.60

0.71 0.70 0.42 0.63 0.36 a 7 sp. 0.63

0.58 0.62 0.38 0.57 o.o8a a 7 sp. 0.44

0.63 0.63 0.49 0.60 0.16 a 6 sp. 0.52

Blue Blue Violet-brown Pink Pink Blue Blue

0.56

0.45

0.48

0.60

0.63

0.44

0.52

Blue

0.57

0.43

0.48

0.57

0.63

0.41

0.51

Violet-brown

0.38 0.54 0.58 0.32 0.29

0.21 0.39 0.46 0.13 0.12

0.37 0.45 0.47 0.47 0.24

0.47 0.54 0.57 0.29 0.16

0.45 0.57 0.61 0.31 0.23

o.18b 0.24b 0.30 o.23b o.04b

o. 41 0.47 0.47 o.35 0.12

Violet-brown Violet-brown Violet-brown Grey-brown Grey-brown

a

Spots. bstreaking spot. Source: By permission of Elsevier Scientific Publishing Co., Amsterdam. Reference:

I. O. Kibwage, E. Roets, and J. Hoogmartens, J. Chromatogr., 256, 164 (1983).

Marcellomycin and related anthracycline antibiotics Antibiotic : Adsorbent: Silica gel plate (Analtech) Toluene-ethyl acetate-methanol ( 3: 1: 1) Solvent: Spotting solution: Pure compounds isolated from fermentation broth UV or color Detection: Cinerubin B , 0. 93; y-pyrromycinone, 0. 90; Rp: e:-pyrromycinone, 0. 77; cinerubin A, 0. 56; marcellomycin, 0.35; pyrromycin, 0.17 J, Bush, W. Bradner, and K. Tomita, J. Antibiot. Reference: (Tokyo), 35, 1174 (1982).

Antibiotic : Adsorbent: Solvent:

Milbemycins D, E, F, G, and H Silica gel 60 F254 (Merck) Dioxane-carbon tetrachloride (15:85)

TLC Techniques and Systems

215

Spotting solution: Pure compounds isolated from fermentation broth Detection : : UV Milbemycin D, 0.46; milbemycin E, 0.61; milbemycin F, RF: 0.22; milbemycin G, 0.86; mibemycin H, 0.88 Reference: Y. Takiguchi, M. Ono, S. Muramatsu, J, Ide, H. Mishima, and M. Terao, J. Antibiot. (Tokyo), 36, 502 (1983).

Antibiotic: Mycoplanecin A Adsorbent: Silica gel (Merck) Solvent: Chloroform-methanol ( 10: 1) Spotting solution: Pure compound isolated from fermentation broth Detection: Iodine or sulfuric acid RF: 0.64 Reference: M. Nakajima, A. Torikata, Y. Ichikawa, T. Katayama, A. Shiraishi, T. Haneishi, and M. Arai, J. An tibiot. (Tokyo), 36, 961 (1983).

Antibiotic : Adsorbent: Solvent: Spotting solution: Detection: RF: Reference:

Mycotrienins I and II Silica gel 1. Chloroform-ethanol (15: 1) 2. Benzene-chloroform-methanol (3: 7: 3) Pure compounds isolated from fermentation broth

UV

Solvent 1, Mycotrienin I , 0. 61; Mycotrienin II, 0. 22 Solvent 2, Mycotrienin I, 0. 78; Mycotrienin II , 0. 67 M. Sugita, Y. Natori, T. Sasaki, K. Furihata, A. Shimazu, H. Seto, and N. Otake , J. Antibiot. (Tokyo) , 35, 1460 ( 1982).

Antibiotic: Adsorbent: Solvent: Spotting solution: Detection: RF: Reference:

Naphthomycins A and B Silica gel 60 F254 (Merck) Ethyl acetate-water-formic acid ( 100: 30: 25), upper phase Pure compounds isolated from fermentation broth

Antibiotic: Adsorbent: Solvent: Spotting solution:

Neplanocin Silica gel n-Butanol-ammonium hydroxide-water (10: 0. 5: 2) Column fractions during purification step from fermentation broth

Detection: RF:

UV

NaphthomycinA, 0.62; naphthomycinB, 0.52 W. Keller-Schierlein , M. Meyer , A. Zeeck , M. Danberg, R. Machinek, H. Zahner, and G. Lazor, J. Antibiot. (Tokyo), 36, 454 (1983).

UV

0.26

216

Chan and Aszalos

Reference:

S. Yaginuma, N. Muto, M. Tsujino, Y. Sudate, M. Hayashi, and M. Otani, J. Antibiot. (Tokyo), 34, 359 (1981).

Antibiotic: Adsorbent: Solvent: Spotting solution: Detection: Rp:

Oligostatins C, D, and E Silica gel 60 (Merck) Ethyl acetate- methanol-water ( 5: 3: 2) Pure compounds isolated from fermentation broth Sulfuric acid Oligostatin C, O. 25; oligostatin D, 0.18; oligostatin E, 0.13 J. Itoh, S. Omoto, T. Shomura, H. Ogino, K. Iwamatsu, and S. Inouye, J. An tibiot. (Tokyo) , 34, 1424 (1981) •

Reference:

Olivanic acids DEAE cellulose 0.1 M NaCl in O. 05 M potassium phosphate buffer, pH 7.0 Spotting solution: Culture filtrate Detection: Bioautography versus B. subtilis Rp: MM 13902 Rp, 0.23; MM 17880, Rp, 0.35 MM 22382, Rp, 0.5; MM 13902, Rp, 0.67 Reference: S. Box, G. Hanscomb, and S. Spear, J. Antibiot. (Tokyo), 34' 600 (1981).

Antibiotic : Adsorbent: Solvent:

Antibiotic : Adsorbent: Solvent: Spotting solution: Detection: Rp: Reference:

OM-173 components Silica gel 60 F254 (Merck) Benzene-ethyl acetate ( 7: 3) Pure components isolated from fermentation broth

UV

OM-173aA, 0.69; OM-173aE, 0.65; OM-173aB, 0.49; OM-173aA, 0.27; OM-173SE, 0.25 Y. Iwai, K. Kimura, Y. Takahashi, K. Hinotozawa, H. Shimizu, H. Tanaka, and S. Omura, J. Antibiot. (Tokyo), 36, 1268 (1983)

Oxytetracycline and its impurities A slurry of 47 ml of 5% (w/v) aqueous disodium EDTA (pH adjusted to 9 with 20% NaOH) and 20 g of Kieselguhr was spread on glass plates to a thickness of 0.25-0.3 mm and air dried for 2 hr at room temperature. Ethylene glycol-water-acetone-ethyl formate-ethyl acetoSolvent: acetate (3:6:45:30:15 v/v) Spotting solution: 1 µl of 1% solution of oxytetracycline in methanol

Antibiotic: Adsorbent:

TLC Techniques and Systems

Reference:

Antibiotic: Adsorbent: Solvent: Spotting solution: Detection: Rp: Reference:

217

UV light at 366 nm Oxytetracycline, O. 31; anhydrooxytetracycline, 0. 83; epioxytetracycline, 0. 05; a.-apooxytetracycline, 0.18; s-apooxytetracycline, o. 90; terrinodide' o. 76 G. J, Willekens, J, Pharm. Sci., 66, 1419 (1977)

Paulomycins Cellulose (Polygram CEL 300) precoated sheets 0.1 M phosphate buffer pH 7. 0 Pure compounds isolated from fermentation broth Bioautography on M. luteus seeded agar or UV Paulomycin A , 0. 14; paulomycin B , 0. 3 2; paulomycin C , 0. 43; paulomycin D , 0. 58 A. Argoudelis, T. Brinkley, T. Brodasky, J. Beuge, H. Meheu, and S. Mizak, J. Antibiot. (Tokyo), 35, 285 ( 1982).

Antibiotic : Adsorbent: Solvent: Spotting solution: Detection: Rp: Reference:

Pyrrolnitrin Silica gel 60 F254 (Merck) Dichloromethane-n-heptane (2: 1) Pure compound isolated from fermentation broth. UV 0.3

K. Gerth, W. Trovitzsch, V. Wray, G. Hofle, H. Irachik, and H. Reichenback, J. Antibiot. (Tokyo), 35, 1101 (1982).

Antibiotic: Adsorbent: Solvent: Spotting solution: Detection: Rp: Reference:

Pyrrolomycin A and B Silica gel Chloroform-methanol ( 10: 1) Pure compounds isolated from fermentation broth UV

Pyrrolomycin A , 0. 73; Pyrrolomycin B , 0. 80 N. Ezaki, T. Shomura, M. Koyama, T. Niwa, M. Kojima, S. Inouye, T. Ito, and T. Niida, J. Antibiot. (Tokyo), 34, 1363 (1981).

Antibiotic : Adsorbent: Solvent: Spotting solution: Detection: Rp: Reference:

Reductiomycin Precoated silica gel plate Ethyl acetate Pure compound isolated from fermentation broth UV or iodine vapor

o. 52

K. Shimizu and G. Tomura, J. Antibiot. (Tokyo), 34, 649 ( 1981).

218

Antibiotic : Adsorbent: Solvent: Spotting solution: Detection: RF: Reference:

Chan and Aszalos

Retrostatin Silica gel Ethyl acetate Pure compound isolated from fermentation broth UV 0.57 M. Nishio, A. Kuroda, M. Suzuki, K. lshimaru, S. Nakamura, and R. Nomi, J. Antibiot. (Tokyo), 36, 761 (1983).

Antibiotic : Rifamycins Adsorbent: Precoated silica gel 60F (Merck) Solvent: Chloroform-methanol-water (80: 20: 25) Spotting solution: 1. 5 mg/ml in chloroform for standard, 15 mg/ml for bulk drug substance in chloroform Detection: UV under short wavelength Rifamycin 0, 0.83 (yellow); rifamycin S, 0.81 (brown); RF: rifampin quinone, 0. 75 (purple); 25-desacetylrifampin quinone, 0.66 (purple); rifampin, 0.60 (orange); 25-desacetylrifampin, O. 41 (orange); 3-formylrifamycin SV, O. 27 (pink); rifamycin B, 0.15 (yellow); rifamycin N-oxide, O. 09 (yellow); 1-amino-4-methylpiperazine, 0. 05 (brown). W. L. Wilson, K. C. Graham, and M. J. Lebelle, Reference: J. Chromatogr., 144, 270 (1977).

Antibiotic: Rubeomycins A, Ai. B and B 1 Adsorbent: Precoated silica gel 60 F254 (Merck) Chloroform-methanol-acetic acid (80: 20: 4) Solvent: Spotting solution: Acetone extraction of mycelial pellet, the extract concentrated in vacuo and re-extracted by chloroform for spotting Detection: UV Rubeomycin A, 0.57; rubeomycin Ai. 0.74; rubeomycin B, RF: 0.34; rubeomycin B1, 0.47 Y. Ogawa, H. Sugi, N. Fujikawa, andH. Mori, J. Reference: An tibiot. (Tokyo) , 34, 938 ( 1981) •

Saccharocin, apramycin, and oxyapramycin Antibiotic : Precoated silica gel 60 F254 (Merck) Adsorbent: Chloroform-methanol-14% ammonium hydroxide ( 1: 2: 1) Solvent: Spotting solution: Pure compounds isolated from fermentation broth Ninhydrin Detection: Saccharocin, 0.26; apramycin, 0.22; oxyapramycin, 0.16 RF: M. Awata, s. Satoi, N. Muto, M. Hayashi, H. Sagai, and Reference: H. Sakakibara, J. Antibiot. (Tokyo), 36, 651 (1983).

TLC Techniques and Systems

Antibiotic : Adsorbent: Solvent: Spotting solution: Detection: Rp: Reference:

Setamycin Silica gel Chloroform-methanol ( 10: 1) Pure compound isolated from fermentation broth UV 0.48 S. Omura, K. Otoguro, T. Nishikiori, R. Oiwa, and Y. Iwai, J. Antibiot. (Tokyo), 34, 1253 (1981).

Antibiotic: Adsorbent: Solvent: Spotting solution: Detection: Rp: Reference:

SF-1623 and SF1623B Silica gel n-Butanol-acetic acid-water ( 2: 1: 1) Pure compounds isolated from fermentation broth Ninhydrin or UV SF-1623, 0.18; SF-1623B, 0.31 S. Inouye, M. Kojima, T. Shomura, K. Iwamatsu, T. Niwa, Y. Kondo, T. Niida, Y. Ogawa, and K. Kusama, J. Antibiot. (Tokyo), 36, 115 (1983).

219

Antibiotic : Siderochelin A Adsorbent: Silica gel Methanol-chloroform ( 1: 19) Solvent: Spotting solution: Pure compound isolated from fermentation broth Detection: UV 0.40 Rp: W. Liu, S. Fisher, J. Wells, C. Ricca, P. Principe, Reference: W. Treyo, D. Bonner, J. Gougoutos, B. Toeplitz, andR. Sykes, J. Antibiot. (Tokyo), 34, 791 (1981).

Antibiotic : Adsorbent: Solvent: Spotting solution: Detection: Rp: Reference:

Spergualin Avicel Cellulose Plate 1-butanol-ethanol-water ( 4: 1: 2) Pure compound isolated from fermentation broth Ninhydrin 0.2 H. Umezawa, S. Kondo, H. Iinuma, S. Kunimoto, Y. Ikeda, H. Iwasawa, D. Ikeda, and T. Takeuchi, J. Antibiot. (Tokyo), 34, 1622 (1981).

SQ 26,180, a monobactam Antibiotic : Silica gel 60 (Merck) Adsorbent: 2-Butanol-acetic acid-water (3: 1: 1) Solvent: Spotting solution: Pure compound isolated from fermentation broth

220

Detection: RF: Reference:

Chan and Aszalos

Rydon-Smith 0.40 W. Parker, W. Koster, C. Cimarusti, P. Floyd, W. Liu, and M. Rathnum, J. Antibiot. (Tokyo), 35, 189 ( 1982).

Antibiotic : Adsorbent:

Sulfazecin Cellulose F (Tokyo Kasei Co.) or DEAE cellulose (Tokyo Kasei Co. ) 1. Cellulose, n-BuOH-acetic acid-H20 ( 4: 1: 2) Solvent: 2. DEAE cellulose, 0.05 M phosphate buffer (pH 6.8) Spotting solution: Pure compound isolated from fermentation broth Ninhydrin Detection: Solvent 1, 0.22; solvent 2, 0.72 RF: Reference: M. Asai, K. Haibara, M. Muroi, K. Kintaka, and T. Kishi, J. Antibiot. (Tokyo), 34, 621 (1981).

Antibiotic: Adsorbent: Solvent: Spotting solution: Detection: RF: Reference:

Antibiotic :

Terrecyclic acid A Precoated silica gel plate 60 GF254 (Merck) Benzene-methanol (80: 20) Pure compound isolated from fermentation broth UV or 2, 4-dinitrophenylhydrazine 0.32 M. Nakagawa, A. Hirota, H. Sakai, and A. Isogai, J. Antibiot. (Tokyo), 35, 778 (1982).

Tetracycline degradation products in tetracycline hydrochloride powders and capsules Adsorbent: Whatman cellulose powder CC41 (30 g) mixed with 0.1 M EDTA disodium salt (65 ml) and applied to glass plates (0.3 mm thick), air dried at room temperature for 1 hr, and heated at 90°C for 20 min Solvent: Plate sprayed with 4 ml of water and then developed with chloroform saturated with 0.1 M EDTA disodium salt Spotting solution: 1% Tetracycline hydrochloride, O. 005%; 4-epianhydrotetracycline hydrochloride and anhydrotetracycline hydrochloride, 0. 04%; 4-epitetracycline hydrochloride and 0. 02% chlorotetracycline in methanol Detection: UV lamp with maximum output at 366 nm 4-Epitetracycline hydrochloride, 0. 035 (yellowish- green RF: spot); tetracycline hydrochloride 0. 06-0. 23 (yellow band); chlorotetracycline hydrochloride, 0.32 (yellow); 4-epianhydrotetracycline hydrochloride, 0. 52 (yellowbrown); anhydrotetracylcine hydrochloride, 0. 92 (yellow brown) A. I. H. Omer, E. A. Grad Karlem, and R. B. Salama Reference: J. Chromatogr., 205, 486 (1981).

TLC Techniques and Systems

221

Antibiotic : Adsorbent: Solvent: Spotting solution: Detection: RF: Reference:

Tetrafungin Precoated silica gel 60 plate (Merck) n-Butanol-acetic acid-water (3: 1: 1) Pure compound isolated from fermentation broth Orthophosphoric acid; heat for 5 min at 100°C 0.37 M. Vega and J. Fabregas, J. Antibiot. (Tokyo), 36, 770 (1983).

Antibiotic: Adsorbent: Solvent: Spotting solution: Detection: RF:

Tetrocarcins Precoated silica gel plates (Merck) Chloroform-methanol (9: 1) Pure compounds isolated from fermentation broth.

Reference:

UV

Tetrocarcin A , 0. 56, tetrocarcin E, 0. 73; tetrocarcin E2, 0.63; tetrocarcin F, 0.67; tetrocarcin F-1, 0.61 T. Tamaoki, M. Kasai, K. Shirahata, and F. Tomita, J. Antibiot. (Tokyo), 35, 979 (1982).

Tetronomycin Antibiotic: Silica gel 60 (Merck) Adsorbent: Solvent: Dichloromethane-acetone ( 9: 1) Spotting solution: Pure compound isolated from fermentation broth Detection: Ceric sulfate ( 0. 2%) in 50% H2SO 4 followed by heating at 130°C producing brown spot 0.36 RF: c. Keller-Juslen, H. D. King, M. Kuhn, H. Loosli, Reference: W. Pache, T. Petcher, H. Weber, and A. Von Wartburg, J. Antibiot. (Tokyo), 35, 142 (1982).

Thiolactomycin Antibiotic: Precoated silica gel plate 60 F254 (Merck) Adsorbent: Benzene-acetone ( 3: 1) Solvent: Spotting solution: Pure compound isolated from fermentation broth Detection: UV 0.3 RF: H. Sasaki, H. Oishi, T. Hayashi, I. Matsuura, K. Ando, Reference: and M. Sawada, J. Antibiot. (Tokyo), 35, 396 (1982).

Antibiotic : Adsorbent: Solvent: Spotting solution: Detection: RF:

Thiotetromycin Silica gel G F254 (Merck) Benzene-acetone ( 7: 3) Pure compound isolated from fermentation broth

UV

0.48

222 Reference:

Chan and Asza!os

S. Omura, Y. Iwai, A. Nakagawa, R. Iwata, Y. Takahashi, H. Shimizu, and H. Tanaka, J. Antibiotic. (Tokyo), 36, 109 (1983).

Trestatins A, B , and C Silica gel F254 (Merck) Chloroform-methanol-25% ammonium hydroxide-water (1:4:2:1) Spotting solution: Pure compounds isolated from fermentation broth Detection: Sulfuric acid Trestatin A, 0.19; trestatin B, 2.9; trestatin C, 0.14 RF: Reference: K. Yokose, K. Ogawa, T. Sano, K. Watanabe, H. Maruyama, and Y. Suhara, J. Antibiot. (Tokyo) , 36, 1157 (1983).

Antibiotic : Adsorbent: Solvent:

Antibiotic : Adsorbent: Solvent:

Trioxacarcins Silica gel plate Ethyl acetate saturated with 0.1 M phosphate buffer (pH 7.0) Spotting solution : Upper layer of acetone-ethyl acetate (1: 1) extraction of whole broth Detection: UV trioxacarcin A , 0. 70; trioxacarcin B , 0. 3; trioxacarcin C , RF: 0.40 Reference: F. Tomita, T. Tamaoki, M. Morimoto, and K. Fujimoto, J. Antibiot. (Tokyo), 34, 1519 (1981).

U-62162 Antibiotic: Silica gel (Analtech) Adsorbent: Solvent: Chloroform-methanol (9: 1) Spotting solution: Fermentation broth and of various fractions obtained during the isolation procedure Detection: Bioautography against Staphylococcus aureus or UV 0.5 RF: Reference: L. Slechta, J. Cialdella, S. Mizak, and H. Holksema, J. Antibiot. (Tokyo), 35, 556 (1982)

Antibiotic : Adsorbent: Solvent: Spotting solution: Detection: RF:

Virantmycin Precoated silica gel plate F254 (Merck) Benzene-acetone ( 2: 1) Column fractions during purification from fermentation broth UV or ferric chloride 0.05

TLC Techniques and Systems

223

Reference:

A. Nakagawa, Y. Iwai, H. Hashimoto, N. Miyazaki, R. Oiwa, Y, Takahashi, A. Hirano, N. Shibukawa, Y. Kojima, and S. Omura, J. Antibiot. (Tokyo), 34, 1408 ( 1981).

Antibiotic: Adsorbent: Solvent: Spotting solution: Detection: RF: Reference:

WF-3161 Silica gel 60 F254 Ethyl acetate Pure compound isolated from fermentation broth Iodine vapor 0.55 K. Umehara, K. Nakahara, S. Koyoto, M. Iwami, M. Okamoto, H. Tanaka, M. Koshaka, H. Aoki, and I. Imanaka, J. Antibiot. (Tokyo), 36, 478 ( 1983).

Antibiotic: Adsorbent: Solvent: Spotting solution: Detection: RF: Reference:

WS-1228 A and B Silica gel Benzene-ethyl acetate (3: 1) Pure compounds isolated from fermentation broth Iodine vapor 0.45 K. Yoshida, M. Okamoto, K. Umehara, M. Iwami, M. Kohsaka, H. Aoki, and H. Imonaka, J. Antibiot. (Tokyo), 35, 151 ( 1982).

QUANTITATIVE ANALYSIS OF ANTIBIOTICS

This section gives examples of the application of TLC for the quantitative analysis of antibiotics from fermentation sources, animal tissue, and pharmaceutical preparations. As can be shown with these examples, even though HPLC is the chromatographic technique of choice as far as quantitative analysis is concerned, quantitative TLC analysis has gained more acceptance recently, especially with the development of high-speed automated TLC scanners, such as the Shimadzu Model CS-910/CS-920.

Antibiotic: Adsorbent: Solvent:

Adriamycin from tissue Precoated silica gel 60 plates (Merck) First developed in chloroform-methanol-acetic acid 93: 5: 2 then developed in chloroform-methanol-acetic acid (76:20:4) Spotting solution: Low-temperature acid-alcohol extraction of tissue homogenate using daunorubicin as internal standards Detection: Fluorescence scanning set at 475 (excitation wavelength) and 580 nm (emission wavelength)

224

Quantitation:

RF: Reference:

Antibiotic : Adsorbent: Solvent: Spotting solution: Detection: Quantitation:

RF: Reference:

Chan and Aszalos

Aminco-Bowman spectrofluorimeter equipped with a thinfilm scanner and an electronic noise filter; measurements linear in the range of 0.05-50 µg when the concentration of adriamycin in tissues is plotted against the fluorescence peak-height ratio between adriamycin and daunorubicin Not reported K. K. Chan and C. D. Wong, J. Chromatogr., 172, 343 (1979).

Carbapenem PS-5 from fermentation broths Precoated silica gel plate (Merck) Acetonitrile-0. 75% acetic acid ( 9: 2) at 5-8°C Extraction of broth filtrate at pH 3 using a 1: 1 mixture of 1-butanol and chloroform; PS-5 standards at various concentrations in phosphate buffer (pH 8. 0) Dipped in Ehrlich reagent, heated at 100°C for 5 min Shimadzu CS-9190 high-speed TLC scanner in the reflectance mode with reading wavelength at 555 nm and reference wavelength at 700 nm, calibration curve linear to 10 µg/spot for PS-5 0.44 M. Okobe, K . Kiyoshima, I. Kojima, R. Okamoto, Y. Fukagawa, and T. lshikura, J. Chromatogr., 256, 447 (1983).

Cephamycin C from fermentation broths Precoated silica gel 60 F254 (Merck) Ethanol-glacial acetic acid-concentrated ammonium hydroxide ( 6: 3: 1); a 10 µl aliquot of glacial acetic acid spotted onto each origin and air dried for 15- 20 min before spotting standards or broth samples Spotting solution: Various concentrations of standard in water or centrifuged fermentation broth samples Detection: UV at 273 nm Quantitation: Shimadzu Model CS-920 high-speed scanner carried out at 273 nm in the reflectance mode; instrument scans the whole plate automatically, with linear range from 0. 2 to 20 µ g I spot of cephamycin Not reported RF: Reference: L. R. Treiber, J. Chromatogr., 213, 129 (1981).

Antibiotic : Adsorbent: Solvent:

Antibiotic: Adsorbent:

3, 6-bis- ( 3-Chloro- 3-piperidinyl)- 2, 5-piperazinedione from fermentation broth Precoated silica gel 60 (Merck)

TLC Techniques and Systems

225

One-dimensional system, n-butanol-acetic-water (13:2:5) 2. Two-dimensional system, chloroform-methanol (2: 1) followed by solvent 1 Spotting solution: Ethyl acetate extraction of the clarified broth at pH 2; broth adjusted to pH 8.6 and extracted three times with ethyl acetate; combined ethyl acetate extracts taken to dryness and residue dissolved in 0.1 ml ethyl acetate; broth spikes with various concentrations of standards worked up in a similar way Dried TLC plates exposed to chlorine vapors and sprayed Detection: with 0-tolidine Quantitation: Schoeffel SD 3000 spectrodensitimeter connected to a Hewlett-Packard 3352A laboratory data system; densitometric measurements made at 425 nm in the reflectance mode; calibration curve indicates linearity between 1 and 15 mg Rp: 0. 4 in the first dimension with chloroform-methanol ( 2: 1) H. J. Issaq, J. A. Chan, and E. W. Barr, J. ChromaReference: togr., 152, 280 (1978). Solvent:

1.

Gentamicin complex components in fermentation broth Precoated silica gel plates (Analtech) Lower layer of a mixture of methanol-chloroform-28% NH40H (1:1:1) Spotting solution: Fermentation broth samples acidified to pH 2 and centrifuges; pH then adjusted to 12 and appropriate dilutions made for spotting Detection: Plates dipped in methanolic 4-chloro-7-nitrobenzo-2-oxa1,3-diazole (NBD chloride), heated for 10 min at 120°C and rechromatographed on methanol in the same direction as the first time A Schoeffel SD 3000 double-beam densitometer used in Quantitation: the reflectance mode; plates scanned using excitation and emission wavelengths of 420 and 520 nm, linear up to 500 mg per spot of sisomicin Rp: C1 > C2 > C1a Reference: P. Rabaskalian, S. Kalliney, and Z. Magatto, Anal. Chem., 49, 953 ( 1977). Antibiotic : Adsorbent: Solvent:

Gentamicins from pharmaceutical sample DC-Alufolien Kieselgel 60 (Merck) Lower layer of chloroform-methanol-concentrated ammonia (1: 1: 1, v/v) Spotting solution: A 10 µl of a 10 mg/ml gentamicin solution and 1-60 mg/ml solution of the gentamicin C1, C1a• and C2 compounds

Antibiotic : Adsorbent: Solvent:

226

Chan and Aszalos

Detection:

Quantitation: Rp: Reference:

Ninhydrin solution made from 1.0 g ninhydrin, 0.25 cadmium acetate in a mixture of 10. 0 ml acetic acid and 50. 0 ml ethanol; plates sprayed and heated at 120°C for 2 hr for optimized quantitative purposes Densitograms recorded on an ERI-165 (Carl Zeiss Jena, Jena, GDR) TLC scanner in the reflectance mode using a 510 nm filter C1 > C2 > C1a I. Torok and T. L. Paal, J. Chromatogr., 246, 356 ( 1982).

Tetracycline standards Precoated cellulose TLC plates without fluorescent indicator 0. 25 M magnesium chloride, 0. 20 M calcium chloride, Solvent: 0. 25 M barium chloride, and 0 .15 M zinc chloride in water Spotting solution: Various amounts of each antibiotic (0.1-2 µg) in methanolic solution ( 0.1-0. 5 mg/ml) Fluorescence detection by exposure to a UV lamp Detection: Quantitation: Vivatron TLD 100/Hg flying-spot densiometer using mercury lamp as light source: UVB filter (240-340 nm) used for fluorescent excitation; calibration curves indicate linearity between 0.25 and 1.25 µg of different tetracyclines Rp x 100 values of tetracyclines on cellulose precoated Rp: plates (Merck, Cat. No. 5716) after development with salt solutions

Antibiotic: Adsorbent:

Developing solution

Tetracycline Tetracycline 4-Epitetracycline Anhydrotetracycline 4-Epianhydrotetracycline Oxytetracycline Chlorotetracycline Demethylchlorotetracycline Methyacycline Doxycycline Minocycline Source:

Magnesium chloride, O. 25 M

Calcium chloride, 0.20 M

Barium chloride, 0.25 M

Zinc chloride, 0.15 M

72 68 29 25 72 62 64 54 53 76

67 63 25 16 68 59 57 47 50 70

63 60 24 17 63 58 54 44 49 60

65 65 22 18 65 52 55 50 48 68

By permission of Elsevier Scientific Publishing Co., Amsterdam.

Reference:

E. Ragazzi and G. Veronese, J, Chromatogr., 132, 105 ( 1977).

227

TLC Techniques and Systems

Antibiotic: Adsorbent:

Tetracyclines from pharmaceutical preparations Precoated silica gel plate (E. Merck 5641), predeveloped with saturated aqueous NA2EDTA solution, air dried at room temperature for 1 hr and activated at 130°C for 2 hr 1. Chloroform-methanol-5% NaiBDTA aqueous solution Solvent: (65: 20: 5), lower layer 2. Isopropanol-ethyl acetate-5% Na2EDTA aqueous solution ( 3: 4: 7) , upper layer 3. Acetone-5% Na2EDTA aqueous solution ( 10: 1) Spotting solution: 1 µl of 10 mg/ml methanol solution A. sample = 360 nm, A. reference = 600 nm for tetraDetection: cycline (TC) oxytetracycline (OTC), chlorotetracycline (CTC), doxycycline (DC), 4-epitetracycline (ETC); A. sample = 450 nm, A. reference = 650 nm for anhydrotetracycline (ATC), 4-epianhydrotetracycline (EATC) Quantitation: Shimadzu CS 910 chromatography scanner in dualwavelength mode; linear scanning in reflectance mode; linear relationship between 0.1 and 1. 0 µg for different tetracyclines Rp:

Antibiotic ATC EATC CTC TC DC OTC ETC

Reference:

1 0.87 0.46 0.44 0.38 0.26 0.12 0.10

2

3

0.83 0.35 0.28 0.28 0.22 0.18 0.11

0.94 0.60 0.56 0.45 0.45 0.38 0.22

H. Oka, K. Uno, K. Harada, Y. Kaneyama, and M. Suzuki, J. Chromatogr., 260, 457 (1983).

TLC CLASSIFICATION IN THE DISCOVERY OF NOVEL ANTIBIOTICS

In a continuing search for new antibiotics with desired biological activity, considerable time can be wasted if no "dereplication" system is set up to rule out known antibiotics. The use of TLC classification in conjugation with bioautography for presumptive identification of antibiotics is well documented [ 46] • No single TLC system can be designed for all known antibiotics originating from fermentation sources. One can approach the problem more effectively by narrowing down the known agents to be dereplicated based on the objective of the screen, for example, whether one is screening for cell wall active, broad-spectrum antibacterial, antitumor, antifungal, and/ or antiviral agents. An example of TLC classification based on specific objective is that published by Issaq et al. [46] for antibiotics exhibiting antitumor properties. TLC classification of antitumor antibiotic standards is achieved according to Figure 1.

1-2

Ac0H:2 Pyridine: 1

(1c)

(1b)

PrOH :8 H20:2

(2b)

AcOH 10 MeOH 20 CHCl 3 70

Acetone (2a)

111-2

11-1

MOVED IN I D

(1d)

Ac0H:1 Pyridine:1

PrOH: 10 H20:8

1-3 Cellulose 11-4

AcOH:1 Pyndine:1

PrOH :8 H20:2

Silica

NONE

II

111-1

(8)

11-3

(3b)

Acetone:95

Me0H:20 CHCl3:76 (2c)

Ill

C,D

(C)

I 111-3

·I

MeOH

(3c)

I111-4

B, C, D

Silica gel plates were used,

(4c) (4b)

IV-3

(4a)

IV-2

I

v

MOVED IN A, B, C, D.

Me0H:8 Benzene:92

IV-1

MOVED IN

I

IV

Me0H:4 Benzene:96

(3d)

Acetone:67

CHCl 3 :33

(0)

MeOH:2 Benzene:98

NH 4 0H :5 Acetonitrile

111-2

MOVED IN

MeOH: 10 CHC1 3: 90

I

NH 4 0H:4

(3a)

AcOH: 10 Et0Ac:90

EtOAc

Figure 1 Solvent system used in classification of 150 antitumor antibiotics by TLC methods. except for 1-3 and 1-4. (From Reference 46.)

(1a)

PrOH :3 AcOH :1

I

1-1

MOVED IN

(A)

f--,

CHCl 3

I

ANTICANCER DRUGS

~

~

rn

0

Q

"'

rn

~

~

Q

;:I

;:I

Q

;:r

(')

Clo

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Actinogan Bleomycin A 1 Bleomycin A 2 Carzinostatin Flammulin Gougerotin Macromomycin Neocarzinostatin Peptinogan Roseolic acid a-Sarcin Trienine 116328

Alanosin , monosodium salt Bluensomycin sulfate Kasugamycin Phleomycin Sancyclin Septacidin Sistomycosin 26697

I-2

I-3

0 0 0 0 0

Asparaginase Mitogillin Mitosper Restrictocin 75603

I-1

A, B, C, D

Name or NSC #

Silica gel

0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0

la

Cellulose

0 0

0

87

+

+ 0 +

0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0

lb

0 50,30 0 0 0 85 80 Os

0 0 0 0 0 42 0 0 0 0 0 0 0

0 0 0 0 0

le

Subgroups of Antitumor Antibiotics That Move with None of the Main Solvents (Main Group I)a

Subgroup

Table 2

ld

40 38 60 25s,82 16 80 77 s

0-20s 0-35s 85 0, 82 0-30s 0 75s,801 80 10 86 0-35s 73 68,90

0 0 0 0 0

Silica gel

'"3

t-:> t-:>

co

Cll

3

Ct>

Cll

.....

'


.o·

;:s

;l'

(')

Ct>

'"3

C".l

I:"'

+

60s 94 21-50s

10

45-50s 83

0 0 0 0 0 0

+

+ 10 + +

0

0

72942

PA 147 Candicidin Copiamycin, acetyl nucleoside fraction of septacidin Oosporin Stendomycin Salicylate

75 + 78 + + + + 0-lOa 82 +

0 0 0 0 Os 0 0 0

0 0 0 0 0 0 0 0

aStreaking from RF to RF = s. RF values expressed as RF x 100. Source: By permission of Elsevier Scientific Publishing Co., Amsterdam.

1-5

+ +

lb

0 0

la

Cellulose

0 0

1-4

A, B, C, D

Silica gel

Actinorubin 3-Amino-2, 36-LHexopyronase HCl Adriamycin Cinnamycin Daunomycin Duramycin Hadacidin lyomycin complex Spectinomycin Zorbamycin

Name or NSC #

(Continued)

Subgroup

Table 2

86 0-75s 88 63,87 45-50s 70

+ 80 + 0 0

+

-50s

87 85 90 80,85,45 64 0-50s 60 99 73 + 77 40 0-30s 23 25,5 0-64s 18

0-4ls,80 82

ld

10s 60

le

Silica gel

t--:>

(/.)

0

Q

N

(/.)

>-

;::! Q.

Q

;::!

Q

;:l"'

(")

w c

231

TLC Techniques and Systems

Table 3 Subgroups of Antitumor Antibiotics That Move with Methanol Only (Main Group II) Silica gel solvent system Subgroup

Name or NSC #

11-1

Azacolution complex Azalomycin F-complex Azotomycin Cytovirin Duazomycin In dole- 3-carboxaldehyde Statolon

0,40 14, 50 86 0,14 s 75 0-33s

0 0 0 0 0 0 0

0 0 0 0 O,s 0 0

0 0 0 0 0,s 0 0

II-2

5-A zacytidine Azaserine DON Formycin B 3H-Indole Pyrazomycin Rubiflavin Sarkomycin, sodium salt

59 s s 76 13 78 10s 68

0 0 0 0 0 0 0 0

29 10 10 31 0 25 0 9,73

0 0 0 0 92 0 58, 98 0

II-3

cAMP Formycin A Palmitoyl citidine Sangivamycin Thiosangivamycin

74 67 94 71 75

28 0 0 0 0

0 29 77 26 42

39 12 33 21 18

II-4

Acrylamide Actinobolin Adenosine L- Lyxo- hexopyranoside Mitocromin

86 36 35 18 79

65,86 0-25s 30 0,22 10

52

Rufochromomycin Steptonigrin

82 86

0-9s,10 21

10 10,18 61 58 100, 71 41,40 0,6,15 21

2a

D

2b

11, 13

21 50 100,95 82,72 68s 15s,63

2c

Source: By permission of Elsevier Scientific Publishing Co. , Amsterdam.

On this basis, the 150 antitumor antibiotics standards were grouped into 5 main and 19 subgroups based on the mobility of the compounds in a solvent system using a certain adsorbent (see Tables 2 to 6). The Rp values are used in the classification system only to indicate movement and are obtained by visualization under UV light or by the appearance of inhibition zones in the bioautographic plate. The method was designed for the fast evaluation of crude antibiotic preparations obtained from fermentation sources, unless the active compound(s) present in the fermentation broth is produced in such a high-titer, crude filtrate cannot be directly spotted onto the TLC plate. Usually, a simple solvent extraction [ 14] at different pH levels (such as n-butanol or ethyl acetate) or small column (Water's Sep-Pak) is

Iyomycin Bl Mithramycin Mithramycin-Mg Sparsomycin

Aureolic acid Amicetin Anisomycin Lagosin

Azastreptonigrin Nebularin Puromycin

III-2

III-3

Name or NSC #

III-1

Subgroup

34 18 15

16 60 15 17

0-10 16 18 16

c

91 71 40

92 80 40 90

0-10 92 92 67

D

23 0 0

0 0 0 0

0 0 0 0

3a

0 29 0-12

0 14-45 13 13

0 0 0 0

3b

0 24 0

96 0 0 0

0 0 0 0

3c

Silica gel solvent system 3d

35,0 0 81

0 0 0 0

0 0 0 0

Table 4 Subgroups of Antitumor Antibiotics That Moved with Methanol and 10% Methanol in Chloroform Solvents (Main Group III)

tll

Q

s""

tll

>

Q.

;:s

Q

;:r Q ;:s

()

~

~ ~

60,50 13 50 47 18 50 44 62,10 0-45 62,25,0

90,0 40 13 0 20,0

11 72,41

88 70 90 95 78 95 70 92 87,77 80

70 70 60 24 80

88 55

By permission of Elsevier Scientific Publishing Co., Amsterdam.

Antibiotic MS-18903 Antibiotic 1037 Olivomycin Chromomycin A 2 Mitomycin C Olivomycin A Pactamycin Rifamycin S V Steptolydigin Steptozotocin HCl

III-5

Source:

Ascomycin Chartreusin- 2-hydrate Cordycepin Fusidic acid Streptozotocin

111-4

Steptonigrin methyl ester Vinblastine, sulfate, hydrate

45 18 26-50 12 90,57 0-20 19

-

32

-

96,0 50,26 17 85 45,0

0-35 0

96 45 98 94 74 93 93 93 55 21

96,0 0 45 0 34,23

0 92

98 75 98 98 75 98 93 98 0-14 0-16

34,0 0-25 21 96 0

0-15 0

25 16 41 48 23 40 78 56 28 11

18-37 0 36 30,20

0 21

~

t.:> t.:>

Cll

(1)

3

Cll ....

'


(1) Cf.)

~

.o·

;:s

;:i"

(1) (")

...,

("')

236

Chan and Aszalos

Table 6 Subgroups of Antitumor Antibiotics That Move with All Main Solvents (Main Group V) Silica gel solvent system A

B

c

D

L-Alanosine Coumermycin Cyclamycin complex

45 0-10 0-14

80 0-23 30,30

75 100 60,80

Kanchanomycin 11254 58987 102810 Bostrycoidin 108408 135015

0-20 96 28 10,20 10 60 0-60

40 10-18 0-12 20,60 0 96 41 91 0-60 88 40

0,60,80 95 77 93 80 100 75

0-24 95 93 91 0-36 85 75

Name or NSC #

Source:

By permission of Elsevier Scientific Publishing Co., Amsterdam.

needed for sample cleanup. Rates of migration (RF values) obtained in TLC systems on crude mixtures tend to be influenced by other constituents of the sample and may not give the same value as those of the pure standards. Therefore, cospotting is frequently found to be useful. However, the main and subclasses for all antibiotics remained identical. A novel antitumor antibiotic, fredricamycin [ 47], was isolated using the above system. During the course of a search for novel bioactive compounds, it is usually very useful if the method of detection use in the prescreen can be easily adapted to bioautography. Such is the case of a biochemical induction assay (BIA) prescreen method utilized by Elespuru and White [ 27] in the search for novel antitumor agents from fermentation sources. The biochemical induction assay is an assay developed for compounds that interact with DNA, which are detected as inducers of bacteriophage ). in a strain of E. coli constructed to maximize sensitivity to inducing agents. Induction is measured as the production of an enzyme, S-galactosidse, in a colorimetric assay. A set of 142 standard antibiotics, composed principally of agents similar to those already described, was tested in the assay. About 25% of the compounds tested were found to be strong inducers. Elespuru and White were also able to adapt the BIA assay for TLC bioautography and found it to be a rapid method in identifying known agents and selecting novel ones. The use of TLC classification and bioautographic systems in conjuction with biological recognition profile patterns (microbiological, selected enzymes, induction test, and resistant organisms) is found to be useful in the early identification of known agents and is used by many laboratories dealing with antibiotic discovery.

TLC Techniques and Systems

237

REFERENCES 1.

2. 3. 4, 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.

J. W. Copius-Peereboom, in Comprehensive Analytical Chemtstt'y, Vol. Uc (C. L. Wilson and D. W. Wilson, eds.), Elsevier, Amsterdam, 1971, p. 77. K. Macek, I M. Hais, J. Kopecky, J. Gaspanic , V. Rabek 1 and J. Churacek (eds.), J. Chromatogr. , Suppl. 2 ( 1972) , Elsevier, Amsterdam, 1973. V. Betina, in Pharmaceutical Application of Thin Layer and Paper Chromatography (K. Macek, ed.), Elsevier, Amsterdam, 1972, p. 503. J, C. Touchstone and M. F. Dobbins, Practice of Thin Layer Chromatography, John Wiley, New York, 1978. B. Fried and J. Cherius, Thin-Layer Chromatography, Vol. 17, Marcel Dekker, New York, 1982. Y. Ito, M. Nawba, N. Negahama, T. Yamaguchi, and T. Okuda, J. Antibiot. (Tokyo) , Ser. A, 17, 218 (1964). V. Betina, in Pharmaceutical Application of Thin-Layer and Paper Chromatography (K. Macek, ed.), Elsevier, Amsterdam, 1972, p. 502. G. H. Wagman and M. J. Weinstein, Chromatography of Antibiotics, Elsevier, Amsterdam, 1973. A. Niederwieser and M. Brenner, Experientia, 21, 105 (1965). K. Randerath, Thin Layer Chromatography, Academic Press, New York, 1964. J. W. Copius-Peereboom, in Comprehensive Analytical Chemistry, Vol. lie ( C. L. Wilson and D. W. Wilson, eds.) , Elsevier, Amsterdam, 1971, p. 5. H. Vink, J. Chromatogr., 18, 25 (1965). E. Stahl, Thin Layer Chromatography, A Laboratory Handbook, 2nd ed., Springer Verlag, New York, 1969. H. Haghizabeh-Nouniza and H. Lamotte, Bull. Soc. Chim. Fr., 1515 (1971). R. Takeshitsa, Chem. Pharm. Bull., 19, 80 (1971). A. V. De Thomas, C. R. De Thomas, R. Lazar, and D. Verrastro, Microchem. J., 16, 52 (1971). G. Zweig and J. Sherma, Anal. Chem., 44, 52R (1972). J. C. Touchstone and N. F. Dobbins, Practice of Thin Layer Chromatography, John Wiley, New York, 1978, p. 327. A. Aszalos, S. Davis, and D. Frost, J. Chromatogr., 37, 487 (1968). S. Ochab and B. Borowiecka, Dis. Pharm. Pharmacol., 21, 359 (1969). E. Stahl and E. Dumont, J, Chromatogr. Sci., 7, 517 (1969). T. Wieland, G. Luben, and H. Determann, Experientia, 18, 4311 ( 1962). E. Randerath and K. Randerath, J. Chromatogr., 16, 126 (1964). Chem. Eng. News, 51, 14, August, 13, 1973. J. Shoji, J. Chromatogr., 26, 306, 1967. A. E. Bird and A. C. Marshall, J. Chromatogr., 63, 313 (1971). R. Elespuru and R. White, Cancer Res., 43, 2819 (1983). J. K. Pauncz, J, Antibiot. (Tokyo), Ser. A, 25, 677 (1972).

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T. Devenyi, J. Bati, J. Kovacs, and P. Kiss, Acta Biochim. Biophys. Acad. Sci. Hung., 7, 237 (1972). H. Struck, H. Karg, and H. Jork, J. Chromatogr., 36, 74 (1968). H. Ganskirt, in Thin-Layer Chromatography, A Laboratory Handbook, 2nd ed. (E. Stahl, ed.), Springer-Verlag, New York, 1969, p. 142. J. F. Martin and L. E. McDaniel, Polyene macrolide antibiotics, in Densitometry in Thin Layer Chromatography (J. C. Touchstone and J. Sherma, eds.), John Wiley, New York, 1979. J. C. Touchstone and J. Sherma (eds.) , Densitometry in Thin Layer Chromatography, John Wiley, New York, 1979. M. Arai, K. Hamano, K. Nose, and K. Nakano, Sankyo Kenkyusho Nempo, 20, 93 (1968). C. Radecka and W. L. Wilson, J. Chromatogr., 57, 297 (1971). J.E. Sinsheimer, D. D. Hong, and J. H. Burckhalter, J. Pharm. Sci., 58, 1041 (1969). L. Lodi, G. Meinardi, and E. Rossi, Fosmaco Ed., Prat., 24, 759 ( 1969). N. N • Lombardi, J. Dobrecky, and D. D. Rosa De Carnevale Bonino, Rev. Ass. Bioquim. Argents., 33, 174 (1968). M. V. Kalinina and E. I. Surikova, Antibiotiki, 13, 112 (1968). E. Stahl and H. K. Mangold, Techniques of thin layer chromatography in Chromatography (E. Heftmann, ed.), Van Nostrand Reinhold, New York, p. 164. M. Lederer, K. Macek, and I. M. Hais (eds.), Reproducibility in Paper and Thin Layer Chromatography, Elsevier, New York, 1968. W. Golkiewicz, Chromatographia, 14, 629 (1981). R. K. Gilpin and W. R. Sisco, J. Chromatogr., 124, 257 (1976). E. Von Arx and M. Faupel, J. Chromatogr., 158, 68 (1978). A. Aszalos and A. Aquilar, J. Chromatogr., 290, 83 (1984). A. Aszalos and H. Issaq, J. Liquid Chromatogr., 3, 867 (1980). R. Misra, R. Pandey, and J. Silverton, J. Amer. Chem. Soc., 104, 4478 ( 1982).

7 High-Performance Liquid Chromatography JOEL J. KIRSCHBAUM Squibb Institute for Medical Research, New Brunswick, New Jersey ADORJAN ASZ ALOS Food and Drug Administration, Washington, D. C.

Aminoglycoside Antibiotics Anthelmintics and Other Antiparasitic Agents Antileprosy and Antituberculosis Agents Antitumor Antibiotics Antiviral Agents S- Lactam Antibiotics Polyene Antibiotics Polypeptide Antibiotics Sulfonamides and Related Drugs Tetracyclines Topical Anti-Infective Agents and Disinfectants Unclassified and Miscellaneous Antibiotics Veterinary Products References

240 245 246 249 254 256 279 281 284 290 295 295

303 306

High-performance liquid chromatography (HPLC) is increasingly becoming the premier choice for analyzing antibiotics and anti-infective agents, according to regulatory agencies and compendia. The advantages of HPLC include simplicity, accuracy, precision, versatility, and, most importantly, selectivity, especially to distinguish between constituents of a multicomponent system. Columns of different polarity can be used to analyze a compound, thus enabling many interfering substances, such as those found in biological matrices, to be reduced or eliminated. HPLC is versatile enough to permit several methods of analysis to be developed starting with almost any type of separating column. The emphasis is on antibiotics and antimicrobial agents comprising the aminoglycosides, penicillins, cephalosporins, and sulfonamide (sulfa) drugs. Compounds like the anti-infective agent phenol and the veterinary antibiotics and anthelmintics are generally limited to one method. When a

239

240

Kirschbaum and Aszalos

pf procedures are available for analyzing important drugs, similar are restricted to one, preferably readily available, procedure. Wheq~vE!r possible, more than one polarity system is given. Assays in body flµ~ds and tissues are emphasized since such methods can generally also qe µ!itad for bulk material. However, except for a few general examples, (ietails of the extractions of drugs from the biological matrices are omitt~d in the interest of saving space. We hope this chapter will also be useq to §peed the development of HPLC assays of new compounds by utilizil\~ succ~r:isful methods for structurally related drugs. Unless ~tated otherwise, the analytical columns are 25-30 cm long and cQntain 10 µ m particles, and the inorganic constituenti; are dissolved in Wfl.ter. Our computer-aided searches of the literature ffiay have missed some [email protected],a,, The authors would be grateful to readers who correct lapses. In several instances, inclusion or exclusion of a compound is based on our judgment; for example, interferon was included because of great interest, although its usefulness as an antiviral agent is controversial. -. The comprehensive review is divided into sections, as follows: aminoglycoside; anthelmintics and other antiparasitic agents; antileprosy and antituberculosis; antitumor; antiviral; S-lactam; polyene; polypeptide; sulfonamide; tetracycline; topical anti-infective; unclassified and miscellaneous; and veterinary (animal health). Some drugs can fit into more than one category, and a drug may have been used as the internal standard to help compensate for inefficient extraction or protein binding in a biological matrix. Comp~risons of antibiotic contents with those obtained by other methods are freq~ently performed. In general, results usually agree within the experimental error expected from the two methods. However, where they do not, assuming good extraction techniques, the cause of discrepant results for HPLC ve~sus such a biological procedure as microbiological assay, often is caused by the greater selectivity of HPLC to separate multiple components, two or m,ore of which may have biological activity. Thus, corrections may be requirec(to compare HPLC results with a less specific method that yields total activity. References 1 and 2 are recent reviews of the HPLC of antibiotics. vari~~y

met~e~~

AMINOGLYCOSIDE ANTIBIOTICS

The first aminoglycoside antibiotic to be used in chemotherapy, streptomycin, was discovered by Waksman and his associates as the result of a planned search begun in 1939. Since then, various other antibiotics related in structure to streptomycin have been discovered. They tend to be poorly adsorbed after oral administration. Their broad-spectrum activity has led to parenteral administration for systemic infections. Serum concentrations must be monitored carefully, since 4-8 µg/ml is the therapeutic dose and 10 µg/ml is the approximate upper limit. Prolonged exposure- to higher concentrations can cause loss of hearing and kidney function, depending in part on the individual and the aminoglycoside. Predictions based on lean body weight and other considerations are often invalid, thus requiring accurate serum assays. HPLC p:povides a sensitive, rapid, and specific method. Derivatization is usually nE!C~l':l§ary to provide a chromophore (see Reference 3 for a review of sample pl'ep~r~tion and properties of fluorescent derivatives). Figure 1 shows the separation of kanamycin, streptomycin, gentamycin, and neomycin using a 5-µin pctadecylsilane (ODS) column (150 x 4.6 mm) and a mobile phase of

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methanol- O. 05 M aqueous camphorsulfonic acid ( 60: 40) flowing at 1 ml/ min through a refractive index detector [ 4) • Amikacin

Amikacin, a semisynthetic derivative of kanamycin, was separated from constituents in serum using a silica column as follows. A disposable Pasteur pipette was plugged with silane-treated glass wool, and silica gel powder was added to a height of 4 cm. The column was washed with 2 ml of 0. 066 M potassium phosphate buffer, pH 9. Phosphate buffer, 1 ml, was added to a 1. 0-ml serum sample containing amikacin and applied to the silica gel column. The serum sample tube was rinsed twice, each time with 1 ml water. These washings were added to the column, eluted with the aid of pressure from a

242

Kirschbaum and Aszalos

rubber bulb, and then discarded. 0-Phthalaldehyde reagent, 1 ml, prepared as follows, was added to the silica gel column. o-Phthalaldehyde (200 mg) was dissolved in 40 ml of 95% ethanol adjusted to pH 10 with 50 µl potassium hydroxide solution ( 450 g /liter), and 0. 4 ml of 2-mercaptoethanol was added. Fresh reagent was prepared weekly. Immediately after adding derivatizing reagent, 2. 0 ml ethanol (95%, pH 10) was added and the elute mixed and immediately heated to 50°C for 5 min. After stirring, it was placed in an ice bath and passed through a Millipore filter ( 0. 6 µ m), stored on ice in the dark, and then chromatographed on an octadecylsilane column using a mobile phase of methanol-water-acetonitrile (65: 30: 5) containing 2 g tripotassium ethylenediamine tetraacetate (EDTA) per liter. The flow rate was 2 ml/min through a filter fluorometer with excitation -350 nm and emission -420 nm [ 5) , to detect as little as 1 µg/ml. A similar system but with a mobile phase of methanolwater ( 70: 30) was used to resolve amikacin from its three isomers in serum [ 6) with excellent linearity. Another serum assay for amikacin utilized an octadecylsilane column with a mobile phase of 0.1 M sodium sulfate, 0.2 M sodium pentanesulfonate, and 0.0174 M acetic acid flowing at 2 ml/min. After derivatization with o-phthalaldehyde, fluorescent detection was at 240 nm excitation/420 nm emission. Tobramycin could also be quantified [ 7), using this precise and accurate method. Pre-column derivatization with l-fluoro-2,4-dinitrobenzene added to plasma or urine gave a stable chromophore absorbing at 360 nm. Separation was on an octadecylsilane column and guard column ( 30 x 4. 6 nm) with a mobile phase of acetonitrile-water (68:32) flowing at 1 ml/min [8]. Responses are linear from 2 to 64 µ g/ml. A similar system, but using an acetonitrile-water ratio of 47: 53 flowing at 2. 5 ml/min coupled with a different extraction method, gave a limit of detection of 1 µ g /ml with samples of 200 µl [ 9) , and kanamycin as internal standard. Here, the aminoglycoside is extracted from serum using a cation exchange column and derivatized using l-fluoro-2, 4-dinitrobenzene (FDNB) as follows. Soak CM-Sephadex C-25 in O. 2 M Na2S04 solution for at least 24 hr at room temperature. Into a Pasteur pipette, with most of the stem removed, place a glass wool plug and then add sufficient slurry to obtain a column height of 1.5 cm. Pipet 200 µl serum into a centrifuge tube, add 20 µl of a 250 mg/liter kanamycin sulfate internal standard, and mix the contents. Transfer the contents to the column, elute with 2 ml of a solution containing 0. 001 M HCl and 0.2 M Na2S04, discard, re-elute with 250 µl of 0.05 M NaOH, and again discard the eluate. Elute with 1 ml of O. 05 M NaOH, collecting the contents in a 5-ml ampule. Add 2. 5 ml of a solution of 3% FDNB in methanol. Mix to dissolve the precipitate, heat-seal the ampule, and place it in boiling water for 5 min. After cooling, break the seal and inject the sample into the liquid chromatograph. Gentamycin Gentamycin (gentamicin) components in 50 µl serum were determined after derivatization with l-fluoro-2, 4-dinitrobenzene using an octadecylsilane column and a mobile phase of 0.1% tris (hydroxymethyl)aminomethane (adjusted to pH 7 with HCl) with acetonitrile (30:70) flowing at 1.5 ml/min through a detector set to 365 nm [ 10). This method was improved and subsequently applied to sisomicin [ 11). After derivatization with o-phthalaldehyde, an octadecylsilane column was used with a mobile phase of either 0. 1 M disodium 1, 2-ethanedisulfonate and O. 005 M sodium octanesulfonate in water-acetonitrile

HPLC

243

(85: 15), adjusted to pH 3. 5 and flowing at 1. 5 ml/min [ 12], or methanolwater-11.4% EDTA (adjusted to pH 7.2 with 5 M KOH) (80:15:5) flowing at 1 ml/min [13]. Fluorescent detection at 340-365 nmex/440-455 nmem was used. Results were similar to those found using microbiological assays. A 5- µ m octylsilane analytical column and precolumn were used to assay the various constituents of gentamycin in serum and urine after derivatization with o-phthalicdicarboxaldehyde, with a mobile phase of 0.1% tris (hydroxymethyl) aminomethane (adjusted to pl:i 4. 3 with 1 M HCl) with acetonitrile ( 30: 70) flowing at 1.5 ml/min. Fluorescence detection was used at 340 nmex/ 418 nmem [ 14] . Although similar results were found using a radioimmunoassay, the HPLC method is more specific. Fluorescamine was used to derivatize the components of gentamycin in body fluids prior to chromatography on a strong cation-exchange column. The mobile phase was acetonitrile-0.5% aqueous phosphoric acid (7:3) flowing at 2 ml/min through a fluorescent detector at 275 nmex I 418 nmem cutoff filter [ 15]. The limit of detection was 1 µg/ml. Using the same column for these assays was recommended by Chiou and colleagues [ 16] . Difficulties in derivatizing gentamycin led to the development of a system using electrochemical detection, which can resolve and quantify the C1a• C2, and C 1 fractions [ 16a] , as well as several unknown minor impurities. An octadecylsilane column was used with a mobile phase of 0. 015 M sodium pentanesulfonate, 0.2 M sodium sulfate, and 0.1% acetic acid flowing at 1.0-1.25 ml/ min into an electrochemical detector set to 1.3 ± 0.1 V versus a Ag/AgCl reference electrode and a glassy carbon electrode. Results are reproducible and linear from 16 to 30 µ g injected, with a correlation coefficient equal to or greater than O. 999 for the three constituents. Kanamycin Kanamycins A and B were separated using a strong cation-exchange column and a mobile phase of 0.1 M EDTA, adjusted to pH 9.0-9.5 with KOH solution, and flowing at 1 ml/min. Detection involved post column in-line reaction with either fluorescamine (375 nmex/480 nmem or o-phthalaldehyde (320 nmex/450 nm em>. Although linearity and recovery studies showed excellent results from 50 to 400 µg/ml, the lower limit of this proc:idure appeared to be 15 µg/ml [ 17]. Neomycin Neomycin A and B can be separated and quantified in formulations after derivatization with 1-fluoro- 2, 4-dinitrobenzene. A 5-µ m silica column was used with a mobile phase of chloroform-tetrahydrofuran-water (600:392:8) flowing at 1 ml/min through a detector set to 350 nm [ 18]. The same mobile phase was used with a silica microbore column (10-µm particles, 500 x 1 mm) to separate neomycin B and C. The flow rate was 200 µI/min [ 19] • Microbore HPLC gave 16-fold greater sensitivity than conventional liquid chromatography. Netilmicin Body fluids are analyzed for netilmicin after forming a fluorescent derivative. After reaction with o-phthalaldehyde, a 5-µm octadecylsilane column was used with a mobile phase of methanol-water-11.4% EDTA, pH 7.2 (80:15:5)

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flowing at 1 ml/min through a fluorescence detector at 340 nm/ 455 nm [ 13]. The lower limit of sensitivity is approximately O. 5 ',.lg/ml using from O. 2 to 0. 5-ml samples. The dansyl derivative was chromatographed using an octadecylsilane column with a mobile phase of acetonitrile-water (95:5) flowing at 1 ml/min [20]. Fluorescence detection was at 220 nmex/470 nm ex (interference filter). Using 0.1 ml of plasma, 1 µg/ml could easily be detected. Sisomicin Sisomicin (sisomycin) in 50 µl of serum was determined using an octadecylsilane column and a mobile phase of water-acetonitrile-acetic acid ( 30: 70: 0. 1) flowing at 1 ml/min through a detector set to 365 nm [ 11]. Recoveries were approximately 85% at 4 µg/ml; 0.5-16 µg/ml gave responses that fell on a straight line. Spectinomycin Spectinomycin, which is stable in a narrow pH range, and related compounds were analyzed using an octylsilane column and precolumn ( 43 x 4. 2 mm) and a mobile phase of 0. 2 M sodium heptanesulfonate, 0. 2 M sodium sulfate, and 0.1% aqueous actic acid flowing at 2 ml/min. Detection involved postcolumn derivatization with o-phthalaldehyde and fluorescent detection at 350 nm/450 nm [ 21] • Results were in good agreement with data obtained using microbiological assays. Spectinomycin and its precursor actinamine were resolved using an amino column ( 100 x 4 mm) and a mobile phase of acetonitrile-water ( 1: 9) flowing at 1 ml/min. Detection was with liquid chromatography-mass spectrometry (LC/ MS) [ 22] as depicted below.

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180

230

280

330

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280

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245

Streptomycin and Dihydrostreptomycin Streptomycin A and B , dihydrostreptomycin A and B , and related compounds were determined using octadecylsilane analytical and guard (250 x 4.6 mm) columns and using a mobile phase of 0. 02 M sodium hexanesulfonate and 0. 25 M aqueous sodium phosphate-acetonitrile ( 90: 10) , adjusted to pH 6. 0 with phosphoric acid. The flow rate was 1 ml/min through an ultraviolet (UV) detector set to 195 nm [ 23]. Microbiological and HPLC results were in good agreement. Tobramycin The lack of a chromophore requires derivatization to quantify tobramycin in body fluid. The following methods used precolumn reaction with o-phthalaldehyde. The limit of detection was generally about 1 µg/ml. Chromatography on an octadecylsilane column used a mobile phase of either 250 ml 0. 5 M tris buffer, plus 10 ml (C 2H5)3N, pH 7.9, with concentrated sulfuric acid and diluted to 11 with methanol [24], or methanol-water (72:26) containing 0.005 M EDTA [ 25] flowing at 2 ml/min. Fluorescent detection was excitation at 360 nm and emission at 430 nm or 418 nm filter cutoff. A cyano column was used with a mobile phase of methanol-water-acetonitrile (62:35.1:2.9) containing 2. 5 g tripotassium EDTA per liter, flowing at 1. 6 ml/min [ 26). Postcolumn derivatization involved chromatography on an octylsilane column (15 cm) and a mobile phase of water-methanol-acetic acid (99. 7: O. 2: 0.1 mole percent) flowing at 1 ml/min [ 27]. Radioimmunoassay was less accurate than LC or microbiological assays (the easiest to perform). HPLC was recommended for research purposes, especially pharmacokinetic studies. An improved method using 20 µl serum was sensitive to 0.2 µg/ml. This procedure [28] utilized an octadecylsilane column and a mobile phase of 0.1 M disodium 1, 2-ethanedisulfonate and 0. 005 M sodium octanesulfonate in a water-methanol mixture (67:33) adjusted to pH 3.5 with acetic acid, flowing at 1.5 ml/min. Postcolumn derivatization was followed by fluorescent detection at the wavelengths already mentioned. Precolumn derivatization with 1-fluoro-2, 4-dinitrobenzene was followed by chromatography on an octadecylsilane column and a mobile phase of water-acetonitrile-acetic acid (34:66:0.1) flowing at 3 ml/min through a UV detector set to 365 nm [29]. The limit of detection is 0.5 µg/ml. 2,4,6Trinitrobenzenesulfonic acid was used to derivatize tobramycin in serum and. the internal standard sisomicin, which was followed by chromatograppy on an octylsilane column at 50°C. The mobile phase was acetonitrile-0. 05 M phosphate buffer, pH 3.5 (70:30), flowing at 2 ml/min through a UV detector set to 340 nm [ 30]. Time per assay is less than 4. 5 min, with a limit of detection below 0.2 µg/ml. ANTHELMINTICS AND OTHER ANTIPARASITIC AGENTS

Parasitism is almost universal in both animals and humans. It is debilitating and frequently disfiguring or fatal. In Africa alone, 1 million children are estimated to die each year from malaria, with 200 million people thought to be infected with schistosomes. The additional food intake required to maintain an infested organism can be equal to the usual daily intake, a quantity often impossible for some victims to maintain. For these reasons, research

Kirschbaum and Aszalos

246 2

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z

5 4

0 c..

(/)

w a: 3 8 6

+

INJECT

I

2

I 4

6

7

8

I 10

I 12

I 14

MINUTES

Figure 2 HPLC of ( 1) oxfendazole, ( 2) thiabendazole, ( 3) cambendazole, ( 4) mebendazole, ( 5) oxibendazole, ( 6) albendazole, ( 7) fenbendazole, and (8) parbendazole. See text for details. (From Ref. 34.)

is continuing to discover more effective drugs. Anthelmintics, antimalarials, antitrichomoniasis, antitypanosamal, and antiprotozoan drugs are considered in the literature: allopurinal [31,32], berberine I 33], cambendazole [34], chloroquine [35-37], ciclobendazole [38], dimetridazole [39], diminazene [40], emetine [ 41] , levamisole [ 42] , ivermectin (avermectin) [ 43, 44] , mebendazole [45,46], mefloquine [47], metronidazole [48-50], ornidazole [51], oxfendazole [ 52] , phenothiazine [ 53] , preziquantel [ 54] , primaquine [ 55] , proquamil [ 56] , pyrentel tartrate [ 57] , pyrimethamine [ 58] , quinine [ 59, 60] , salicylhydroxamic acid [ 61], tetramisole [ 62] , thiabendazole [ 32, 63, 64], and tinidazole [65,66]. Figure 2 shows the separation of eight benzimidazoles after injecting 5 µ g /ml of each compound [ 34] •

HPLC

247

ANTILEPROSY AND ANTITUBERCULOSIS AGENTS

The principal antileprosy drugs are dapsone ( 4, 4'-diaminodiphenylsulfone, DDS), rifampicin, and clofazimine. Secondary drugs are ethionamide and prothionamide. Since rifampicin and isoniazid are the two major antitubercu losis agents and pyrazinamide is a secondary drug, these two groups are considered together. Rifampicin is also an antitumor agent. Clofazimine Clofazimine, a drug administered to leprosy victims resistant to other drugs, was quantified in plasma using a 5- µ m octylsilane column with a mobile phase of 0.042 M phosphoric acid, pH 2.4-methanol (19:81) flowing at 1.5 ml/min [ 67]. Detection was at 285 nm with a limit of detection of 10 ng/ml. Dapsone Dapsone, the drug of choice in the treatment of leprosy for more than 30 years, and its major metabolite, 4-acetamido-4 1 -aminodiphenylsulfone (monoacetyldapsone) were analyzed with a minimum concentration of 2 nmol/ml using an octadecylsilane column, a mobile phase of acetonitrile-water ( 20: 80) flowing at 2 ml/min, and detection at 260 or 295 nm [ 68] • Dapsone, administered by injection, and its metabolite, were quantified using a mobile phase of either acetonitrile-1.5% aqueous acetic acid (26: 74) [ 69] or water-acetonitrile-acetic acid ( 100: 300: 25) [ 70] flowing at 2 ml/min through a 280-nm detector. As little as 20 ng/ml can be detected using an ODS column. A microbore silica column, 60 x 0. 77 cm, was used with a mobile phase of ethyl acetate flowing at the unusually high rate of 3. 75 ml/min into a fluorescent detector (285 nm/375 nm) to quantify dapsone and its major metabolite as well as the derivative 4, 4'-diacetamidodiphenylsulfone [ 71]. Sensitivities are 2 ng/ml. A 5-µm silica column, 100 x 4.6 mm, was used with a mobile phase of diisopropyl ether-methanol-21% aqueous ammonium hydroxide (96: 4: 0.1) flowing at 2 ml/min through a 254 nm detector [ 72] to assay dapsone in combination with pyrimethamine. The minimum concentrations that could be reliably measured were 10 ng/ml for pyrimethamine and 5 ng/ml for dapsone and monoacetyldap sone. Dapsone and its synthetic precursors could be separated [ 73] using either a 5-µm ODS column, 150 mm, and a mobile phase of acetonitrile-water (16: 64) flowing at 1 ml/min or a silica column and a mobile phase of isopropyl alcohol-ethyl acetate-acetonitrile-n-pentane ( 1: 1: 1: 7) flowing at rates up to 3 ml/min. Ultraviolet detection at 295 or 254 nm and fluorescence detection at 285 nm/340 nm were used simultaneously. The respective limits of these two detectors were 250 and 10 pg. Ethionamide Concentrations of ethionamide in plasma and urine as low as 0.01 µg/ml were determined using a silica column with a mobile phase of diethyl ether-methanol ( 96: 4) flowing at 1. 3 ml/min. Detection was at 295 nm. The column was periodically washed with dry acetonitrile. Prothionamide was used as internal standard [ 74].

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lsoniazid Isoniazid in formulations was assayed using a cyano column with a mobile phase of 0.01 M acetate buffer, pH 3.5-acetonitrile (95:5) pumped at 1.5 ml/min through a 254 nm detector. The lactose reaction product 1-isonicotinyl-2lactosylhydrazine could also be quantified. Periodically, the column had to be regenerated with 1% aqueous HCl [ 75]. Isoniazid and the lactose hydrazine could also be assayed in tablets using a silica column and a mobile phase of methylene chloride (or chloroform)-methanol-2-propanol-water (85: 5: 10: 0. 5) flowing at 1. 2 ml/min into a 254 nm detector [ 76]. Isoniazid and pyridoxine hydrochloride were quantified in a combination product using an octadecylsilane column with a mobile phase of methanol-water (60:40) containing 0.01 M dioctyl sodium sulfosuccinate (adjusted to pH 2. 5 with sulfuric acid). The flow rate was 2.5 ml/min, and detection was at 293 nm to equalize peak responses [ 77]. To analyze isoniazid and its acetyl derivative in plasma and urine, this ODS system was used with the mobile phase flowing at 2 ml/min and detection at 266 nm [ 78] • The respective limits of detection were 200 and 50 ng. The assay was extended to include the hepatotoxin acetyl hydrazine (which is generated in vivo from acetyl isoniazid) by utilizing an octadecylsilane column with a mobile phase of methanol-0.1 M potassium phosphate buffer, pH 6. 9 (5:95) flowing at 2 ml/min through a 254 nm detector (79]. The limit of detection of isoniazid was 0.5 µg/ml; the therapeutic dose is 1-3 µg/ml. Prothionamide Prothionamide in body fluids was determined by the same procedure used for ethionamide. Linearity of response is 0.5-5 µg/ml (74]. Pyrazinamide After oral administration, pyrazinamide is hydrolyzed to pyrazinoic acid, which was determined at concentrations of 1-10 µg/ml in 300 µl of serum with its metabolite, 5-hydroxypyrazinoic acid (80]. An amino column and precolumn, 40 x 4.6 mm, were used with a mobile phase of 0.01 M ammonium phosphate buffer, pH 4.2-acetonitrile (30:70) flowing at 2 ml/min through a 254 nm detector. Rifampicin Rifampicin is related to the rifamycin group of antitumor agents. Rifampicin, its quinone form, and the dihydro and tetrahydro derivatives were quantified during hydrogenation studies using an amino column with a mobile phase of chloroform-methanol (97: 3) flowing at O. 2-0. 7 ml/min into a detector set to 334 nm (81]. Rifampicin and its metabolites could be resolved from clofazimine using an octadecylsilane column and a mobile phase of tetrahydrofuran-0.05% acetic acid (40:60) flowing at 1.5 ml/min into a detector set to 287 nm. Recoveries average 101%, as 10-500 pmol were analyzed (82]. Rifampicin and its metabolites can be determined in body fluids using a wide range of polarities of columns. Rifampicin and its metabolites in serum were analyzed using an octadecylsilane column and a mobile phase of 0. 01 M sodium acetate buffer, pH 7.0-acetonitrile (62:38) flowing at 1.5 ml/min through a detector set to 340 nm [ 83] •

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An octylsilane column was used to assay rifampicin and its principal metabolite, 25-desacetylrifampicin, in plasma with a mobile phase of 0.1 M phosphate buffer, pH 3. 5-acetonitrile ( 62: 38) flowing at 2 ml/min through a 254 nm detector. The limits of detection are 0.2 µg/ml [84]. Rifampicin and three of its metabolites in human plasma, urine, and saliva were quantified using a 5-µm silica column ( 100 x 7. 5 mm) with a mobile phase of dichloromethane-isooctane-ethanol-water-acetic acid ( 36. 6: 45: 16. 8: 1.65:0.002) flowing at 3 ml/min through a 254 nm detector. For each compound the limit of detection is 0.05-0.1 µg/ml [85]. Combination assays in body fluids utilized octadecylsilane columns. Rifampicin, desacetylrifampicin, isoniazid, and acetylisoniazid could be simultaneously assayed in human serum extracts, polymorphonuclearleukocytes, and alveolar macrophages using a mobile phase of O. 05 M ammonium formate-methanol (35: 65), adjusted to pH 7. 3, flowing at 2 mlfmin through a 254 nm detector. The respective limits of detection are 17, 10, 95, and 85 ng/ml [86]. Clofazimine and rifampicin and their main metabolites were quantified in serum using tetrahydrofuran-0.5% acetic acid (40:60) flowing at 1.5 ml/min through a detector set to 287 nm [87]. Respective minimum detectable limits are 40 and 15 pmol. ANTITUMOR ANTIBIOTICS

Cancer chemotherapy is a concern of many people today-laboratory scientists, clinicians, cancer victims, and news reporters. Their hopes for a cancer cure are always raised when a new chemotherapeutic agent is discovered in the laboratory and shows some promising characteristics toward clinical application. Some of the agents that have been discovered are of natural origin and are complex structures of unknown composition; others are enzymes or antibiotics. The antibiotics are obtained mostly from fermentations but are also derived from plants or marine animals. Considerable efforts are being made in laboratories to purify these antibiotics to homogeneity and to determine their structure, mode of action, toxicity, and applicability to clinical cancer chemotherapy. HPLC is being used with increasing frequency to analyze these antitumor antibiotics in different media. Rifampicins were discussed in the previous section. Aclacinomycin The fermentation of aclacinomycin (an anthracycline type of antibiotic) and seven related compounds in broths was followed using a silica column with a mobile phase of chloroform-methanol-acetic acid-water-triethanolamine (68: 20: 10: 2: 0.1, v/v) flowing at 1 ml/min into a detector set to 436 nm to give equal molecular absorbance of all constituents [ 86] • Aclacinomycin and three metabolites were assayed in plasma using a phenyl column and acetonitrile-0.03 M ammonium formate, pH 5.0 (1:1), flowing at 1 ml/min into a fluorescence detector (435 nmex/505 nmem>· The limit of sensitivity is 20 ng/ml [87]. References 88 and 89 discusses difficulties in analyzed for the drug in pharmaceutical preparations. Pharmacokinetic studies of aclacinomycin in biological fluids involved an octadecylsilane column with a mobile phase of acetonitrile-methanol-waterphosphoric acid (20:45:30:0.75) flowing at 2 ml/min into a 254 nm detector

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[90]. Recoveries averaged 85%, linearity was found from 25 ng to 5 µg/ml, and the minimum detectable concentration appeared to be 5 ng/ml. Actinomycin

These closely related antibiotics, which differ only in amino acid composition, were resolved into Ci. c 2 , and C3 using either a phenyl or an octadecylsilane column with a mobile phase of water-acetonitrile ( 1: 1) flowing at 1 ml/min into a detector set to 254 nm [ 91]. The actinomycin D content of bulk preparations was quantified using an octadecylsilane column and a mobile phase of acetonitrile-water ( 6: 4) flowing at 2. 5 ml/min into a 254 nm detector [92]. Anthracyclines

The two most important members of this group of structurally related antibiotics are adriamycin (doxorubicin) and daunorubicin (daunomycin). Bulk adriamycin was analyzed using an octadecylsilane column and a mobile phase of water-acetonitrile ( 69: 31) , adjusted to apparent pH 2 with phosphoric acid. The flow rate was 1. 5 ml/min into a 254 nm detector [93]. Daunorubicin hydrochloride in various pharmaceutical preparations was analyzed using a variant of the preceding system [ 94] • The water-acetonitrile ratio was changed to 62: 38 with an apparent pH of 2. 2. Adriamycin, daunorubicin, and the possible aglycone impurities could be assayed using an octadecylsilane column with a mobile phase of methanol0. 005 M 1-heptanesulfonic acid, pH 3.5 (62.5:37.5), flowing at 1-2 ml/min through a 254 nm detector [ 95]. Good separation could be obtained for adriamycin, daunorubicin, and a third anthracycline antibiotic, carminomycin, when acetonitrile-0. 025 M camphorsulfuric acid, pH 3. 8 ( 1: 1) flowing at 1.1 ml/min was used as solvent with a column of 5-µm octylsilane [ 96] and detection at 254 nm. Adriamycin and daunorubicin were quantified in fermentation broths after extraction at pH 1. 5, using an octadecylsilane column, acetonitrile-potassium phosphate, citric acid buffer, pH 3 (7:18), flowing· at 1 ml/min into a 254 nm detector [ 97] . These agents and some related anthracyclines were separated in broths using an octadecylsilane column and a water (adjusted to pH 2 with phosphoric acid)-methanol (35:65 to 40:60) mobile phase flowing at 2 ml/min into a 254 nm detector [98]. Pharmaceutical preparations were assayed after dissolution in 0. 2% 2-naphthalene sulfuric acid in water-acetonitrile ( 6 2: 38) adjusted to apparent pH 2. 2 with phosphoric acid. Both anthracyclines and their hydroxylated metabolites could be resolved using 5- µm ethylsilane, octylsilane, or octadecylsilane and a mobile phase of 20-90% acetonitrile in water. The optimal system was octylsilane, 0.01 M phosphoric acid in water-acetonitrile (80: 20), flowing at :::: 1. 5 ml/min into a detector set to 254 nm [ 99]. Body fluids were typically deproteinized, adjusted to pH 8. 5, extracted with 5 volumes of chloroform-isopropanol ( 2: 1) , evaporated to dryness, and the residue dissolved in methanol prior to injection. Adriamycin, adriamycinol, and the two aglycones were resolved using a 5-µm octadecylsilane column with a mobile phase of acetonitrile-0.01 M phosphoric acid, pH 2.3 (40:60), flowing at 1 ml/min into a fluorescence detector at 465 nmex/580 nmem·

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Daunorubicin was used as an internal standard [ 100]. The same detection system was used with a 5- µm silica column and a mobile phase of chloroformmethanol-ammonium hydroxide-water (855: 130: 10: 5) flowing at 1.1 ml/min to determine adriamycin and six related compounds in plasma. The limits of detection are 2-4 ng/ml adriamycin and 10-20 ng/ml aclacinomycin [101]. Cellular uptake and metabolism of daunorubicin in leukemic cells were followed by HPLC using a silica column, a mobile phase of chloroform-methanolacetic acid water ( 720: 210: 35: 30) flowing at 1 ml/min, and fluorescence detector at 480 nm ex I 560 nm em. Adriamycin was used as internal standard; good linearity was found for daunorubicin, daunorubicinol, and the aglycone. The limit of detection was 1. 5 ng/ml [ 102]. Daunorubicin and its main metabolite daunorubicinol were assayed in plasma from leukemia patients using a 5- µm ethylsilane column and a mobile phase of acetonitrile-water-0.1 M phosphoric acid (25:65:10) flowing at 0.8-1 ml/min into a detector set to 254 or the less sensitive wavelength 500 nm [ 103]. Precision was better than 2% within the range of 20 ng/ml. Recoveries of each compound was approximately 100%. Bleomycins Using an octadecylisilane column with gradient elution, 10 related glycopeptides of the bleomycin group can be separated. A linear gradient of 10-40% methanol and 0.005 M 1-pentanesulfonic acid in 0.5% acetate buffer, pH 4.3, with mixing time 60 min and flowing at 1. 5 ml/min into a 254 nm detector [ 104] was used. Bleomycins in urine were purified by passage -through a Sep-Pak C18 cartridge, which was successively washed with water, acetone, water, and methanol. The bleomycins eluted with 2 ml of O. 02 M sodium heptane sulfonate in methanol. Portions were analyzed using an octadecylsilane analytical column and guard column ( 3. 9 x 60 mm) and a mobile phase of methanolacetonitrile-0. 0085 M aqueous sodium heptane sulfonate-acetic acid ( 30: 10: 59: 1) flowing at 2 ml/min into a 254 nm detector [ 105]. Deflectins The initial chromatographic methods used to isolate a crystalline product led the investigators to believe that they obtained a homogeneous antibiotic. However, HPLC indicated the presence of five major and several minor components in the original crystalline isolate. Since different fermentations produced deflectin complexes of different compositions, the crude products of these fermentations were analyzed using an octadecylsilane column, a mobile phase of methanol-triethylammonium formate buffer, pH 6. 0 ( 8: 20) , and a detector set to 339 nm [ 106] • Etoposide and Teniposide These drugs could be quantitated in serum at concentrations as low as 50 ng/ml using an octadecylsilane analytical column and precolumn and a methanolwater ( 60: 40) mobile phase flowing at 1 ml/min into a fluorescence detector with excitation 215 nm and emission 328 nm [ 107]. An automated system for these two antitumorigenic drugs in serum, plasma, or urine used an octadecylsilane analytical column and precolumn with a mobile phase of methanol-water (1: 1), with or without 1% glacial acetic acid, flowing at 1 ml/min into a fluorescent detector at 230 nmex/328 nmem·

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Calibration curves were linear from 0.01 to 25 µg/ml, with detection limits in plasma of etoposide of 8 ng. Recoveries of spiked plasma samples averaged 100%, with the option of using aglycone as internal standard [ 108]. S-Fluorouracil

5-Fluorouracil was resolved from 1-hexylcarbamoyl-5-fluorouracil, 1-w-carboxypentylcarbamoyl- 3-fluorouracil and 1-w -carboxypropylcarbamoyl- 5-fluorouracil in pharmacokinetic studies to obtain an optimal drug administration schedule and minimize the concentrations of the last two compounds, which are associated with side effects. A silica column was used with a mobile phase of watertetrahydrofuran-acetonitrile ( 50: 35: 15) flowing at 1 ml/min into a 254 nm detector [109]. Fredericamycin

Constituents of this antibiotic complex were resolved using either an octadecylsilane column with methanol-acetic acid-water ( 70: 1: 30) flowing at 2 ml/min or a silica column with chloroform-methanol-acetic acid (87:3:3) flowing at 1 ml/min into a 254 nm detector. Fractions were collected for biological assays [ 110]. Gilvocarcin

Constituents of the gilvocarcin atltitumor antibiotic complex from fermentation broths were resolved using an octadecylsilane column and a mobile phase of methanol-water ( 70: 30) flowing at 1. 5 ml/min into a 254 nm detector [ 111]. Reference 112 details a preparative scale LC system used to separate individual members of the complex. Mitomycin

Mitomycin A and B and other related compounds could be resolved from the antineoplastic agent mitomycin C using two silica columns ( 61 x 0. 2 cm and 30 x O. 4 cm) with a mobile phase of chloroform-methanol ( 9: 1) flowing at 1 ml/min into a detector set to 245 nm [ 113] • Mitomycin C in biological fluids was first extracted into ethyl acetate, and after evaporation of this layer, was redissolved in methanol. It could be quantified at concentrations as low as 40 µg/ml using an octadecylsilane column, a mobile phase of methanol-water ( 35: 65) flowing at 1 ml/min, and a detector operated at 365 nm [ 114]. Responses were linear from 1 to 25 µg mitomycin injected. This system was applied to pharmaceutical preparations using a 254 nm detector [ 115]. Nanomolar (nmol) quantities of different metabolites and derivatives of mitomycin C were resolved after adsorption on Amberlite XAD- 2 resin, water washing, and elution by methanol, using an octadecylsilane column with a mobile phase of acetonitrile-0.03 M potassium phosphate buffer, pH 6 (1:7), flowing at 2 ml/min into a detector set to 204 nm; see Fig. 3 [116]. Pharmacokinetic studies of mitomycin C were performed with the aid of electrochemical detection (mercury drop electrode 0. 6 versus a Ag I AgCl reference electrode) and UV detection at 360 nm [117]. A silica column was used with a mobile phase of ethyl acetate-methanol-water-dichloromethane (97: 2: 1: 1).

253

HPLC 6

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Figure 3 Separation profile of mitomycin and its metabolites by the method of Tomasz and Lipman. (1) Mixture of 1,2-cis- and 1,2-trans-2,7-diaminomitosene 1-phosphate; (2) 1, 2-trans-1-hydroxy-2, 7-diamino-10-decarbamoylmitosene and 1,2-trans-1-hydroxy-2, 7-diaminomitosene; (3) 1, 2-cis-1hydroxy-2, 7-diamino-10-decarbamoylmitosene; ( 4) 2, 7-diamino-10-decarbamoylmitosene; (5) 1, 2-cis-1-hydroxy-2,7-diaminomitosene; (6) 2,7-diaminomitosene and 10-carbamoylmitomycin C; ( 7) mitomycin C. Quantities are in the 1-10 nmol range. (From Ref. 115.) Mitomycin C, two mitosomes, and twelve mitosene derivatives were characterized using an octadecylsilane analytical column ( 100 x 8 mm) and guard column (70 x 2.1 mm, 37-µm particles) and gradient elution starting from 100% 0.01 M potassium phosphate buffer, pH 7.0, to 100% methanol in 13 min. The flow rate was 3 ml/min, and detection was at either 313 and 365 nm or by electron impact mass spectrometry (especially for acetylated mitosenes) [ 118] • The assay was sensitive to 5 pmol injected and provides definitive evidence of mitomycin C products generated by chemical and metabolic means. Neocarcinostatin

Neocarcinostatin is a chromoprotein that was initially extracted with 0.1 M acetic acid or 0.1 M HCl in methanol or glacial acetic acid prior to chromatography and was separated into different forms using an octadecylsilane column with a concave gradient run of 50 min of 56-84% methanol in 0. 01 M ammonium acetate, pH 4, flowing at 2 ml/min into a 254 nm detector [ 119]. Paulomycins

The constituents of the paulomycin complex isolated from broths were resolved using an octadecylsilane column (100 x 4.6 mm) and a mobile phase of acetonitrile-0. 5 M potassium phosphate buffer, pH 7. 0 ( 38: 62) , flowing at 2 ml/min into a detector set to 329 nm [ 120].

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Streptozotocin Unchanged streptozotocin in serum was measured, after deproteinization with ethanol, lyophilization of pH 4 supernatent, and redissolution in methanolacetone (3: 1), using a nitrile column with a mobile phase of hexane-isopropanol (3: 1) flowing into a 254 nm detector [ 121]. V inca Alkaloids All 26 related vinca alkaloids can be separated using an octylsilane column with a mobile phase of acetonitrile-0. 01 M ammonium carbonate ( 47: 53) flowing at 1. 5 ml/min into a detector set to 298 nm [ 122] . Structural assignments were aided by UV data obtained at lower wavelengths. Gradient elution was used to assess the composition of crude Vinca rosea plant extracts into an acid-water mixture. An octadecylsilane analytical column and precolumn (10 cm) was used with a mobile phase of 50-85% methanolwater, both containing 0.1% ethanolamine (no gradient time specified), flowing at 2 ml/min into a detector set to 280 nm [ 123]. Monomeric and dimeric alcohols could be distinguished by the number of theoretical plates N needed for resolution. To help identify the individual vinca alkaloids, two octadecylsilane columns connected in series were used with acetonitrile-0.01 M sodium phosphate buffer, pH 7. 4 ( 1: 1), flowing at 1. 0 ml/min into a 254 nm detector [ 124]. Fractions were collected and analyzed by radioimmunoassay. Metabolites of these vinca alkaloids were separated after modifying the techniques to one column and a linear gradient of 20-80% acetonitrile-0.001 M potassium phosphate buffer, pH 7. 5, flowing at 2. 5 ml/min into a 254 nm detector [ 125]. ANTIVIRAL AGENTS Acyclovir Acyclovir and its urinary metabolites were analyzed using an octadecylsilane column and precolumn ( 70 x 2.1 mm) with a mobile phase of 2% ethanol in 0.05 M phosphate buffer, pH 3.3, flowing at 0.5 ml/min. Ultraviolet absorption was monitored at 254 and 280 nm [ 126] . The results were used to determine the kinetic and metabolic disposition of the drug. For assays in tissues, a strong anion-exchange column was used with a linear gradient of O. 0151. 0 M KH2P04.-pH 3.5, flowing at 0.5 ml/min with detection at 254 nm [127]. Sensitivity was 0.01 mol/mg tissue. Tissues were removed and immediately frozen in liquid nitrogen. After weighing and grinding within a cold mortar, the powder was extracted with 2 volumes of ice-cold 15% perchloric acid and allowed to thaw before the addition of 3 volumes of ice-cold deionized water. After centrifugation, the supernatant was neutralized with a freshly prepared, saturated KOH solution, recentrifuged, evaporated to dryness under reduced pressure, and the residue redissolved in deionized water prior to injection. Amantadine Amantadine was derivatized with fluorescamine and chromatographed using a 5-µm octadecylsilane column with a mobile phase of methanol-0.067 M phosphate buffer, pH 7.0 (575:425), flowing at 1.3 ml/min. Fluorometric detection was at 395 nm, excitation and 485 nm, emission [ 128], in these feasibility studies.

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Figure 4 HPLC of derivatized amantadine (see text for details).

After derivatization with phthalic anhydride, to diminish noncovalent interactions and provide a convenient chromophore, amantadine hydrochloride was chromatographed using an octadecylsilane column and a mobile phase of methanol-water-85% phosphoric acid (60:40:1) flowing at 1 ml/min through a 254 nm detector [ 129] . Figure 4 shows a typical chromatogram. Although HPLC peaks frequently are sharp, with as many as 30, 000 theoretical plates (using distillation terminology), here we calculated only several hundred theoretical plates, but related compounds are resolved, and the linearity and reproducibility were excellent. Ari I done

Arildone and its metabolites in tissues were determined using either an anionexchange, octylsilane or octadecylsilane column, using acetonitrile-water-98% formic acid ratios of 1000:20:2, 10:90:0.5, or 0:100:0.1, respectively, flowing at 2 ml/min through a detector set to 280 nm [ 130]. Sensitivity was .::: 6 ng/ml of the parent compound. Enviroxime

Enviroxime in body fluids was separated from its less active synoxime isomer zinviroxime and quantitated [131] using a 6-µm octylsilane analytical column and octadecylsilane guard column and a mobile phase of methanol-0.14 M sodium acetate (65:35) with 3 mg disodium EDTA added per liter. The flow rate was 0.9 ml/min through an electrochemical detector set to +0.85 versus a Ag/AgCl3 M NaCl reference electrode. Some assays required column switching techniques. The limit of detection was 4 ng/ml plasma.

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Interferon

Active fractions were separated using a diol column equilibrated with 0.1 M sodium acetate, pH 7.5 (0.01% thiodiglycol)-n-propanol (20:80) flowing at 1.3 ml/min. Elution was with a linear gradient of n-propanol changing from 72. 5 to 50% in 4 hr, at a flow rate of O. 25 ml/min [ 132] • At least three peaks were found after diverting 1% to a fluorescence detection system [ 133] involving derivatization with fluorescamine. Vidarabine

Bulk vidarabine was assayed using an octadecylsilane column and a mobile phase consisting of 2. 2 g sodium docusate in 10 ml glacial acetic acid, plus 500 ml methanol, which is finally diluted to 1 liter with water. Detection was at 254 nm [ 134]. Vidarabine and its metabolite, arabinosylhypoxanthine, were assayed in biological fluids using a 5-µm octylsilane column at 40°C, with a mobile phase of 0.005 M sodium pentanesulfonate buffer, pH 7.2-acetonitrile (20:480) flowing at 1 ml/min through a detector set to 250 nm [135]. The limit of detection is 0. 5 µg/ml in serum and cerebrospinal fluid and 2. 5 µg/ml in urine. Virantmycin

Virantmycin, in fermentation broths, was analyzed using an octadecylsilane column with a mobile phase of methanol-water-acetic acid (80: 20: 5) flowing at 1 ml/min through a detector set to 300 nm [ 136]. 13-LACTAM ANTIBIOTICS

The modern era of the treatment of disease can best be considered to have begun with the discovery of penicillin by Sir Alexander Fleming in 1929 and its meaningful application to therapy by Florey and Chain in 1940. Penicillin contains the 13-lactam ring, a four-membered cyclic amide, which is responsible for interfering with cell wall synthesis by acylating a D-alanine transpeptidase and thus preventing the formation of peptide cross-links between two linear peptidoglycan chains. A second major class of 13-lactam-containing antibiotics, the cephalosporins, appears to have a similar mode of action. HPLC has been used in every step of 13-lactam research and production, from examining fermentation broths of natural antibiotics and reaction mixtures of synthetic and semisynthetic ones and testing stability and physical and chemical properties of bulk and formulated drugs, to quantifying contents in body fluids. First, assays capable of chromatographing several 13-lactam antibiotics then methods suitable for one to three such compounds are described. Most of these methods involve reversed phase HPLC, typically using an octadecylsilane column with a mobile phase of methanol or acetonitrile-water or aqueous buffer and UV detection. Purified material is usually dissolved in a methanol or methanolic mobile phase prior to injection. Most formulations can be extracted or dissolved into similar solvents. 13-Lactam antibiotics in body fluids are usually analyzed after deproteinization (typically by ultrafiltration or precipitation), dilution with buffered mobile phase, addition of an internal standard, and then injection.

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257

Penicillin Combination Assays

Side-chain diastereoisomers of commercial penicillins were separated using an octylsilane column and a mobile phase of methanol-water-0.2 M phosphate buffer, pH 7, flowing at 1 ml/min through a detector set to 254 nm. Next to the phosphate buffer content fixed at 5% (v/v), are the penicillins and percentages of methanol, phenethicillin, 37%; propicillin, 45%; clometocillin, 50%; carbenicillin, 5%, ticarcillin, 5%; ampicillin, 25%; amoxicillin, 10%; and azidocillin, 40%. Gas chromatography verified results for the first five penicillins [ 13 7] • Mecillinam (amdinocillin), ampicillin, and amoxicillin were chromatographed using an octadecylsilane column and a mobile phase of acetonitrile-0. 01 M potassium phosphate buffer, pH 5 ( 15: 85), flowing at 2 ml/min into a detector set to 220 nm. Mecillinam in urine had a sensitivity limit of 50 µg /ml [ 138]. Figure 5 shows the separation of ampicillin, methicillin, penicillin G, penicillin V, and oxacillin using a 5- µm octylsilane column and a mobile phase of methanol-0.01 M sodium dihydrogen phosphate (35:65) flowing at 1 ml/min

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258

Kirschbaum and Aszalos

through a detector set to 225 nm [ 4]. If an octadecylsilane column was used, the elution order was ampicillin, penicillin G, methicillin, penicillin V, and oxacillin. Antibiotic sensitivity disks were analyzed using an octadecylsilane column and mobile phase of acetonitrile- 0. 01 M sodium phosphate buffer, pH 5. The buffer-acetonitrile ratios were ampicillin, 90: 10; carbenicillin, 97: 3; and mecillinam (amdinocillin), 85: 15. Microbiological assays verified the HPLC results [ 139] . Penicillinase-resistant penicillins were analyzed in serum using an octadecylsilane column and a mobile phase of water-acetonitrile-0. 2 M ammonium acetate, pH 5. 6 ( 62: 28: 10). Flow rates were 1 ml/min for oxacillin, cloxacillin, methicillin, and nafcillin, and 3 ml/min was used for dicloxacillin. Drug concentrations as low as O. 5 µg/ml could be measured with linearity from 0 to 128 µg/ml. Serum (0.4 ml) was mixed with an equal volume of acetonitrile, mixed for 10 sec, slowly shaken for 15 min, and then centrifuged for 10 min at 3000 g. The supernatant fluid was decanted into a screw-top test tube, and 4.0 ml of CH2Cl2 was added. After mixing for 10 sec, shaking for 10 min, and recentrifugation, a portion of the upper (aqueous) layer was injected. Results were verified using microbiological assays [ 140] • The isoxazolyl penicillins oxacillin, cloxacillin, flucloxacillin, and dicloxacillin and their active 5-hydroxymethyl derivatives were quantified in serum and urine. A 5- µm octylsilane analytical column was used with an octadecylsilane guard column ( 50 x 2.1 mm) and a mobile phase composed of 0. 02 M sodium acetate (pH 5.5)-methanol (10:8) or 0.02 M ammonium acetate (pH 6.6)acetonitrile ( 100: 34) flowing at 1. 2 ml I min through a detector set to 220 nm. As little as O. 4 µg penicillin per milliliter plasma could be assayed accurately. The acetate-methanol mobile phase was also useful for analyzing the penicilloic acids [ 141] . The metabolism and pharmacokinetics of aminopenicillins were determined using human urine. An octadecylsilane analytical column and ethylsilane precolumn ( 50 x 1. 5 mm) were used as follows. For ampicillin and its prodrugs, hetacillin and talampicillin, methanol-water ( 5: 8) containing O. 01 sodium n-heptylsulfonate, 0. 005 M NaH2P04, and 1.3% 0. 5 M HCl (pH 2. 7) was used at a flow rate of 0.8 ml/min, with detection at 218 nm. The same conditions were used for amoxicillin except methanol-water (2: 5), an ion-pair concentration of 0.0085 M, 0.001 M NaH2P04, and monitoring at 228 nm. Cyclacillin needed a methanol-water ratio of 295:500, 0.01 M ion pair reagent, and 0.001 M NaH2P04 with monitoring at 210 nm. The major metabolites, penicilloic and penamaldic acids, could also be determined [ 142]. Amoxicillin, carbenicillin, ampicillin, epicillin, cyclacillin, benzylpenicillin, mecillinam (amdinocillin), phenoxymethylpenicillin, and azidocillin could be chromatographed using a 5- µm octylsilane column with a mobile phase of phosphate buffer (I= 0.1, pH 8)-methanol (7:3) flowing at 1 ml/min. Imidazole and mercuric chloride were used to derivatize prior to detection at 310 nm. Ampicillin and amdinocillin (mecillinam) contents in plasma, whole blood, urine and lymph were determined at concentrations as low as 100 ng/ml. This procedure may be generalized to cephalosporins [ 143] . Precolumn derivatization with an imidazole-mercuric chloride reagent was followed by chromatography using a 5- µm octadecylsilane analytical column and guard column ( 4 x 10 mm) with a mobile phase of aqueous 0. 01 M NaH2P04 and 0.01 M EDTA initially containing 16.5% acetonitrile and increasing to 31.5% in 16 min [ 144]. The flow rate was 2 ml /min with detection at 325 nm of penicillin G, V, and X and methicillin in a variety of biological media, including fermentation broths. Novel penicillins may be detected by this reagent.

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259

Penicillins and Cephalosporins The biological properties of the penicillins and cephalosporin epimers vary greatly. The D epimers of ampicillin , cephalexin, and cephaloglycin are con siderably more active than their corresponding L isomers. Conversely, the D isomer of a.-phenoxyethylpenicillin is more active than the L isomer, but when the two isomers are mixed, biological activity of the combination is similar to that of the more active isomer. Diasteroisimers were resolved using an octadecylsilane column and detection at 254 nm. A flow rate of 1. 5 ml /min was used with the following ratios of 0.1 M phosphate buffer-methanol: (95:5) at pH 3.5 for cephalexin and ampicillin and 80:20 (pH 5.5) for a.-phenoxyethylpenicillin [ 145). An octadecylsilane column was used with a mobile phase of water-methanol (55:45) containing 30 mM of the crown ether 18-crown-6 at pH 2.5. Detection was at 220 nm for penicillin and cephalosporins to detect ampicillin, amoxicillin, cyclacillin, ben zylpenicillin, carbenicillin, oxacillin, cloxacillin , dicloxacillin , cephalexin, cephaloglycin, cephradine, and cephaloridine [ 146) . Effects of varying crown ether, crown ether concentration, and pH were studied. Partition coefficients of carbenicillin, dicloxacillin, floxacillin, cloxacillin, oxacillin, propicillin, phenethicillin, penicillin V, penicillin G, cyclacillin ampicillin , amoxicillin , sulbenicillin, cephaloridine, cephalothin , cephaloglycin , cephalexin, cephradine, and cephazolin (in order of decreasing k') were determined using an octadecylsilane column and a mobile phase of 0.35 Mammonium chloride-30% methanol in water, adjusted to pH 7. 4, flowing at an ungiven rate through a detector set to 254 nm [ 147). Amoxicillin, ampicillin, etacillin, oxacillin, cloxacillin, flucloxacillin, and cicloxacillin eluted in this order, as did cephaloridine, cephacetrile, cephazolin, cephapirin, cephradine, and cephalothin, using an octadecylsilane column and a mobile phase of 0.1 M borate buffer, pH 8.5-n-propanol, containing O. 6% cetyltrimethylammonium bromide, flowing at 1 ml/min into a detector set to 252 nm [ 148). The stability of 2% solutions of antibiotics in saline and aqueous dextrose were studied using a phenyl column and the following conditions: for carbenicillin, 0.01 M ammonium acetate in water flowing at 1.6 ml/min (245 nm); for cephazolin, 0. 01 M ammonium acetate in water-methanol ( 70: 30) flowing at 1. 3 ml/min (254 nm); for cephalothin, the previous solvent flowing at 2. 2 ml/min ( 254 nm); for nafcillin, aqueous methanol ( 1: 1) flowing at 2 ml/min ( 280 nm); and for ticarcillin, 0. 01 M ammonium acetate in water flowing at 1. 5 ml/min ( 245 nm). Results were verified using colorimetric assays [ 149] . A 5-µm octadecylsilane analytical column (150 x 4.6 mm) and precolumn ( 45 x 4. 6 mm) were used with a mobile phase of water-acetonitrile-0. 2 M ammonium acetate, pH 5. 0, flowing at 1 ml/ min to separate the following an tibiotics, with the values in parentheses the ratios of mobile phase constituents [ 150): benzylpenicillin, mezlocillin, and cloxacillin ( 65: 24: 10), ampicillin (82:8:10), and cefotaxime (78:12:10). Ampicillin , cephalexin , cephradine, and amoxicillin were determined in plasma using a 5- µm octadecylsilane column at 55°C and a mobile phase of methanol-water (3:2). Detection utilized fluorescence at 345 nmexl 420 nmem• except for amoxicillin, for which the methanol-water ratio was 55: 45 and the respective wavelengths 355 and 435 nm. Limits of detection were 0. 5 ng/ml for ampicillin, 2 ng /ml for cephalexin, and 10 ng /ml for amoxicillin and cephradine [ 151) .

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Cephalosporins An octadecylsilane column was used with a mobile phase of 0.1 M acetate buffer, pH 4. 66-methanol ( 78: 22) flowing at 0. 8 ml/min into a detector set to 206 nm. Cephacetrile, cefuroxime, cephalexin, cephaloridine, cephradine, and cephalothin [ 152] were resolved. The separation of cephacetrile, cephalexin, cefazolin, cephradine, cephaloglycin, and cephalothin is shown in Figure 6. A 5-µm octylsilane column (150 x 4.6 mm) was used with a mobile phase of methanol-0.01 M sodium dihydrogen phosphate flowing at 1 ml/min through a detector at 254 nm [ 4]. The same type of column was used with 0. 01 M tetrabutylammonium hydroxide (adjusted to pH 7.4 with H3P04)-methanol (90: 10) flowing at 1.1 ml/min through a 254 nm detector to monitor cephalosporin starting materials, which are often poorly resolved without an ion-pairing agent. Desacetylcephalosporin C, cephalosporin C, desacetoxycephalosporin C, 7-ADCA, and 7-ACA were resolved. Semisynthetic cephalosporins in biological fluids were extracted and chromatographed using 0.01 M acetate buffer, pH 4.8-methanol (15:85) flowing at 1.5 ml/min using the following wavelengths: cefuroxime, 254 nm; cefoxitin, 245 nm; cefotaxime, 234 nm; cefazolin, 275 nm; cefamandole, 270 nm; cephalotin, 240 nm; and cefoperazone, 240 nm, in order of increasing elution time.

2.0 µg

4.2 µg 3.1 µg 2.8 µg

0.5 µg .5µg c:

a N

.!3? CIJ

(.)

c:

·c:; >

'i5> 0

'°

~

"ECIJ u

CIJ

. gave a limit of detection of 2.5-5 µg/ml for amoxicillin and the penicilloic acid metabolite [ 168] . HPLC and microbiological assays agree.

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Amoxicillin and clavulanic acid, the components of Augmentin, were assayed in biological fluids [ 169] using an octadecylsilane analytical column, precolumn, and mobile phase that varied with the body fluid but generally was 0.1 M potassium phosphate buffer, pH 3.5-methanol (95:5) flowing at 2.5 ml/min into a detector set to 227 nm. The limit of detection of amoxicillin in plasma was 0. 5 µ g /ml. As expected, HPLC and microbiological assays gave similar results. Another method also involved an octadecylsilane column but with a mobile phase of 0.01 M tetra-n-butyl ammonium bromide plus 0.006 M NaH2P04 plus 0.004 M Na2HP04 in water-methanol (10:1), pH 7.02, flowing at 1. 5 ml/min through a detector set to 220 nm [ 179]. Ampicillin

A variety of octadecylsilane analytical columns were used with an octadecylsilane guard column (100 x 4.6 mm) to analyze the sodium, trihydrate, and anhydrous forms of ampicillin. The mobile phase consisted of 10 ml of 1 M phosphate potassium dihydrogen and 1 ml of 1 M acetic acid in 920 ml water plus 80 ml of acetonitrile flowing at 2 ml/min into a detector set to 254 nm. Responses were linear from 0. 7 to 36 µg [ 171]. Ampicillin and its penicillenic and penicilloic degradation products can be analyzed in formulations using an anion-exchange resin and a mobile phase of 0.02 M sodium nitrate in 0.01 M sodium borate, pH 9.15, flowing at 0.5 ml/min into a 254 nm detector. The method is sensitive to 20 ng of sample injected [ 172] , which could be lowered to 1 ng using a mass spectrometer to obtain positive- and negative-ion spectra [ 1731 • Mezlocillin could also be detected in this manner. Ampicillin in fermentation broths can be quantified using an octadecylsilane column and a mobile phase of 300 ml methanol, 700 ml water, and 20 ml of O. 25 M tetrabutylammonium phosphate flowing at 1 ml/min into a 254 nm detector [174]. Polymers were resolved using a column packed with Sephadex LH-60, and a mobile phase of dioxane-water ( 7: 3) flowing at 0.1 ml/min into a detector set to 254 nm. Polymers of amoxicillin and pipericillin could also be separated [ 175]. Alternatively, ampicillin was resolved from its polymers [ 176] using an octylsilane column and gradient elution. Mobile phase A was 0.01 M phosphate buffer, pH 7-acetonitrile (9:1) and B was 9:2. Phase A was used for 2 min, and then B was added following the formula %B = SO(t/15)0.5, where t is the elapsed time up to 15 min. Detection at 254 nm gave minimum detectable limits of the di-, tetra-, and hexamers of O. 2-0. 5%, with found contents of 0.6-1.6%, 0.3 to 2.3%, and 0.1-0.4%, respectively. Ampicillin was determined in body fluids using a 5- µm octylsilane column and a mobile phase of 0. 06 M potassium phosphate buffer, pH 4. 6-methanol (425:75) flowing at 1.2 ml/min. Detection at 225 nm [177] gave precision of 100 ± 2% with limits of detection Of O. 5 µg /ml urine, saliva, or plasma. Apalcillin

A 5-µm octadecylsilane column was used with a mobile phase of 0.01 M acetate buffer, pH 3.15-acetonitrile (80:20) flowing at 2 ml/min through a detector set to either 254 or 310 nm [ 178]. A gradient method was developed to separate metabolites in body fluids.

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Asparenomycin Asparenomycins A, B, and C, which contain a substituted ethylidene side chain, were chromatographed in fermentation broths and crude powders using an octadecylsilane column, a mobile phase consisting of either 0.02 M phosphate buffer, pH 7, or buffer-methanol ( 95: 5) flowing at 2 ml/min and detection at 254nm[179]. Azlocillin The ureidopenicillins azlocillin and mezlocillin, in plasma, were separately chromatographed using an octadecylsilane column, a mobile phase of acetonitrile-0. 05 M phosphate buffer, pH 7 (27:73), flowing at 2 ml/min, and detection at 220 nm. Recoveries were 92 and 102%, respectively, and limits of detection were 1. 3 and 1. 5 µg/ml [ 180] . Azlocillin and its penicilloate were quantified in urine using an octadecylsilane column and gradient elution with, for phase A, 350 mg KH2P04 and 650 mg (NH4)2C03 dissolved in 11 water and, for phase B, acetonitrile. From the initial time to 5 min, 5% B: 95%A; from 5-15 min, phase B increases to 50% and then A diminishes to 5%. The flow rate was 3 ml/min, and detection was at 220 nm [181] to achieve a minimum detection limit of 1-2 µg/ml. Aztreonam Bulk aztreonam can be analyzed on a variety of polarity columns. It may be resolved from related compounds using either a silica or a diol column and a mobile phase of O. 01 M phosphate buffer, pH 2-acetonitrile ( 1: 2) flowing at 1 ml/min through a detector set to 206 nm [ 182] , an amino column at 31°c with a mobile phase of 0.1 M phosphate buffer containing 0.3% tetrabutylammonium hydrogen sulfate plus O. 33% ammonium sulfate, and adjusted to pH 4. 5methanol ( 95: 5) flowing at 1 ml/min through a detector set to 210 nm [ 183] , or the reversed-phase systems described as follows. This monocyclic S-lactam was quantified in blood, urine, and induced blister fluid using an octadecylsilane column and a mobile phase of watermethanol-acetic acid (86.5:12.5:1) flowing at 2.5 ml/min into a 254 nm detector. At least 0. 5 µg could be detected per milliliter [ 184]. A similar column with a mobile phase of 0. 005 M aqueous tetrabutylammonium hydrogen sulfate adjusted to pH 3 with 1 M dipotassium hydrogen sulfate and O. 005 M ammonium sulfate-acetonitrile (80: 20) flowing at 2 ml/min into a detector set at 293 nm gave a limit of detection of 5 µg/ml urine and 1 µg/ml serum. Relative standard deviations were 3- 5% [ 185] • These investigators performed a large number of recovery, linearity, and microbiological studies to validate this procedure. Bacmecillinam Bacmecillinam and pivmecillinam are the 1' -ethoxycarboxyloxyethyl and pivaloyloxymethyl esters, respectively, of amdinocillin (formally known as mecillinam). Although bacmecillinam is 30% hydrolyzed in whole blood within 1 min at 37°C, it was quantified using a 5-µm octadecylsilane filled, glasslined, steel column (100 x 4 mm) and a mobile phase of phosphate buffer pH 6 (ionic strength= 0.05)-acetonitrile (6:4) containing 0.001 M N-hexyl-Nmethylamine [ 186] . Detection was at 230 nm. Recoveries averaged 98% after administration to humans. The limit of detection was 600 pg/ml.

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Carbenicillin Carbenicillin and its indanyl sodium and phenyl sodium prodrugs were determined using an octadecylsilane column and a mobile phase of 20% acetonitrile0. 001 M ammonium acetate flowing at 2 ml/min through a detector set to 254 nm. Stability and degradation products were studied [ 187] • Carbenicillin and its penicilloic acid and penilloic acid degradation products were determined using a 5-µm octadecylsilane column with a mobile phase of methanol-0.05 M phosphate buffer, pH 4.35 (37:63), containing 0.1% tetrabutylammonium bromide flowing at 1. 2 ml/min into a detector set to 220 or 254 nm [ 188]. Penicillin G and its degradation products could also be resolved. The stability of unit doses of carbenicillin, penicillin G, and ticaracillin was studied after reconstitution and storage at room temperature using an octadecylsilane column with a mobile phase of methanol-0. 05 M ammonium carbonate (1:3) flowing at 2 ml/min into a 254 nm detector. Polymerization may occur within 24 hr, resulting in the appearance of a new peak. Refrigeration or recent reconstitution is recommended [ 189]. Cephacetrile Cephacetrile was quantified in bulk form and in pharmaceutical formulations using an octadecylsilane column and then a mobile phase of methanol-0. 01 M acetate buffer, pH 4. 2 ( 1: 19) , for the first 5 min after injection , which in creased to 30% methanol in 7 min. The flow rate was 2 ml/min into a 254 nm detector [ 190] • Cefazolin was used as internal standard, and the desacetyl and lactone impurities could be resolved. Cefaclor Cefaclor was analyzed in plasma and urine using an octadecylsilane column and a mobile phase of methanol-water-acetic acid ( 20: 80: O. 5) flowing at 2 ml/ min through a detector set to 254 nm [ 191]. Responses were linear between O. 5 and 30 µg/ml, with recoveries averaging 98%. Minimum detectable limit was O. 5 µg/ml body fluid for both cefaclor and cephaloglycin. Contents in serum were determined using an octylsilane column with a mobile phase of methanol- 0. 01 M sodium acetate buffer, pH 5. 2 ( 30: 70) , flowing at 1 ml/min into a detector adjusted to 266 nm [ 192]. The limit of sensitivity was 0. 2 µg/ml. Cefaclor and cephradine were also analyzed using an octylsilane column with a mobile phase of 0.05 M sodium citrate buffer, pH 4-methanol-dioxane (80:13:7) flowing at 2 ml/min into a 254 nm detector [193]. Recoveries were 94-105%, the lower limit of detection was 1 µg/ml, responses were linear from 1 to 24 ;µ g /ml, and results were similar to those obtained by microbiological analyses. Cefadroxil The kinetics of degradation of cefadroxil were studied using an octadecylsilane column and a mobile phase of 0. 01 M ammonium acetate-acetonitrile (98:2 or 97:3) flowing at 2 ml/min through a 254 nm detector [194]. Degradation was shown to proceed by three pathways to various products depending on the pH of the dissolution solvent. Drug in plasma was quantified using the sarr.e type of reversed-phase column with a mobile phase of 0.01 M acetate

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267

buffer, pH 4.8-methanol (95: 5) flowing at 1. 5 ml/min into a detector set to 240 nm [ 195]. The limit of detection was 150 ng/ml. Cephalexin

Cephalexin, a derivative of cephalosporin C, was analyzed in bulk material and formulations using an octadecylsilane column, a mobile phase of 0. 03% ammonium carbonate-methanol (92:8) flowing at 0.9 ml/min, and detection at 254 nm [ 196]. As little as 0. 5 µg cephradine can be analyzed using cephaloglycin as internal standard in this system, in addition to resolving cephalosporin C and cefazolin. Cephalexin, formulated with lysine, could be quantified after precolumn derivatization with o-nitrophenol, using an amino column at 50°C, with a mobile phase of 1% citric acid in methanol-water ( 5: 40) flowing at 0. 8 ml/min through a detector set to 425 nm. The lower limit of detection was 0.3 µg/ml [ 197]. Cephalexin in cephradine formulations was analyzed using either an octadecylsilane column with a mobile phase of methanol-0.07 M sodium phosphate buffer ( 25: 75) flowing at 1 ml/min or a silica column at 40°C and a mobile phase of acetonitrile-water (80:20) containing 1 x 10-9% ammonia, flowing at 1. 5 ml/min. Detection was at 254 nm [ 198]. HPLC methods were compared with thin-layer chromatography (TLC) and showed similar results. Cephalexin could be resolved from flucloxacillin using an octylsilane column at 55°C and a mobile phase of O. 5 M phosphate buffer, pH 6.6-methanol (60: 40) flowing at 1 ml/min into a detector set to 220 nm [ 199]. Results were verified using spectrophotometric and microbiological assays. Contents of drug in plasma, urine, and saliva were assayed using an octadecylsilane column and a mobile phase of water-methanol-acetic acid (83: 17: 0. 5) flowing at 2. 0 ml/min into a detector set to 254 nm [200]. Responses were linear from 0. 2 to 50 µg /ml. Recoveries averaged 99% from plasma, 97% from urine, and 98% from saliva. This method was also suitable for cephaloglycin. This system appears based on a previously published method [ 201] for cephalexin in urine. Cephalexin in 20 µl serum was analyzed using an octadecylsilane column and a mobile phase of 30% aqueous acetonitrile solution containing 0. 005 M 2-propanesulfonate (adjusted to pH 3 using acetic acid) flowing at 2 ml/min into a 254 nm detector [ 202]. Responses were linear between 2 and 30 µg/ml. The effect of various counterions on the chromatography was studied. Cephaloglycin

Cephaloglycin and its metabolite desacetylcephaloglycin were determined in urine using an octadecylsilane analytical column and guard column with a mobile phase of water-methanol ( 4: 1) containing 0. 01 M aqueous ammonium dihydrogen phosphate and 0. 004 M sodium n-heptylsulfonate. The flow rate was 1 ml/min into a 254 nm detector [203]. Kinetics and pharmacology were discussed. Cephaloridine

Bulk and formulated cephaloridine was analyzed using an octylsilane column and a mobile phase of 0. 01 M sodium monobasic phosphate-methanol ( 70: 30) flowing at 1 ml/min into a 254 nm detector [ 204]. HPLC assays were faster

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and more accurate than microbiological. This same system was used to resolve impurities using a mobile phase ratio of 87: 13 [205]. Cephaloridine in serum and tissue was quantified also using a phenyl column with a mobile phase of methanol- 0. 2 M aqueouSi :ammonium acetate ( 20: 80) flowing at 2 ml/min into a 254 nm detector [ 206]. Recoveries of drug added to tissue homogenates averaged 103% and from serum averaged 98%. Responses were linear between 2 and 100 µg/ml serum. Cephalosporin C A 5-µm silica analytical column and precolumn were used with a mobile phase of 2% aqueous adipic acid flowing at 1 ml/min through .11 detector set to 250 nm. Desacetoxycephalosporin C and desacetylcephaloaporin C were also resolved [ 207]. The drug was assayed in fermentation broths using an octadecylsilane analytical column and precolumn and a mobile phase of 20% methanol-0. 01 M tetrabutylammonium hydroxide in water adjusted to pH 7. 0 using acetic acid. The flow rate was 1-1.5 ml/min, depending on the age of the column. Detection was at 254 nm [ 208]. Postcolumn derivatization. with o-phthaldialdehyde was also used to determine cephalosporin C in broths. An octylsilane analytical column and guard column were used with a mobile phase of 0.01 M NaH2P04 containing O. 2% acetic acid-acetonitrile ( 920: 80) plus added tetra-n-butylammonium hydroxide to a final concentration of 0. 01 M [ 209]. Fluorescent detection at 352.nmex/452 nmem was used to achieve a minimum limit of detection of 1 µg /ml. Recoveries averaged 99%. Cephamycin C and penicillin N could also be analyzed using this method. Cephalothin (Cephalotin) Chromatographic properties of bulk cephalothin were studied using an octadecylsilane column at 55°C and a mobile phase of 0. 001 M tetrabutylammonium hydrogen sulfate, 0. 001 M tetrabutylammonium hydroxide, and 7. 5% methanol in 0.01 M sodium phosphate buffer, pH 7.5, flowing at 0.5 ml/min into a 254 nm detector. The influence of temperature, organic modifier, and ion -pairing agent was studied. Cephapirin, desacetylcephapi:dn, cefazolin and 7-aminocephalosporanic acid (7-ACA) can be satisfactorily resolved by this method [210]. Hydrolysis and aminolysis were monitored using a strong anion-exchange column with a mobile phase of 0.01 M sodium phosphate buffer, pH 8.5, flowing at 1 ml/min (adjusted for optimal peak shape) into a 254 nm detector [ 211]. Reactions with aqueous amino acids were studied. Pharmacokinetics of cephalothin and desacetylcephalothin were investigated using an ocitadecylsilane column and a mobile phase of O. 01 M sodium acetate buffer, pH 3.8-methanol (65:35) flowing at 1 ml/min into a detector set to 237 nm [212]. No details of method validation were given. A similar column but with 1% acetic acid-methanol ( 60: 40) mobile phase flowing at 2. 5 ml/min and detection at 254 nm [ 213] gave a minimum limit of detection of 1 µg/ml serum. ·Responses were linear from 1 to 60 µg/ml serum. Recoveries averaged 99 and 100% for cephalothin and the desacetyl metabolite, respectively, added to serum. Contents in serum and urine were quantified using a strong anion-exchange column ( 900 x 2 mm) at 50°C and a mobile phase of O. 01 M sodium dihydrogen phosphate containing 0.01 M sodium nitrate, pH 4.8, flowing at 0.43 ml/min

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into a 254 nm detector. Recoveries of added drug were 81% from serum and 103% from urine. Linear responses were found from 8 to 350 µg/ml, with a minimum detectable limit of 0. 5 µg/ml serum. Microbiological results were invalid due to the inability to distinguish between drug and active metabolites [ 214] . Cefamandole The stability of cefamandole nafate in solution was investigated using a phenyl column and a mobile phase of methanol-0. 01 M ammonium acetate solution ( 40: 60) flowing at 1. 6 ml/min into a 254 nm detector. Cefamandole can be resolved from nafate [ 215]. Decreases in drug content due to degradation were reflected by increases in impurity peaks. No validity data were given. Cefoxitin was analyzed by the same system using a mobile phase ratio of 15: 85 and a flow rate of 2.5 ml/min. Solution stability was also investigated [216] using an octadecylsilane column with a mobile phase of water-acetonitrile-acetic acid ( 78: 20: 2) flowing at 1. 7 ml/min into a detector set to 260 nm. Sophisticated statistical methods were applied to incomplete studies to provide a semblance of validation. Cefamandole in serum and urine was analyzed using an octadecylsilane column and a mobile phase of methanol- 0. 2 M sodium acetate, pH 5. 2 ( 60: 40) , flowing at 2 ml/min into a detector adjusted to 270 nm. Cephalothin was used as internal standard. The minimum concentration detectable was 0. 3 µg /ml [ 217]. Cephapirin Concentrations in plasma and urine were determined using an octadecylsilane column and a mobile phase of 0.1 M phosphate buffer, pH 7. 5-acetonitrile ( 85: 15) flowing at 1. 5 ml/min through a 254 nm detector. Using cephaloridine as internal standard, calibration curves were constructed using concentrations of 2-250 µg /ml to investigate pharmacokinetics [ 218]. Cephapirin in plasma was quantified using an octadecylsilane column with a mobile phase of acetonitrile-dilute acetic acid, pH 2.8 (13:87), flowing at 1.5 ml/min into a detector set to 270 nm [ 219] . Recoveries averaged 100% between 200 and 1 µg /ml. Cefotaxime and cefoxitin could also be quantified by this method. Cefatri zine Cefatrizine was quantified in serum and urine using an octadecylsilane column with a mobile phase of 0.03 M sodium phosphate buffer, pH 5-methanol (80:20) flowing at 1 ml/min through a 254 nm detector [ 220]. The limit of detection was approximately 1-2 µg/ml serum or urine using cephradine as internal standard. These same investigators improved the detection system by using postcolumn derivatization with fluorescamine and fluorescent detection at 387 nm ex I 480 nm em [ 221]. This modification gave a 10-fold increase in sensitivity, with excellent linearity in the ranges of 0.1-1 µ g/ml and 10-100 µg/ml. Another chromatographic system used an octadecylsilane column with a mobile phase of 0.01 M acetate buffer, pH 4, flowing at the extraordinarily high rate of 4 ml/min through a 254 nm detector to determine [14c]cefatrizine in urine and feces and to study pharmacokinetics [ 222] .

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Cefazedone [ 14c]-Cefazedone was analyzed in body fluids and tissues using two coupled octylsilane columns and gradient elution starting with O. 05 M sodium citrate buffer, pH 4-acetonitrile (95:5) and finishing with a ratio of 20:80. The flow rate was 1 ml/min into a UV detector adjusted to 278 nm and a glass scintillator radioactivity detector. Recoveries were 81-100% from urine, 74-89% from serum, and 70-96% from tissues. At least 0.4 µg/ml serum could be detected [ 223] • Cefazolin The effect of surfactants on the aqueous stability and solubility of cefazolin was studied using an octadecylsilane column with a mobile phase of acetonitrile0. 02 M ammonium acetate (10:90) flowing at 1 ml/min into a 254 nm detector. The limit of detection is about 10 µg/ml. Propicillin and penicillin V were also investigated [ 224]. Contents in serum and urine were determined using a similar chromatographic system but with a mobile phase of acetonitrile-acetic acid-1 M triethylammonium acetate-water (12.5:1.4:2.7:84.6) to determine the degree of binding to proteins and pharmacokinetics [ 225] at 254 nm. Cefazolin in serum was quantified using a phenyl column and a mobile phase of methanol-1% aqueous acetic acid (12: 88) flowing at 2 ml/min into a 254 nm detector. Responses were linear between 10 and 200 µg/ml. Recoveries averaged 104% using HPLC and 87% ( 72-106%) by a microbiological method [226]. Pharmacokinetics were studied using an octadecylsilane column at 40°C with a mobile phase of methanol-water ( 5: 95) flowing at 1. 5 ml/min into a 254 nm detector [227]. Responses from 5 to 200 µg/ml were linear. Cefmenoxime Cefmenoxime in plasma and urine was quantified using a phenyl column and a mobile phase of acetonitrile-0.2% phosphoric acid (14:86) flowing at 2 ml/min into a 254 nm detector [ 228] • Recoveries averaged 99% using p-anisic acid as internal standard. Lower detection limits were O. 2 µg/ml plasma and 5 µg/ml urine. The drug was assayed in plasma modifying this method using an octadecylsilane column with a mobile phase of acetonitrile-0. 2 M acetate buffer, pH 5.3, flowing at 2 ml/min into a 254 nm detector [229]. The minimum limit of detection was O. 05 µg/ml using the same internal standard, and linearity of response was found from 0.5 to 200 µg/ml. Recoveries averaged 99%. Another modification exists [ 230] • Cefmetazole Cefmetazole was assayed in serum using an octadecylsilane column with a mobile phase of 10-15% acetonitrile in 0.005 M citrate buffer, pH 5.4, flowing at 1 ml/min into a 254 nm detector. Responses were linear between 0.4 and 100 µg/ml, the minimum limit of detection was O. 4 µg/ml, and recoveries averaged 100% [ 231]. Cefopera zone Most procedures utilize an octadecylsilane column with detection at 254 nm. With a mobile phase of 2. 4 ml of 1 M triethylamine, 5. 6 ml of 1 M acetic acid, and 240 ml of acetonitrile plus 752 ml of water, pharmacokinetics were deter-

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mined without additional analytical data [ 232]. Cefoperazone in cerebral spinal fluid was assayed using a similar mobile phase but with half the quantity of acetonitrile and ior:i-pairing agent and a flow rate of 2 ml/min. Responses were linear from 1. 25 to 5 µg /ml [ 233]. Using a phenyl guard column and a mobile phase of 1. 2 ml triethylamine, 2. 8 ml of 1 M acetic acid, and 120 ml acetonitrile diluted to 1 liter with water flowing at 2 ml/min, assays were performed on 0. 1 ml plasma, urine, or cerebral spinal fluid [ 234] . Hydrochlorothiazide was used as internal standard, with standard curves linear from 25 to 200 µg /ml (plasma), 100 to 800 µg /ml (urine), and 1. 25 to 10 µg/ml ( CSF) ranges. Respective recoveries averaged 100, 99, and 99%. A mobile phase of 0.1 M sodium phosphate, pH 6.0-acetonitrile (84:16) flowing at 2.5 ml/min was used to obtain a linear response for 2-100 µg/ml serum [235]. Recoveries ranged from an average of 100% in serum to 93% in tissue. Microbiological results showed a low bias of 6-40%, apparently due to the drug binding to protein. Cefoperazone was assayed in serum and muscle tissue using a phenyl analytical column and a C 18 guard column with a mobile phase of 0. 00625 M aqueous tetrabutylammonium phosphate-acetonitrile (80: 20) flowing at 2. 7 ml/min into a 254 nm detector [ 236] • Responses were linear from 5 to 150 µg/ml, the lower limit of detection was 1 µg/ml, and recoveries from sera averaged 99% of added drug. Cefoxitin and moxalactam can also be analyzed by this method. Cefoperazone in human serum and urine was quantified using an octadecylsilane column with gradient elution. Mobile phase A was composed of 0.0012 M triethylamine and 0.042 M acetic acid in water. Mobile phase B was mobile phase A and acetonitrile (24:76). The flow rate of 1.5 ml/min A and 0.5 ml/ min B was adjusted during 15 min to 1. 2 ml/min mobile phase A and 0. 8 ml/min mobile phase B [ 237]. Cefoperazone A, B, D, and E are resolved from cefoperazone, as well as from ampicillin and potassium penicillin G. Recoveries average 98%, and responses were linear from 0.3 to 1. 2 µg/ml. Good agreement was found with a microbiological assay. Cefotaxime

Ce.fotaxime and four degradation products in bulk material were resolved using an octadecylsilane column and a mobile phase consisting of O. 0044 M potassium monobasic phosphate plus 0.02 M sodium dibasic phosphate, pH 7. 6-methanol ( 83: 17) flowing at 1. 5 ml/min into a detector monitoring at 235 nm. Results were verified using TLC. Limits of detection were approximately 6 ng [ 238]. Comparative pharmacokinetics of cefotaxime and desacetylcefotaxime were studied in serum using an octadecylsilane column with a mobile phase of 0.01 M sodium acetate buffer, pH 4.0-acetonitrile (95:5) flowing at 1. 5 ml/min into a 254 nm detector [ 239]. Alternatively, 0. 02 M NaH2P04methanol-acetonitrile (83: 7: 10) could be used with an octadecylsilane-filled guard column [ 240] • Linear responses were found for 0. 5-100 µg cefotaxime per milliliter and 1. 25-100 µg desacetylcefotaxime per milliliter. Respective limits of detection were O. 5 and 1 µg/ml. Results were verified using a microbiological assay. Cefotaxime and desacetylcefotaxime were quantified in serum , urine , and other biological fluids using a 5-µm octadecylsilane column and a mobile phase of methanol-water-glacial acetic acid (12:87:1) flowing at 1.1 ml/min into a detector adjusted to 262 nm. Recoveries average 96-111%, with a limit of detection of 0.5 µg/ml serum and 5 µg/ml urine. Other columns were studied. Lactone metabolites in which the S-lactam ring was opened were also resolved [ 241].

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Drug in urine was quantified using a 5-µm octylsilane column and a mobile phase of 0.002 M aqueous phosphoric acid-methanol (72:28) flowing at 1 ml/min into a detector set to 310 nm. The claimed limit of detection was 0.1 1.1g/ml. Results were verified by microbiological assays. Pharmacokinetics of the desacetyl metabolite were also determined [ 242] • Transplacental passage of cefotaxime and desacetylcefotaxime was studied using a SC-02 gel with a mobile phase of O. 2% ammonium acetate solutionmethanol (5: 1) flowing at 1 ml/min into a 254 nm detector. Respective limits of detection were 0.1 and O. 2 µg/ml. A microbiological assay was used to verify the results [ 243] • Cefotetan Cefotetan in plasma and urine were determined using an octadecylsilane column ( 150 x 4 mm) with a mobile phase of 0. 1 M sodium dihydrogen phosphate, pH 3-acetonitrile (92: 8) flowing at 1 ml/min through a detector set to 280 nm [ 244, 245] • Occasionally, a precolumn ( 50 x 2 mm) was used at 45°C; the limits of detection were 0. 7 µg/ml plasma and 1 µg/ml urine. Pharmacokinetics were compared with cefazolin. Cefotiam Cefotiam and cefsulodin in serum and bone marrow samples were quantified using an octadecylsilane column with a mobile phase of O. 005 M tetrabutylammonium pposphate-methanol ( 65: 35) flowing at 1. 5 ml/ min into a detector set to 280 nm [ 246] • Recoveries averaged 100%. Cefoxitin Solution stability was measured using an octadecylsilane column with a mobile phase of acetonitrile-water-acetic acid (20:80:1) flowing at 0.5 ml/min into a 254 nm detector [247 ,248]. No validation data were given. Cefoxitin in serum was quantified using an octadecylsilane column and a mobile phase of acetonitrile-acetic acid-0. 005 M potassium dihydrogen phosphate (25:0.5:74.5) flowing at 2 ml/min into a detector set to 235 nm [249]. The limit of detection was 1 µg/ml. Results were confirmed by a microbiological assay. Alternatively, a mobile phase of methanol-0. 03% ammonium carbonate ( 15: 85) flowing at 2 ml/mih into a detector adjusted to 238 nm [ 250] was used. Recoveries averaged 93%, the response was linear from 1 to 100 µg/ml, and the limit of detection was 0.1 µg/ml. Another octadecylsilanebased method used a mobile phase of acetic acid in water (:::: 1. 5 ml/liter), pH 2.8-acetonitrile (13:87) flowing at 1.5 ml/min, with detection at 270 nm [ 251]. Pharmacokinetics were studied in serum and urine using a strong anionexchange column at 50°C with a mobile phase of acetate buffer, pH 5, flowing at 1. 5 ml/min into a 254 nm detector. The lower limit of detection was 1 µg/ml [225]. Cephradine Cephradine bulk and formulated material was analyzed using an octadecylsilane column and a mobile phase of methanol-0.03% ammonium carbonate

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solution (8:92 to 5:95) flowing at approximately 0.9 ml/min into a 254 nm detector. Drug in urine was quantitated at mobile phase ratio of 2:98 and a flow rate of 1. 3 ml/min.1 Cephaloglycin was us.ed as internal standard. Cephalosporin C, cephalexin, cefazolin, and cephalothin also showed a good retention time [ 252']'. Cephradine in serum and urine was analyzed using an octadecylsilane cdlumn and a mobile phase of methanol-0.01 M phosphate buffer, pH 6. 8 ( 20: 80), flowing at 2 ml/min into a detector [ 253]. Responses were linear between 0 and 100 µg /ml. Cephradine in serum was analyzed using an octylsilane column with O. 05 M sodium citrate buffer, pH 4-methanol-dioxane (80: 13: 7) flowing at 2 ml/min into a 254 nm detector [ 196] . The response was linear between 1 and 24 µg I ml, recoveries ranged from 94 to 105%, and the results correlated with microbiological data. Cefroxadine Cefroxadine was analyzed in serum using a 5-µm octylsilane column (150 x 4. 6 mm) and a mobile phase of 0. 002 M phosphoric acid-methanol ( 72: 28) flowing at 1 ml/min into a detector set to 280 nm [ 254]. The lower sensitivity limit was 0.3 µg/ml, with an average of 94% recovered from sera containing added drug. The correlation with the microbiological assay results was 0. 991. This method appears to be based on a previous one for plasma that also used an octylsilane column, but also a C8-precolumn and detection at 254 nm [ 255]. Urine required the same mobile phase constituents but at a ratio of 65: 35. The minimum concentration that could be determined with a relative standard deviation of 10% was 0.5 µg/ml plasma and 20 µg/ml urine, with a minimum detectable limit of 0.1 µg/ml plasma. Cefsulodin This antipseudomonal cephalosporin was analyzed in vials using an octylsilane analytical column and precolumn with a mobile phase of 0. 02 M ammonium acetate solution ( 1. 54 g + 4 ml glacial acetic acid)-methanol-acetonitrile (950: 35:15) adjusted to pH 4.1, flowing at 1.5 ml/min~.into a 254 nm detector. Five related compounds could be resolved. Responses were linear from 29 to 145 µg/ml [258]. Cefsulodin in plasma was analyzed using an oc:tadecylsilane column with a mobile phase of acetonitrile-0.02 M ammonium acetate, pH 4.2 (4.5:95.5), flowing at 2 ml/min into a 254 nm detector. Recoveries averaged 99%, responses were linear between O. 78 and 100 µg/ml, and the results correlated excellently with microbiological data [ 257] . Ceftazidime Ceftazidime in plasma and urine was quantified using a 5-µm octadecylsilane column ( 100 x 5 mm) and a mobile phase consisting of O. 05 M ammonium dihydrogen phosphate containing 6% acetonitrile and 0. 01% formic acid for urine ( 7% acetonitrile and 1 ml/min for serum) flowing at 1. 6 ml/min into a detector adjusted to 257 nm. Recoveries from both fluids are about 100%. The minimum detectable limit is 0.6 µg/ml [258].

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Cefti zoxime An octadecylsilane column was used with a mobile phase of acetic acid added to water up to pH 2. 8-acetonitrile ( 87: 13) flowing at 1. 5 ml} min through a detector set to 270 nm [259]. No validation data were given. A phenyl column and precolumn ( 50 x 2mm) were used with a mobile phase of either O. 03 M phosphate buffer, pH 2. 6-acetonitrile (87: 13) for serum, a ratio of 87: 11 for bile or 1% acetic acid-acetonitrile (87: 13) for urine, flowing at 2 ml/min through a detector set to 280 nm [ 260] • Recoveries of drug added to biological fluid averaged 100% for serum, 101% for bile, and 99% for urine (area measurements). Responses were linear for respective concentrations from 0.2 to 100 µg/ml, 100 to 500 µg/ml, and 100 to 4000 µg}ml. The low concentrations represented the detection limits. Ceftizoxime was also determined in body fluids using an octadecylsilane column and a mobile phase of 0.6% potassium phosphate-0.2% sodium phosphateacetonitrile (1: 1: O. 22) flowing at 2 ml/min into a 254 nm detector [ 261]. Responses were linear from 50-600 µg/ml of urine and 2-10 µg}ml serum. The minimum detectable limit was at least 0.1 µg/ml. Ceftriaxone Ceftriaxone in body fluids was analyzed using an octadecylsilane column and a mobile phase of acetonitrile-1% hexadecyltrimethylammonium bromide-0.1 M phosphate buffer (pH 7), 60:30:10 for plasma and 44:35:21 for urine, flowing at 2 ml/min into a detector set to 280 nm [ 262] . The linear range was 2-300 µg/ml, and at least 0. 5 µg/ml could be detected. A similar system but using 0. 58% tetraoctylammonium bromide and 2% 1 M phosphate-acetonitrile ( 600: 400) as mobile phase aided in quantifying drug in urine [ 263] • Responses were linear between 6 and 167 µg/ml. To analyze drug in body fluids, an amino analytical column was used with a silica guard column (100 x 4 mm, packed with 37-µm particles) and a mobile phase of acetonitrile-water-10% ammonium carbonate (70:26:4) flowing at 1.5 ml/min through a detector adjusted to 274 nm [46]. For saliva, 5% ammonium carbonate was substituted. Respective detection limits for plasma, urine, and saliva were 0.4, 3, and 0.03 µg}ml. Recoveries averaged 98%from 0.6 to 225 µg /ml plasma [ 264] • Cefuroxime Bulk drug was chromatographed using 5-µm octyl- or hexylsilane columns ( 100 x 4. 5 mm) with a mobile phase of 0. 01 M sodium acetate buffer, pH 3. 4-acetonitrile (90: 10) flowing at 2 ml/min into a detector set to 273 nm [265]. Responses were linear in the range studied, 12.5-75 µg/ml. Partially degraded samples were assayed by HPLC and microbiological procedures with the results in excellent agreement. There were no interfering peaks. Cefuroxime in urine and serum was quantified using an octadecylsilane column and a mobile phase of methanol-0.01 M sodium acetate (20:80) flowing at 1. 5 ml/min into a detector set to 270 nm [ 266]. At least O. 5 µg/ml serum and 20 µg/ml urine could be detected. The HPLC results agreed well with microbiological data. Cefuroxime in serum was determined using an octadecylsilane column and a mobile phase of acetic acid-water-methanol (1: 79: 20) flowing at 2 ml/min into a 280 nm detector. Recoveries averaged 98% between 1 and 20 µg/ml, with a limit of detection of 1 µg/ml [267]. A 5-µm octylsilane

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column ( 150 x 4. 6 mm) was used to assay drug in plasma with a mobile phase consisting of O. 067 M KH2P04-methanol ( 425: 75) flowing at 1. 2 ml/min into a detector adjusted to 278 nm [268]. The minimum detectable concentration is 0.5 µg/ml, recoveries averaged 100% (with relative standard deviation of 2%), and responses were linear from 5 to 100 µg/ml. Clavulanic Acid

A previously developed method for this drug formulated as the conjugate with amoxicillin [ 167] was modified to increase sensitivity when measuring urinary excretion by treatment with pH 12 solution at 60°C, to give a fluorescent product at 375 nmex/445 nmem [269). There are no other analytical details. Cloxacillin

Cloxacillin in oral dosage forms was quantified using an ethylsilane column with KH2P04, pH 4. 5-acetonitrile (80: 20) mobile phase flowing at 1. 5 ml/min ( 30°C) through a 254 nm detector [ 270] • The other isoxazole penicillins, oxacillin and dicloxacillin, were also resolved from each other, along with cloxacillin degradation products, cloxalloic acid and two minor unknown peaks. Responses for cloxacillin were linear from 0.11 to 0.65 mg/ml. Cloxacillin in serum was determined using an octadecylsilane column and a mobile phase of 0. 01 M phosphate buffer, pH 7-acetonitrile ( 76: 24) flowing at 2 ml/min into a detector adjusted to 195 nm (nafcillin can be analyzed by this procedure using 218 nm). 5-(p-Hydroxyphenyl)-5-phenylhydantoin was used as internal standard to obtain relative standard deviations of 15, 12, and 10% at 2, 8, and 20 µg/ml, respectively. The minimum detection limit is 0.080 µg/ml [271). Assays in serum and urine involved an octylsilane column ( 100 x 4. 6 mm) and a mobile phase of 0. 04 M NaH 2PO 4, pH 4. 5-acetonitrile ( 6. 2: 20) or phosphate-methanol ( 41: 59) flowing at 1. 6 ml/min through a detector set to 210 nm [ 272). Nafcillin was used as internal standard. Recoveries of cloxacillin averaged 83% from serum and 91% from urine, with a minimum quantitative limit of 0.05-0.3 µg/ml. Cyclacillin

HPLC was used to monitor the titration of cyclacillin to investigate stoichiometric analysis. An octadecylsilane column was used with a mobile phase composed of 3 ml concentrated ammonium hydroxide, 3 ml concentrated phosphoric acid, and 150 ml acetonitrile added to 900 ml water, flowing at 1 ml/min into a detector adjusted to 205 nm [ 273) . Hydroxylamine reacts to give more than one product, as verified with ampicillin using the same system and 6-aminopenicillanic acid using a strong anion-exchange column and a mobile phase of 500 ml 0.1% NaH2P04 in water with 50 ml acetonitrile. Moxalactam

Moxalactam is a ~-lactam antibiotic in which an oxygen replaces the sulfur atom in the cephem ring. The R and S epimers of the bulk drug were resolved using an octadecylsilane column and a mobile phase of methanol-0. 5 M potassium phosphate solution, pH 6.5 (5:95), flowing at 2 ml/min into a 254 nm detector. Typical ratios of R epimer are 1.11 and 1. 05 to 1 of S-epimer

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[274]. The mobile phase was modified to methanol-0.005 M tetra-n-butylammonium phosphate adjusted to pH 6.0 with phosphate buffer (25:75) to quantify the epimers in urine. The linear range studied was a meager O. 50.1 µg/ml. There appears to be no interconversion in vivo between epimers. Moxalactam epimers were quantified in body tissues using an octadecylsilane column and a mobile phase of 0.1 M sodium phosphate-methanol (84: 16) at pH 3. 2 flowing at 2 ml/min into a 254 nm detector. Responses were linear from 0 to 100 µg/ml, with recoveries averaging 98%. The R epimer may have a greater affinity for tissue [ 275]. Drug was determined in plasma and urine using an octadecylsilane column with a mobile phase of acetonitrile-phosphate buffer, pH 7 (171: 822), containing 25 g CaCl2 and 8 ml of tetra-n-butylammonium hydroxide per liter flowing at 2 ml/min into a 280 nm detector. The limit of sensitivity was 50 ng/ml [ 276] . Epimers were resolved in serum, urine, and cerebrospinal fluid using a similar column and a C18 guard column with a mobile phase of methanol-0.01 M ammonium phosphate (adjusted to pH 6.5 by the addition of phosphoric acid, 4: 96) flowing at 1 ml/min into a detector monitoring 230 nm [ 277]. Responses were linear between 1 and 50 µg/ml, recoveries averaged 102%, and the ratio of R to S was 54-46%. Results of microbiological and HPLC assays correlated highly (r = O. 99). Another assay for the epimers also utilized an octadecylsilane column and a mobile phase of acetonitrile-0. 05 M ammonium acetate ( 3: 97) flowing at 1. 5 ml/min into a detector set to 275 nm [ 278]. Overall recovery of both isomers was 73-81%, with a linear range from 2. 5 to 50 µg/ml and a lower limit of detection for each isomer of 0. 5 µg/ml plasma or urine. Both isomers degrade in plasma at similar rates. A trimethylsilane column was used to assay epimers in body fluids using a mobile phase of O. 005 M aqueous n-heptylamine-methanol ( 89: 11) adjusted to pH 6 with phosphoric acid and flowing at 1. 4 ml/min into a 280 nm detector [279]. Detection limits were 1. 5 µg/ml of plasma and 7.5 µg/ml of urine. HPLC assays showed a negative 3% bias compared with microbiological plasma assays, but the precision is superior. A phenyl column was also used to determine R-S isomer ratios in serum and tissue using a mobile phase of acetonitrile-0.1 M ammonium acetate, adjusted to pH 5 with acetic acid (2:98), flowing at 1. 5 ml/min into a detector set to 270 nm [ 280]. The limit of detection was 1 µg/ml for each epimer with a linear response from 5 to 20 µg/ml. Clearance of R is higher than that of S-moxalactam. Nafcillin

Nafcillin in serum, urine, and protein-free filtrate was analyzed using a diol column and a mobile phase of O. 05 M aqueous diethylamine hydrochloride flowing at 1 ml/min into a detector set to 293 nm [ 281] . Responses were linear from 2 to 100 µg/ml, recoveries averaged 95%, and the miminum detectable quantity was lower than 1 µg/ml urine and 0.1 µg/ml serum and filtrate. Oxacillin

Oxacillin is a isoxazolylpenicillin used in treating diseases resistant to penicillin G. It can be quantified in body fluids after oral administration using an octadecylsilane analytical column and an ethylsilane guard column with a mobile phase of 0.03 M acetate buffer, pH 5.6-methanol (2:1) flowing at 1.5 ml/min through a 254 nm detector [ 282]. Responses to oxacillin and its active 5-hydroxymethyl derivative were linear in urine from 83 to 1620 µg/ml and

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74 to 920 µg/ml, respectively, as well as to the corresponding penicilloic acids from 20 to 200 µg/ml. Penicillin G

The cationic and anionic moieties of penicillin G (benzathine, penicillin G itself, and procaine benzylpenicillin) can be resolved. Benzathine penicillin utilized an octylsilane column at 50°C and a mobile phase of O. 067 M phosphate buffer plus O. 1% triethylamine, pH 5. 5-methanol ( 7: 3) flowing at 2. 4 ml/min into a detector set to 215 nm [ 283] • Procaine penicillin requires a pH of 7. 37. 5. Degradation products elute prior to penicillin G. Responses were linear between 0.1 and 1 mg/ml. Benzathine, procaine, and benzylpenicillin were separated in this order using an octadecylsilane column and a mobile phase of methanol-phosphate buffer, pH 3 (30:70), containing 0.005 M tetra-n-butylammonium phosphate flowing at 1. 5 ml/min into a 254 nm detector. Mixtures of the three salts in formulations were assayed with excellent agreement between found and expected values. Relative standard deviations were 2% [ 284] • Potassium penicillin G was resolved from its degradation products using an octadecylsilane column with a mobile phase of 1% monobasic potassium phosphate-acetonitrile ( 4: 1; adjusted to an apparent pH of 4.15 with HCl), flowing at 1. 3 ml/min into a 254 nm detector [ 285]. A similar system was used to study degradation by irradiation with 60Co, except for a mobile phase composition of acetonitrile-water-0. 2 M ammonium acetate buffer (20:70:10) adjusted to an apparent pH of 6.0 and flowing at 1 ml/min [286]. No effect was found. A 5-µm octadecylsilane column (150 x 4.6 mm) was used to resolve penicillin from penicilloic, penilloic, and penicillic acids using O. 008 M tetrabutylammonium chloride in O. 006 M phosphate buffer-acetonitrile ( 70: 30), pH 6-7.5, flowing at 1.5 ml/min into a 254 nm detector. Penilloic acid was partly resolved into two peaks, which may represent the R and S diastereomers [ 287]. Degradation in acid media was investigated using an anionexchange column with a mobile phase of 15.4 ml of 0.1 M citric acid and 7.0 ml of 0. 2 M disodium phosphate diluted to 650 ml water (pH 3. 8) flowing at 0. 7 ml/min into both refractive index and 254 nm UV detectors. Benzylpenicillenic acid, benzylpenilloic acid, penicillamine, benzylpenillic acid, and benzylpenamaldic acid were resolved [ 288]. The first compound was detected after 4 hr. and the last four were still present after 48 hr. Penicillin N

Penicillin N, which is a cephem derivative, can be assayed in reaction mixtures of cell-free microbial preparations using an octadecylsilane column with a mobile phase of 0.05 M phosphate buffer, pH 4-methanol (95:5) flowing at 2 ml/min through a detector set to 220 nm [ 289]. Isopenicillin N is not resolved from penicillin N unless a mobile phase of methanol-acetonitrile-acetic acid-water (36:7:2:55) is used flowing at 1.2 ml/min into a 254 nm detector after precolumn reaction with 2, 3, 4, 6-tetra-o-acetyl- S-D-glucopyranosylisothiocyanate [ 290] • Penicillin N could also be chromatographed from purified fractions using an octadecylsilane column and a mobile phase of pyridine-acetic acid-water (0.4:0.4:99.2) flowing at a rate of 1 ml/min into a refractive index detector [ 291] •

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Contents in fermentation broths were determined using an amino column with a mobile phase of acetic acid-methanol-acetonitrile-water (2:4:7.5:86.5) flowing at the unusually high rate of 4. 0 ml/min into a 254 nm detector [ 292]. Cephalosoporin C and desacetylcephalosporin C could also be resolved. Penicillin V

The benzathine portion can be resolved from the phenoxymethylpenicillin moiety using an octylsilane column at 50°C, and a mobile phase of O. 067 M phosphate buffer, pH 4. 5-methanol (3: 7) flowing at a rate of 1.8 ml/min into a detector set to 215 nm. p-Hydroxypenicillin V was also separated [283]. Penicillin V, p-hydroxypenicillin V, p-hydroxyphenoxyacetic acid, and phenoxyacetic acid were determined in broth using a 5- µm octadecylsilane column with a mobile phase of 1. 5% phosphoric acid adjusted to pH 3. 0 with ammonium hydroxide-acetonitrile (70:30) flowing at 1 ml/min into, first, a low-volume ( 2. 4 µl) ultraviolet detector adjusted to 220 nm and then an electrochemical detector monitoring at +0.8 V with respect to a Ag}AgCl reference electrode [ 293] • The UV detector measured penicillin V and phenoxyacetic acid, and the electrochemical detector helped determine the hydroxylated compounds. Responses were linear. Bulk drug and oral suspensions were analyzed using an octylsilane column and mobile phases of methanol-0.05 M phosphate buffer, pH 3.5 (53:47), flowing at 1 ml/min through a detector set to 274 nm [294]. Also resolved prior to V were p-hydroxyphenoxymethylpenicillin and the penicilloic, penilloic, and penicillenic acids, in this order. This type of kinetic data could be manipulated using a computer L295J with a BASIC program. Nonisothermal degradation was studied using this system to predict shelf life. An ion-pair system involved an octadecylsilane column with a mobile phase of methanolwater (67:33) containing 0.005 M hexanesulfonic, heptanesulfonic, or octanesulfonic acid in glacial acetic acids (Waters Associates, PIC reagents) flowing at 1. 5 ml/min through a 254 nm detector [ 296]. Piperacillin

Piperacillin was determined in plasma and cerebrospinal fluid using an octadecylsilane column with a mobile phase of methanol-0. 033 M phosphate buffer (40:60), adjusted to an apparent pH of 6.5, flowing at 1.5 ml/min into a 254 nm detector. Responses were linear between 10 and 60 µg/ml, with a limit of detection of 0.1 µg/ml [ 297]. Plasma contents were quantified using an octadecylsilane column and a mobile phase of methanol-0.01 M phosphate buffer, pH 4.8 (40:60), flowing at 1 ml/min into a 254 nm detector. Linearity was found for concentrations between 0.25 and 100 µg/ml with a minimum detectable limit of 0.05 µg}ml. Recoveries averaged 62% [ 298] . Pharmacokinetics in serum and urine were studied [299] using an octadecylsilane column and guard column (300 x 4 mm) with a mobile phase of 0.02 M phosphate buffer, pH 3. 5-acetonitrile ( 3: 1) flowing at an ungiven rate into a detector set to 210 nm. Although data for drug in body fluids were given, validating data for the assay were omitted. Pivmecillinam

Pivmecillinam, the prodrug of amdinocillin, can be analyzed in tablets together with the hydrolysis product and related compounds using an octadecylsilane

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column with a mobile phase of acetonitrile-0. 01 M sodium phosphate buffer, pH 3 (60: 40), flowing at 2 ml/min through a detector set to 220 nm [300). Recoveries averaged 100%, with excellent agreement found with results obtained by a spectrophotometric method. Arndinocillin and other related compounds could also be resolved. Sulbenicillin

Sulbenicillin in urine was determined using an octadecylsilane column and a mobile phase of methanol-0.01 M aqueous tetra-n-butylammonium bromide ( 4: 7) flowing at 3 ml/ min through a 254 nm detector. Responses were linear from 0.01 to 9 mg/ml of urine. Diastereomers were not separated [301). Ticarcillin

Assays in serum and urine utilized an octadecylsilane column (10 x 0.8 cm) with a mobile phase of O. 06 M sodium biphosphate-acetonitrile ( 100: 50. 5) adjusted to pH 2.05 with phosphoric acid, flowing at 1.5 ml/min through a detector set to 210 nm. Carbenicillin was used as internal standard [ 302) •

POLYENE ANTIBIOTICS

Polyene antibiotics contain three to eight conjugated double bonds and often show similar antifungal activity. They are classified according to the number of conjugations, according to the type of sugar moiety attached to the molecule or the absence of sugar, according to ring size, and according to the biological activity. These antibiotics present challenging analytical requirements because of the chemical sensitivity of the molecules and because of the difficulty in purifying them. As seen below, the use of HPLC for separation of the different members of these antibiotics for purification and for the quantitation of these antibiotics are described. The utility of HPLC in chromatographing polyene antibiotics is illustrated by the resolution of a single TLC spot obtained from the antibiotic AB- 315 into eight components [ 303) using a TSK-GEL LS410K column, and a mobile phase of methanol-formic acid-water (200:0.84:80) flowing at 0.5 ml/min into a detector. Filipin , eurocidin, nystatin, candidin, amphotericin B , hamycin , fungimycin, trichomycin, mediocidin, and candicidin eluted in this order [ 304) using an octadecylsilane column and a mobile phase of water-methanol-tetrahydrofuran ( 420: 90: 75) flowing at 0. 5-1. 0 ml/min into a detector adjusted to 350 nm. The aromatic heptaene antibiotics partricin, ayfactin, hamycin, trichomycin, aureofungin, and candimycin were chromatographed using an octadecylsilane column and a pH gradient: solvent A, acetonitrile-0.05 M citrate buffer, pH 5.3 (35:65); solvent B, acetonitrile-0.05 M citrate buffer, pH 6 (32:68), Ultraviolet detection was at 350 nm [ 305). The aromatic heptaene antibiotics candicidin and trichomycin were studied using an octadecylsilane column ( 1 m x 21.1 mm) at 55°C, with an initial mobile phase of O. 05 M phosphate buffer, pH 7-methanol ( 8: 2) changing to 100% methanol in a concave gradient of 33 min duration. Pure methanol was pumped for an additional 5 min before returning to the initial mobile phase. Detection varied between 325 and 425 nm. Candicidin and levorin appeared to be identical in this system [ 306) •

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Amphotericin B

Amphotericin B in serum and cerebrospinal fluid was quantified after mixing 1.0 ml with 3 volumes of methanol, storage at room temperature for 10 min, centrifugation at 2000 g for 10 min, and filtration through 0. 5-µm porosity filters prior to injection. The system used an octadecylsilane column and a mobile phase of methanol-aqueous EDTA (8: 2) flowing at 2. 5 ml/min into a detector adjusted to 405 nm [307). At concentrations of 0. 2-1.0 µg/ml, the relative standard deviation was 3. 6% using peak height measurements. The results agreed well with a more time-consuming microbiological assay. Amphotericin B could be separated from amphotericin A using an octadecylsilane analytical column and precolumn ( 100 x 4. 6 mm) with a mobile phase of 0.05 M sodium phosphate buffer, pH 2.6-methanol (3:7) flowing at 2 ml/min into a detector set to 313 nm [308) to accentuate the A content. During these studies, a previously undescribed heptaene was also detected. Candicidin

Candicidin was resolved into several components using a 5-µm octylsilane column and a mobile phase of acetonitrile-0.05 M ammonium acetate, pH 4.6 ( 37. 5: 52. 4), containing O. 1% dimethylsulfoxide, flowing at 1 ml/min into a detector adjusted to 355 nm. The individual constituents of candicidin are unstable in solution [309). Fungichromin

Fungichromin was chromatographed using an octadecylsilane column and a mob.He phase of methanol-water (6:4) flowing at 1.5 µl/min into a detector set to 245 nm [310). Hamycin

The constituents of the multicomponent heptaenic antibiotic hamycin were resolved using a 5 µm octadecylsilane column and a mobile phase of methanoltetrahydrofuran-0.1% ammonium carbonate ( 25: 25: 50) flowing into a detector operated at 380 nm (311). After assigning a potency factor to each component, linear algebraic equations were used to obtain biological activity. The potency estimates of a preparation by HPLC agreed well with a lengthy microbiological assay. Natamycin

Natamycin in cheese extracts was analyzed using an octylsilane column with a mobile phase of methanol-water ( 54: 35) and detection at 303 nm. The limit of detection was 20 ng per injection [ 312). Other systems were described [ 308) for natamycin using octadecylsilane columns and mobile phases of either methanol-water-acetic acid ( 48: 32: 1), or methanol-water-tetrahydrofuran (440:470:20, containing 1% ammonium acetate) flowing at 2 ml/min into a detector set to 303 nm [313). Nystatin

Nystatin in impure and purified pharmaceutical preparations was resolved into various constituents using a 3-µm octadecylsilane analytical column ( 100 x 4 mm)

HPLC

281

(bl

(a)

I

I

I

I

I

I

I

I

0 10 20 30 40 Min

I

I

I

I

I

I

I

I

I

I

I

0 10 20 30 40 50 Min

Figure 7 HPLC of nystatin: (a) impure sample; (b) purified preparation. (See text for details of the chromatography.) (From Ref. 315.)

and 5-µm precolumn (35 x 4 mm) and a mobile phase of 0.005 M sodium acetate buffer, pH 5. 8-methanol gradient [ 314) • Initially, the methanol content was increased from 55 to 58. 75% in 15 min, held constant for 10 min, and then increased to 67% in 10 min and maintained for 15 min. The flow rate was 1. 0 ml/min into a detector set to 304 nm. Figure 7a shows a chromatogram of the impure sample and Figure 7b the purified preparation [315). Unfortunately, these results did not correlate with microbiological assays. Using the LC method described, amorphous nystatin contents (determined from peak height) showed a good relationship with microbiological assays. Inexplicably, crystalline nystatins gave no such correlation, although these samples were 90-95% of one component based on countercurrent distribution studies. Octadecylsilane columns were used with a mobile phase of either methanol-water-tetrahydrofuran (35: 43: 22) or methanol-0. 05 M phosphate buffer, pH 2. 3 ( 75: 25), flowing at O. 4-1. 5 ml/min into a detector adjusted to 313 nm [316]. The latter solvent was also used to dissolve nystatin in many of these studies. POLYPEPTIDE ANTIBIOTICS

Some of the most powerful antibiotics known possess polypeptide structures. These polypeptide antibiotics are of limited use, however, at the present time, due to undesirable side effects, such as renal toxicity. Bacitracin

More than 22 components of bacitracin were resolved [316) using an octadecylsilane column ( 100 x 2.1 mm) and convex gradient elution starting with methanol-water-0.01 M potassium phosphate buffer, pH 4.5 (5:75:20), and, after 40 min, flushing with methanol-acetonitrile-water-0.1 M phosphate buffer ( 40: 20: 20: 20). Samples were usually dissolved in water. The flow rate was 2 ml/min into a 254 nm detector [ 317). Contents of bacitracins from various vendors and from different lots agreed well with microbiological

Kirschbaum and Aszalos

282 A

a, x

0

4

B

tt

u

H

N

H

n

TIME (Min)

H

~

"

u

n

~

"

Figure 8 HPLC of bacitracin showing the separation of a large number of components. (From Ref. 318.)

assays. Using a uniform octadecylsilane packing, this group improved the assay to increase resolution by using gradient elution of methanol-water-0.1 M phosphate buffer, pH 4. 5 ( 5: 8 5: 10) for A, and for phase B , methanolacetonitrile-water- 0. 1 M phosphate buffer (50:20:20:10), using y = [191/ (1 + e0.438 - 0.67X] - 78.5, from 1.5 min to 60 min, where x =time in minutes and y =percentage B with respect to A. The flow rate was 1 ml/min [318]. Figure 8 shows a typical separation. This system was also used to analyze polymyxin, circulin (to resolve circulin A and B from a mixture of components), and colistin (also resolving colistin A and B from related compounds). Zinc and methylene disalicylate bacitracin in feeds were determined using an octylsilane column and a mobile phase of acetonitrile-methanol-buffer (13.6 g KH2P04 and 2.5 g EDTA per liter)-water (24:7:20:49), adjusted to apparent pH 6. 8 with NaOH. The flow rate was 2 ml/min into a 254 nm detector [ 319]. Average recovery was 102%, relative standard deviation of repetitive injections of standard was about 1%, and resolution and peak slopes were described as excellent. Cyclosporin Cyclosporin A is a cyclic undecapeptide that is no longer of interest as an antimicrobial agent but is used as an immunosuppressive drug. It was quantitated using an octadecylsilane column at 70°C, with a mobile phase of acetonitrilewater ( 72: 28) flowing at 1. 7 ml/min into a detector set to 210 nm [319a]. Cyclosporin D was used as internal standard. The limit of detection was 20 ng/ml body fluid using a column switching technique for sample purificatior., and responses are linear from 20 to 5000 ng/ml. Gramicidin Gramicidins were resolved using a 5-µm octadecylsilane column at 60°C, with a mobile phase of 0.005 M ammonium sulfate-methanol (26:74) at a flow rate of approximately 1 ml/min into a detector monitoring at 220 nm [ 320]. The elution order is, in increasing lipophilicity, [Val]-Gdin C + [Ile]-Gdin C + [Val]-Gdin A + [Ile] -Gdin A + [Val] -Gdin B + [lle]-Gdin B, with a typical lot containing 78% A, 14% C, and 8% B (wfw). Results are in excellent agreement with those calculated on the basis of amino acid analysis.

HPLC

283

Linear gramcidins were separated using a phenyl column with a mobile phase of methanol-water ( 75: 25) flowing at 1. 2 ml/min into a 254 nm detector. Elution sequence is C + A + B. For preparative chromatography, a larger column was used [ 321] . Gramicidin content in tyrothricin and gramicidin was determined using a silica column with a mobile phase of n-hexane-ethanol (75: 25) flowing at 2 ml/min into a detector adjusted to 282 nm [322]. The results are in good agreement with those obtained by TLC using the one large peak observed. Polymyxin The polymyxins have a general structure of a cyclic heptapeptide moiety, a side chain consisting of a tripeptide with a fatty acyl residue and five or six 2, 4-diaminobutyric acid residues. A total of 17 members were resolved using a 5-µm octadecylsilane column with a mobile phase of 0. 005 M tartrate buffer, pH 3.0, containing 0.005 M sodium 1-butanesulfonate and 0.05 M sodium sulfate-acetonitrile (77.5:22.5) flowing at 1 ml/min into a detector set to 220 nm. Retention times increased in the order of fatty acyl residues, if their polypeptide portions were identical. Retention time of the polypeptides are in order of hydrophobicity [ 323] • Octapeptins can also be resolved by this method. Polyrr:yxins A, B , D, E, K, M, and P were separated into individual components using a porous poly(styrene-divinylbenzene) column with a mobile phase of methanol- 0. 2 M potassium chloride-hydrochloric acid buffer, pH 2 (1:1), flowing at 0.5-1 ml}min into either a refractive index, UV (210 nm) detector or, after reaction with o-phthalaldehyde, a fluorescence detector at 360 nm ex/450 nm em (cutoff filter). Various resins were investigated [ 324] . The curculin and colistin antibiotics can also be resolved into individual members using this procedure. Polymyxins B and E (colistin) were separated into 10-13 components using a 5-µm octadecylsilane column ( 150 x 4. 6 mm) and a mobile phase of buffer, 0.023 M phosphoric acid, 0.01 M acetic acid, and 0.05 Min sodium sulfate and triethylamine-acetonitrile (78:22 or 77:23) flowing at 0.9 ml/min into a detector adjusted to 220 nm. With the help of this system modified to largescale separations, three new components were identified; polymyxin 11, Oi. and L2. Elution order depends, in part, on the nature of the fatty acid residue [ 325] . Polymyxin B sulfate was resolved into B 1 and B 2 and colistin A and B using a 5-µm octadecylsilane column with a mobile phase of O. 05 M triethanolammonium phosphate buffer, pH 2. 2, containing O. 025 M 1-butanesulfonic acid and 25% acetonitrile flowing at 1 ml/min into a detector adjusted to 220 nm. Preparative HPLC was also performed [326]. Polymyxins Bl• B 2, and B 3 and related components were separated from polymyxin B sulfate using a 5-µm octadecylsilane column with a mobile phase of 0.1 M tribasic sodium phosphate-acetonitrile ( 77: 23) adjusted to pH 3 with phosphoric acid. The flow rate was 1 ml/min into a detector set to 200 or 185 nm. The resolution factor for B 2 and B 3 was 1. 5, indicating in complete separation [ 327] . Repetitive injections gave relative standard deviations of the major constituents of about 1%. Polymyxins B1 and B2 were resolved into a large number of components using a 5-µm octadecylsilane column and a mobile phase of 20-30% acetonitrile in O. 2% sulfuric acid and 0. 5% tetramethylammonium chloride flowing at 1. 1-1. 2 ml/min into a detector adjusted to 220 nm [ 328] . Responses of

284

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total area of polymyxins B 1 and B 2 are linear with concentration; the correlation coefficient was 0. 9999. Tyrothricin Tyrothricin, a mixture of 60-80% decapeptides and gramicidins, in tablets, was analyzed using an octadecylsilane column with a mobile phase of watermethanol (15:85) flowing at 1 ml/min into a detector set to 212 nm [329]. In lozenges, a similar system was used with a 5- µm column, the mobile phase ratio modified to 20: 80, and detection at 220 nm. The limit of detection was 60 ng tyrothricin injected [ 330]. Thiostrepton Thiostrepton was quantified using a 5-µm octylsilane column with a mobile phase of water-tetrahydrofuran-methanol ( 40: 40: 20) flowing at 2 ml/ min into a 254 nm detector [ 331] • Responses were linear with concentration, and the relative standard deviation of a series of injections of the same concentration was 0. 9% SULFONAMIDES AND RELATED DRUGS

Because sulfonamide antimicrobial agents have often been used to demonstrate the selectivity of hPLC, a large number of these agents can be assayed simultaneously. Thus, the analyst has a considerable choice of methods for a bulk drug. Figure 9 illustrates the separation of sulfanilic

1 . 81

-t U'.O 17.0 17.2 45. 0 24. 5 24.2 23.8

17.5 Baseline sep. Baseline sep. 17.5

11.0 Baseline sep.

Baseline sep. 11. 0 19. 5

9. 5:

sep. Baseline sep. Bas~ne

~etection was at 380 nm. buffer-acetonitrile (65: 35) flowing at 1. 3 ml/m!,n. Alternatively, 0.1 M citric acid-acetonitrile ( 76: 24) can be used with a flow rate of 0. 33 ml/min into a detector set to 350 nm to separate TC, methacycline, OTC, demeclocycline, and deoxycycline [383). A 10-µm octylsilane column was used with this flow rate and detection system and a mobile phase of O. 5% tetrabutylammonium hydrogen sulfate in water-acetonitrile (92: 8) or water-acetonitrile (80:20) to separate oxytetracycline and related compounds [ 384) . A general LC separation method for nine commercially important tetracyclines can be used for bulk or formulated drug [ 385) usi~g an octadecylsilane column and a mobile phase of 35-15% methanol in 0.0~01 M EDTA (pH 6. 6) fl.owing at 1-2 ml/min into a detector set to 380 nm. Retention times are shown .in Table 3. Bulk material usually dissolved in mobile phase. Formulati9ns generally are extracted with methanol and then diluted with mobile phase. Tetr~cycline, chlorotetracycline (CTC), OTC, doxycycline, and minocycline and !,:\1.eir possible impurities, 4-epitetracycline (ETC), epianhydrotetracycline (Et}J'!J), and anhydrotetracycline (ATC) could be resolved using an octadecylsilane column and a gradient system of 10 ml of 0.1 M tetra-ammonium ethylenediaminetetracetate, 50 ml of 1 M diethylamine, pH 7. 3 (adjusted with

HPLC

Minocycline

293

4-Epi-TC

Doxycycline

O~-TC

4-Epianhy.-TC

Anhy.TC

8.5

3.8

6.8

3,6

3.9

5.0

14.0

4.2

10.0

4,3

4.6

9.3

17.5

4.7 4.3

11.5

4.5

5.0

10.5

21. 0

4.7

15.0

5.7

5.8

28.0

37.0

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23.2

11, 0 11, 0

15.4

38.0

7. 2 Baseline sep • Baseline sep. 27.5 27.9 4.5 44.0 44.0

4.5 4.5 4.5 4.5 Base.line sep •

23.0 22.0 Baseline sep.

10. 0 4.5 4.2 4.8

47. 0 24.4

15.7

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5.5 13.0 6.5 6.7

38.0 8.5

26.0 26.0

8.5

26.3 48.0 26.5 26.8

85% phosphoric: acid), and 20-100 ml isopropanol flowing at 1. 5 ml/min into a 254 nm dete~t~r [386]. These compounds a~d demecycline, 6-epidoxycline, methacycline, epidemethylchlortetracycline, CTC, isochlortetracycline, epianhydrochlortetracycline, and anhydrochlortefracycline (ATC) [ 387] were investigated using an octylsilane column with a solvent of 0.1 M sodium phosphate and of 0.2 M of N,N-dimethyloctylamine (adjusted to pH 8 with NaOH)acetonitrile (70: 30) flowing at 1 ml/min in a detector set to 280 nm. Impurities in TC, OTC, doxycycline, CTC, methyacycline, demeclocycline, and ATC were 0.1-8. 4%. · Tetracycline, OTC, and CTC were analyzed in plasma and urine using an octadecylsilane column with a mobile phase of O. 01 M sodium phosphate buffer, pH 2. 4-acetonitrile (70: 30 or 60: 40) flowing at 1 ml/min into a detector set to 355 nm. Respective sensitivities in the two body fluids are 1 µg /ml, with a relative standard deviation of 5% [ 388]. The e..&raction procedure was subsequently modified to improve recoveries [389):" , Tetracycline, OTC, and 4-epitetracycline ~an. b~ qµa,~titated in honey using a phenyl or octylsilane column and either 0. ~~!VI phosphoric acid-acetonitrile ( 9: 1 or 95: 5) increasing in acetonitrile conte~t l:lY 5% mi~: 1 tp 40% [ 390]. The limit of detection is 1 µg I g honey.

294

Kirschbaum and Aszalos

Tetracycline The impurity contents of 2 9 tetracycline preparations were studied using an octadecylsilane column and a mobile phase of water-acetonitrile-70% perchloric acid (76. 2: 22: 1. 8) for TC and ETC and 61. 2: 37: 1. 8 for ATC and EATC flowing at 2 ml/min into a 254 nm detector [ 391]. These impurities plus CTC could be analyzed in a formulation using an octadecylsilane column and gradient elution with acetonitrile-water- O. 2 M phosphate buffer, pH 2. 5 (1: 8: 1), going to a ratio of 6: 3: 1 in 15 min and flowing at 1 ml/min into a detector operated at 380 nm. Phosphate ions promote epimerization so samples cannot be dissolved in mobile phase [ 392]. An assay for capsulated tetracycline products [ 393] utilizes an octadecylsilane column and a mobile phase of acetonitrile-water-ethanolamine-DMF (240: 760: 5: 60, v /v) containing 5. 2 g ammonium phosphate and adjusted to pH 2.5 with phosphoric acid flowing at 2.5 ml/min. The possible impurities are also resolved. Tetracycline in fermentation broths was assayed using an octadecylsilane column and a mobile phase of methanol-water-0. 2 M phosphate buffer, pH 2. 5 ( 30: 60: 10), changing in 15 min to the final solvent of methanol-acetonitrile-water-0.2 M phosphate buffer, pH 2.5 (50:20:20:10). Cofermented impurities can be separated and quantified [ 394]. Tetracycline in serum was extracted by adding 1 ml serum dropwise to 1 ml of 20% trichloroacetic acid in methanol, centrifuging, and filtering. It was assayed using an octadecylsilane column with a mobile phase of methanol-EDTA (3: 7) flowing at 2. 8 ml /min into a detector set to 355 nm. Recovery is close to 100%; sensitivity is O. 3 µg/ml [ 395]. This system was extended [ 396] to the analysis of lumecycline in plasma and urine using the same conditions except for a mobile phase of 0.01 M sodium phosphate buffer, pH 2.2-acetonitrile ( 84: 16). Linearity was found from 0.1 to 4 µg /ml. Tetracycline in urine was assayed using an octylsilane column with a mobile phase of isopropranol-1 M diethylamine buffer (adjusted to pH 7. 3 with phosphoric acid)-tetra-ammonium EDTA-water (11:5.1:83) flowing at 2 ml/min into a detector set to 365 nm [397]. The limit of detection was 0.1 µg/ml. HPLC assays were recently renewed [397a].

13-Acetotetrine S-Acetotetrine (2-acetyltetracycline) was determined in plasma and urine using a phenyl column and a mobile phase of O. 025 M potassium phosphate, 0.1% EDTA, pH 7.8-ethanol (93:7) flowing at 0.6 ml/min into an electrochemical detector: thin-layer carbon paste at an applied potential of +O. 58 V versus a standard calomel electrode [ 398]. The limit of detection is O. 25 pmol; linearity was found from 0. 5 to 10 µg/ml.

Doxycycline Doxycycline in serum was quantified using an octylsilane column with a mobile phase of acetonitrile- 0. 1 M citric acid (24: 76) flowing at O. 5 ml/min into a detector set to 350 nm [ 399]. This system can also separate OTC, TC, demeclocyline, methacycline, and a metabolite of doxycycline. The limit of detection is 50 ng /ml.

HPLC

295

TOPICAL ANTI-INFECTIVE AGENTS AND DISINFECTANTS

Topical anti-infective agents generally comprise antiseptics, which are applied to living tissues, and disinfectants, which are put on inanimate objects and rapidly produce a lethal effect. Representative agents include benzoic acid [ 400], benzalkonium chloride [ 401], chlorobutanol [ 402], clioquinol [ 403], ethacridine [ 404], gentian violet (hexamethylparosanaline chloride) [ 405], hexachlorophene [ 406], hexylresorcinol [ 407], iodochlorhydroxyquin (clioquinol) [ 408, 409], merulinic acids [ 410] , mildiomycin [ 411] , miloxacin [ 412, 413] , parabens [ 414] , perfloxacin [ 415] ; phenols [ 416, 417] , pipemidic acid [ 418] , propylene phenoxetal [ 419] , resorcinol [ 420] , salicylic acid [ 421] , thimersol [ 422] , and trichlorocarbanilide [ 423]. Figure 11 shows separations of methyl, ethyl, propyl, and butyl parabens (p-hydroxybenzoic acid esters) in less than 5 min using a conventional 30-cm octadecylsilane column packed with a 10-µm particles and separation in less than 30 sec using 3-µm octadecylsilane particles in a 4-cm column [ 414]. In addition, the ease of chromatographing the various parabens using 60: 40 to 40: 60 acetonitrile or methanol-water mobile phases have made them commonly used internal standards, with hundreds of literature citations. UNCLASSIFIED AND MISCELLANEOUS ANTIBIOTICS

Antimycin A

Antimycin A in tissues was chromatographed using a 5-um octadecylsilane column and precolumn (50 x 4 mm) and a mobile phase of O. 25 M acetate buffer, pH 5-methanol (25: 75). Detection of this antifungal agent was either electrochemical (using a glass carbon electrode at +l.00 V versus Ag/AgCl), fluorescence at 365 I 418 (after derivatization with dimethylaminonaphthalene-5sulfonyl chloride, or UV at 254 nm. The respective minimum detectable quantities were approximately O. 5, 0. 5, and 5 ng [ 424]. Chloramphenicol Chloramphenicol is an antibacterial and antirickettsial agent. Hydrolysis products were resolved using an octadecylsilane column, a water-methanolacetic acid (55: 45: 1) mobile phase flowing at 1 ml/min, and detection at 254 nm [ 425]. Cloramphenicol was separated from its six possible synthetic contaminants using an amino column and a mobile phase of tetrahydrofuran-cyclohexane ( 60: 40) flowing at 2 ml/min through a 254 nm detector [ 426]. It was analyzed in plasma with its succinate ester prodrug using an octadecylsilane column and a mobile phase of methanol-water-acetic acid (37: 62: 1) flowing at 1. 5 ml/min through a 254 nm detector. The respective limits of detection were 0.5 and 0.2 µg/ml, with a linear response of 0.5-60 µg/ml and an average of 99% recovery [ 427]. A similar LC system using O. 05 M acetate buffer, pH 5.3-acetonitrile (80:20) as mobile phase and detection at 278 nm was slightly less sensitive (0.5 and 1 µg/ml, respectively) but equally efficient with respect to extraction efficiency (101%) [428]. A micromethod for 0. 2 ml or less of plasma utilized a similar reversed-phase system and a

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Injection: 1 µI Sample: @Methyl @Ethyl @Propyl @Butyl parabens in order of elution

Injection: 10 µI Samples: @Methyl @Ethyl @'ropyl @Butyl parabens in order of elutlon

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Detector: UV-100, 280 nm. 0.05 sec Time Constant. 1.0 AUFS Flow: 6 ml/min

Detector: UV-100. 280 nm, 1.0 AUFS

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Solvent: 45% ACN, 55% H 2 0

Solvent: 45% ACN, 55% H 2 0 Flow: 2 ml/min

Instrument: Varian 5000 LC Column: MicroPak®-SP C 18 -3 µm, 4 cm x 4.6 mm

Column: MicroPak® MCH-10, 30 cm x 4 mm

(b)

Instrument: Varian 5000 LC

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Figure 11. Comparison of standard HPLC methyl, ethyl, propyl, and butyl parabens using a column 30 cm x 4 mm packed with 10-µm particles with short column, "fast" LC using a column 4 cm x 4.6 mm packed with 3-µm particles. (From Ref. 414.)

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HPLC

297

mobile phase of 95% ethanol-0.05% phosphoric acid, pH 2.5 (2:8). It was as sensitive as the previous' method [ 429] • After chloramphenicoi was allowed to react with liver microsomes, a number of metabolites were formec\ that could be resolved using a 5-µm octadecylsilane column and a mobile phase. ~f water-methanol-acetic acid ( 75: 25: O. 5) flowing at 1 ml/min through a detec£tpr set to 280 nm [ 430]. An ethylsilane column was used with~ia mobile pha.se· of 0. 01 M monobasic potassium phosphatemethanol ( 58: 42) flowing at L 5 ml/min and detection at 254 nm [ 431]. Chloramphenicol and its 111onosuccinate ester were separated in plasma using an octylsilane column, with a mobile phase of methanol-0. 05% aqueous phosphoric acid ( 4: 6) flowmg_,at 2. 5 ml/min through a detector set to 280 nm [ 432]. Ethyl paraben was useti(·as. inter~h>tandard. Extraction efficiencies were 51 and 45% , respectively. A 5-µm octyls:µane ilOlumn was used to resolve its nitro degradation products, directly o.~ in biologisial material, using a mobile phase of methanol-0. 01 M perchlotlc acid (plu~ 0.0005 M sodium 1-pentane sulfonate 15: 85) flowing at 2. 2 ml/~in and a ppJ.arographic detector-mercury electrode at -0. 5 V versus a Ag/AgC\ referena~ electrode and a Pt auxiliary electrode [ 433]. The linear dynami~ range wa~._2 ng to 20 µg, with a limit of detection of 1 ng. A cyano column was useci with a mobile phase of 20% methanol in water flowing at 1. 7 ml/min and detection at 280 nm [ 434]. The limit of detection was 2 µg/ml. Figure 12 shows a chromatogram of chloramphenicol in plasma using a octadecylsilane column with' a mobile phase of water-methanol ( 70: 30) flowing 0

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UV 970A VARIABLE WAVELENGTH Attenuation: .005 AUFS 0

8

16

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UV 965 PHOTQ-CONDUCTIVITY Attenuation: 10 x 10 8

16

Figure 12 Chromatography of chloramphenicol in plasma using two types of detector. (From Ref. 435.)

298

Kirschbaum and Aszalos

at 1 ml/min into a 254 nm detector. The chromatogram at the left was obtained with 50 ng/ml. To the right is the response obtained using a photoconductivity detector using 214 nm. The limit of detection is 10 µg/ml [ 435]. Clindamycin Clindamycin was quantified using an octadecylsilane column with a mobile phase of 1 g sodium dioctylsulfosuccinate and 1 ml formic acid in 125 ml water diluted to 500 ml with methanol [ 436] , and a refractive index detector. The related compound lincomycin can also be separated. Clindamycin, clindamycin B, and 7-epiclindamycin can be resolved using a octadecylsilane column with a mobile phase of methanol-0. 005 M 10-sodium camphor sulfonate-acetic acid (600: 400: 2) flowing at 1 ml/min into either a refractive index or a UV ( 214 nm) detector [ 437]. Clotrimazole Bulk and formulated clotrimazole, an antimycotic agent, was analyzed using an octadecylsilane column with a mobile phase of methanol-0. 025 M K2HP0 4 (3: 1) flowing at 1 ml/min into a 254 nm detector. Recoveries ranged from 99. 5 to 100. 0%, responses were linear from 2 to 40 µg injected, and relative standard deviations for material assayed on different days ranged from 0. 6 to 1. 8% [ 438]. The (o-chlorophenyl)d1phenylmethanol hydrolysis product was also resolved. Econazole Econazole in plasma was determined using an octadecylsilane column, with a mobile phase of 0. 01 M potassium dihydrogen phosphate-methanol (30: 70) adjusted to pH 4. 5 and flowing at 2 ml/min. Detection was at 220 nm using miconazole as internal standard [ 43S]. Responses for this antimycotic drug were linear from 0.2 to 5.0 µg/ml, recoveries averaged 85%, and the limit of detection was approximately O. U4 µg/ml. Erythromycin Commercial preparations of this drug were analyzed for erythromycin A (the major and most active antimicrobial component), B, and C, and related compounds, such as anhydroerythromycin A and dihydroerythromycin A, using an octadecylsilane column with a mobile phase composed of acetonitrile-methanol0. 2 M ammonium acetate-water (45:10:10:35), adjusted to pH 7.0-7.8 flowing at 1 ml/min into a detector set to 215 nm [ 440]. The relative standard deviation of this assay for erythromycin A was O. 6%, with good agreement found with results of microbiological assays. This procedure was modified to quantify fermentation broth contents [ 441]. Erythromycin was determined using an octadecylsilane column with a mobile phase of methanol-water-ammonium hydroxide (80: 19. 9: 0.1) flowing at 1. 4 ml/min into a refractive index detector [ 441a]. Ammonium hydroxide was used to suppress ionization of the basic group. Erythromycins A and B were determined in fermentation broths after extraction in isoamyl acetate using an octadecylsilane analytical column with an octylsilane precolumn ( 40 x 4 mm, 5-20 µm particle) and a mobile phase composed of methanol-water-ammonia (80:19.9:0.1) flowing at 1.5 ml/min into a detector adjusted to 215 nm. Microbiological assays gave approximately 20%

HPLC

299

higher results than did HPLC, perhaps due to the presence of erythromycins C to F. Recoveries of erythromycin A and B added to broth were 97 and 96%, respectively [ 442] • A new erythromycin, designated F, was isolated on a preparative scale [ 443] from mother liquors using an octadecylsilane column with a mobile phase of O. 025 M dihydrogen potassium phosphate-acetonitrile-triethylamine(600: 400: 1). Eryothromycin in biological fluids was quantified using an octadecylsilane column with a mobile phase of acetonitrile-methanol-0. 2 M sodium acetate, pH 6. 7 (40: 5: 55), flowing at 1 ml/min into a dual-electrode electrochemical electrode. The applied potential of the screen electrode was set at + O. 7 V and the sample electrode at 0. 9 V, to minimize responses of undesirable components. Recovery of the erythromycin A was nearly 100%, the responses were linear from 2. 5 to 50 µg I ml, and the detection limits in plasma were 510 ng/ml [444]. Other related erythromycins and degradation products were also resolved. Fusidic Acid

Fusidic acid is an antibiotic with high antistaphylococcal activity. In the form of bulk material or as the sodium salt in pharmaceutical preparations, it was quantified using an octadecylsilane column and a mobile phase composed of 0.01 M KH2P04, pH :::6.6-methanol (3: 1), flowing at 2 ml/min into a 254 nm detector [ 445] . Responses were linear between 50 and 1000 µg /1. An assay in serum, which also resolved metabolites, utilized a silica column with a mobile phase of hexanes-methylene chloride-ethanol ( 69: 25: 6) flowing at 1.8 ml/min and detection at 254 nm. Recoveries averaged 76% between O. 5 and 100 µg /ml. The limit of detection was 250 ng in 500 µl plasma [ 446]. Griseofulvin

This antifungal agent was assayed in bulk material and in formulations using a cyano column and a mobile phase of methanol-water (3: 2) flowing at 1 or 2 ml/min into a 254 nm detector [447]. Also resolved are griseofulvic acid, dechlorogriseoful vin , isogriseoful vin , and tetrahydrogriseoful vin. The 4and 6-demethylgriseofulvin metabolites can also be separated. A series of methods for assaying griseofulvin in plasma using reversed-phase octadecylsilane columns were reported. Using a mobile phase of 0.045 M potassium phosphate buffer, pH 3. 0-acetonitrile ( 55: 45) flowing at 2. 5 ml/min into a detector set to 295 nm , 0. 2- 5 µg of drug could be analyzed, with recoveries of 94% [ 448] • With a 100 x 3 mm column and a mobile phase of acetonitrile-water ( 40: 60) flowing at 1.2 ml/min into a fluorescence detector (260 nmex/389 nmem), linearity of response was found between 0.1 and 100 µg/ml [ 449]. Recoveries of griseofulvin added to plasma ranged from 94 to 100%. Using an acetonitrilewater ratio of 1: 1 and a flow rate of 2 ml/min and the same detector setting, a different extraction gave a linear range of O. 05-3. 0 µg}ml with 50-µl injection volumes [ 450]. Griseofulvin and its primary metabolite, 6-demethylgriseofulvin, were resolved using a mobile phase of methanol-0.5% aqueous acetic acid (61.5:38.5) flowing at 2.2 ml/min into a fluorescence detector (300 nmex/418 nmem>· Concentrations as low as 0.10 µg/ml could be quantitated using warfarin as internal standard. Recoveries from plasma averaged 91% [ 451] •

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Griseofulvin, 6-demethylgriseofulvin, and griseofulvic acid were quantified in biological fluid using a mobile phase of 0.1 M acetic acid-acetonitrile (55:45) flowing at 1 ml/min into a fluorescence detector (280 nmex/389 nmem) and a UV monitor set to 290 nm. 4- Demethylgriseofulvin could be resolved but was present in almost negligible quantities. The minimum detectable limits were 0.05 µg/ml. Recoveries of all compounds averaged 98% [452]. Lavendamycin

Lavendamycin is a new antibiotic related to streptonigrin. To monitor the purification from fermentation broths, an octadecylsilane column was used with a mobile phase of 0. 01 M potassium phosphate buffer, pH 8-acetonitrile ( 80: 20) [ 453] • No other details were given. Li neomycin

Lincomycin A and B (which are related to clindamycin) were quantified in fermentation broths using octylsilane analytical and guard columns at 45°C, with a mobile phase prepared by dissolving 2. 9 g of sodium dodecylsulfate and 10 ml of concentrated phosphoric acid in 660 ml of water, and then adding 330 ml acetonitrile. The apparent pH was adjusted to 6. 0 with concentrated ammonium hydroxide. The flow rate was 2 ml/min into a detector set to 214 nm [ 454]. Responses were linear from 1. 76 to 35. 2 µg lincomycin A per milliliter and 0.29-5.82 µg B per milliliter, and relative standard deviations from repeatability studies were 1. 2 and 6. 6%, respectively. Nalidixic Acid

Nalidixic acid is used intravenously to treat systemic infections, especially those of the urinary tract. To determine nalidixic acid and its active metabolite, hydroxynalidixic acid, in plasma, an octadecylsilane column was used with a mobile phase of water-methanol-cetrimonium bromide (cetrimide) ( 50: 50: O. 12) flowing at 1. 5 ml/min into a detector set to 313 nm [ 455]. The limit of detection of 1 µg/ml is well below the therapeutic range of 20-50 µg/ml. Recoveries are approximately 90%. Contents in plasma and urine were assayed using an amino-cyano column and a mobile phase of methanol-0.1 M citrate buffer, pH 3 (95:15 [sic]), flowing at 1.6 ml/min. Detection at 254 nm gave limits of detection of 0.08 µg/ml plasma and 0.42 µg/ml urine [456]. Nalidixic acid and its two metabolites were quantified in plasma and urine using an octylsilane column and a mobile phase of 7. 5 g/liter KH2P04 and 2.5 g/liter Na2HP04, adjusted to pH 8.2 with 1 M sodium hydroxide, plus 0.2% N,N,N-trimethylacetylammonium bromide-methanol (45:55) flowing at 0.6 ml/min into a detector set to 254 nm [457]. Limits of detection were below O. 5 µg/ml. The linear range was 1-50 µg/ml plasma and 100-200 µg/ml urine. Nifuroxazide

Nifuroxazide is used in the treatment of acute bacterial diarrhea. It was assayed in plasma using a 5-µm octadecylsilane column (150 x 4.6 mm) and a mobile phase of water acidified with phosphoric acid to pH 2. 5-acetonitrile (70:30) flowing at 1 ml/min through a detector set to 362 nm [458]. Responses were linear from 2 to 200 ng/ml. The relative standard deviation at 2 ng/ml

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was 15%, with 90% recovered; at 200 ng/ml, 100% was recovered with a relative standard deviation of 3%. Nimorazole Nimorazole, which is structurally related to metronidazole, was assayed in blood using an octadecylsilane column with a mobile phase of methanol-water (30: 70) containing 5 mM heptanesulfonic acid per liter and adjusted to pH 3. 2 with acetic acid. The flow rate was 2 ml/min, and detection was at 313 nm [ 459]. As little as 5 µl of blood could be analyzed by this method. Between 5 and 1000 µg/ml the extraction efficiency was 100%. The lower limit of sensitivity was 5 ng. Nitrofurantoin Nitrofurantoin is used in treating infections of the urinary tract. This drug and its active 4-hydroxylation ring metabolite were determined in urine using an octadecylsilane column and a mobile phase of methanol-0. 05 M phosphate buffer, pH 5 (30:70), flowing at 0.8 ml/min into a detector set to 320 nm [ 460] • Nitrofurantoin in plasma and urine was assayed using an octylsilane column with a mobile phase of water-ethanol ( 95: 5) flowing at 1. 6 ml/min through a detector set to 370 nm [461]. The limit of detection was 0.02 µg/ ml. Recoveries averaged 92%. As little as 20 µl fluid was needed. Nitrofural, nitrofurazolidone, metronidazole, and hydroxymethylnitrifurantoin could be resolved by this LC system. Nitroxoline Nitroxoline is used in the treatment of urinary tract infections of gram-positive and gram-negative microorganisms. It was quantified in plasma using an octadecylsilane column with a mobile phase of 8-hydroxyquinoline-0. 2 M aqeuous phosphate buffer-methanol-water (0.1:8:35:57, w/w) flowing at 1. 5 ml/min into a detector adjusted to 436 nm [ 462]. The limit of detection was 80 ng/ml plasma, with 95% recovery. The calibration curve was linear between 0. 08 and 40 µg/ml. At 0. 2 µg/ml, reproducibility studies showed a relative standard deviation of 8%; at 20 µg/ml, the relative standard deviation was 3%. Norfloxacin Norfloxacin is a broad-spectrum antibiotic structurally related to nalidixic acid. It was assayed in serum and urine using an anion-exchange analytical and guard column with a mobile phase of 0. 05 M phosphate buffer, pH 7acetonitrile (80:20) flowing at 1.2 ml/min into a detector set to 273 nm [463]. Limits of sensitivity were 0.1 µg/ml serum and 1 µg/ml urine. Standard curves were linear from 0.1 to 20 µg/ml serum and from 1 to 550 µg/ml urine. Respective recoveries from body fluids were 88 to 95%. Novobiocin Novobiocin is an antibiotic produced by Streptomyces. It was quantified in a bovine mastitis product using an octadecylsilane analytical column and precolumn (50 x 2 mm) with a mobile phase of 0.005 M 1-heptanesulfonate, sodium

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salt-methanol (20:80) flowing at 1 ml/min through a 254 nm detector. Recoveries averaged 101%. Novenamine, hydroxynovobiocin, novobiocic acid, desmethyldescarbamylnovobiocin, dihydronovobiocin, isonovobiocin, descarbamylnovobiocin, and two other unknown impurities can be resolved by this system [ 464] . Isomers and degradation products can be separated using an ECTEOLAcellulose ion-exchange resin (ET-41) with a column temperature of 70°C and a mobile phase of 0.002 M perchlorate-0.01 M phosphate buffer, pH 6.8 [465]. Oligostatin

The three component oligostatin complex was resolved from fermentation broths using an amino column with a mobile phase of acetonitrile-water ( 60: 40) flowing at 1 ml/min through a refractive index detector [ 466] • Pepleomycin

Pepleomycin sulfate (which is a glycopeptide related to bleomycin) and its metabolites were determined in body fluids and tissues using a 5- µm silica column ( 330 x 2 mm) and a mobile phase of acetonitrile-methanol-20% aqueous ammonium acetate-acetic acid (500:500:50:0.5) flowing through a detector set to 292 nm [ 467] . Rosaramicin

Rosaramicin, which is a macrolide related to erythromycin, has activity against both gram-positive and gram-negative bacteria. It was resolved from related compounds using an octadecylsilane column, and a mobile phase consisting of acetonitrile- 0. 01 M acetate buffer, pH 4 ( 3: 1) , flowing at 1. 3 ml/min. The limit of detection at 254 nm is about 0.015 µg/ml serum. Recoveries average 86% [ 468]. Rosoxacin

Rosoxacin was determined in serum, urine, and prostatic tissue using an octadecylsilane column with a mobile phase of acetonitrile-0. 2 M aqueous phosphoric acid (37.5:82.5 for serum and 32:68 for urine and tissue) flowing at 1. 5 ml/min into a detector set to 280 nm [ 469]. Cinoxacin was used as internal standard. Spiramycin

The 16-membered macrolide antibiotics spiramycin, turimycin, maridomycin, and propionylmaridomycin were chromatographed using an octadecylsilane column ( 100 x 3. 2 mm) maintained at 50°C with a mobile phase of acetonitrilewater-diethylamine (500:499.5:0.5) flowing at 0.8 ml/min into a detector set to 232 nm. The spiramycin content in bulk, tablet, and suppository formulations was determined [ 470]. Doubling the diethylamine content and diminishing the wavelength to 203 nm and the flow rate to 0. 5 ml/min resolved the various maridomycins. The spiramycin content could also be quantified using a silica column and a mobile phase of dichloromethane-propanol-water-diethylamine (956:40:4:0.002) flowing at 1 ml/min into a detector set to 232 nm. The turimycins could be resolved using a silica column as a gradient system

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303

of, initially, diisopropyl ether-isooctane-water ( 500: 500: 0. 004) and, finally, diisopropyl ether-methanol-water (500:497.4:2.6). The flow rate was 1 ml/ min into a detector adjusted to 232 nm. Sulconazole

The antimycotic agent sulconazole nitrate, in plasma, was analyzed using an octadecylsilane column and precolumn (4 x 80 mm) with a mobile phase consisting of acetonitrile-0. 01 M sodium phosphate buffer, pH 8 ( 66: 34) , flowing at 2 ml/min through a detector set to 229 nm [ 471] • Responses were linear from O. 5 to 5 µg/ml. Tunicamycin

The glycopeptide tunicamycin was resolved into 16 components, with four major fractions, using a 5-µm octadecylsilane column ( 40°C) and a mobile phase of methanol-water (68: 32 or 80: 20) flowing at 1 ml/min into a detector adjusted to 260 nm [ 472]. Vancomycin

Vancomycin is a bactericidal glycopeptide antibiotic active against a variety of gram-positive and some gram-negative cocci. It was quantified in serum using an octadecylsilane column and a mobile phase of 0. 05 M phosphate buffer, pH 6-acetonitrile (91: 9) flowing at 2 ml/min into a detector set to 210 nm. Recoveries averaged 99%, with a linear range of 2-128 µg/ml [473]. Another LC system was similar but with a mobile phase of 0.01 M 1-heptanesulfonic acid-acetonitrile (88: 12) and a wavelength of 280 nm [ 474]. This system could not quantify the antibiotic at concentrations below 5 µg/ml. However, microbiological and HPLC results agreed within experimental error. VETERINARY PRODUCTS

Some antibiotics used for animal health were discussed previously. In general, hereditary transmission of resistance factors by microorganisms across species, especially to humans, is causing fewer antibiotics to be used for animal health purposes. Amprolium

The poultry coccidiostat amprolium was analyzed in feeds using a 5-µm silica column with a mobile phase consisting of 8 g ammonium nitrate and 40 ml of 3% aqueous ammonium hydroxide in 1000 ml methanol {apparent pH 8. 1) flowing at 2 ml/min into a detector set to 270 nm [ 475]. A minimum of 5 mg/kg could be analyzed. At a concentration of 125 mg/kg, recoveries averaged 101% with a relative standard deviation of 3%. Aprinocid

The anticoccidial agent aprinocid was determined in animal feed at concentrations between O. 0045 and O. 0080% using a 5-µm silica column with a mobile phase of methanol-water-chloroform (containing 1% ethanol) in the ratio 3: O. 2: 97. Detection at 254 nm [ 476] showed an average recovery of 101%.

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Decoquinate The coccidiostat decoquinate was analyzed in animal feed using a 5-µm octadecylsilane column (150 x 4. 5 mm) and a gradient mobile phase of Amethanol-0. 001% aqueous acetic acid plus 1% magnesium sulfate heptahydrateand B-methanol. Initially, the ratio of A to B was 5: 95. It was changed to 80: 20 in 10 min using a convex gradient. During the next 5 min, a linear gradient changed the proportion to 95: 5, and then, in the next 5 min, decreased to 5: 95. The system was equilibrated for 10 min at the same ratio of 5: 95 before injecting the next sample [ 477]. The flow rate was maintained at 1 ml/min through a fluorescence detector at 314 nmex/390 nmem· Recoveries averaged 93% at concentrations as low as 1 mg/kg. Dinsed Dinsed and sulfanitran are almost always formulated together in animals as coccidiostats. They were separated and quantified using an octadecylsilane analytical column and precolumn ( 200 x 2 mm) and a mobile phase of acetonitrile-water ( 45: 55) flowing at 1 ml/min through a 254 nm detector [ 478]. Recoveries of sulfanitran at 0.03% averaged 102% and of 0.02% dinsed averaged 100%. 3, S-Dinitro-0-Toluamide 3, 5-Dinitro-o-toluamide (DOT) is a coccidiostat added to poultry feeds. It was analyzed using a 5-µm silica column, a mobile phase of acetonitriledichloromethane ( 1: 1) flowing at 2 ml/min, and detection at 270 nm [ 279]. Recoveries averaged 100% between 20 and 400 mg/kg, with good agreement with an existing colorimetric assay. Ethopabate The coccidiostat ethopabate was assayed in animal feeds using an octadecylsilane analytical column and precolumn and a mobile phase of acetonitrilewater (30: 70) flowing at 1.4 ml/min through a detector adjusted to 280 nm [ 480] • The limit of detection was 2 ng, and the linear range studied was 4-33 ng. Fu razoli done In feeds, furazolidone, an antibacterial agent against livestock diseases, and the related compounds carbadox, nitrofurazone, and ethopadate can be quantitated using an octadecylsilane analytical column and precolumn ( 100 x 2 mm) with a mobile phase of acetonitrile-1% aqueous acetic acid (20:80) flowing at 1.5 ml/min through a detector set to 365 nm (280 nm for ethopabate). Typical contents of 0.005% were assayed in good agreement with a colorimetric method [ 481]. In body tissues , a strong anion -exchange column preceded by a guard column ( 100 x 2. 1 mm) containing octylsilane ( 30- 40 µm) was used with a mobile phase of acetonitrile-water (25: 75) flowing at 1. 75 ml/min. Detection was at 360 nm [ 482]. Recoveries ranged from 89 to 98%. The limit of detection was 50 ng/mg.

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305

Halofuginone The anticoccidial drug halofuginone was analyzed in animal feed and tissues using an octadecylsilane column with a mobile phase of acetonitrile-0. 25 M acetate buffer-water (5:3:12) adjusted to an apparent pH of 4.3. The eluate flowing at 2 ml/min was monitored at 243 nm [ 483]. The limit of detection in feed is 1 µg/mg and in tissues is 1 ng/g. Respective recoveries averaged 93 and 84%. Lasalocid Lasalocid is one of a series of polyether antibiotics incorporated into feeds as a coccidiostat. Concentrates were analyzed using a silica column with a mobile phase of heptane-methanol-acetic acid (97. 6: 2: 0. 4) flow:ing at 1. 5 ml/min into a 254 nm detector [ 484] • Fluorescence detection was used ( 310 nm exl 418 nmem> to analyzed feeds containing 0.008%-0.0120%, with recoveries averaging 100% [ 485]. Monensin The anticoccidiosis and growth-promoting agent monensin, a polyether, was assayed in animal feed at high concentrations of 13.2% using an octadecylsilane column and a mobile phase of methanol-water ( 90: 10) flow:ing at 2 ml/ min through a refractive index detector. Recoveries averaged 103%. The responses are linear from 1 to 4 mg/ml [ 486]. Monensin A was separated from B using an octylsilane column with a mobile phase of methanol-water (88:12) flow:ing at 1 ml/min into a detector set to 215 nm. Minor components were also resolved. Preparative scale chromatography was also described [ 487] • Nifurpirinol Nifurpirinol in fish was analyzed using a 5- µm octadecylsilane colurru1 and a mobile phase of acetonitrile-water (44:55) containing 2.5 x 10-5 M acetic acid. Detection was at 390 nm [ 488). Recoveries averaged 85%, with a linear calibration curve from 25 to 150 ng. The minimum detectable quantity was 1. 25 ng. Nifursol Nifursol, which is added to feeds to prevent histomoniasis in turkeys, was analyzed us:ing a strong anion-exchange column and a mobile phase of O. 4% aqueous sodium perchlorate-acetonitrile ( 1: 1) flowing at 3 ml/min through a detector set to 365 nm [ 489) • Recoveries averaged 100. 6% over the range 25-100 µg I g, with a relative standard deviation of 3. 5%. Niridazole In addition to anthelmintic and antischistosomiasis activity, niridazole possesses immunosuppressive properties. It was analyzed in biological fluids using a 50- µm octadecylsilane column and a mobile phase of methanol-water ( 4: 1) flowing at O. 5 ml/min. Although the absorption maximum is 380 nm, a wave length of 254 nm can be used, with diminished sensitivity. The limit of detection is 50 ng /ml, and linearity was from 50 ng to 100 µg/ml [ 490).

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Nitrovin Nitrovin in animal feeds was analyzed using a 5- µm octadecylsilane column with a mobile phase of acetonitrile-dimethylformamide-0. 22 M sulfuric acid (30:30:40) flowing at 1.5 ml/min into a detector set to 375 nm [491]. Recoveries averaged 103% at concentrations of 5 and 15 mg/kg. Robenidine Robenidine hydrochloride is added to poultry feeds to prevent coccidiosis. It can be resolved from impurities and degradation products using a column packed with controlled pore glass (Corning) and a mobile phase of methanolglacial acetic acid-methylene chloride ( 90: 10: 900) flowing at 1 ml/min through a detector set to 280 nm [492]. Recoveries average 101%. A 5-µm silica column could probably be substituted, with minor changes in mobile phase composition. Tiamulin Bulk tiamulin, an animal antibiotic, was assayed using an octadecylsilane column with a mobile phase of methanol-acetonitrile-2 M aqueous ammonium chloride-10% ammonia ( 10 ml 30% ammonium hydroxide solution diluted to 100 ml in water; 700:200:40:80) flowing at 1 ml/min through a 254 nm detector [ 493]. Responses were linear between 2 and 8 µg /ml. Ammonium acetate can be substituted for ammonium chloride, we found in this laboratory. ACKNOWLEDGMENTS The authors thank Dr. Glenn Brewer, Dr. E. Meyers, Mr. Solomon Perlman and Dr. T. Platt for their many helpful comments and suggestions, the staff of the library at the Squibb Institute for Medical Research for obtaining a profuse number of references and searches, and Mrs. Connie Saloom and Ms. Lynn Mendenko for typing this manuscript. Literature was surveyed through May 1984 with the invaluable aid of Mrs. Muriel George. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.

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HPLC

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HPLC

190. 191. 192. 193. 194. 195. 196. 197. 198. 199. 200. 201. 202. 203. 204. 205. 206. 207. 208. 209. 210. 211. 212. 213. 214. 215. 216. 217. 218. 219.

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HPLC

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Kirschbaum and Aszalos

Sj. van der Wal and J. F. K. Huber, J. Chromatogr., 135, 287 ( 1977). J. Itoh, S. Omoto, T. Shomura, H. Ogino, K. lwamatsu, S. Inouye, and H. Hidaka, J. Antibiot., 34, 1424 (1981). H. Takayama, M. Itoh, S. Mizuguchi, H. Abuki, M. Ishibashi, and H. Miyazaki, J. Antibiot., 31, 895 (1978). C. Lin, H. Kim, D. Schuessler, E. Oden, and S. Symchowicz, Antimicrob. Agents Chemother., 18, 780 ( 1980). K. M.-E. Jensen, R. B. Patel, P. G. Welling, and P. 0. Madsen, Prostate, 3, 523 ( 1982). G. A. Bens, Verh. K. Acad. Geneeskd Belg., 44, 131 (1982). M. Fass, B. Zaro, M. Chaplin, and S. Matin, J. Pharm. Sci., 70, 1338 ( 1981). W. C. Mahoney and D. Duksin, J. Chromatogr., 198, 506 (1980). J. R. Uhl and J. P. Anhalt, Therapeut. Drug Monitor., l, 75 ( 1979). J. B. L. McClain, R. Bongiovanni, and S. Brown, J. Chromatogr., 231, 453 (1982). G. B. Cox and K. Sugden, Analyst, 101, 738 (1976). D. W. Fink, J. Ass. Offic. Anal. Chem., 64, 973 (1981). A. Hobson-Frohock, Analyst, 107, 1195 (1982). K. L. Eaves, B. M. Colvin, A. R. Hanks, and R. J. Bushway, J. Offic. Agr. Chem., 60, 1064 (1977). I. W. Burns and A. D. Jones, Analyst, 105, 509 (1980). L. R. Schrank, B. M. Colvin, A. R. Hanks, and R. J. Bushway, J. Ass. Off. Agr. Chem., 60, 1048 ( 1977). V. A. Thorpe, J. Ass. Offic. Anal. Chem., 63, 981 ( 1980). G. F. Ernst and A. van der Kaaden, J. Chromatogr., 198, 526 ( 1980). A. Anderson, E. Goodall, G. W. Bliss, and R. N. Woodhouse, J. Chromatogr., 212, 347 ( 1981). R. B. Hagel, J. Ass. Offic. Anal. Chem., 61, 2070 (1978). M. Osadca and M. Araujo, J. Ass. Offic. Anal. Chem., 61, 1074 ( 1978). T. D. Macy and A. Loh, J. Ass. Offic. Anal. Chem., 66, 284 ( 1983). M. Beran, J. Tax, V. Schon, z. Vanke, and M. Podejil, J. Chromatogr., 268, 315 (1983). K. Otsuka, K, Horibe, A. Sugitani, and F. Yamada, Shokuhin Eiseigaku Zasshi, 23, 373 ( 1982), CA, 98, 84435e ( 1983). G. M. George, L. J. Frahm, and J. P. McDonnell, J. Ass. Offic. Anal. Chem. , 64, 969 ( 1981). J. J. Miller, B. M. Jones, P. R. Massey, and J. R. Salaman, J. Chromatogr., 147, 507 ( 1978). M. J. Gliddon, C. Gordon, and G. M. Parnham, Analyst, 108, 116 ( 1983). J. B. Zagar, P. P. Ascione, and G. P. Chrekian, J. Ass. Offic. Anal. Chem. , 58, 822 ( 1975). D. Bergin, personal communication, 1984.

8 Thermal Analysis HAROLD JACOBSON

E. R. Squibb & Sons, Inc., New Brunswick, New Jersey

Instrumentation and General Principles Thermogravimetric Analysis Studies of Compatibility in Antibiotic Formulations Characterization of Different Polymorphic and Pseudopolymorphic Forms Assessing Stability Purity Analysis General Characterization and Compilation of Thermal Analytical Data of Antibiotics References

324 326 327 330 332 336 336 338

This chapter is limited to the discussion of differential thermal analysis (DTA), the variation called differential scanning calorimetry (DSC), and thermogravimetric analysis (TGA) as applied to materials science problems in the field of antibiotics research. Thermal analysis [ 1- 3] has found wide acceptance in the last two decades for characterizing materials of very diverse natures and has found perhaps its greatest utility with polymers. The relative ease of recording data, because of the availability of many excellent and readily operated instruments, and the meaningfulness of the characteristic information developed makes these tools an indispensable part of a laboratory's armamentarium. Too, the physical phenomena of interest are almost always associated with the absorption or evolution of calories or heat. Thermal analysis readily detects these changes. Applied to physical states, it will monitor the presence of crystalline polymorphs and pseudopolymorphs; it can detect a solid-solid polymorphic transformation; it can detect disassociation phenomena (for example, of aggregated forms); so-called glass transitions (relaxation phenomena) are observable, as well as the common transformations of melting, freezing, and vaporization. It will also furnish data related to chemical changes: a common usage is the detection of desolvation; it will detect thermal degradation

323

Jacobson

324

(pyrolytic or explosive); chemical reactions with other species, either in the solid state or in solution, can be studied; and other phenomena associated with a flow of calories are observable. Because of the foregoing aspects of thermal analysis, it is well suited for constructing phase diagrams. Also, it has been used to measure purity, both qualitatively and, albeit with limiting restrictions, quantitatively. INSTRUMENTATION AND GENERAL PRINCIPLES

The essential features of a differential thermal analysis instrument are comprised of three parts as follows (Figure 1): 1.

2. 3.

An oven, provided with a heating element that can be controlled to give chosen linear heating rates, generally from less than a degree per minute to about 100 degrees per minute, Two sample containers within the oven's environment that will accommodate the sample of interest and a reference material. Typically, these containers require less than 10 mg of material. A detection system: In the DTA system, thermocouples are used for measuring the temperature of the sample and of the reference, and because they are wired in series opposition, they provide the temperature difference between the sample and the reference.

Another mode of differential thermal analysis exists that is referred to as differential scanning calorimetry (Figure 2). With some instrumental designs,

4T + +

Heating Block

w l!J

Sample

Reference

Figure 1 Essential features of a differential thermal analysis system.

325

Thermal Analysis

Silver Ring

Heating Block

Heater

....-'V"'-1• '·----Radiation Shield Bell Jar

Thermoelectric Disc

Convection Shield

Heating Block Gas Purge Coolant Vacuum,

Alumel Wire

CELL CROSS SECTION

Figure 2 Basic features of a differential scanning calorimeter cell (Du Pont).

this terminology refers to refinements in the design of DT A systems so that data can be obtained in more quantitative and reproducible ways. The earlier use of the DSC terminology, which is still in use, referred to a system in which the calories required to maintain a constant temperature difference between a sample and a reference were directly measured. This was accomplished by measuring the amperage flowing into small heaters next to the sample, the flow of amperes so controlled as to be proportional to developing temperature differences. In this manner a direct measurement of the calories involved was obtained. In a typical DTA or DSC measurement, a linear heating rate is maintained that is ordinarily plotted on the abscissa as the temperature of the sample. Sometimes it is the temperature of the oven that is plotted. The former is used more frequently and is best suited for arrangements in which small pans are used to contain the sample and reference. In another configuration, where the sample and reference materials are contained in melting-point capillaries, which in turn are embedded in cylindrical holes drilled into the oven block, it is the temperature of the oven at a point close to the sample that is monitored. In that instance, the oven and sample temperatures are nearly equal because the mass of the oven is great relative to that of the sample and its container. With capillaries, the difference in temperature between the sample and reference is measured via thermocouples imbedded directly into the sample and reference substances. Given on the ordinate scale of the DTA plot is the temperature difference between the sample and reference or, in a DSC, the differential heat flow between the sample and reference. Figure 3 illustrates a generalized thermogram. In typical thermoanalytical measurement, the temperatures of the sample and reference rise together according to a selected (programmed) heating rate. These temperature, ideally, are equal. In actual practice, a constant difference develops because generally the heat capacities and heat conductivities of the sample and reference materials do not match exactly, nor do the heatflow resistances of the interfaces between the pans and heaters. The

Jacobson

326 E (Exo)

c 4T

(Endo) Temperature,

•c

Figure 3 Generalized example of a differential thermogram. Peaks A, B, and D are endotherms caused by the absorption of heat. Peaks C and E and exotherms representing the evolution of heat.

plot, thereby, exhibits a sloping baseline or a baseline offset from what would be obtained if sample and reference properties were exactly matched. Most instruments include an electronic compensation circuit such that a sloping baseline can be made to approximate a horozontal one. An offset baseline can sometimes be used for measuring heat capacity. In the absence of a thermal event, a baseline continues to be recorded. When a thermal event or transition takes place, such as melting, the sample becomes isothermal; the calories entering the sample are absorbed in the transition process (for example, melting) and do not cause a rise in temperature. Concurrently, the calories entering the reference cause the temperature of the reference to rise. The result is the development of a temperature difference that is generally recorded on the ordinate. When the transition is complete, the sample temperature quickly reaches that of the reference because of the relatively massive heat source proximal to both reference and sample. The recorder tracing returns to baseline. The absorption of calories results in what is called an endotherm; the release of calories results in an exotherm. The latter can occur, for example, when a sample oxidizes in the presence of air or if a solid-solid transformation occurs to a less energetic crystalline form. THERMOGRAVIMETRIC ANALYSIS

The technique of thermogravimetric analysis [ 1, 3, 4] is that of constantly weighing a sample as its temperature is either held constant or linearly raised. Typically, the amount of sample is of the order of 5-30 mg. The atmosphere surrounding the sample can be controlled as to the type of gas (air, oxygen, and nitrogen, for example), the humidity of the gas, and whether the gas is static or flowing at a specific velocity. Several different designs are available. In one design, a small oven is moved into a position nearly surrounding the sample; in another design, the balance, including the sample, is moved on a track so that the sample is enclosed within an oven. Both types appear to be suitable. The balances used are electronic microbalances of differing designs but all of high quality. Details of the different types will not be described here.

Thermal Analysis

327

Suffice it to say that all have electronic tares and that their controls allow weight suppression and greatly expanded sensitivities. Thermogravimetric analysis is an excellent tool for measuring the total voltailes content of a material that itself does not readily volatilize. In general, it can give a measure of the volatility, but care must be excerised that subliming solids do not condense on cooler parts of the balance beam. It will provide information about whether an exotherm or endotherm that may be observed in a DTA measurement is coincident with weight losses or gains. The most significant drawback to TGA is the danger of not taking a representative sample, because the sample weight is often very small. Another observed problem is that, occasionally, occuluded volatile material cannot be forced out of a solid mass before the latter decomposes, which is generally also accompanied by weight losses. The technique, however, remains a complement to DTA, and in the process of characterizing drug materials, the two techniques are often employed. In what follows, specific examples illustrating the employment of thermal analysis in the field of antibiotics will be given. Most, if not all, of the examples can be used as a basis for studying similar problems with other antibiotics and, for that matter, with most other materials. STUDIES OF COMPATIBILITY IN ANTIBIOTIC FORMULATIONS

In general, thermal analysis will provide information when solid-state interactions take place. The extension of this idea to the study of formulations of tablets or capsules and of parenterals is clear [ 5, 6]. For example, in an unstable capsule formulation of sodium dicloxacillin monohydrate, the ingredient responsible for the instability was identified [ 7]. The formulation was composed of about 70% antibiotic, 23% lactose, 4% magnesium stearate, and 3% stearic acid. For the study, differential thermal patterns were taken of the nominally pure antibiotic; of a mixture of the antibiotic and the lactose; of a mixture of these two items plus magnesium stearate; and, finally, of a mixture of all the components. The results are shown in Figure 4, where details of the compositions are also given. The preparations were made using laboratory conditions that, as closely as possible, approximated those used in preparing the original formulation [ 7] • In Figure 4, thermogram 1 is that of the nominally pure sodium dicloxacillin monohydrate. The endotherm at 178°C represents the dehydration of the antibiotic, and the exotherm at about 223°C represents oxidative and thermal degradation. Thermogram 2 of Figure 4 is that of a mixture of the cited dicloxacillin and lactose, one of the excipients used in this formulation. The salient characteristic here is the retention of thermal features of each of the two components. Pure USP lactose shows endotherms at 148 and 218°C, with decomposition beginning at the latter temperature. The combined thermogram thus shows the endotherms due to the lactose, the dehydration of the sodium dicloxacillin nonhydrate, and finally an exotherm that, although of a lower temperature than the nominally pure dicloxacillin, is characteristic of the dicloxacillin. The depression of the temperature of the exotherm of the dicloxacillin is probably caused by the presence of the lactose degrading in this temperature region. The significant conclusion that can be made from thermogram 2 is that no interaction took place between the two materials.

328

Jacobson

--c:

( .) 0

50

100 150 200 250 Temperature, °C

Figure 4 Thermograms of sodium dicloxacillin and mixtures thereof. Key: ( 1) dicloxacillin; ( 2) 76% dicloxacillin + 24% lactose; ( 3) 73% dicloxacillin + 23% lactose + 4% magnesium stearate; ( 4) 71% dicloxacillin + 22% lactose + 4% magnesium stearate + 3% stearic acid; ( 5) 93% dicloxacillin + 7% stearic acid.

The addition of magnesium stearate to the two components already present also showed no interaction (thermogram 3). Pure magnesium stearate gives a melting endotherm at 125°C, and an endotherm at this temperature is just barely discernible in the mixture. It is small because of its low concentration in the mixture. Thermogram 4 is of the mixture now including stearic acid, and a significant effect is observed. The endotherms present are those due to stearic acid (50°C) and lactose (148°C); those due to the sodium dicloxacillin monohydrate have disappeared. To confirm the effect of stearic acid, a mixture of it and dicloxacillin was made and thermogram 5 was recorded. Again, the disappearance of those features associated with the dicloxacillin is noted. It is clear that stearic acid interacted with the dicloxacillin and that it was the cause for the instability of the original formulations. Equally interesting is that this incompatibility could be predicted. Thus to extend this concept, the influence of stearic acid upon the stability of sodium oxacillin monohydrate, potassium penicillin G, and ampicillin trihydrate was examined [ 7] • The thermograms are shown in Figure 5. Each system was made up of 95% of the penicillin of interest plus 5% stearic acid. The two ingredients were mixed in a mortar and pestle and thence slugged using a 1 inch die under 5000 psi pressure, and finally reduced through a number 24 screen. The thermograms clearly indicate that, similar to what had been observed with dicloxacillin, an interaction has taken place between the stearic acid and the oxacillin as well as with the penicillin G. However, no interaction took place with the ampicillin trihydrate. The stability profiles of these mixtures as well as those of the pure penicillins were studied in capped vials at 50°C for 8 weeks. The results showed that the oxacillin and penicillin G in mixture with stearic acid lost about 10%

329

Thermal Analysis A >.

1

~ ,.....------.-_ -=c

.c ~

0

Microbiological Assay

363

they form zones of inhibition by themselves, as in Reference 134, it is not satisfactory [ 9, 20] because of the possibilities that the interferences may add to, synergize, or antagonize the activity of the measured antibiotic. Recent experience with this approach is shown in Table 12. Generally, unless the test organism supplies an enzyme capable of specifically degrading the interfering activity, or when the mode of action of the interferent excludes activity against the test organism, the approach will not succeed. By contrast, removal or blocking the interfering activity in a mixture can be more successful, as shown in Table 13. Use of S-lactamase to remove S-lactams, blockage of aminoglycoside activity with salts, detergents, and lower pH values, blockage of sulfonamides with PABA ( 50 mg /liter of medium) and imidazoles with thymidine (5 mg/liter of medium) are the usual methods of coping with these kinds of interference. The presence in the mixture of S-lactam antibiotics resistant to S-lactamase can render this method ineffective, however, unless an effective S-lactamase is used, as described for the usually resistant moxalactam [ 22] , or conditions are selected to render it effective as described for aztreonam. These procedures also require knowledge of likely interferences. This approach should always be used with caution because of the possible intrusion of an interference not covered by the interferant measures employed. It appears as a general technique, that use of a resistant culture together with Slactamase to inactivate S-lactams, blocking of tetracycline activity with divalent cations, and performing the assay at the lowest possible pH to reduce aminoglycoside interference are the most effective measures. In addition, where the interferant has relatively little effect under the test conditions, modest dilution often will suffice to eliminate the interference [ 75, 148] , providing the interference is not synergistic. Protein Binding

The long-recognized influence of body fluids on antibiotic activity [ 1] is usually ascribed to protein binding [ 149] • Body fluids usually, but not always [ 1] , cause a decrease in apparent activity in vitro compared to that found when the same concentration of antibiotic is tested in water or phosphate buffer. This is shown for aztreonam in Figure 1 by the method described later. Binding is inversely correlated with protection against in vivo bacterial challenge [ 150]. The effect of sample sometimes is more than a simple offset, as the dose response of the antibiotic in a body fluid may be nonparallel to that in water or buffer. Traditionally, microbiological assays, although they measure only free, unbound antibiotic, have been corrected for sample binding to give results in terms of total activity. There are four ways in which this problem has been handled: (1) using control, antibiotic-free fluid as nearly the same as the sample in composition for diluting the standards and sarr.ples and keeping the proportion of body fluid constant in all test solutions, ( 2) performing the test at a dilution sufficient to avoid sample interference, (3) separation of interfering constituents of the sample from the antibiotic, and ( 4) combinations of methods 1 and 3 with method 2. The first method is most frequently used with serum because of the relatively constant composition of this type of sample and the ready availability of control serum. Caution should be exercised with respect to the use of commercially available pooled normal human serum as control fluid and substitution of animal serum for human serum because differences in

Gentamicin with

Gentamicin with

Gentamicin with carbenicillinresistant K. pneumoniae Amikacin with multi-resistant Gentamicin with K. edwardsii NCTC 10896

Cancer chemotherapeutic agents

Various antimicrobials

Carbenicillin

Penicillins

Penicillins, cephalosporins, erythromycin, imadazoles sulfonamides

E. coli

K. pneumoniae ATCC 27799

K. pneumoniae ATCC 27799

Aminoglycosides with K. pneumoniae ATCC 27799

Erythromycin

Assay

Not tested in mixture

Gross interference by penicillins

30% High bias

Interference by colistin , polymyxin B , nalidixic acid, amikacin, tobramycin

Interference by 5-fluorouracil Slight interference by doxorubicin, methotrexate, vinblastine

No interference

Result

20

9

9

19

136

135

Reference

Specificity Imparted to Microbiological Assay by Test Organisms Resistant to Possible Interfering Antibiotics

Interfering antibiotics

Table 12

0:.

c..

"l:l

S' ..... .....

""'

137 137 137 137 80 138

Little or no interference Good agreement with HPLC Good agreement with HPLC Good agreement with HPLC Good agreement with HPLC Good agreement with HPLC No interference No interference

Cefotaxime with E. coli Tobramycin with K. edwardsii NCTC 10,896 Tobramycin with S. aureus Alkmaar Tobramycin with S. aureus Alkmaar Tobramycin with S. aureus Alkmaar Cefotaxime with P. aeruginosa Aminoglycosides with Enterobacter cloacae Benzylpenicillin with Neisseria meningitidis, carbenicillin with Proteus mirabilis

Desacetylcefotaxim e

Clindamycin

Tetracycline

Chloramphenicol

Amoxicillin

Desacetylcefotaxim e

Penicillins

Clindamycin

139

78

100

Interference observed

Ketoconazole with C. pseudotropicalis

5- Fluorocytosine

100

No interference

Ketoconazole with C. pseudotropicalis

Amphotericin B

w 0) c.n

'


e.

c:;·

'Cl

dO' s· s

c:;·

E::

142 Greatly reduced interference

0. 6-1. 0% Sodium polyanethol sulfonatc in the assay medium O. 6-1. 0% Sodium polyanethol sulfonate in the assay medium 6% NaCl in the medium

75 mM CaClz/liter of medium

13-Lactams, macrolides, tetracycline in presence of aminoglycosid es

Moxalactam and cefotaxime in presence of aminoglycosides

13- Lactams and erythromycin in presence of aminoglycosides

Assay of clindamycin in presence of aminoglycosides

Blocked gentamicin activity, no effect on clindamycin

141 Eliminated carbenicillin

13-Lactamase

Tobramycin in presence of carbenicillin

143

135

77

141,46

Cellulose phosphate, 50 mg/O. 5 ml serum sample

140

Reference

Carbenicillin in presence of aminoglycosid es

Low bias for penicillins; high bias for cephalsporins

Result

Cellulose phosphate, 50 mg I 0. 5 ml serum sample

Means

Removal of Interfering Antibiotics from Mixtures

13- Lactams in presence of aminoglycosid es

Assay

Table 13

w

""Cl

s ..... .....

°'°'

147 146

Penicillins removed satisfactorily Good agreement with HPLC

Blocked tetracycline activity versus Sarcina lutea

Penicillinase, 1-2 x 105 KU/ml serum What man S-lactamase , 1 U I ml, + resistant test organism, S, aureus Alkmaar 5% magnesium sulfate in assay medium S- Lactamase 6% NaCl in medium plus rifampin resistant B, sub tilis PABA, 100 mg/liter in assay medium

50 mg cellulose phosphate/ml serum

Gentamicin in presence of carbenicillin or piperacillin

Tobramycin in presence of cefuroxime, cefamandole, cephalothin, azlocillin

Benzylpenicillin in presence of tetracycline

Ceftriaxone in presence of ampicillin

Vancomycin in presence of rifampin and aminoglycosides

Trimethoprim in presence of sulfamethoxazole

Piperacillin or carbenicillin in presence of gentamycin

Penicillins recovered

Ampicillin activity eliminated

Moxalactam interference removed

S-Lactamase II

Tobramycin in presence of of moxalactam

119

89

144

137

146

22

145

Thymidine, 5 mg /liter, Whatman S- Iactamase

144

Aminoglycosides in presence of imidazoles, penicillins

Tetracycline activity blocked

5% MgSO 4 in the medium

Benzylpenicillin in presence of tetracycline

""O:l

~

'
Cl.>

:;i,..

p

-

c:;·

c.q

0

c;·

tr

c

"":

~

c:;·

368

Platt

100

~

;:. u · (ii

40

(])

a:

20

0

20

40

60

80

100

Serum Content of Test Solutions, %

Figure 1 Effect of serum content of test solutions on the relative activity of aztreonam determined by agar diffusion assay.

antibiotic binding will be reflected in the values reported for sample potencies; the greater the antibiotic binding of the control lot compared with binding by samples, the greater is the apparent potency of the samples measured against standards prepared in the contr9l lot. Careful comparisons of proposed new lots of control° fluids to assure tli,at they bind antibiotic activity to the same degree as the previously used lot'are required to maintain consistent results. This can best pe determined by comparing recovery of antibiotic from the spiked control fluids with standards in a non binding fluid, such as phosphate buffer. Simp~ comparison of zone sizes is not satisfactory because the previously noted logarithmic relation between zone sizes and antibiotic concentration will tend tq mask the differences. When all precautions are observed, excellent agreement between antibiotic activity in serum and radioactivity from administration of radioactive antibiotic can be achieved, as shown, for example, in Figure 2 for cephradine. The second and third methods are appropriate for excretory sample types, such as urine, bile, and feces, where samples are 11pt constant in composition and thus the control material has little actual value afi a control. Whether dilution or separation of antibiotic from the sample matrix is best will depend on the antibiotic concentration in the sample and the type of sample. Dilution cim be a means of removing assay bias caused by abnormal sample composit~on , for example in the assay of cefuroxime in serum from renally impaired patients [ 42} . Dilution almost always is adequate for urine, but bile and feces usually require more than simple dilution. Feces require sterilization q~ separation from native, resistant microflora when low dilutions are tested. Tqus, the third metho.d, that is, to free as much as possible of the antibiotic from the sample matrix, is preferred. Acetonitrile or methanol deproteinizatiqp. usually will nearly free the antibiotic from serum binding as well as effect practical sterilization. An example pf the fourth method is the development of two methods for aztreonam in fierum. At dilutions of serum of 1:20 and above, most of the

369

Microbiological Assay 50

10

E ..... Cl

5

.,:I.c

:c

~

.:;;;

a a.

1.0 0.5

0.1

0

2

3

4

5

6

Time (Hr)

Figure 2 Comparison of total cephradine in serum measured by radioactivity and microbiological assay corrected for serum binding.

65% binding by serum disappears (Figure 1). However, since the therapeutic concentration extends below sensitivities obtainable at a 1: 20 dilution, 0. 4 µg/ml, a second sample preparation was developed employing methanol deproteinization to extend sensitivity to O. 06 µ g/ml. Antibiotics in Tissue

Unlike most other drugs, which act on the homeostatic mechaniSms of the host, antibiotics act on the agent of infection within the infectious site. Except for rarely occurring bacteremia, blood concentration does not bear directly on the infectious process. Thus, although tissue concentrations can be predicted from blood concentrations [ 149] , the more usual approach is td measure actual antibiotic concentration in tissue. Penetration of antibiotic to these sites is diminished by protein binding and inactivation by enzymatic action and is affected by the physicochemical characteristics of the antibiotiC; that is, pKa , lipid solubility, and by the site itself, local pH, and presence of active transport [ 151] . Methods of assaying antibiotics in solid tissues are inore complex than those for body fluids because antibiotic in tissue can be ioeated in the blood portion of the tissue, in the interstitial space, and in or bn the tissue cells [ 152]. Freezing of tissues for storage or sample preparation may alter the distribution of antibiotics among these spaces, but is a common operation in most tissue work. Since the object of this type of assay is td measure antibiotic transfer to the site of infection, the interstitial fluid; gross assay of whole tissue as performed for regulatory purposes [ 153] clearly will be

370

Platt

inaccurate because it will include antibiotic in the blood portion of the sample. Washing the outside of the sample to remove blood cannot remove blood from within the tissue and has the potential for removing extravascular antibiotic, too. Four approaches have been used for assay of tissue antibiotics, and they achieve the desired measurement of interstitial antibiotic to varying degrees. 1. Blood-free interstitial fluid is collected in skin windows, implanted tissue cages or fibrin clots, blisters (reviewed in Reference 154) , or implanted threads [ 155] • Microbiological assay of the fluid or threads is performed by conventional agar diffusion techniques. This procedure is the most direct method for interstitial fluid but has analytical and practical difficulties that prevent its application to all tissues, for example, internal human organs. The principal problem is the required operations to develop blisters or implant devices in the test subject. In addition, adequate amounts of control fluid for construction of standard curves must be found. However, the exact composi tion of most interstitial fluids is not known and is different depending on the method used to collect it [ 76, 154] . For cantharides-induced blister fluids, standards in 70% serum [ 33] and 75% serum [ 156] were used to match the protein content of blister fluid. Suction-induced blister fluids were reported to contain 60% of serum total protein [ 157] . 2. Small pieces of washed tissue are embedded in or laid on the assay agar [42,43,95,155,159]. Results from diffusion of the bioactivity can be converted to total activity by use of standards made from control tissue of the same type that has been allowed to imbibe known amounts of the antibiotic [ 42] or, probably much less accurately, by substitution of other fluids for control tissue. Assay of thin disks of eye tissue directly applied to the test agar for [14c]benzylpenicillin, cefamandole, or gentamicin was found to yield 85-90% recovery versus standards applied on paper disks using radioactivity diffused into the agar as the marker for total antibiotic [ 159] • Some of the antibiotic in the tissue sample must diffuse from the blood portion, and thus this method is not an unequivocal measure of interstitial fluid antibiotic. 3. Tissue is digested with crude proteolytic enzyme mixtures before assay [ 160, 161] • Antiibiotic activity is determined by agar diffusion using standards prepared in control tissue and subjected to the same enzyme treatment as the samples. This method measures total antibiotic in the tissue sample in the manner of the regulatory tests. 4. The great majority of the preparation methods involve extracting antibiotic from minced, ground, or pulverized tissue and removing the solids by centrifugation, as in the recent references listed in Table 14. Assay is by agar diffusion, usually with standards prepared in control tissue. Dilution of samples with four parts of extractant is most often used. This ratio must be maintained, if further dilution is required, by dilution in control tissue extract. Correction for antibiotic in samples contributed by blood in the samples is performed using hemoglobin as the blood marker [ 82] • Unless this correction is made, serious error is possible in estimation of antibiotic in the extracellular tissue compartment [ 162]. Calculation of extravascular extracellular tissue antibiotic is most accurately calculated [ 163]: Tissue = antibiotic

(

gross ~ntibiotic)

m tissue

-

(antibiotic contributed) ( 1-fraction) by .;of blood tissue blood in tissue

371

Microbiological Assay

Table 14 Recent References to Extraction of Antibiotics from Minced, Ground, or Pulverized Tissue Antibiotic

Tissue

Reference

Aorta

Clindamycin, cefazolin, cefoxitin

48

Adipose, gallbladder, muscle

Piperacillin

102

Atrial appendage

Cefonicid

125

Bone

Cefuroxime

202

Bone

Cephalothin, cefamandole

203

Bone

Metronidazole, clindamycin, cefoxitin, ticarcillin, chloramphenicol, moxalactam

189

Bone

Cefamandole

145

Bone

Cefoxitin, cephalothin

143

Bone

Tobramycin, azlocillin

113

Bone

Cefamandole

103

Bone

Rifamycin

71

Bone

Cefoxitin, cephalothin

41

Bone

Cefotaxime

35

Bone, skin , tendon

Ketoconazole

116

Brain

Nafcillin, methicillin, cefazolin

155

Gallbladder

Cefoperazone

135

Gallbladder

Kanamycin, amikacin

181

Heart valve

Methicillin

123

Heart valve, liver, muscle

Methicillin

144

Heart, liver, kidney, lung

Fosmidomycin

Histological components

Ampicillin

Muscle

Amoxicillin, clavulanic acid

95

Prostate

Moxalactam

61

Soft tissue

Cephamycin

185

86 141

Platt

372 EXAMPLES OF DETAILED METHODS

Cylinder Plate Method-Aztreonam Assay of over 20,000 samples of serum, urine, and other fluids, as well as tissues, for aztreonam have been performed by the cylinder plate method given below [ 164]. The method correlates well with HPLC (correlation coefficient, O. 990) and has good precision (CV = 4. 7) and sensitivity ( 0. 06 µg /ml of serum). If other S-lactam antibiotics are present in the samples, the samples should be treated with S-lactamase. However, because aztreonam is a S-lactamase inhibitor [ 165] large amounts of enzyme activity are required. With the larger amounts of enzyme some reduction of aztreonam activity is also observed. When the samples contain a penicillin, deposit solutions of penicillinase, 105 KU /ml (Difeo or BBL) in approximately 5 µl volumes of pH 6 phosphate buffer to the outside of each cylinder at the agar-cylinder interface. Capillary effects distribute the enzyme in a band at this junction, thus ensuring the treatment of the sample with a relatively constant amount of enzyme. The reference solution cylinders on each plate also must be treated to compnesate for the 10-15% loss of aztreonam activity found on treatment of the sample cylinders. When the samples contain first- and second-generation cephalosporins, treat them in the same way with Miles Laboratories cephalosporinase at 50 units/ml. Third generation cephalosporins and low-reactionrate penicillins will interfere even after this treatment. Thus, moxalactam, cefoxitin, cefapirin , ceftriaxone , cefazolin, cloxacillin, and dicloxacillin interfere. No other interferences are known. Inoculum Maintain the test culture, Escherichia coli Squibb Culture No. 12155, on slants of medium 1 (media key, Tables 2-6). Prepare the inoculum from cultures on the same medium in Roux bottles incubated for 24 hr at 37°C. Use 50-60 ml saline per bottle to suspend the growth, and determine the absorbance at 530 nm in a 1 cm cell. Adjust the cell concentration to 7 absorbance units/ml. Store the suspension at 0-5°C. It is usable for 1-2 weeks. Plates

Adjust the pH of medium 1 to 7. 9 after sterilization, cool it to 48- 50°C, inoculate it with cell suspension equivalent to a total of about 200 AU /liter of medium, and promptly dispense 7 ml into sterile, flat-bottom, 100 mm Petri dishes. Allow to solidify for about 20 min on a level surface. Store at 0-5°C for up to 2 weeks in plastic bags. These plates become increasingly sensitive with time of storage, as noted. The unusually high pH of the assay medium for assay of a S-lactam antibiotic is used because it yields well-defined zone edges and a dose-response slope of 3 or more. Preparation of Test Solutions Sensitivity, 0. 4 µg/ml serum: For routine work where sc.nsitivities better than 0.4 µg/ml are not required, dilute samples 1:20 in 1% phosphate buffer, pH 6. O. Dilute those samples with potencies exceeding 2. 5 µg/ml further with buffer-pooled normal human serum (95: 5) to give zone diameters within the limits of the standard curve. Prepare standards (from the Squibb Institute for Medical Research) at 2.5, 2.0, 1.5, 1.0, 0.6, and 0.4 µg/ml in the bufferserum mixture.

Microbiological Assay

373

Sensitivity, O. 06 µgjml Serum: Dilute serum samples with equal volumes of methanol, mix well, allow to stand at room temperature for 10 min, and clarify by centrifugation. Further dilute with an equal volume of the buffer. Prepare the standards for these test solutions in pooled normal human serum at concentrations of 0.06, 0.1, 0.2, and 0.4 µg/ml and process them in the same manner as the samples. Other Body Fluids, Except Bile: Peritoneal, pleural, cerebrospinal, bronchial, and synovial fluids, as well as protein-free filtrate of serum and urine, can be assayed versus a standard curve in 1% phosphate buffer, pH 6. 0, providing the samples are diluted 1: 5 or greater in the buffer. Standard concentrations are 0.02, 0.03, 0.05 (reference concentration), 0.08, and 0.13 µg/ml of buffer. Store control fluids supplemented with aztreonam together with the samples at -70°C or lower until assayed. Determine recovery from these control fluids with each assay run for each of the fluids. Recoveries range from 80 to 90%, and the dose responses are parallel to those of the phosphate buffer curve. Control cerebrospinal fluid can be obtained commercially (Fisher Scientific). Protein-free filtrate of serum can be made in the laboratory by ultrafiltration (Amicon CF 50A Cones, Amicon Corp. , Danvers, Massachusetts). Pleural and peritoneal fluids are produced by injecting saline into the appropriate cavities of cows and harvesting the fluid, after a period of hours, yield 10-20% (Agrilab, Bridgewater, New Jersey). Other fluids must be obtained from participating hospital laboratories. Nominal potencies found for the samples are normalized to 100% recovery using the recoveries of the appropriate control fluids. Bile: Bile cannot be assayed by simple dilution and application to plates as are the other fluids because recovery at low concentrations is very poor. Instead, dilute 1 ml of bile sample with 10 ml of saturated NaCl solution and suspend the mixture in 4 ml of methylene chloride to remove the bile salts. Add 1 ml of O. 6 N citrate buffer, pH 3. 7, to 8 ml of the clarified aqueous layer and pass it through a C 18 Sep-Pak cartridge (Waters Associates, Milford, Massachusetts). Wash the cartridge with 8-10 ml of water, and elute it with 3. 8 ml of methanol-1% phosphate buffer, pH 6 ( 4: 6). Dilute the eluate with 1. 2 ml of the pH 6 buffer to bring the methanol content to 30%. Assay it against a standard curve in methanol-pH 6 buffer (30: 70) with aztreonam concentrations of 0.0125, 0.025, 0.025, and 0.05 µg/ml, using the plates described. This procedure recovers about 96% of the aztreonam from bile at concentrations of O. 2 µ g/ml and above with a CV of 5%. Assay Design and Calculations

The conventional six cylinder Grove and Randall [ 8] small-plate assay is used. Set six stainless steel cylinders 10 mm high, 6 mm inner diameter, 8 mm outer diameter on each plate about 60° apart on a 2.8 cm radius. A cylinder setting machine (Farmer Machine Co., Trenton, New Jersey) allows this operation to be performed rapidly. Fill each of three alternate cylinders on each plate with reference solution and the remaining three with a sample or standard solution. The reference solution of aztreonam is 0.05 µg/ml of 1% phosphate buffer, pH 6.0. Use one to three plates for each sample dilution. The reference cylinders on each plate must be filled within a minute or two of filling the test solutions. As noted earlier, it is important to deliver uniform, if not exactly known, volumes to each cylinder. Experienced technicians can perform this operation by

374

Platt

complete filling of each cylinder with syringe-controlled Pasteur pipettes. Otherwise, air displacement pipettes set to deliver 250-280 µl should be used. Incubate the plates at 37°C for 18-24 hr, remove the cylinders, and determine the sizes of the zones of inhibition. Determine the average size of the reference solution zones and test solution zones on each set of plates. Determine the average reference solution zone size on the standard curve plates. To each average test solution zone size for each of the sets of plates containing individual standards or test solutions, algebraically add the difference between the average reference zone size on the set and the average reference zone size of the standard plate sets to yield a corrected zone size for each test solution. Plot a least-squares standard curve of ln standard concentrations versus corrected zone size, using a hand-held calculator, such as a Texas Instruments 55, and calculate potencies of test solutions using their corrected zone sizes. Turbidimetric Assay-Tetracycline in Urine Although turbidimetric assays are only occasionally used [ 166-168] for body fluids and tissues because of the laborious pipetting required, the inconveniently short incubation time, and the interference by color or turbidity at low dilutions, the method is capable of excellent precision [ 169] and semiautomatic equipment is available for performance of the test (AutoTurb, Elanco, Indianapolis, Indiana). The method given here was developed at Squibb [ 170] , has a sensitivity of O. 5 µg/ml with a relative 95% confidence limit of 6% for the mean value from four tubes. Inoculum

The test culture is Streptococcus faecium ATCC 10,541. It can be maintained by monthly transfer in stabs of Micro Inoculum Broth (Difeo) containing 1. 5% agar. Incubate 24 hr at 37°C and store at 0- 5°C. Prepare assay inoculum by growth of the culture in the assay medium (Penassay Broth, Difeo, supplemented with 1% glucose, 0.5% yeast extract, and 1% tryptone) under microaerophilic conditions for 18 hr at 37°C. This inoculum can be used for 5 days when stored at 0- 5°C. Alternatively, adjust the pH of the culture to 6. 8, dilute with sterile glycerol to provide 5. 9% glycerol in the inoculum, and store at -20°c in 3. 3 ml volumes. Inoculum stored in this way is usable for at least 3 years. Inoculate 2 liters of the assay medium, with 2 ml of inoculum or with a vial of cryogenic inoculum, thawed at 37°C. Hold the inoculated medium in ice while filling assay test tubes. Test Solutions

Urine samples should be kept cold and buffered to pH 4. 5 during collection. This can be done by adding 10 ml of 1 M phosphate buffer, pH 4. 5, to each collecting vessel and storing the samples during collections at 0-5°C. At the end of the collection period, the volume is corrected for the buffer and a small portion stored at -20°C until assayed together with buffered "control" urine supplemented with known amounts of the antibiotic. On the day of assay, thaw the samples and controls at room temperature. Sterilize them by filtration through sterile disposable membrane filters, 0.45 µm (Nalgene), and dilute in 0.1 M phosphate buffer to give test solution concentrations in the range O. 5-3 µg/ml.

Microbiological Assay

375

Assay Procedure and Calculations

Pipet 0.1 ml portions of the test solution into each of four test tubes and add 10 ml of inoculated medium. Include a complete set of staIJ.dard test solutions in each rack of test tubes. Incubate the racks of tubes at 37°C for 3 hr. Continue incubation but check the turbidity difference between the highest and lowest standard concentrations. When the difference is about O. 3 AU at 580 nm, transfer the racks to a 80°C water bath for 3 min. Cool the tubes to room temperature, and read the responses at 580 nm. Using a calculator, plot standard concentrations versus their average absorbance and calculate the correlation coefficient. If it is O. 9910 or above, indicating that the curve concentrations fall on a straight line, calculate sample concentrations from their average absorbance values. If the correlation coefficient is below 0. 9910, try plots of ln standard concentration versus percentage transmission and 1n standard concentration versus absorbance. If none of these variations provide a straight line, evaluate the individual tube readings for outliers and consider dropping the highest or lowest concentration standard from the standard curve. Disk Method-Antibiotics in Serum

Radial diffusion of antibiotics from disks dates from the earliest reports of antibiotic assay [ 1]. The method is favored when sample is limited, precision is not so critical, and equipment and procedure complexity need to minimized. An excellent example of this type of assay was outlined by Peterson et al. [ 171] using three test cultures and three assay media to provide methods for 27 individual antibiotics. The most sensitive of these assays are shown in Table 15. Inoculum

Grow Micrococcus luteus ATCC 9341 and Staphylococcus aureus ATCC 6538P overnight at 37°C in Trypticase Soy Broth (BBL, Cockeysville, Maryland) on a shaker with 3 mm glass beads. Maintain the culture by storing some of this culture on glass beads at - 70°C. Prepare assay inoculum by overnight culture started from some of the -70°C beads under these conditions. Dilute the cultures 1: 10 in sterile Trypticase Soy Broth. Dilute a stock spore suspension (BBL) of Bacillus subtilis ATCC 6633 1: 6 in sterile water. Plates

Inoculate 1 liter of sterile assay media at 50°C with 1 ml of diluted inoculum. Pour 6 ml of inoculated medium into 100 mm sterile, plastic, flat-bottom Petri dishes, and allow them to solidify on a level surface. Store the plates at 4°C. They are usable for 14 days. Preparation of Test Solutions Dissolve antibiotic standards to produce 1000 µ g /ml solutions and dilute to 40 µg/ml of pooled normal human serum. Then serially dilute with the serum in twofold steps provide standard solutions of 0.078-20 µg/ml. Prepare erythromycin standards in the same way except make the 1000 µg/ml solution in acetone

376

Platt

Table 15 Disk Assays of Antibiotics Antibiotic Aminoglycosides Amikacin Gentamicin Kanamycin Neomycin Streptomycin Tobramycin Penicillins Ampicillin Benzylpenicillin Cloxacillin Dicloxacillin Methicillin Nafcillin Oxacillin Cephalosporins Cefaclor Cefamandole Cefazolin Cefoxitin Cephacetrile Cephaloridine cephalothin Cephapirin Cephradine Other antibiotics Chloramphenicol Clindamycin Tetracycline Vancomycin Erythromycin

Test culturea

Mediumb

Sensitivity (µg/ml serum)

6 6}13 6 6/13 6 6/13

5 5/17 5 5/17 5 5/17

0.312 0.312 0.625 0.312 0.625 0.312

1 1}6}13 13 13 1 13 1

1 1 1 1 1 1/5 1/5

0.078 0.078 0.312 0.312 0.625 1. 25 0.625

1 l/ 6/ 13 6/13 13 6 13 13 6 6 1

1 1 5 1/5117 1 1 115117 l/ 5/ 17 115/ 17 1

0.312 0.312 0.312 0.625 2.5 0.625 0.078 0.625 0.312 0.156

1 17 1/ 5/ 17 1 1 17

5.0 5.0 0.312 0.312 1.25 0.078

1 6 1 6 6 1/6

aSee Key to Assay Organisms for Tables 2 to 6. bsee Key to Assay Media for Tables 2 to 6. Source: From Peterson et al. [68].

Microbiological Assay

377

Dilute samples with potencies exceeding the highest standard concentration in the pooled normal human serum so that their zone diameters fall within the standard curve. Assay Design and Calculations

Arrange six pairs of 6.35 mm filter paper disks (Schleicher and Schull, Inc., Keene, New Hampshire) on a wire screen and deposit on three pairs of them 20 µl volumes of three standard test solutions. As standard test solutions use the sensitivity concentration for the antibiotic shown in Table 15 and the next two higher serial concentrations. On the remaining three pairs of disks deposit 20 µl of each of three serum samples. Transfer the disks to a pair of plates, alternating standard disks with sample disks on a radius of about 2. 8 cm at 60° relative to each other to provide a complete standard curve and one disk of each sample on each plate. Incubate the plates at 37°C for 24 hr. Read the diameters of the zones of inhibition to the nearest 0.1 mm. Calculate the potency of the sample test solution from its average zone diameter from a plot of ln standard concentration versus average standard zone diameters on the two plates. Agar Well Method This method was originally applied to 14 antibiotics using four test organisms in a large plate assay [ 133] • In the 1980-1983 survey shown in Tables 2-6, 25% of the papers using agar well methods still cited this 1966 paper. The assay medium is nutrient agar, 2. 7 mm thick, with eighty-one 4. 5 or 5. 5 mm wells. Since the paper was published, sterile, plastic assay plates, 23 x 23 cm, have become available from Denmark (NUNC, Vlihgard International, Neptune, New Jersey) and the method as described below was adapted to this more convenient plate. Inoculum

Maintain the test cultures, Sarcina lutea ATCC 9341 (M. luteus ATCC 9341), Bordetella bronchiseptica ATCC 4716, and Bacillus cereus ATCC 11778, by monthly transfers on nutrient agar slants. Prepare inoculum as shown in Table 16. Store the inoculum suspensions in 2 ml volumes at 4°C. Use each vial only once. The inocula are stable for 4 weeks. Plates

Add the volumes of inocula suspensions shown in Table 16 to melted nutrient agar adjusted to a temperature of 50°C, mix gently, and pour the mixture into a NUNC dish. Allow the medium to solidify on a level surface, and cut wells in a uniform 6 x 6 or 7 x 7 pattern using the mouth-suction operated steel-cylinder cutters described in the original paper or one of the commercially available cutters (Saxby Ltd., Liverpool, England, Tom-Tee Orange, Connecticut). The assay also can be successfully performed in conventional plastic Petri disches (83 mm effective inner diameter) using 15 ml of medium and the wells cut with a Grafar punch (Gr afar, Detroit, Michigan). Prepare the plates required for the antibiotics to be tested as shown in Table 17.

0.2 0.5

0.1 0.3

Tryptose phosphate broth (Difeo) culture, 37°C/16 hr Surface culture , blood agar, 30°C/16-20 hr

Bordetella bronchiseptica

Source:

From Reference 133.

cereus ATCC 11,778

0.3

(2 vials/100)

Difeo Spore suspension

B. subtilis ATCC 6633

B,

0.3

Inoculum used/100 ml assay agar (ml)

0.47

Inoculum concentration dilute in tryptose phosphate broth (absorbance at 550 nm)

Surface culture, nutrient agar, 37°CJ24 hr

Produce by

Inoculum Preparation for Agar Well Assays

S. lutea ATCC 9341

Inoculum

Table 16

w

"O

s ..... .....

Oo

"'I

379

Microbiological Assay

Table 17 Agar Well Antibiotic

Dilute standards to ( µg/ml)

o. 5,

Well size (mm)

Test culture

4.5

B.

1, 2, 4, 8, 16

4.5

B. subtilis

Kanamycin

1, 2, 4, 8, 16

5.5

B. subtilis

Cephalothin

0.625, 1.25, 2.5, 5, 10

4.5

B. subtilis

Methicillin

1, 2, 4, 8, 16

4.5

B.

subtilis

Benzylpenicillin

0.25,

1, 2, 4

4.5

B.

subtilis

Penicillin

0.25, 0.5, 1, 2, 4

4.5

B.

subtilis

Vancomycin

0.5, 1, 2, 4, 8

5.5

B.

Cloxacillin

1, 2, 4, 8, 16

5.5

Dicloxacillin

3, 6, 12, 24, 48

5.5

Nafcillin

0.35, 0.7, 1.4, 2.8, 5.6

5.5

s. s. s.

subtilis

Oxacillin

1, 2, 4, 8, 16

5.5

s.

Tetracycline

0.25, 0.5, 1, 2, 4

5.5

B. cereus

Colistin methane-sulfonate

0.5, 1, 2, 4, 8

5.5

B.

Ampicillin

0.25,

Cephaloridine

o. 5,

1, 2, 4

subtilis

lutea lutea lutea lutea

bronchiseptica

Source: From Reference 133.

Preparation of Test Solutions As specified in the original paper, prepared standards in pooled normal serum at the concentrations shown in Table 17. Dilute serum samples in the same manner, so that their responses fall within the limits of the standard curve. Assay Design and Calculations

Fill wells to the top with samples and standards; about 64 µl per 5.5 mm is required. The most satisfactory design from a statistical point of view would be a Latin square. However, with five standards, this would be very inefficient since it would allow only one or two sample test solutions to be tested or plated, depending on whether a 6 x 6 or 7 x 7 well pattern is used. The most efficient design still appears to be that specified in the original paper, assignment of code numbers to each test solution, and application of the solutions in the code number order. For the plates specified here, 11 samples can be tested on two of the 7 x 7 plates and 7 on two of the 6 x 6 plates to give replication equivalent to the original method (three zones per plate for each test solution). Incubate the plates at 30°C. Read

380

Platt

the sizes of the zones of inhibition, and determine average zone sizes for each of the test solutions on each plate. Determine antibiotic concentrations of the samples from a point-to-point plot of In standard concentration versus average zone size. It should be noted that considerable curvature of the standard dose-response lines occurs as noted in the original paper and seems to be caused by the zone sizes of the extreme upper and lower standards.

ACKNOWLEDGMENTS The author thanks Mrs. Muriel George and her assistants in the Squibb Library for aid in surveying the literature and Mrs. Connie Saloom for typing the manuscript.

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Microbiological Assay

19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45.

381

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( 1980). D. F. Mahoney, T. F. Butler, and J, R. Turner, Antimicrob. Agents Chemother., 19: 934 ( 1981). R. P. Harvey, R. A. Isenberg and D. A. Stevens, Rev. Infect. Dis. 2:559 (1980). C. Brass, J. N. Galgiani, T. F. Blaschke, R. Defelice, R. A. O'Reilly, and D. A. Stevens, Antimicrob. Agents Chemother., 21:151 (1982). J. H. Jorgenson, G. A. Alexander, J. R. Graybill, and D. J. Drutz, Antimicrob. Agents Chemother., 20: 59 ( 1981). T. K. Daneshmend, D. W. Warnock, A. Turner, and C. J, C. Roberts, J. Antimicrob. Chemother. , 8: 299 ( 1981). F. Notten, A. Koek-Van Oosten, and F. Mikx, Antimicrob. Agents Chemother., 21:836 (1982). R. Yogev, C. Melick, and W. J. Kabat, Antimicrob. Agents Chemother., 19: 993 ( 1981). A. P. Ball, P. G. Davey, A. M. Geddes, I. D. Farrell, and G. R. Brooks, Lancet, 1:620 (1980). U. B. Schaad, P. A. Casey, and D. L. Cooper, Antimicrob. Agents Chemother. , 23: 252 ( 1983). K. A. DeSante, K. S. Israel, G. L. Brier, J. D. Wolny, and B. L. Hatcher, Antimicrob. Agents Chemother., 21: 58 ( 1982). D. Adam, I. DeVisser, and P. Koeppe, Antimicrob. Agents Chemother., 22: 353 (1982). S. Bennett, R. Wise, D. Weston, and J. Dent, Antimicrob. Agents Chemother., 23:831 (1983). R. M. Brown, R. Wise, J. M. Andrews, and J. Hancox, Antimicrob. Agents Chemother., 21: 565 (1982). L. Kager, L. Liljeqvist, A. Malmborg, C.-E. Nord, and R. Pieper, Antimicrob. Agents Chemother., 22: 208 ( 1982). G. Foulds, J. P. Stankewich, D. C. Marshal, M. M. O'Brien, S. L. Hayes, D. J. Weidler, and F. G. McMahon, A ntimicrob. Agents Chemother., 23:692 (1983). A. Arancibia, J. Guttmann, G. Gonzalez, and C. Gonzalez, Antimicrob. Agents Chemother., 17: 199 ( 1980). R. R. Tight and A. C. White, Antimicrob. Agents Chemother., 17: 229 (1980). R. D. Smyth, M. Pfeffer, D. R. Van Harken, A. Cohen, and G. H. Hottendorf, Antimicrob. Agents Chemother., 19: 1004 ( 1981). J. M. Mylotte, T. R. Bates, K. A. Sergeant, R. E. Matson, and J. R. Beam, Jr., Antimicrob. Agents Chemother., 20: 81 (1981). I. A. J. M. Bakker-Woudenberg, P. DeBos, W. B. Van Leeuwen, and M. F. Michel,, Antimicrob. Agents Chemother., 19: 76 (1981). 0. Cars and S. Ogren, J. Antimicrob, Chemother., 8:39 (1981). o. Cars, Scand. J. Infect. Dis., 13:283 (1981). S. Trottier and M. G. Bergeron, Antimicrob. Agents Chemother., 19: 761 ( 1981). H. Kourtopoulos, S. E. Holm, and S. R. Norrby, J, Antimicrob. Chemother., 11:251 (1983).

382 46. 47. 48. 49. 50.

51. 52. 53. 54.

55. 56. 57. 58. 59. 60.

61. 62. 63. 64.

65. 66. 67. 68. 69. 70.

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D. J. Miner, D. L. Coleman, A. M. M. Shepherd, and T. C. Hardin, Antimicrob. Agents Chemother., 20: 252 ( 1981). 0. V. Martinez, J. U. Levi, A. Livingstone, T. I. Malinin, R. Zeppa, D. Hutson, and N. Einhorn, Antimicrob. Agents Chemother., 20: 231 ( 1983). G. R. Aronoff, R. S. Sloan, S. A. Mong, F. C. Luft, and S. A. Kleit, Antimicrob. Agents Chemother., 19:575 (1981). E. H. Estey, S. S. Weaver, D. W. Ho, and G. P. Bodey, Antimicrob. Agents Chemother., 19:639 (1981). W. K. Bolton, W. M. Scheld, D. A. Spyker, T. L. Overby, and M. A. Sande, Antimicrob. Agents Chemother., 18:933 (1980). A. Leroy, G. Humbert, and J. P. Fillastre, Antimicrob. Agents Chemother., 19: 965 ( 1981). K. A. De Sante, K. S. Israel, G. L. Brier, J. D. Wolney, and B. L. Hatcher, Antimicrob. Agents. Chemother., 21: 58 (1982). R. M. Bannatyne and R. Cheung, Antimicrob. Agents Chemother., 16: 43 ( 1979). G. H. McCracken, J. D. Nelson, and L. Grimm, Antimicrob. Agents Chemother., 21:262 (1982). C. B. Walker, J. M. Gordon, H. A. Cornwall, J. C. Murphy and S. S. Socransky, Antimicrob. Agents Chemother., 19:867 (1981). P. J. Thompson, K. R. Burgess, and G. E. Marlin, Antimicrob. Agents Chemother., 18: 829 ( 1980). P. D. Kroboth, A. Brown, J. A. Lyon, F. J. Kroboth, and R. P. Juhl, Antimicrob. Agents Chemother., 21:135 (1982). P. Patamasucon, S. Kaojarern, H. Kusmiesz, and J. D. Nelson, Antimicrob. Agents Chemother., 19: 736 (1981). T. Murakawa, H. Sakamoto, S. Fukada, T. Konishi, and M. Nishida, Antimicrob. Agents Chemother., 21: 224 ( 1982). P. Iverson, O. S. Nielson, K. M. E. Jensen, and P. 0. Madsen, Antimicrob. Agents Chemother., 23: 338 ( 1983). D. G. Delgado, C. J. Brau, and C. K. Avent, Antimicrob. Agents Chemother., 17: 286 (1980). J. M. Gordon, C. B. Walker, J. M. Goodson, and S. S. Socransky, Antimicrob. Agents Chemother., 17: 193 (1980). E. M. Anifantakis, J. Dairy Sci. 65:426 (1982). S. R. Norrby, K. Alestig, F. Ferber, J. L. Huber, K. H. Jones, F. M. Kahan, M. A. P. Meisinger, and J. D. Rodgers, A ntimicrob. Agents Chemother., 23: 293 ( 1983). H. Kropp, J. G. Sundelof, J. S. Kahan, F. M. Kahan, and J. Birnbaum, Antimicrob. Agents Chemother., 17:993 (1980). A. W. Chow, K. R. Finlay, H. G. Stiver, and C. L. Carlson, Antimicrob. Agents Chemother., 23: 634 ( 1983). P. Patamasucon and G. H. McCracken, Antimicrob. Agents Chemother., 21: 390 ( 1982). C. N. Walker, Antimicrob. Agents Chemother., 17:730 (1980). G. Czegledi and G. Danko, Acta Vet. Acad. Sci. Hung., 28: 121 ( 1980). S. Faine and D. C. Knight, Lancet, 2:375 (1968). S. A. Smith and S. E. Smith, Brit. J. Clin. Pathol. 3(2):341P (1975).

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C. M. Kunin, Clin. Pharmacol. Therapeut. 16: 251 (1974). M. Barza, J. Antimicrob. Chemother., 8(Suppl. C):7 (1981). J. E. L. Corry, M. R. Sharma, and M. L. Bates, in Antibiotics: Assessment of Antimicrobial Activity and Resistance (A. D. Russell and L. B. Quesnel, eds.), Academic Press, London, 1983. S. E. Holm, Scand. J. Infect. Dis. (Suppl.), 13:47 (1978). D. M. Ryan and U. Mason, J. Antimicrob. Chemother., 5:116 (1979). R. Wise, A. P. Gillett, B. Cadge, S. R. Durham, and S. Baker, J. Infect. Dis., 142:77 (1980). A. Schreiner, K. B. Hellum, A. Diganes, and I. Bergman, Scand. J. Infect. Dis. (Suppl.), 14:233 (1978). M. C. Kolczun, C. L. Nelson, M. C. McHenry, T. L. Gavan, and P. Pinovich, J. Bone Joint Surg., 56-A: 305 ( 1974). P. M. Young, M. Barza, A. Kane, and J. Baum, Arch. Opthalmol., 97: 717 (1979). R. H. Fitzgerald, P. J. Kelly, R. J. Snyder, and J. A. Washington, II, Antimicrob. Agents Chemother., 14:723 (1978). M. Barza, A. Kane, and J. Baum, Am. J. Opthalmol., 86:121 (1978). D. M. Ryan and 0. Cars, Scand. J. Infect. Dis., 12:307 (1980). A. G. Itkin, personal communication, 1980. E. A. Swabb, M. A. Leitz, F. G. Pilkiewicz, and A. A. Sugerman, J. Antimicrob. Chemother., 8(Suppl. E): 131 (1981). K. Bush and R. B. Sykes, J. Antimicrob. Chemother., 11:97 (1983). J. L. Trudel, D. D. Mutch, P. R. Brown, G. K. Richards, and R. A. Brown, Surg. Forum, 33:26 (1982). W. L. Greaves, J. H. Kreeft, and R. I. Ogilvie, Antibiot. Agents Chemother., 19: 253 ( 1981). D. Mutch, G. Richards, R. A. Brown, and D. S. Mulder, Surgery, 86:1068 (1982). F. W. Kavanagh, J. Pharm. Sci., 63:1463 (1974). c. Fassbender, J. Gentile, and H. Weisblatt, personal communication, 1983. L. R. Peterson, D. N. Gerding, C. E. Fasching, and C. CostasMartinez, Minnesota Med., May: 321 (1983).

10 Assay of Antibiotics in Mammalian Cell Culture ALINE L. GARRETSON

Gaithersburg, Maryland

Protein Determination Materials Assay Procedure Calculation of Results Quality Control Interpretation of Results Cell Enumeration Equipment and Supplies Assay Procedure Agar Diffusion Disk Assay Materials Assay Procedure Quality Control Interpretation of Results Bioautography Materials Assay Procedure Human Tumor Stem Cell (Clonogenic) Assays Materials Assay Procedure Alternative Medium Additives Modulation of Macrophage Tumoricidal Capability Materials Assay Procedure Results References

389 389 391 392 392 392 394 394 394 395 395 396 398 398 400 400 400 400 402 406 407 407 407 409 409 409

387

388

Garretson

Mammalian cells may serve as targets in a wide variety of pharmacological tests designed to assess the capability of a chemical agent to produce general toxicity or changes in macromolecular biosynthesis, differentiated function, or morphology. Cell cultures may also be employed in more complex mechan isms of action studies, as well as in the chronic toxicity tests for mutagenicity and carcinogenesis. A review of all the methods workable in mammalian cell systems for the evaluation of antibiotics is beyond the scope of this chapter and methods useful for defining the mechanism of action of drugs too numerous and too limited in application to present in this chapter. Whatever the course of investigation followed for the characterization of a compound, the methods cited here provide a sound foundation for secondary studies. The most practical and widely used assays for antibiotics are those based on nonspecific effects characterized as growth inhibitory or acutely lethal. Two methods that measure cellular proliferation as either protein content or cell number are presented in this chapter as the first two assay methods. The protein determination method, which has been well characterized during its long usage, is the best standardized assay available. This method requires no unique or extraordinary equipment or supplies and is quantitative, reproducible, simple, and relatively inexpensive compared with in vivo assays. Furthermore, the basic procedure for drug exposure and cell culture may serve as a foundation for assays measuring different responses by substitution of the protein analysis with other analytical techniques, such as DNA or RNA determinations. With monolayer cultures, gross morphological changes may be observed as criteria of toxicity. Similarly, some of the steps required in the assays are duplicated, but detailed only for the cell enumeration method. The reader attempting to perform assays found in later sections is advised to review this method. The agar diffusion disk assay described in this chapter is a useful screening tool suitable for testing small amounts of compounds inhibitory to dehydrogenase activity. It is a simple and rapid tissue culture assay but discriminates against low concentrations of some compounds active in growth inhibition assays performed with KB or P388 cells. This assay is particularly useful in the fractionation and purification of crude products because of its simplicity and rapidity and when used in conjuction with antimicrobial assays can iden tify antimicrobial substances that are also cytotoxic to mammalian cells. Bioautography with paper or thin-layer chromatograms is another application of the agar diffusion assay and is particularly useful in the separation and purification of biologically active substances. Recently, the human tumor clonogenic assay emerged as the tissue culture system offering the most promise for the discovery of substances active against human tumors. Its application to large-scale screening is still in the developmental stage. However, its usefulness as a predictive test for the chemotherapeutic treatment of cancer patients has met with some success in a limited number of tumor types. Although it is not expected that this assay will replace well-standardized methods already established for potency and other routine cytotoxicity evaluations, or most mechanisms of action studies, it may have a place in defining activities heretofore not seen in cytotoxicity studies using continuous cell lines. Although an assay for determining the modulation of macrophage tumoricidal capability will not have widespread application for the biological evaluation of antibiotics, its value in the study of polyene antibiotics and other substances interacting with cell membranes warrants inclusion in this chapter.

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389

PROTEIN DETERMINATION

The cytotoxicity of antitumor agents as determined by growth inhibition of mammalian cells, which is quantitated by measurement of protein concentration, was described initially by Eagle and Foley [ 1] • In this procedure, the Oyama and Eagle modification [ 2] of the Lowry method [ 3] employing a phenol reagent for the development of color is utilized for the protein determination. A further simplification of the Eagle-Foley assay, which reduced the assay period from 7 to 3 days and eliminated intermittent medium changes, subsequently was reported by Smith et al. [ 4]. The protocol utilized for many years by screening contractors and devised by the Division of Cancer Treatment (DCT) at the National Cancer Institute (NCI) for the screening and evaluation of potential antitumor agents in KB cell culture [ 5] encompasses the best features of the procedures previously cited. The cytotoxicity assay that follows is similar to that of Smith, Lummis, and Grady, which employs the Oyama-Eagle method for protein determination and includes the essential features of the NCI protocol. This assay may be performed without further modification using any mammalian cell line grown either in monolayer or suspension culture providing the cell line has a growth potential equal to that of the KB cell line and a monodispersed cell suspension is readily attainable. With slower dividing cells the assay must be modified by prolonging the incubation period and, if necessary, also replacing the medium during the incubation period to obtain the appropriate number of cell divisions for statistical validation. Materials Reagents

Protein Assay: 1.

2. 3. 4. 5. 6.

Solution A: Dissolve 20 g Na2C03, 4 g NaOH, and O. 2 g NaK tartrate to obtain a final volume of 1 liter. Solution B: Dissolve 5 g CuS04 •5H20 in a final volume of 1 liter. Solution C: Mix 50 parts of solution A with 1 part of solution B prior to using. Folin-Ciocalteau's reagent (Fisher Scientific Co., 2 N): Dilute to 1 N. Bovine serum albumin, crystallized: Prepare a stock solution of 200 µg/ml in distilled water. Organic solvents: Reagent grade as required for solubilizing test compounds, including ethanol, dimethylsulfoxide, acetone, methanol, and other solvents of known toxicity.

Cell Line: Cultures of mammalian cells should be maintained in the exponential growth phase for several passages prior to assay. If the KB cell line is used, it can be obtained from the American Type Culture Collection (ATCC CCL 17). Culture Medium and Solutions: 1. 2.

Balanced salt solution: Use a solution compatible with the growth medium and without bicarbonate for washing cells. Complete growth medium: Use a medium providing a maximum growth rate with 5-10% calf or horse serum.

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Garretson

3.

Dispersing agent: Select the agent best suited for the cell line utilized.

Test Compound Preparation

Compounds for assay should be prepared using aseptic techniques and sterile diluent and equipment. Although pure products need not be sterilized, those known to contain microbial contaminants, such as crude natural products, should be sterilized by filtration through bacteriological filters. Watersoluble compounds may be dissolved initially in distilled water or saline. Dissolve water-insoluble compounds in a suitable solvent, such as ethanol, methanol, acetone, dimethylsulfoxide, or dimethylformamide. Final concentration of these solvents should not exceed 1000 µg/ml. Solvents of unknown toxicity must be pretested and cytotoxic end points determined. When it is necessary to adjust the pH of acidic or basic test solutions to within the range of 6.5-7.5, add 0.25 N HCl or NaOH. Prepare a series of 10-fold dilutions ranging from O. 2 to 2000 µg/ml in complete growth medium for each test compound. When diluted with cell suspensions in the subsequent step, the resulting test concentrations will span the range of 0.01-100 µg/ml. Also include a positive control reproducibly active within the same range of activity of the substances being tested. Cell Suspension Preparation

Monolayer cell cultures may be collected by mechanical scraping or digestion with trypsin or versene. The choice of method depends on the cell line in use and the ease of dispersion of cell clumps. Uniformly dispersed cells with minimal clumping is essential for obtaining replicate cultures. After collection, cells are resuspended in fresh growth medium to give an estimated concentration of 10-20 µg cell protein per milliliter. For the KB cell line this amount of protein is equivalent to 15,000-30,000 cells per milliliter [2]. Suspension cultures also should be resuspended in fresh growth medium even if dispersal of cells by chemical means is not required. The dilute cell suspension is kept in suspension by constant stirring until delivered to culture tubes using a continuous pipetting device. Baseline values for protein are determined on the seeding cell suspension. Aliquots are transferred into centrifuge tubes and the suspending medium removed after centrifugation. The cells are washed twice with a balanced salt solution (minus bicarbonate) and the concentration of protein determined using the procedures given in steps 2 and 7-12 of the assay procedure. Supplies and Equipment

Incubator, 37°C Reciprocating shaker to accommodate test tube racks, or other mechanical shaking device Screw cap culture tubes, 16 x 150 mm Test tube racks, slant and nonslant Spectrophotometer at 660 nm Centrifuge Cornwell syringes or other automatic dispensers with delivery tubes

Mammalian Cell Culture

391

Assay Procedure 1. Add O. 2 ml of each dilution of test sample to duplicate culture tubes. Use a slant rack with a 10° slope for monolayer cultures. Culture tubes containing suspension cultures are incubated upright. 2. Determine the number of baseline and growth controls to be included in each assay from statistical analysis of prior runs. For the KB cell line the formula N = 21il may be used, where n =number of test substances [5]. Add 0. 2 ml of complete growth medium to the number of tubes calculated for growth controls. 3. Dispense 3.8 ml of the cell suspension into the tubes using an automatic pipette. Agitate the tube racks gently to distribute the contents evenly. 4. Prepare a set of five tubes containing 4. O ml of complete growth medium as growth medium controls. 5. Incubate the cultures and controls for 72 hr or until a 6-12 fold increase in protein content is reached in the growth controls. With cell lines of unknown or differing growth rates, the incubation period as well as seeding density should be predetermined as described previously. 6. At termination of the growth period the medium is decanted from monolayers by inversion of the culture tubes. When suspension cultures are used, transfer aliquots to centrifuge tubes, sediment the cells, and decant off the medium. Maintain tubes at 37°C until digestion is completed in step 9. 7. Wash the cultures twice with 5 ml of prewarmed ( 37°C) balanced salt solution (minus bicarbonate), being careful not to disturb the cell sheet in monolayer cultures. Allow these cultures to drain 15-20 min over an absorbent pad. Suspension cultures are sedimented by centrifugation and also washed twice. 8. Prepare a set of protein standards with final concentrations within the range of 0-200 µg protein per milliliter distilled water. Prepare triplicate tubes containing 1 ml for each dilution. Also prepare five reagent controls containing 1 ml of distilled water only. 9. Add 5 ml of solution C to all assay tubes. Incubate at 37°C for 1 hr or heat to 56-60°C for 5 min to dissolve the cell material. Shake for 5-15 min on a reciprocal shaker to complete the dissolution of cells. The intensity of the biuret color produced by the alkaline copper sulfate tartrate (solution C) with the protein is an indication of the amount of protein solution that can be used to react with the Folin~Ciocalteau reagent to produce a reading within the range of the spectrophotometer. Appropriate dilutions of cell solutions showing a pale to deep violet color should be made and further diluted with solution C to a final volume of 5 ml as outlined in the table:

Color intensity

Treatment of cell solution

None

Pale violet

Deep violet

Predilution

None

None

1/2-1/3

Cells (ml)

5

0.5

0.5

SolutioTl C (ml)

None

4.5

4.5

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10. Add 1 ml of distilled water to each sample or its dilution in the test sample series and experimental controls to give a final volume of 6. 0 ml. 11. Treat all tubes in the test with 0. 5 ml of 1 N Folin-Ciocalteau reagent using the Cornwall syringe and appropriate delivery tube to jet in the reagent. It is important to mix the reagent rapidly with the tube contents. 12. Allow 30 min to 1 hr for the color to develop, but read before 2 hr has elapsed. Absorbance is read at 660 nm with the instrument adjusted to zero using the reagent control for reading the protein standards. The instrument is readjusted to zero against the average of the digests of the growth medium controls before reading the test samples. This compensates for the residual growth medium protein contained in the test cultures. Calculation of Results Plot protein concentration versus absorbance for the protein standards and convert the absorbancy values of the test samples into micrograms of protein by extrapolation. Calculate the mean values for replicate tubes. Growth ratios are determined using the following formula: th t . _ Protein cone. treated (final - initial) G . •t•ia1) . cone. con t ro1 (f.inal - in1 row ra io - p ro t em The concentration of test compound effecting 50% inhibition of growth may be interpolated from a log dose-response curve obtained by plotting the percentage of growth against the logarithm of the concentration. The slope is the difference in growth ratios for a one-log difference in concentration of test substance. Quality Control Growth in the control cultures should be consistent with the growth pattern for the cell line studied and the drug response uniform and reproducible. The Drug Evaluation Branch of DCT, NCI, requires that certain criteria be met for the validation of the KB assay [ 51 : 1. 2.

Control growth must attain at least a sixfold increase. Absorbancy values of duplicate tubes should not differ by more than

3.

Growth ratios should not reverse between consecutive dose levels by more than 0. 10. The ED50 value (concentration calculated to give 50% growth inhibition) for the positive control ( 6-mercaptopurine) included in odd-numbered experiments should remain within the limits of 0.05-0.5 µg/ml.

4.

0.10.

Interpretation of Results The slope of the dose-response curve for certain pure compounds remains constant with changes in concentration and reflects the type of inhibition. The presence of other inhibitory agents or cell growth stimulants may alter the slope of the dose-response curve [ 41 . Time and temperature of exposure and the composition of the assay medium have been shown to effect the sensitivity of the assay to certain inhibitory agents [ 6] . In the KB assay the medium supplement lactalbumin hydrolysate at 3 mg /ml reportedly

393

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increases the yield of total cell protein without stimulating the rate of growth and increases the sensitivity and reproducibility with streptovitacin A [ 4) • The use of a positive control whose cytotoxic effect is altered by chemical or physical variations in assay conditions is therefore recommended. Cytotoxic end points (ID50) obtained with the KB cell line after exposure for 72 hr to a variety of antibiotics are given in Table 1. The end points are for the inhibition of cell multiplication as determined either by protein con tent [ 4, 7, 8) or by cell enumeration using an electronic counter [ 9) • Except

Table 1 Relative Cytotoxicity of Various Antibiotics for KB Cells Method of evaluationb

Antibiotic

References

Actinomycin C

>10- 6 6. 0 x 10-5

PD PD

8

Actinomycin D

1. 2 x 10-5

PD

7

Carzinophilin

0.005

PD

8

Daunomycin

0.005

PD

7

Xanthomycin

0.005

PD

8

Tubercidin

0.02

PD

8

Mitomycin C

0.025

PD

8

Streptovitacin A

0.035

PD

4

Cycloheximide

0.10

PD

4

7

Chloramphenicol

25.0

PD

8

Cytochalasin B

25.0

PD

7

Tetracycline •HCI

33.0, 38.5

PD, CE

8, 9

Chlortetracycline •HCI

50.0

PD

8

Clindamycin •HCI

102.0

CE

9

Erythromycin

117.0

CE

9

)500.0

PD

8

733.0

CE

9

1471. 0

CE

9

Kanamycin Ampicillin, trihydrate Lincomycin •HCl

aCalculated concentration for 50% inhibition of cell growth after 72 hr exposure to antibiotic. borowth inhibition determined using the Oyama-Eagle method for protein determination (PD) or by cell enumeration using the Coulter Counter (CE).

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for clindamycin, compounds with an ID 5o at 100 µg/ml or less also exhibit antitumor activity in in vivo tumor systems. Between 79 and 85% of the compounds active in vivo are also active in the Eagle- Foley assay system at concentrations ~ 100 µg/ml [ 6] • CELL ENUMERATION The counting of cell populations with the aid of a hemocytometer and microscope has had wide application in tissue culture; for small numbers or volumes of samples it is the method most commonly used. This method has the distinct advantage over electronic enumeration, as quantitative information on cell viability may be obtained simultaneously by the incorporation of a vital stain into the diluting medium. However, this method quickly becomes tedious in the counting of large numbers of samples, a factor likely to increase the existing counting error, estimated at 10% [ 10] • Automated electronic counters, which have been widely used in recent years for studies on growth and population dynamics, offer greater rapidity and accuracy and their application to mammalian cells is well defined [ 11] • With sufficienty large populations of cells that can be readily dissociated in the absence of cell damage or debris formation, it is clearly the method of choice for quantitative growth studies. The assay procedure for protein determination just described may be modified by replacing the measurement of protein with cell enumeration using the Coulter Counter (Coulter Electronics Inc. , Hialeah, Florida) or a similar electronic counting device. Cytotoxicity assays with L1210 or P388D1 cells grown in suspension culture have been described (7 ,12] and employed as prescreens for the evaluation of natural products as antitumor agents [ 13] • Equipment and Supplies In addition to the supplies and equipment previously listed for the protein assay, the following materials are required. 1.

2. 3.

Select a cell line grown either in suspension or monolayer culture, which is capable of being rapidly dissociated without cell damage or the formation of debris. An electronic cell counter, such as a Coulter Counter or a similar type, equipped with an orifice for use with mammalian cells. A cell diluent, such as Isoton (Coulter Electronics Inc., Hialeah, Florida), is required for the preparation of the cell suspension for counting in electronic counters.

Assay Procedure The basic procedure for the protein assay .is modified by making the following substitutions: 1.

Prepare cell suspensions to contain 10, 000- 50, 000 cells per milliliter and add 4 ml of the diluted suspension to 16 x 150 mm screw cap tubes. The number of cells seeded depends on the growth characteristics of the cell line employed. As in the protein assay, a 6- to 12fold increase in cell number should be obtained in the control cultures.

Mammalian Cell Culture

2. 3. 4. 5.

395

Add aliquots of test sample to each tube to be treated in a volume of 0.2-0.25 ml. Incubate cultures for 48 hr for those seeded at 50, 000 cells per milliliter, 72 hr for those seeded at 10, 000-20, 000 cells per milliliter, or until the desired number of cell divisions has been achieved. Collect cells as described previously, and dilute cells in counting solution according to directions provided by the manufacturer of the instrument to be utilized. Determine growth ratios as described previously.

AGAR DIFFUSION DISK ASSAY

The inhibition of dye-reducing activity of mammalian cells suspended in agar has been applied to the preliminary screening of cytotoxic substances by a number of investigators [ 14-20]. All these methods rely on the inhibition of dehydrogenase activity of cells visually detected by the lack of reduction of a redox indicator. The assay method that follows was developed for use with suspension-grown cultures of P388 and L1210 cells and differs from other diffusion assays in one or more respects, such as cell line, nutrient media, incubation conditions, or redox agent. The method was modified further through the use of square plastic plates (23 x 23 cm), which provided agar layers of more uniform thickness and also permitted plate inversion during the incubation period, resulting in more accurate and better defined zones of activity. Although this method has not been described in detail previously, its use as a prescreen for the detection of antitumor agents in fermentation products has been reported [ 21] . This method has proven to be particularly useful in the isolation and purification of cytotoxic substances from mixtures of unidentified antimicrobial products. Both agar disks saturated with chemical extracts and thin-layer chromatograms (TLC) may be utilized in this assay system. Although growth inhibition assays are more sensitive, dehydrogenase inhibition in agar plates has distinct advantages, namely, ease of operation, rapidity, large amounts of test substances are not required, and suitability for bioautography. Materials Reagents

Cell Line: Cell cultures suitable for use in the assay are those that can adapt to growth in suspension, such as P388D 1 , a lymphoma cell line available from the American Type Culture Collection, CCL 46 L1210, a murine lymphoctyic leukemia also available from the American Type Culture Collection, CCL 219 Other cell lines may be utilized, but selection should be based not only on their suitability as target cells to provide relevant experimental data, but also on their ability to grow exponentially at high viability levels in sufficient quantity for assay. The cells must also metabolize normally when suspended in an agar medium.

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396 Growth Medium: 1.

2. 3. 4. 5.

Fischer medium modified for leukemic cells, lX and lOX for propagation and agar assay, respectively (Grand Island Biological Co., Grand Island, New York) Sodium bicarbonate, 7. 5% Horse serum, not heat inactivated, sterile filtered Gentamicin sulfate, sterile solution of 50 mg I ml diluted 1: 1000 in growth medium propagation of cell line Oxoid agar No. 1 (K. C. Biological, Inc., Lenexa, Kansas) or a washed agar of equivalent purity

Diffusion Assay: 1.

2. 3.

Sodium 2,6-dichlorophenol-indophenol (Fisher), individual lots pretested for quality of response to reduction by cell suspensions Tubercidin, positive control, prepared as a solution in phosphatebuffered saline at a concentration of 200 µg/ml Phosphate-buffered saline, adjusted to pH 7.2-7.4 with 5 N NaOH

Equipment and Supplies

In addition to equipment mentioned in previous sections, the following equipment and supplies are required: Porta-trace light box with Plexiglas top Screw cap flasks, wide bottom and narrow neck C02 incubator humidified Nunc-Bioassay plates, 23 x 23 cm (Vangard International, Inc., Neptune, New Jersey) or 100 mm petri plates (glass or plastic) Magnetic stirrers Water baths, adjusted to 48 and 37°C Antibiotic assay disks, 12. 5 mm paper disks (Schleicher and Schuell Inc. , Keene, New Hampshire) Assay Procedure Preparation of Suspension Cultures

Suspension cultures of either P388 or L1210 may be produced in roller bottles [ 22] , on a rotating shaker [ 23] , or in spinner flasks [ 24] . Cells are propagated in Fischer medium, lX, supplemented with 10% horse serum and gentamicin sulfate at 50 µg /ml. Supplemental L-glutamine may also be added at a 2 mM concentration. Cells are harvested while in the logarithmic growth phase and collected by centrifugation. Resuspension is in prewarmed ( 3 7°C) complete growth medium (Fischer medium, lX, with 10% horse serum and no antibiotics). The cell suspension is then diluted to contain 107 viable cells per milliliter after determining the viable count using the trypan blue dye exclusion test. Stock solutions needed for the next step should be prepared and ready to use before the cells are collected to minimize trauma to the cells. Preparation of Agar-Suspended Cells

For each 220 ml of agar-suspended cells to be plated, prepare an agar solution by dissolving 1. 9 g of agar in 144 ml of distilled water in a 500 ml

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397

screw cap flask with a stir bar. Heat to dissolve, and autoclave 15 min. Allow to cool at room temperature before placing in a 48°C water bath. Agar solutions should be prepared as needed and should not be reheated. After a minimum of 30 min equilibration at 48°C, the melted agar is placed on a magnetic stirrer set at low speed. The remaining components of the agar medium are mixed rapidly in a second screw cap flask. Stock solutions and cells are added in the order and amounts shown: Fischer medium (lOX), 16. 0 ml Horse serum, 16. 0 ml NaHC03 (7. 5%), 2. 4 ml Cell suspension ( 107 /ml), 44. 0 ml All the stock solutions are prewarmed to 37°C, with the exception of the NaHC03 solution, which is at room temperature. The melted agar and the remaining ingredients are combined before a significant loss in temperature occurs. The nutrient and cell mixture is slowly poured into the flask containing the melted agar. Care should be taken to avoid frothing and bubble formation in the agar. Stir until the cells are uniformly dispersed, and immediately pour the agar mixture into sterile plates using the entire formulation of 220 ml for a single 23 x 23 plate. Use 25 ml for 100 mm petri plates. Distribute the agar evenly over the plate and allow to cool on a level surface. Application of Test Material

Samples need not be sterilized by filtration; however, aseptic techniques should be employed in the solubilizing and diluting process using sterile equipment and diluents. Final dilutions are made in phosphate-buffered saline. Agar plates should be utilized immediately after preparation. Therefore, all test dilutions should be made well in advance. When using 13 mm disks, apply 100 µl of test sample. Add 25 µl to 8 mm disks, but use a fourfold more concentrated test solution to obtain a zone of inhibition of similar size. When solvents exceed a concentration .of 1 part per 100, the disks must be air dried prior to placement on the agar surface. Place the treated disks evenly over the surface of the agar not exceeding 64 disks (13 mm) per Nunc-bioassay plate, or 5 per 100 mm plate. Use caution in positioning the disks so as not to scar the agar surface. Include a positive control on each plate. Tubercidin at 20 µg per 13 mm disk is a suitable inhibitor for this assay. Invert the plates, and incubate overnight at 36°C in a humidified C02 incubator. Regulate the C02 at 10% and humidity at maximum saturation. Without disturbing the agar surface, remove the disks and immediately initiate the staining procedure. Staining Procedure

Prepare a 0.05% (w/v) solution of 2,6-dichlorophenol-indophenol, sodium salt in distilled water, and filter through a Whatman No. 1 filter to remove undissolved particles. Apply 40 ml of the stain to an elevated corner of the 23 cm square dish permitting the stain to flow across the entire surface. Alternately rock the plate toward each outside edge, and allow the stain to diffuse for 1 min. It is essential that the stain be distributed evenly. Use 5 ml of the stain for the 100 mm plates.

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Tilt the plate, pour off the stain, and remove any remaining solution by aspiration. Be certain that all free solution is removed. Isolated droplets remaining on the agar surface will overwhelm the capacity of the dehydrogenases, resulting in a localized false response. In larger amounts the staining solution may produce a streaming effect. Incubate the plates for 30 min in the inverted position at 36°C in the humidified C02 incubator. This should be sufficient time for the uninhibited cells to enzymatically reduce the stain to the colorless form. In actual practice, the background fades from intense blue to a pale blue, leaving the zones of inhibition unchanged in color. Record the results quickly, as the reduced stain soon becomes oxidized. Determine the size of the zones of inhibition using a millimeter ruler or caliper with the plates illuminated on a light box. Quality Control It is critical that exponential-phase cultures be used in the seeding of plates

and that viability be monitored during the cell production phase. For the P388 cell line, viabilities of ~95% are attainable and high viability of cell suspensions will give consistent results; cell suspensions with low viabilities result in weak dehydrogenase activity. Although clarity of the zones of inhibition is reduced with decreasing numbers of cells, the physiological state of the cells appears to have a more pronounced effect on enzyme activity. The inclusion of a positive control whose activity falls within the range of activity of the substances tested is recommended. Table 2 contains a number of known antibiotics whose activity can be accurately defined in this assay. Zone sizes of less than 18 mm produced by crude fermentation broths loaded on 13 mm disks are often not reproducible [ 21] ; therefore, the level of activity acceptable should be ;.:is mm for crude preparations. Activity criteria for more potent pure compounds is individually determined based on a linear dose-response curve. Interpretation of Results The edge of the zone separating enzyme activity and inhibition may vary from very sharp to a gradation over a large distance. Repetitive assays revealed that zone definition and clarity characteristically varied with the type of in hibitor at cell concentrations optimal for stain decolorization. Dose-response lines constructed by plotting the diameter of the zone of inhibition against logarithmic concentration give a variety of slopes and ranges of activity for selected antitumor agents. Zones of inhibition produced by these compounds and the range of concentrations giving linear dose-response lines are shown in Table 2. Table 2 Inhibition of Dehydrogenase Activity of P388 Cells

Antibiotic Actinomycin C

Concentration (µg/ml)

Zone of inhibition (mm)a

Linear doseresponse range (µg/ml)

200.0 100.0 50.0

26.0 25.0 23.0

3.12-200

Table 2 (continued) Concentration ( µg/ml)

Zone of inhibition (mm)a

Actinomycin c (continued)

12.5 6.25 3.12

20.0 18.0 17.0

Actinomycin D

200.0 100.0 50.0 25.0 6.25 1. 56

30.0 28.0 27.0 24.0 21.0 18.0

1. 56-200

28.5 25.5 24.0 21. 5 19. 5 17.0

0.156-5

Antibiotic

Borrelidin

5.0 2.5 1. 25 0.625 0.312 0.156

Linear doseresponse range (µg/ml)

1000.0 200.0 100.0 50.0

23.0 19.5 18.0 16.0

50-1000

Cycloheximide

200.0 25.0 20.0 10.0 5.0 2.0

56.0 40.0 38.0 34.1 27.5 20.0

2-200

Mithramycin

200.0 150.0 100.0 50.0 10.0

30.0 28.0 26.3 24.0 17.0

10-200

Olivomycin A

200.0 100.0 50.0 25.0 12.5 6.25 3.125

31. 0 29.0 27.5 26.0 24.0 22.5 20.5

3.125-200

Toyocamycin

200.0 100.0 50.0 25.0 12.5 6.25 3.12

41.3 36.2 30.2 27.0 23.0 21.0 18.0

3.12-20

Cordycepin

a

Average values from two or more assays. 399

400

Garretson

BIOAUTOGRAPHY

The detection of biological activity on chromatograms using indicator microorganisms has had wide application over the last three decades in the identification and isolation of antibiotic substances [ 25] • Analogous methods have been devised using mammalian cells for the identification of cytotoxic substances [26-28]. The agar diffusion assay for P388 cells previously described was adapted for use with paper and thin-layer chromatography. The bioautography of samples arising from studies on the classification and identification of antitumor agents from crude fermentations was perfected using the chromatographic separation techniques described previously for microbial systems [ 29] . Materials In addition to the materials described in the previous section, the following supplies are needed: P388 seeded agar plates, 23 x 23 cm as prepared in the previous section Thin-layer chromatograms, developed in near neutral solvents and dried according to Reference 29 Filter paper sheets, Whatman No. 1, cut to loosely fit agar plates and sterilized by autoclaving Assay Procedure As soon as agar plates are solidified, a filter sheet is placed gently on the agar surface. This is accomplished by gradually unrolling the sheet over the agar surface in a manner to prevent air entrapment or wrinkling of the paper. After the paper sheet is saturated with moisture absorbed from the agar, place the dried chromatogram face down on the filter sheet. The plates are then incubated without inversion in a humidified C02 incubator for 1-2 hr to permit the diffusion of substances from the chromatogram in to the agar. After the diffusion period has elapsed, the TLC plates and filter paper are removed, the plates inverted, and returned to the incubator. After overnight incubation, the plates are stained and the location and character of the inhibitory zones noted. HUMAN TUMOR STEM CELL (CLONOGENIC) ASSAYS

Considerable interest and activity in the potential application of human tumor clonogenic assays to clinical medicine has been evident since the initial report of Hamburger and Salmon describing human tumor stem cell colony growth in soft agar appeared in 1977 [ 30] • Subsequent studies also have reported on the successful growth of various tumor types in soft agar [31-38] and the high correlation of in vitro and in vivo chemosensitivity of ovarian carcinoma and multiple myeloma [31, 38-40]. Von Hoff and coworkers extended the chemosensitivity assays to include a wider variety of human tumors [ 41]. The suggested applications of this bioassay to include not only the measurement of chemosensitivity or radiosensitivity for selected human therapy, but its

Mammalian Cell Culture

401

use in new drug screening and for diagnostic and prognostic purposes as well, has not gone without criticism [ 42] . According to a stem cell model of human tumor growth [42,43], the total tumor cell population is made up of (1) nonproliferating, differentiated (end) cells, (2) proliferating, nonrenewing (transitional) cells, and (3) proliferating, self-renewing (stem) cells. In this model, stem cells, which are the only cells capable of self-renewal, must be inactivated or lost to effect a cure, as these cells are responsible for tumor regrowth. Not all clonogenic cells have the stem cell property of self-renewal, and the fraction of clonogenic cells that are stem cells may be small and varies from patient to patient. Since the ability of clonogenic assays to quantitate stem cells has not been adequately evaluated, it is expected the effectiveness of predictability of long-term effects from clonogenic assays will be highly variable [ 43]. The sensitivity of clonogenic cells to chemotherapeutic agents, in light of this model, would appear to be predictive of short-term effects that may be generated by destruction of end cells or transitional cell populations. In addition to the conceptual limitations, a number of technical limitations are recognized; procurement of specimens, number of cells yielded per specimen, low plating efficiencies, low success rates in colony formation, and assay periods of 10- 21 days [ 38, 42] • Furthermore, the method is tedious in both preparation and counting of cell colonies, although promise of an automated image analyzer may remove the difficulties and improve accuracy in colony counting [ 44]. The utilization of cell lines possessing characteristics of the tissue of origin may prove useful and overcome the difficulties encountered with fresh tumor specimens. In spite of these and other limitations, this assay may prove useful for either the identification of a limited number of selected agents that show potential as antitumor agents in this system but lack effectiveness in the traditional murine tumor screens [ 45] or the evaluation of a novel mechanism of action of compounds also demonstrating activity in other biological test systems. The ability to grow human tumor colonies in culture is perhaps more significant for studies of human tumor biology, and its most important contribution to cancer research will most probably be in this area. The search at the National Cancer Institute for an in vitro screening assay predictive for clinical activity in recent years also has been focused on the application of the human tumor stem cell assay [ 45-47] • However, the feasibility for de novo screening against primary cultures of human tumors is recognized as being limited to specimens from breast, colorectal, kidney, lung, melanoma, and ovarian cancer patients [ 47] • The complexities and costs of human tumor colony-forming assays have been sufficiently high to discourage its application to large-scale screening [ 48]. The most widely cited procedure for bioassay of human tumor cells in soft agar is that developed by Salmon and coworkers [30,38], and the stepwise procedure included in this chapter is basically their bioassay method. In this procedure, single-cell suspensions are exposed to drugs for 1 hr prior to seeding the cells in an agar overlay. The use of a 1 hr exposure period allows the selection of test levels that approximate the pharmacokinetic con ditions in vivo, where most drugs have short half-lives and are rapidly cleared from plasma [ 49] . However, for most experimental agents pharmacokinetic information will not be available and an alternative continuous exposure method using standard test concentrations would be more convenient. The basic procedure may be modified further to accommodate the growth of certain tumor types requiring additional growth factors or conditioning media.

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According to Hamburger and Salmon, myeloma and lymphoma cells require the presence of a conditioned medium prepared by growth of adherent spleen cells of mineral-oil-primed BALB/c mice in the base layer together with 2mercaptoethanol in the overlay for maximum colony development [50,51]. In the cloning of lymphoma cells, the requirement for conditioned medium also may be met using spent medium from human B lymphocyte cell cultures (RPMI 1788) [ 51] • Other tumor types do not have these requirements, and the con ditioned medium and mercaptoethanol may be omitted [ 38]. A potential improvement in methodology would be the substitution of penicillin and streptolycin solutions with gentamicin sulfate at O. 05 mg/ml in the specimen and cell suspension bathing fluids and O. 02 mg /ml in culture media. Gentamicin sulfate has fungicidal and antimycoplasmal properties, in addition to being an effective antibacterial agent with low cytotoxicity for mammalian cells in culture. The use of higher seeding cell concentrations might also improve the growth response rate of tumors that usually have poor growth rates in vitro. Additional modifications to the basic procedure may be desirable, depending upon the tumor or type specimen. Some suggested changes have been noted in the appropriate sections of the bioassay procedure. Materials As in all tissue culture procedures, the use of sterile equipment and solutions together with aseptic techniques is essential for the protection of cultures from environmental contamination. It is equally imperative that laboratory personnel be fully safeguarded from the danger of infection with communicable diseases, particularly when working with human tissue specimens. It is a common practice in research laboratories engaged in cell culture studies to observe safety precautions aimed at the protection of both cultures and personnel from cross contamination. These procedures should be strictly observed at all times. Also, the rights of individuals donating tissue to research laboratories must be recognized and informed consent obtained from the donor.

Reagents Stock Solutions and Media Additives: 1.

2. 3. 4. 5. 6. 7. 8. 9. 10.

Heparin, sodium heparin injection, 1000 USP U /ml, no preservatives. Fetal bovine serum (FBS), heat inactivated at 56°C for 60 min in a water bath. Penicillin-streptomycin solution (PS), lOOX stock solution containing 10,000 IU/ml penicillin and 10,000 µg/ml streptomycin. Sodium bicarbonate solution, 7. 5% Dextran in saline, commercially available as 6% dextran in saline from Pharmachem Corp. (Bethlehem, Pennsylvania) or as Gentran 6% from Travenol Laboratories (Deerfield, Illinois). Hanks' balanced salt solution (HBSS), lOX without divalent cations in NaHC03, for the preparation of storage and rinsing solutions. HBSS90FBS10: Dilute lOX HBSS to lX with 10% FBS (final concentration) and purified water, and add PS at 1% Horse serum. L-serine solution: Prepare as a stock solution of 21 mg/ml. L-glutamine solution, lOOX, 200 mM.

Mammalian Cell Culture

11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.

403

Sodium pyruvate solution: Prepare a stock solution containing 22 mg/ml, and pretest in tissue culture. McCoy's medium 5A, lX, with glutamine. CMRL 1066, lX, with glutamine Calcium chloride solution: Analytical reagent, anhydrous, prepare as 100 mM solution •. Insulin, 100 U /ml. L-asparagine solution: Prepare a stock solution containing 6.6 mg anhydrous L-asparagine per milliliter. DEAE-dextran solution, molecular weight of 500, 000 (Pharmacia Fine Chemicals, Piscataway, New Jersey). Prepare a 50 mg/ml solution, and sterilize by autoclaving. Bacto-Agar (Difeo Laboratories, Detroit, Michigan) : Prepare 3% suspension in purified water, and sterilize by autoclaving. Vitamin C, L-ascorbic acid, 30 mM solution, must be stored at -20°C or below. Tryptic soy broth (Difeo Laboratories, Detroit, Michigan): Prepare a 3% stock solution, and sterilize by autoclaving. 2-Mercaptoethanol, a O. 5 x 10-3 M stock solution is prepared weekly.

Many of these media ingredients may be purchased from a supplier as sterile solutions without added antibiotics. Solutions prepared in the laboratory are made up with purified water (tissue culture grade) and sterilized by autoclaving or filtration through 0. 22 µm membrane filters. Liquid Media Formulations: Enriched McCoy's Medium 5A: McCoy's medium 5A, 100 ml Horse serum, 5 ml FCS, 10 ml Sodium pyruvate solution, 1 ml L-serine solution, 0. 2 ml L-glutamine, lOOX, 1 ml PS, 1 ml Enriched CMRL 1066, minus asparagine, DEAE-dextran: CMRL 1066, 100 ml Horse serum, 15 ml CaCl2 solution, 4 ml Insulin, 2 ml Vitamin C , 1 ml PS, 1 ml L-glutamine, 1 ml Plating Media Formulations: For the base layer, molten agar (cooled to 48-50°C) is gently mixed with the other medium ingredients {prewarmed to 37°C) in the proportions given. Care should be taken to mix the ingredients uniformly without excessive aeration. Immediately distribute 1 ml of the agar mixture to each 35 mm tissue culture dish, and allow to solidify on a level surface. After the agar has solidified, the plates may be used immediately or stored under refrigeration for 1 week, depending on the stability of the ingredients.

404

Garretson

Partial substitution of the enriched McCoy's medium 5A may be made with conditioned media found stimulatory for the growth of certain tumor types at a final medium concentration of 25%. Other growth factors may be added to improve clonogenicity. The medium ingredients are mixed in the following proportions: Enriched McCoy's medium 5A, 40 ml Tryptic soy broth solution, 10 ml Asparagin solution, 0. 6 ml DEAE-dextran solution, 0. 3 ml Agar solution, 6.1 ml For the top layer, the following ingredients are added to the liquid formulation of enriched CMRL 1066: Enriched CMRL 1066, 40 ml Asparagin solution, O. 6 ml DEAE-dextran, 0.3 ml 2-Mercaptoethanol (optional), O. 4 ml Agar and cells are added last in the sequence specified for drug treatment (see assay procedure). Tumor Cell Suspensions: Pleural or ascitic fluids are collected by Thoracentesis or paracentesis into 500 ml vacuum plasma bottles to which 5 ml of sodium heparin solution has been added. When adding the heparin, use a small-gauge needle to avoid breaking the vacuum. The quantity of fluid withdrawn from a single donor will vary, but may be as much as 2 liters or more. During the collection process, the bottle should be gently agitated to prevent clotting. All effusions are stored at 4°C and used within 24 hr of collection. In the laboratory, the following procedure is followed to obtain single-cell suspensions. Insert an airway needle into the vacuum bottle to break the vacuum and remove the rubber stopper. Distribute the fluid into 50 ml centrifuge tubes, and centrifuge at 150 g for 10 min. Cells attached to the wall of the transport container may be removed mechanically by gentle scraping or enzymatic procedures. Examine the pellet for the presence of red blood cells (RBC). If the pellet is predominately white, proceed to the next step without further treatment. Large amounts of RBC may be removed by hemolysis by the addition of 10 volumes of distilled water, rapid mixing by pipetting for up to 2 min, and the subsequent addition of 1 volume of lOX HBSS90FBS10. The hemolyzed suspension is centrifuged and the cells resuspended in the same washing formulation. Wash the cells twice with HBSS90FBS10. and resuspend in the top layer formulation or in McCoy's 5A plus 10% FCS. Determine cell viability using the trypan blue dye exclusion method, and calculate the cell yield. Adjust the suspension to contain 4 x 106 viable cells per milliliter in the medium for the 1 hr drug exposure or the top layer formu lation (see assay procedure). Bone marrow specimens obtained by sternal or iliac puncture are aspirated into a heparinized syringe and mixed 5: 1 with 6% dextran in saline to sediment RBC. After 45 min at room temperature, the supernatant fluid containing the bone marrow cells is removed by aspiration. The cells are washed twice in the

Mammalian Cell Culture

405

washing solution (HBSS90FBS1o> by centrifugation at 150 g for 10 min. The washed cells are then resuspended at 4 x 106 viable cells per milliliter in the appropriate medium (see assay procedure). Solid tumor specimens obtained from primary tumor sites, nodules, or lymph nodes are placed in a jar with sufficient growth medium to cover the specimen and promptly transported to the laboratory. McCoy's 5A medium supplemented with 10% FCS and antibiotics may be used for bathing the specimen. The best results are obtained with specimens processed within 2 hr after excision, but some tumor specimens may remain viable at 40°C for 24 hr. The tumor mass should be separated from extraneous tissue and necrotic areas and washed two to three times in HBSS 90FBs 10 • 1. 2.

3. 4. 5.

Transfer the washed tumor tissue to a glass petri dish, add 5-10 ml of growth medium, and mince the tissue using a pair of scalpels to 1 mm fragments. Transfer the tissue fragments and medium to a 50 ml centrifuge tube. Pipette the tissue suspension repeatedly to break up the clumps and free the cells from the tissue slices. Allow the larger fragments to settle for 2-3 min, and decant the supernatant fluid over a sterile gauze pad draped over a small funnel. Pipette the filtered cell suspensions to break up the remaining clumps of cells. Progressively pass the cell suspensions through needles of decreasing size from 18 to 25 gauge to obtain a single cell suspension. Collect the cells in a 50 ml centrifuge tube. Wash the cells twice in growth medium at 150 g for 10 min, and resuspend at 4 x 106 cells per milliliter in the appropriate growth medium (see assay procedure).

Test Compounds

Experimental drugs are solubilized as described previously in an analogous section under Protein Determination. Antitumor agents available as intravenous formulations and other solubilized test compounds are diluted to a final lOX concentration in buffered saline for each test concentration. All compounds should be tested initially at a minimum of three dose levels at 10 log intervals within the range 0.1-10. 0 µg /ml. Since most chemotherapeutic agents have molecular weights of 100-1000, this dose range will approximate the pharmacologically achievable concentrations of 0.1-10 µM. When tumor cells are obtained in sufficient quantity, the concentration range should be extended to O. 01 and 100 µg/ml to add a negative and positive control and to better define the lower limits of activity for the more cytotoxic substances. It should be noted that, in the continuous exposure method, the concentration of test compound will be reduced by 50% by diffusion into the base layer. Supplies and Equipment

Vacuum plasma bottles, 500 ml Syringes, 5 and 10 ml Syringe needles, 18 to 25 gauge Airway needle, 17 gauge Pipettes, large orifice, 5 and 10 ml Bard-Parker handles and blades (B-P 10 or 22)

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Sterile gauze pads, 4 x 4 inch squares (Johnson and Johnson, New Brunswick, New Jersey) Centrifuge tubes, conical, 40 or 50 ml, graduated, screw cap Specimen jars, screw cap , 8 ounce Screw-capped test tubes, 16 x 100 mm Petri dishes, 100 x 15 mm, glass Tissue culture dishes, 35 x 10 mm, disposable, grid Inverted phase microscope Laminar flow biohazard safety cabinet Humidified C02 incubator Water baths, 37 and 50°C Centrifuge Filter units with 0. 22 µm membrane filters Water purification system Assay Procedure Suspension of Cells in Top Agar Layer Drug Exposure of 1 hr: Mix in a screw cap culture tube O. 5 ml of tumor cell suspension (4 x 106 cells/ml), 0.2 ml of lOX drug solution, and 1.3 ml of McCoy's 5A medium supplemented with 10% FCS (no antibiotics) for a total volume of 4 ml. Simultaneously prepare a single mixture of 1. 5 ml of the medium and 0. 5 ml of tumor cell suspension for seeding the controls. Incubate the control and treated cell mixture for 1 hr at 37°C. Wash the cells twice with the same medium by centrifugation at 150 g for 10 min. Resuspend the cell pellets in 3. 6 ml of enriched CMRL 1066 (top layer formulation). Add 0. 4 ml of molten agar, and mix gently. Distribute 1 ml of the agar mixture to each of three plates containing a 1 ml base layer. Continuous Drug Exposure (Optional Method): Incubate the cell suspension for 1 hr at 37°C in the absence of any other additives. Combine in a screw cap tissue culture tube 0.5 ml of cell suspension, 0.4 ml of lOX test compound, 2. 7 ml of enriched CMRL 1066 (top layer formulation), and 0. 4 ml of 3% agar for a total volume of 4. 0 ml. Mix gently to distribute the contents evenly. Distribute 1 ml of the agar mixture to each of three plates containing a precooled base layer. Incubation of Plates

Place the plates in a 37°C humidified (maximum saturation) incubator flushed with 5- 7. 5% CO 2. The actual amount of CO 2 used should be preestablished as the minimum amount of CO 2 required to maintain the desired pH of the medium using identical equipment and experimental conditions. Examine the cultures biweekly with an inverted phase microscope at 30- lOOX magnification. Cell clumps and debris are scored before colony growth is evident. When colonies reach maximum size, usually within 7-21 days, the experiment is terminated and colonies containing 30 or more cells are scored.

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Results

In vitro sensitivity to each concentration of test compound (T /C) is calculated as the average percentage of survival of drug-exposed colonies (X colonies treated) divided by the average number of colonies in the controls (X colonies control). An arbitrary end point defining an acceptable level of activity may be established from log dose- survival curves as the con centration of drug effecting 50% survival. The arbitrary selection of a minimum-sized colony for inclusion in the surviving colony count may not exclude small abortive colonies of cells whose continued growth may have been interrupted by a slow-acting cytotoxic compound. The incorporation of an appropriate drug standard at concentrations spanning the entire range of activity would be useful in identifying artifactual plateaus in the dose-response curves and defining the limits of assay sensitivity in terms related to minimum colony size. Alternative Medium Additives Preparation of Conditioned Media

Adherent spleen cell populations comprised predominately of macrophages produce in culture soluble factors that support the growth of human myeloma and lymphoma in soft agar [ 50, 51] • Conditioned medium incorporated at 25% in the feeder layer is prepared from primary cultures of spleen cells derived from BALB}c mice primed 8-12 weeks previously with 0. 2 ml mineral oil injected intraperitoneally. The culture fluid from adherent cells grown for 3 days is harvested, centrifuged to remove cells and debris, and passed through O. 22 µm membrane filters. For a more detailed procedure for splenic cell cultures, consult References 38 and 50. All culture fluids selected as conditioning medium in the colony assay should be obtained from actively metabolizing cell cultures, pretested for growth stimulation and the absence of microbial contamination, and stored at

-20°c.

Use of Growth-Modulating Factors The use of hormonal growth promoters, such as insulin, thyroxin , steroids, and pituitary factors, might well be evaluated for the support of growth of specific tumors, particularly those that are hormonally responsive in vivo. In addition , growth factors elaborated by host cell populations, such as human macrophages, and purified growth factors available commercially, including epidermal growth factor and nerve growth factor, may prove to significantly support clonal growth and are worthy of investigation. MODULATION OF MACROPHAGE TUMORICIDAL CAPABILITY Materials

In an assay developed for demonstrating macrophage-mediated cytotoxicity, amphotericin B and nystatin show an enhancing effect on macrophage tumoricidal potential toward tumorigenic 3T 12 target cells [ 52] • Among the polyene class of antibiotics this activity appears to be limited to the two large, sugarcontaining amphoteric polyenes, as other sterol-binding polyenes with smaller

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ring structure, filipin and pimaricin, are without activity. Nystatin also activates macrophages in vivo to become tumoricidal for Moloney murine leukemia cells in vitro [ 53] • Although the relationship of biological activity to alterations in macrophage membranes is not known, membrane perturbation effects were observed by electron spin resonance measurements after nystatin treatment of peritoneal macrophages [ 54] • The assay method that follows is essentially that of Chapman and Hibbs [ 52], described in detail in earlier publications [ 55- 57] .

Reagents All sera utilized in this assay must be endotoxin free and heat inactivated ( 56°C for 30 min). Test substances active in this system should also be analyzed for the presence of endotoxin by the Limulus amoebocyte lysate assay (Microbiological Associates, Bethesda, Maryland). Chemicals and Stock Solutions: 1. 2. 3. 4.

5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

Adult bovine serum Fetal bovine serum HEPES buffer Penicillin-streptomycin, lOOX stock solution containing 10,000 IU/ml of penicillin and 10, 000 µg /ml streptomycin Dulbecco's modified Eagle's Medium (DMM), lX stock solution Dulbecco's modified Eagle's medium, fortified (DMMF), DMM fortified with 0.1% glucose, 1% PS, and 0.02 M HEPES buffer 3T12 growth medium, DMMF suplemented with 10% FBS Assay medium, DMMF supplemented with 10% ABS Bacille Calmette-Guerin, BCG, Paris strain Nystatin (ICN Pharmaceuticals, Cleveland, Ohio) Phosphate-buffered saline Absolute methanol Giemsa stain Heparin, sodium salt, without preservatives, 1000 USP U/ml

Laboratory Mice: Female mice, 8-12 weeks old, are obtained from C3H/ HeN, C56BL/6, BALB/c, or other pathogen-free colonies of pure-bred mice. Animals are housed and fed according to standard animal care procedures. Cell Cultures: Monolayer cultures are prepared using nontumoricidal activated macrophages taken from mice i.p. injected with 0.2 mg BCG, 17-22 days before initiating the assay. Approximately 5 x 107 cells are obtained from six to eight mice. Peritoneal exudates are collected from control and treated mice by .i.p. injection of 4.0 ml of warm PBS containing 2 U/ml heparin followed by paracentesis 5 min later. The peritoneal washings are pooled, washed twice with PBS, and diluted to about 4 x 106 viable cells per milliliter in the assay medium. The 3T12 cell line (ATCC CCL 164, American Type Culture Collection, Rockville, Maryland) is maintained in 3T 12 growth medium by weekly passage of T-15 flask cultures seeded with 2-4 x 105 viable cells per flask. The yield is approximately seven- to eightfold. Cells are harvested and resuspended to contain 3 x 104 viable cells per milliliter.

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Supplies and Equipment

Humidified C02 incubator, 5% C02 in air Inverted microscope, 300X magnification Microtest II tissue culture plates, Falcon No. 3040 or equivalent (Falcon Plastics, Oxnard, California) Assay Procedure Each experimental culture plate should be set up with control rows of eight chambers containing ( 1) normal macrophages alone, ( 2) BCG-activated macrophages alone, and (3) 3T12 cells alone. The remaining rows are utilized for macrophage activation by experimental substances and cytotoxicity evaluations of test substances for BCG-activated macrophages. The concentrations of compounds inducing tumor cell killing by macrophages should be noncytotoxic to macrophages. Macrophage cells are added to the culture plates by delivering 0.1 ml of the cell suspension to the appropriately labeled chambers. Macrophages are allowed to adhere to the chamber floor for 1 hr at 37°C in a humidified co 2 incubator. The adherend cells are then washed twice with warm PBS using gentle suction. Care should be taken not to disturb the monolayer during the washing procedure. Final dilutions of test compounds are made in the assay medium, and 0.1 ml of each test dilution is added to the macrophage containing chambers in triplicate. Controls receive 0.1 ml of assay medium only. Plates are reincubated for 2 hr. Wash the monolayers twice with PBS, add 0.2 ml of the 3T12 cell suspension to each chamber of treated macrophages, and seed a row of 3T 12 cells for controls. Incubate for 60 hr, and observe cytostatic and cytocidal effects at 300X magnification after fixing in methanol and staining with Giemsa stain. Results The control 3T 12 cells attached to the macrophage-free chamber surface are compared with the experimental cultures and tumor cell kill rated using an arbitrary rating system suitable for the purposes of the investigation. Tumor cell killing effect is rated positive when less than five 3T 12 cells are observed in a single microscopic field at 300X magnification. Both amphotericin B and nystatin produce this response at 0. 5-1. 5 µg /ml and 50-500 U /ml, respectively [ 52) . Gradations intermediate between the positive and negative responses may also be assigned on the basis of the condition and number, or percentage of target cells remaining per microscopic field. REFERENCES 1. 2.

H. Eagle and G. E. Foley, The cytotoxic action of carcinolytic agents in tissue culture, Am. J. Med., 21:739 (1956). V. I. Oyama and H. Eagle, Measurement of cell growth in tissue culture with a phenol reagent (Folin-Ciocalteau), Proc. Soc. Exp. Biol. Med., 91:305 (1956).

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6. 7. 8. 9. 10. 11.

12. 13.

14.

15. 16. 17. 18.

Garretson O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193:265 (1951). C. G. Smith, W. L. Lummis, and J. E. Grady, An improved tissue culture assay. I. Methodology and cytotoxicity of antitumor agents, Cancer Res., 19:843 (1959). R. I. Geran, N. H. Greenburg, M. M. Macdonald, A. M. Schumacher, and B. J. Abbott, Protocols for screening chemical agents and natural products against animal tumors and other biological systems, Cancer Chemother. Rep. Part 3, 3: 1 ( 1972). G. E. Foley and S. S. Epstein , Cell culture and cancer chemotherapy, in Advances in Chemotherapy (A. Goldin and F. Hawking, eds.), Academic Press, New York, 1964, p. 175. P. S. Thayer, P. Himmelfarb, and G. L. Watts, Cytotoxicity assays with L1210 cells in vitro: Comparison with L1210 in vivo and KB cells in vitro, Cancer Chemother. Rep. Part 2, 2:1 (1971). C. G. Smith, W. L. Lummis, and J. E. Grady, An improved tissue culture assay. II. Cytotoxicity studies with antibiotics, chemicals, and solvents, Cancer Res., 19: 847 ( 1959). L. H. Li, S. L. Kuentzel, K. D. Shugats, and B. K. Bhuyan, Cytotoxicity of several marketed antibiotics on mammalian cells in culture, J. Antibiot., 30: 506 (1977). K. K. Sanford, W. R. Earle, V. J. Evans, H. K. Waltz, and J. E. Shannon, The measurement of proliferation of tissue cultures by enumeration of cell nuclei, JNCI, 11: 773 (1951). M. Harris, Electronic enumeration and sizing of cells. B. Tissue culture cells, in Tissue Culture Methods and Applications (P. F. Kruse, Jr., and M. K. Patterson, Jr., eds.), Academic Press, New York, 1973, p. 400. H. H. Buskirk, Assay of cytotoxic agents with L1210 cells, Proc. Tissue Culture Ass., 20: 23 ( 1969). J. Douros and M. Suffness, The National Cancer Institute's Natural Products Antineoplastic Development Program, in Recent Results in Cancer Research, New Anticancer Drugs (S. K. Carter and Y. Sakurai, eds.), Springer-Verlag, Berlin, 1980, p. 21. S. Yamazaki, K. Nitta, T. Hikiji, M. Nogi, T. Takeuchi, T. Yamamoto, and H. Umezawa, Studies on antitumor substances produced by Actinomycetes. XII. Cylinder plate method of testing the anti-cell effect, J. Antibiot. Ser. A, 9:135 (1956). S. Miyamura, A determination method for anticancer action of antibiotics by the agar plate diffusion technique, Antibiot. Chemother., 6: 280 (1956). J. A. DiPaolo and G. E. Moore, An evaluation of ascites tumor cell plating for screening chemotherapeutic agents, Antibiot. Chemother., 7: 465 (1957). S. Miyamura and S. Niwayama, An agar plate diffusion method using HeLa cells for antitumor screening, Antibiot. Chemother., 9: 497 (1959). M. Abe, K. Miyaki, D. Mizuno, N. Narita, T. Takeuchi, T. Ukita, and T. Yamamoto, The cell agar plate screening for antitumor cell effect. I. Screening results of 1300 organic compounds, Jpn. J. Med. Sci. Biol., 12:175 (1959).

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22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36.

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D. M. Schuurmans, D. T. Duncan, and B. H. Olson, An agar plate assay for anticancer agents utilizing serially cultured Sarcoma 180 (Foley), Antibiot. Chemother., 10: 535 ( 1960). D. Perlman, W. L. Lummis, and H. J. Geiersbach, Differential agardiffusion bioassay for cytotoxic substances, J. Pharm. Sci. , 58: 633 ( 1969). A. L. Garretson, R. K. Elespuru, I. Lefriu, D. Warnick, T. Wei, and R. J. White, In vitro prescreens for the detection of antitumor agents, in Developments in Industrial Microbiology, Vol. 22 (L. A. Underkofler and M. L. Wulf, eds.), Society for Industrial Microbiology, Arlington, Virginia, 1981, p. 211. F. Klein and R. T. Ricketts, Uniform production and bulk storage of P388 murine lymphoma cells for antitumor assay, Proc. Soc. Exp. Biol. Med., 163: 406 (1980). D. Perlman, J. B. Semar, G. W. Krakower, and P. A. Diassi, Structure-cytotoxicity relations of some corticosteroids, Cancer Chemother. Rep. , 51: 255 (1967). P. s. Thayer, Spin filter device for suspension cultures, in Tissue Culture Methods and Applications, (P. F. Kruse, Jr. , and M. K. Patterson, Jr., eds.), Academic Press, New York, 1973, p. 345. M. J. Weinstein and G. H. Wagman (eds.), Antibiotics: Isolation, Separation and Purification, Elsevier Scientific, New York, 1978. J. E. G!.'ady, W. L. Lummis, and C. G. Smith, Tissue culture bioautographic system, Proc. Soc. Exp. Biol. Med., 103: 727 (1960). P. Siminoff and V. S. Hursky, Determination of mammalian cell (strain HeLa) inhibition by an agar diffusion technic. II. Paper chromatographic methods, Cancer Res. , 20: 618 (1960). D. M. Schuurmans, D. T. Duncan, and B. H. Olson, A bioautographic system employing mammalian cell strains, and its application to antitumor antibiotics, Cancer Res. , 24: 83 (1964). H. L. Issaq, E. W. Barr, T. Wei, C. Meyers, and A. A. Aszalos, Thin-layer chromatographic classification of antibiotics exhibiting antitumor properties, J. Chromatogr., 133:291 (1977). A. W. Hamburger and S. E. Salmon, Primary bioassay of human tumor stem cells, Science, 197:461 (1977). A. W. Hamburger, S. E. Salmon, M. B. Kim, J. M. Trent, B. J. Soehnlen, D. S. Alberts, and H. J. Schmidt, Direct cloning of human ovarian carcinoma cells in agar, Cancer Res., 38: 3438 (1978). V. D. Courtenay, P. J. Selby, I. E. Smith, and M. J. Peckham, Growth of human tumor cell colonies from biopsies using two softagar techniques, Br. J. Cancer, 38:77 (1978). M. L. Rosenblum, D. A. Vasquez, T. Hoshino, and C. B. Wilson, Development of a clonogenic cell assay for human brain tumors, Cancer, 41:2305 (1978). P. M. Kimball, M. G. Brattain, and A. M. Pitts, A soft-agar procedure measuring growth of human colonic carcinomas, Brit. J. Cancer, 37: 1015 (1978). R. F. Ozols, J. K. V. Wilson, K. R. Grotzinger, and R. C. Young, Cloning of human ovarian cells in soft agar from malignant effusions and peritoneal washings, Cancer Res., 40: 2743 (1980). D. D. Von Hoff, J. Casper, E. Bradley, J. M. Trent, A. Hodach, c. Reichert, R. Makuch, and A. Altman, Direct cloning of human neuroblastoma cells in soft agar culture, Cancer Res., 40: 3591 (1980).

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38. 39.

40.

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42. 43. 44.

45.

46. 47. 48. 49.

50. 51.

Garretson V. P. Pavelic, H. K. Slocum, Y. M. Rustum, P. J. Creaven, C. Karahousis, and H. Takita, Growth of cell colonies in soft agar from biopsies of different human solid tumors, Cancer Res. , 40: 4151 (1980). S. E. Salmon (ed.), Cloning of Human Tumor Stem Cells, Alan R. Liss, New York, 1980. S. E. Salmon, A. W. Hamburger, B. Soehnlen, B. G. M. Durie, D. S. Alberts, and T. E. Moon, Quantitation of differential sensitivity of human-tumor stem cells to anticancer drugs, N. Engl. J. Med., 298: 1321 (1978). D. S. Alberts, H. S. G. Chen, B. Soehnlen, S. E. Salmon, E. A. Surwit, L. Young, and T. E. Moon, In vitro clonogenic assay for predicting response of ovarian cancer to chemotherapy, Lancet, 2: 340 (1980). D. D. Von Hoff, J. Casper, E. Bradley, J. Sandback, D. Jones, and R. Makuch, Association between human tumor colony-forming assay results and response of an individual patient's tumor to chemotherapy, Am. J. Med., 70: 1027 (1981). P. Selby, R. N. Buick, and I. Tannock, A critical appraisal of the "human tumor stem-cell assay," N. Engl. J. Med. , 308: 129 (1983). W. J. Mackillop, A. Ciampe, J. E. Till, and R. N. Buick, A stem cell model of human tumor growth: Implications for tumor cell clonogenic assays, JNCI, 70:9 (1983). B. E. Kressner, R. A. Roger, A. E. Morton, A. E. Martens, S. E. Salmon, D. D. Von Hoff, and B. Soehnlen, Use of an image analysis system to count colonies in stem cell assays of human tumors, in Cloning of Human Tumor Stem Cells (S. E. Salmon, ed.), Alan R. Liss, New York, 1980, p. 179. R. H. Shoemaker, M. K. Wolpert-DeFilippes, and J. M. Venditti, Application of a human tumor clonogenic assay to screening for new antitumor drugs, in Proc. XIII Int. Cong. Chemother., Vienna, Austria, 1983. R. H. Shoemaker, M. K. Wolpert-DeFilippes, R. W. Makuch, and J. M. Venditti, Application of the human tumor clonogenic assay to drug screening, Stem Cells, 1:308 (1981). R. H. Shoemaker, M. K. Wolpert-DeFilippes, R. W. Makuch, and J. M. Venditti, Use of the human tumor clonogenic assay for new drug screening, Proc. Am. Ass. Cancer Res. , 24: 311 (1983). J. M. Venditti, The National Cancer Institute antitumor drug discovery program, current and future perspectives: A commentary, Cancer Treat. Rep., 67: 767 (1983). D. S. Alberts, H. S. G. Chen, and S. E. Salmon, In vitro drug assay; pharmacologic considerations, in Cloning of Human Tumor Stem Cells (S. E. Salmon, ed.), Alan R. Liss, New York, 1980, p. 197. A. W. Hamburger and S. E. Salmon, Development of a bioassay for human myeloma colony-forming cells, in Cloning of Human Tumor Stem Cells (S. E. Salmon, ed.), Alan R. Liss, New York, 1980, p. 23. A. W. Hamburger, S. E. Jones, and S. E. Salmon, Soft-agar cloning of cells from patients with lymphoma, in Cloning of Human Tumor Stem Cells (S. E. Salmon, ed.), Alan R. Liss, New York, 1980, p. 43.

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53. 54.

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H. A. Chapman, Jr., and J. B. Hibbs, Jr., Modulation of macrophage tumoricidal capability by polyene antibiotics: Support for membrane lipid as a regulatory determinant of macrophage function, Proc. Natl. A cad. Sci. USA, 75: 4349 (1978). D. L. Klein, A. Aszalos, and J, W. Pearson, Macrophage cytostasis and T and B cell blastogenic transformation in mice treated with nystatin, J. Immunopharmacol., 2: 367 (1980). A. Aszalos, A. Doshi, R. A. Zaldivar, and D. L. Klein, Studies on the mode of action and macrophage activity modulating ability of the polyene antibiotic nystatin, in Proc. XII Int. Cong. Chemother., 12: 1 (1981). J. B. Hibbs, Jr., R. R. Taintor, H. A. Chapman, Jr., and J, B. Weinberg, Macrophage tumor killing: Influence of the local environment, Science, 19 7: 279 ( 1977). H. A. Chapman, Jr. , and J. B. Hibbs, Jr., Modulation of macrophage tumoricidal capability by components of normal serum: A central role for lipid, Science, 197: 282 (1977). J. B. Weinberg, H. A. Chapman, Jr. , and J. B. Hibbs, Jr., Characterization of the effects of endotoxin on macrophage tumor cell killing, J. lmmunol., 121: 72 (1978).

11 Immunological Approaches DEBORAH E. DIXON,* SUSAN J. STEINER, and STANLEY E. KATZ

Cook College, New Jersey Agricultural Experiment Station, Rutgers University, New Brunswick, New Jersey

The Immunological Assay Definitions Antigen Antibody Hap tens Haptenic Groups Agglutination Passive Agglutination Precipitation Complement Production of Antibodies Immunological Approaches to the Analysis of Antibiotics The Agglutination Assay Complement Fixation Assay Immunodiffusion Radioimmunoassay Nonisotopic Immunoassays Fluoroimmunoassays Enzyme Multiplied Immunoassay Technique Enzyme- Linked Immunosorbent Assay Overview References

416 417 417 417 417 417 418 418 418 418 418 419 419 421 421

422

425 425 426 427 429 429

Traditionally, the analysis of a wide variety of antibiotics, regardless of the structures and matrices involved, has been performed using microbiological assay methods. In 1945 [ 1), the basic protocols for antibiotic analyses were established; these protocols for inhibition or diffusion procedures remain as the basis of the analytical science involved [ 2) • The microbiological assay procedures, whether based upon the diffusion of turbidimetric systems, all

*Present affiliation: Michigan State University, East Lansing, Michigan. 415

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suffer from a basic flaw: the procedures are nonspecific. Many of the commonly used procedures utilize only a purification by dilution prior to analysis. Also, the organisms used in the assays respond to more than one antibiotic family, as well as other nonspecific inhibitory materials. Hence there are, inherent in the microbiological assay systems, sources of analytical bias and inaccurate results. On balance, however, the sensitivity of the assay organisms to the antibiotic measured is usually sufficient to minimize many of the aforementioned problems. The lack of specificity remains a serious flaw. Reasonably elaborate separations are necessary to separate the various antibiotic families prior to analysis. This problem is not especially serious when the formulation being analyzed is known. Only in the case of unknown samples or materials of dubious lineage can this be a serious problem. This is not unique to microbial methods alone, although the problem can be acute in microbial procedures. Chromatographic procedures for antibiotics are increasing in number and in the ability to measure low levels. These procedures have excellent potential since there is a reasonably good agreement between the chromatographic and microbial assay results. The chromatographic systems have been extended to the analysis of biomass products with promising results. Although few chromatographic procedures have been extended successfully to the analysis of antibiotics in the parts per million range, there are indications that such extensions will be more commonplace in the future. Chromatographic procedures are not without problems. In complex matrices, extensive cleanup procedures are required. All chromatographic procedures are limited in the sense that chromatography is only a method of separation, not identification. Quantitation is the forte of chromatographic procedure, not identification. Spectral identification would be required if structural confirmation is necessary [ 3) • Because of these problems, immunological approaches have significant merit. Such procedures have the potential of possessing reasonable sensitivity, accuracy, precision, and a significant degree of specificity. THE IMMUNOLOGICAL ASSAY

The basis of the immunological assay is somewhat different than microbiological assays. Immunological assays measure a structural component of the molecule. In contrast, microbiological assays measure the ability to inhibit the growth of microorganisms. Microbial assays measure the active antibiotic as well as microbiologically inhibitory metabolites but do not measure inactive metabolites. Immunological assays can measure all moieties, microbiologically active or not, that have a structure that can evoke the antigenic response. Immunological assays should not be affected by antibiotics from families other than that being assayed or nonspecific inhibitory sources. The basis of the immunoassay for antibiotics depends upon the binding of the antibiotic with a specific antibody in a reversible reaction: Ag

+

Ab

Ag:ab

antibiotic + antibody =r antigen: antibody (complex) (antigen) The reaction is an equilibrium reaction and continues until the concentration of antigen in both the free state and the complex are constant [ 4) • The

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antiserum is one of the most important reagents in the immunoassay. It determines the specificity and to some extent the sensitivity. Because most antibiotics are relatively small molecules (molecular weights less than 400), they must be complexed with carrier proteins, such as bovine serum albumin, to create an immunogenic reagent. This complex is used to invoke the immune response and the development of antibodies. The specificity of the antibodies developed is dependent upon the haptenic structure. In general, immune reactions are highly specific. Any given population of antibody molecules will (usually) have different affinities for those ligands, whose structures differ in a subtle fashion. As a rough rule of thumb, the ability of antibodies to distinguish molecules is comparable to the specificity of enzymes. DEFINITIONS

Prior to any discussion, it is necessary to define commonly used terms and to explore common concepts. Antigen

Antigens have two common properties: ( 1) the ability to stimulate the formation of antibody and (2) the ability to react specifically and essentially exclusively with these antibodies. Specificity relates to the highly selective fashion with which the antigen reacts with the corresponding antibody, not with antibodies produced by other antigens. Antibody

These are proteins that are formed from the response to an antigen and react specifically with the antigen. All antibodies belong to a group of proteins, the immunoglobulins. Although the definition implies that antibodies are formed only in response to antigen, sera may contain immunoglobulins that react specifically with certain antigens although there was no known exposure. These are called natural antibodies. Haptens

These are substances that are not immunogenic in their own right but react selectively with antibodies of appropriate specificity. Haptens are usually relatively small molecules with a molecular weight less than 1000 and are not immunogenic unless covalently linked to proteins (in vitro or in vivo). Haptenic Groups

Proteins with substituents covalently linked to the side chains (sometimes covalently linked to amino acid residues) are haptenic groups. Although the haptenic group is part of the antigenic determinant, there is no definitive information as to the extent it is represented in the determinant. Some antibodies against a haptenic-protein conjugate seem to react only with the haptenic group; others, unfortunately, exhibit carrier specificity, requiring not only the haptenic group and the amino acid attachment residue but neighboring unsubstituted residues of the immunogen.

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Agglutination

This is the clumping of cells that occurs following a union of antigen and antibody. A more general definition is an aggregation of particles not in solution. It is assumed generally that the term agglutination refers to cells, including bacteria and red blood cells. Antigen-coated particles, such as polystyrene latex particles or bentonite, are included in the definition. The antibodies are directed against antigenic determinants located on red blood cells or bacterial cell surface . Passive Agglutination

This is the agglutination of particulates by antibodies directed against soluble antigen attached to the surface of the materials. The attachment of antibody or immunoglobulin has been referred to as reserve passive agglutination, since the antigen in the sample binds to the antibody. Inhibition of passive agglutination is used for antigen determination. Agglutination is inhibited by the addition of free antigen that competes for the antibody. Precipitation

The phenomenon occurs when antibody connects with antigen to form large polymericlike structures that become insoluble because of the modifications of the polar groups responsible for solubility. One of the most common uses of this phenomenon is the agar diffusion, double diffusion, or Ouchterlony reaction. Here, antibody and antigen diffuse through agar, and where they meet, in concentrations meeting the equivalence zone of the precipitin reaction, a band will form. The precipitin line will form closest to the lower concentration of either the antibody or antigen source and is dependent upon which reagent is in excess. Complement

This is a labile factor that is a protein although not an immunoglobulin. This protein factor is a collection of proteins, up to 20, that reacts with antigenantibody complexes and is capable of lysing sensitized cells or causing other abberations of cells. Red cell lysis provides the basis for the complement fixation assay. This collection of proteins is highly unstable and sensitive to heat, storage, and even violent agitation. Complement can be inactivated by acids, baaes, solvents, proteolytic enzymes, tissue cells, or extracts, as well as many unsensitized bacteria [ 5] . PRODUCTION OF ANTIBODIES

An immune response is initiated when an antigen, carrying a number of antigenic determinants, is introduced into an animal's body. Lines of B cells mature into plasma cells, and each secretes an immunoglobulin molecule that fits a single determinant or a portion of the determinant [ 6].

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Antibodies in conventional antisera are polyclonal proteins since they are also directed against other components in the antigen preparation, rather than to the antigen alone. It is virtually impossible to separate the different antibodies, a problem that has led to major drawbacks in the use of polyclonal antibodies in biology and medicine. Myeloma proteins are referred to as monoclonal proteins, since they are produced from a single clone. Kohler and Milstein [ 7] exploited this knowledge and revolutionized immunology by demonstrating that mortal (not capable of growing in culture) antibody-producing cells, namely, spleen cells, could be immortalized by fusion with malignant mouse myeloma cells and could produce individual clones called hybridomas. These cells secrete only a single species of antibody [ 8] • The immunization schedule of a normal spleen donor varies depending upon the nature of the immunizing antigens. Cell surface antigens usually are highly immunogenic when administered on intact cells. A typical protocol involves priming with 2 x 107 cells intraperitoneally and boosting with the same dose 3 weeks later [ 9] . Many soluble proteins are often very poorly immunogenic in aqueous solution. Therefore, adjuvants are often used to enhance the immunogenic capabilities. There are two desired goals from immunization: ( 1) expansion of desired clones to increase the chance of obtaining relevant hybrids, and (2) to cause B cells to divide and differentiate into cells that will fuse and form useful hybrids [ 6] • In the last 8 years, the use of monoclonal antibodies has spread into almost every field of biology, medicine, biochemistry, and chemistry [ 10-12] • Monoclonal antibodies have been employed to prove the fine structure of proteins, as reagents for radioimmunoassays, enzymeimmunoassays for hormones and drugs, tumor localization and classification, immunotherapy, histocompatability testing, purification of molecules by affinity chromatography, and identification of the causative agents in microbial, parastic, and viral diseases. IMMUNOLOGICAL APPROACHES TO THE ANALYSIS OF ANTIBIOTICS

The Agglutination Assay

The prirciple and the ultimate sensitivity of the passive hemagglutination test is based upon the least amount of soluble antigen necessary to inhibit agglutination. Analytically, this is the amount of antigen in the last tube that will give a wide ring agglutination pattern. Tubes containing less antigen than this tube will allow an agglutination pattern to be formed. Hence, a competitive effect is not found between the antigen-sensitized cells, antiserum, and antigen-containing sample. The antigen on the cell will bind the antiserum, and an agglutination pattern will be found [ 13]. Methods can be based upon the agglutination reaction and would be semiquantitative assay procedures. Since it is common to use a twofold dilution scheme, the procedures can only yield results that reflect the dilution sequence. The greater the interval between concentration levels, obviously the greater is the inherent error; conversely, the narrower the range, the more accurate is the assay.

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In general, blood from sheep is collected and mixed with an anticoagulant, such as heparin or Alsever's solution. The anticoagulant-treated blood should be stored at 4dc for at least 24 hr or preferably 5 days before use. The erythrocytes are separated and treated with tannic acid. The treated erythrocytes are exposed to antibiotic in physiological saline and heated for 37°C for 1 hr. The suspension is centrifuged, the supernatant discarded and the cell concentraHon is adjusted to approximately 2% with diluent.

Titration of Antiserum Titrations are necessary to standardize the reagents and to remove any nonspecific interferences (lack of hemagglutination). In suitable test tubes a fixed volume of the 2% cell suspension is added to a fixed volume of antiserum. The antiserum and the cells are incubated at room temperature for 30-60 min and the hemagglutination patterns observed.

Interpretation of Results of Hemmagglutination Assay Results from hemagglutination assays are interpreted in the following manner [ 13] : +4 Partial agglutination: the ring is light in color and wide in appearance and approaches mat formation or total agglutination. +3, +2 Partial agglutination: the ring is clearly visible in the bottom of the tube. +1 Little or no agglutination: compact ring in the bottom of the tube. M Mat, total agglutination: no ring in the bottom of the tube. b button, no agglutination: cells are precipitated in the bottom of the tube. e Edge, slight ring formation around edge of tube: usually read as total agglutination. FI Fold-in: ring pattern formation will fold in from interfering substances.

Antibiotic Assay System for Gentamicin Assays Using Hemagglutination The aminoglycoside antibiotic, gentamicin sulfate, was utilized as a model antibiotic to study the feasibility of hemagglutination as an approach for the analysis of antibiotics in different matrices. The literature was and remains essentially devoid of research reports utilizing this approach for antibiotic analysis. The fundamental procedure using hemagglutination to measure gentamicin content was very simple. A measured volume of gentamicin -treated red cell suspension was added to gentamicin-supplemented matrices, such as urine, blood serum, acid-precipitated milk extract, and pH 8. 0 buffer extracts of animal feeds. A fixed volume of anti-gentamicin sulfate was added and the mixture incubated at room temperature for 30-60 min. The hemagglutination reactions were observed, and the concentration of antibiotic capable of being measured was determined. The sensitivity of the hemagglutination assay system was 0. 4 µg gentamicin per milliliter chicken serum, urine, and feed extract, and 1. 9 µg/ml for milk and human serum. These levels, although

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not especially sensitive, compare quite favorably with microbiological assay systems for other aminoglycoside antibiotics, the streptomycins, in similar matrices. The streptomycins, by comparison, can be measured by the cylinder-plate diffusion system at levels of 0.125 µg/ml, 0.2-0.4 µg/g muscle tissue, and 0.15 µg for feed extracts [14,15]. Although the levels of detection for gentamicin are similar to its familial counterparts, the streptomycins, the hemagglutination procedure offers two distinct advantages, speed of analysis and specificity. The hemagglutination assay can be completed within 1 hr after extraction with little or no interference from other antibiotics [ 13]. Complement Fixation Assay The complement fixation assay is based upon the phenomenon of immune hemolysis. The assay is performed in two stages. In stage 1, the antibody and the antigen are mixed in equivalent amounts in the presence of carefully measured amounts of complement. If one of these, either antibody or antigen, is lacking, there will be no fixation of comp le men t. In stage 2, if the an tibody-antigen complexes are formed and complement is fixed, hemolysis will not occur. If hemolysis does occur, complement remains and an effective antibody-antigen reaction did not occur. Hence, hemolysis or the degree thereof becomes the end point measurement. The assay system requires a standardization of hemolysis. All reagents are used in constant quantities except for the unknown. For the unknown, a series of dilutions is utilized. Since 100% lysis of the red cells is approached asymptotically, it is convenient to define the unit of complement as that amount that lyses 50% of the sensitized cells. The 50% level is preferred because, between 30 and 70% hemolysis, the hemolysis curve is essentially linear and the amount of complement can be titrated more accurately than using other levels. Hence, the highest dilution of the unknown that can produce a predetermined level of hemolysis represents the measured concentration of the unknown. The complement fixation assay requires a large number of controls that are essential for successful assays. In addition to the antigen and antibody, the assay requires sheep red blood cells, rabbit antibodies to the sheep cells, and fresh guinea pig serum as the source of the complement. The antigen and the antibody must be tested individually to determine that they are not anticomplementary, that is, they do inactivate the complement. It is also necessary to ascertain that the red blood cells will not lyse spontaneously and that the complement protein survives stage 1 in the absence of an authentic antibody-antigen reagent. Because the complement fixation assay requires so much care and is subject to both reagent and matrix interferences, its use for the analysis of antibiotics is very limited. Obviously, the complement fixation assay would lend itself to the assay of either very pure materials or to high-potency products. In these two instances, interferences should be minimal, because of the formu lation or through the purification by dilution. I mmunodiffusion

The diffusion procedure, the basis of the classic microbiological procedures that have been used to measure antibiotics, can be used to assay antibiotics

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using the antigen-antibody reaction. Radial immunodiffusion is quite similar to the agar well diffusion procedure. Agar containing antibody is poured onto a suitable glass surface, and wells are cut. The solution containing the antigen (the antibiotic) is pipetted into the well. After a period of diffusion, at a constant temperature, a ring of precipitin forms. The diameter of the ring is proportional to the concentration of the antigen. As in the classic microbial diffusion assay, a standard curve is prepared and the concentration of the unknown is determined from the curve [ 8) • The r.adial immunodiffusion assay offers no overall advantages over the classic microbial diffusion procedures. The time element for immunodiffusion is not shorter (ranging from several hours to days), the accuracy of the measurement of the zones is no more precise, and the sensitivity of the technique is usually considerably less than can be achieved by microbial diffusion assays. The only advantage of the immunodiffusion technique is specificity. When sensitivity of detection is not a problem, specificity is a requirement, and antibody is plentiful, immunodiffusion could be a useful approach. Radioimmunoassay Radioimmunoassay (RIA) was described by Berson and Yalow [ 16) for measurement of serum insulin concentrations. Any substance, whether an antigen or hap ten, can be measured by RIA. The assay [ 17) is based on the competition for antibody between a radioactive indicator antigen and its unlabeled counterpart in the test sample. As the amount of unlabeled antigen in the test sample increases, less labeled antigen is bound. The concentration of antigen in the test sample can be determined from comparison with a standard calibration curve prepared with known concentrations of the purified antigen. The assay can be extremely sensitive since antisera can be selected with great specificity for the antigen. The sensitivity is limited primarily by the amount of radioactivity that can be introduced into the radiolabeled antigen [ 8) . It is possible to detect levels as low as 1 ng when carrier-free radioactive iodine 125I is used as an extrinsic label. Precipitates are usually not evident, because extremely low concentrations are used. However, several procedures can be used to separate free and bound indicator antigen. In the general method, which is widely used, complexes of antibody bound to radiolabeled antigen are precipitated with antiserum prepared against the antibody moiety. A second anti-species antibody can be prepared by using the donor species as the immunizing antigen, because it is usually immunogenic in other species. Therefore, the second anti-species antibody reacts with essentially all antibodies of the donor species, regardless of their antigenbinding specificities. A number of solid-phase assays eliminate the requirement for the second anti-species antibody. In one widely used method, antibodies to the antigen are absorbed to the walls of plastic (such as polystyrene) tubes. Radiolabeled antigen binds specifically to the adsorbed antibodies and can be counted. When competing unlabeled antigen is also present, less radiolabeled antigen is proportionally bound. The remaining unbound fraction can be decanted. The method is inexpensive, rapid, and highly sensitive. For example, it is possible to detect less than 0. 001 µg of antigen when a tube is coated with 1 µg of antibody [ 8) • Aminoglycosides, of which gentamicin is the best known example, are of great clinical importance, but they are also potentially toxic. Ototoxicity

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and nephrotoxicity occur when serum levels rise above 12 µg/ml; levels below 4 µg/ml may not be adequate for bacterial control [ 18). Therefore, it is necessary to have a rapid, sensitive, and precise method for monitoring gentamicin serum levels of patients. Lewis et al. [ 19] described the first RIA for tritium-labeled gentamicin. Tritium is a S emitter, a type of isotope that has a number of analytical problems associated with its use. The assay requires expensive vials and liquid scintillants, the sample preparation is somewhat tedious, counting times can be 10 min or longer, and other problems can arise when biological samples are assayed [ 20) . 1251 is most widely accepted as the isotopic label of choice for routine RIA, because counting is much simpler, requires less expensive counting vials, and is less tedious. Assays have been developed for 1251-labeled gentamicin [ 18, 21) , tobramycin [ 22) , sisomicin and netilmicin [ 23] , and hygromicin B [ 24] • Gentamicin contains no reactive groups that can be iodinated. Therefore, the drug must be conjugated first to a protein to produce a complex for this purpose. Glutaraldehyde was first used to conjugate gentamicin to lysozyme [ 25]. [ 125I]lysozyme conjugates, although easy to prepare, require second antibody separation methods [ 23]. Watson et al. [ 21] conjugated gentamicin to lysooyme via glutaraldehyde. Aliquots were iodinated by the chloramine-T method [ 26] and purified using Sephadex column chromatography. Recoveries of 92-110% were found with known supplementations of gentamicin in serum over the range 0.5-16 µg}ml. Broughton and Strong [ 18] described a RIA for gentamicin in which iodinated gentamicin was prepared using an 1251-labeled acylating agent, 3-(4-hydroxyphenyl)propionic acid N-hydroxysuccinimide ester, which spontaneously reacts with gentamicin to produce an iodinated conjugate. The sensitivity of the assay was 80 pg. Kanamycin and neomycin, two aminoglycoside antibiotics, did not cross-react with gentamicin until their concentrations were at least 105 greater than the blood levels of gentamicin. The radioimmunoassay developed for tobramycin [ 22] has advantages over both the microbiological and radioenzymatic assays previously developed. The RIA is more highly specific and much more rapid than the microbiological assay; the use of 1251 in RIA alleviates the need for liquid scintillation counting required in radioenzymatic assays. Levels as low as 280 pg tobramycin can be detected, and only minor cross-reaction occurs with other aminoglycoside antibiotics. Watson and Wenk [ 23) described a method for measuring sisomicin and netilmicin. The aminoglycosides were conjugated to fluorescein isothiocyanate (FITC) to produce fluorescein thiocarbamyl (FTC)-aminoglycoside conjugates, which were then iodinated by the chloramine-T method

[ 26).

Sisomicin was measured using a gentamicin antiserum, which cross-reacted 75% with sisomicin. The assay was acceptable for routine use, and it also provided greater sensitivity for pharmacokinetic studies [ 23) . Gentamicin antiserum can be used to measure netilmicin for routine clinical purposes, but the antiserum does not provide sufficient sensitivity for pharmacokinetic studies. Instead, an antiserum was prepared by immunizing New Zealand rabbits with netilmicin coupled to bovine serum albumin. The sensivivity of the RIA was then greatly increased. Levels as low as 300 pg/ml netilmicin can be measured. Foglesong and LeFeber [24) developed a RIA for determination of hygromycin B in feeds. Recoveries of 97-103% of hygromycin B were made from

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various feed mixtures. There was no significant cross-reactivity with the other antibiotics usually added in combination with hygromycin B. Monensin, tylosin, bacitracin, streptomycin, and chlortetracycline all showed less than 0.1% cross-reactivity with the hygromycin B antibody. The procedure of Fogelsong and Lefeber [ 24] for the measurement of hygromycin B in feed illustrates the relative speed, efficiency, and ease of the RIA technique. The following outline describes the application of the technique: 1.

2. 3. 4. 5. 6.

Prepare two tubes containing a specified volume of PBS (volume X); these tubes are used to count any nonspecific radiation. Prepare two tubes containing XJ 2 volume of PBS; these tubes are used to measure total binding. Prepare two tubes XJ2 volume of dilute standard solution and two tubes containing X/2 volumes of the sample to be measured. Add XJ 2 volume of rabbit antihygromycin B reagent to all tubes, except the tubes used to measure the nonspecific radiation. Add X/2 volume of [125I]hygromycin B to all tubes plus an empty tube used for the total count. Mix all tubes well, except for the tube used for the total count. Add X /2 volume of sheep anti-rabbit precipitating antibody to all tubes except the tube used for the total count. Mix all other tubes well, preferably with a vortex mixer.

Allow all the tubes to react at room temperature for 20 min. Centrifuge all tubes, except the tube used for the total count, for 10 min at 2000 g. Remove the supernatant solution of all tubes, except the tube for the total count, by aspiration, being careful not to remove any of the pellet (precipitate). Place all the tubes into screw-cap y counting tubes, and count all tubes to 10, 000 counts with a suitable scintillation counter. Determine the average counts per minute for the duplicate tubes. Calculate the percentage of bound radioactivity: Average cpm standards or sample corrected for % Bound nonspecific radioactivity (cpm) = ~~~~~~--~~~~~~~--'-'--"'-""-~'--~~~x 100 Total cpm corrected for nonspecific radioactivity radioactivity (cpm) By plotting the percentage of [ 125I]hygromycin B bound versus the concentration of hygromycin B using Logit-log paper, a response line can be prepared. Determining the concentration of the samples can be performed by comparisons with the standard response line. The advantages of using RIA for measurement of antibiotics include the requirement for a small sample size, only a few microliters, the high degree of specificity and accuracy, and the ability to obtain results within an hour after the sample has been received in the laboratory [ 20] . The disadvantages of RIA appear to outweigh the advantages. There is increasing legislative as well as personal bias in many countries against the use, transport, and disposal of radioisotopes. A potential health hazard exists for those associated with the production as well as the use of 1251labeled antibiotics. The labeled reactant is unstable. The relatively short shelf life of the 1251 label leads to increased costs for quality assurance and distribution. The great sensitivity may require considerable dilution

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prior to assay, and antibody-bound fractions must be separated from free fractions prior to counting [ 20] • Nonisotopic lmmunoassays A number of nonisotopic immunoassays have been developed that avoid the problems encountered when working with radioimmunoassays. Nonisotopic immunoassays only differ from the isotopic immunoassays in terms of the type of label used, the means of end point detection, and the possibility of circumventing a separation step. There are two types of nonisotopic immunoassays: fluoroimmunoassays, which employ fluorophore-labeled antigen, and enzymoimmunoassays, in which the antigen is labeled with an enzyme [20]. These types of assays can be further subdivided, depending on whether the method requires a separation step. The nonseparation nonisotopic immunoassays include polarization fluoroimmunoassays, direct quenching fluoroimmunoassay, substrate-labeled fluoroimmunoassay, and nonseparation enzymoimmunoassay. Fluoroimmunoassays The first nonseparation immunoassay for an antibiotic was described by Watson et al. [21]. When fluorescent molecules are excited by a beam of polarized light, the extent of polarization of the emitted light varies with respect to the molecule's size. There is little polarization in the emission from a small molecule that exhibits a great deal of rotational motion. However, when the small fluorescent molecule is bound to a fairly large particle, such as an antibody molecule, its rotational movement is restricted and the polarization of its fluorescence is increased. Therefore, the proportion of antigen molecules bound by antibodies can be determined from the polarization of fluorescent emission. This procedure can be useful for small antigens and haptens that possess fluorescence spectra distinctly different from those of the antibodies [ 8] • Watson et al. [ 21] used fluorescein-labeled gentamicin for the routine determination of gentamicin levels in serum. The method is rapid, reproducible, and economical, and the reactants have an excellent shelf life. The incubation time is only 2 min, and only 1. 25 µl serum is required. Although polarization fluoroimmunoassays depend upon antibody binding to enhance the signal, quenching fluoroimmunoassays depend on a decrease in signal from the bound fraction, which is attributed to the antibody's ability to impair the excitation, or the emission from the labeled antibiotic [ 20] • The method developed for several aminoglycosides enables the use of a simple fluorin:eter [ 27] • Shaw et al. [28] described the continuous-flow automation of a quenching fluoroimmunoassay for the measurement of gentamicin in serum. The sample was first mixed with fluorescein-labeled gentamicin, followed by antiserum. Following a short incubation time of 6 min for each sample, the fluorescence was measured. Correction for the intrinsic blank of each sample was accomplished during single passage through the system by pumping antiserum discontinuously. This was done to divide the final stream into sections corresponding to conventional separate assay and blank mixtures. Substrate-labeled fluoroimmunoassays for gentamicin, tobramycin, and amikacin [20] require the drug to be labeled with umbelliferyl-8-D-galactoside to produce nonfluorescent conjugates. The enzyme 8-galactosidase is

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added, and the fluorescent products are released from the labeled antibiotic. The antibody in the bound fraction inhibits the enzyme from acting on its fluorogenic substrate. The differential in fluorescence is proportional to the concentration of antibiotic. Burd et al. [ 29] compared the results obtained with this method with results using RIA. Good correlations were obtained. Care must be taken to achieve accurate timing between enzyme addition and fluorescence measurement. The care necessary may cause problems in busy laboratories. The advantages of using the fluorescent immunoassay include the lack of the need for separation of antibody bound and free drug-dye conjugate; the rapid and minimal manipulation of the small sample (1 µl); stable, nonisotopic reagents; sensitivity, specificity, and precision comparable to those of RIA; and the ability to be totally automated. Nonseparation fluoroimmunoassays may be affected by endogeneous fluorophores, such as bilirubin, and by light scattering in turbid samples [ 20] • These problems can be circumvented by extensive predilution of the sample or using appropriate blanks. These extra steps are time consuming and also may lead to a decrease in the accuracy and precision of the assay. A step is now commonly included in the fluoroimmunoassays for the aminoglycoside antibiotics, to separate bound and free fractions, as well as to remove all the interfering factors before fluorescence is measured [ 20] • It is possible to link the antibodies covalently to magnetizable particles. The need for separation by centrifugation is eliminated, and it also enables the measurement of antibiotic levels in whole blood. Enzyme Multiplied Immunoassay Technique

This technique employs enzyme-labeled antibiotics, which react in a fashion analogous to polarization fluoroimmunoassays and to quenching fluoroimmunoassays, in that a reduction of enzyme activity is attributed to antibody binding. As the concentration of unlabeled drug increases in the test sample, less enzyme-labeled drug will be bound to antibody. To perform the EMIT assay, five reagents are necessary: (1) the sample matrix, which could be either urine, serum, milk, tissue extract, or feed extract; (2) the substrate-antibody reagent, which could be S-NAD (Snicotinamide adenine dinucleotide) and L-malate (L-malic acid), S-NAD and glucose-6-phosphate, or S-NAD and peptidoglycan, mixed with the antibody; (3) the enzyme reagent, which could be malic dehydrogenase or glucose-6phosphate dehydrogenase or lysozyme coupled to the antibiotic; standard antibiotic solution, which covers a wide range such as 0.01-1.00 µg/ml antibiotic; borate solution, usually 0.10 M. The sample to be assayed is added to a plastic test tube. The substrateantibody reagent is added, and the tubes and contents are heated to 37°C using a water bath. The corresponding enzyme reagent is added at timed intervals and the reaction stopped after precisely 10 min with the borate solution. The intensity of the resulting color is measured at 340 nm. An assay of this type was describ ;:l

c:n

>I:>.

"
!>.

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Table 2 Factors Influencing the Design of Antiviral Chemotherapy Experiments Virus: species, type, strain, infecting col).centration, infection route Host: cell, organ culture , ariimal Test substance: quantity available, concentrations used, lability, solubility, administration route, vehicle, rate of metabolic breakdown In vitro evaluation parameter In vivo evaluation parameter Timing considerations relative to virus exposure: parameter examination, toxicity observation, treatment Controls: treated uninfected, untreated uninfected, untreated infected, placebo-treated infected, known positive-treated infected Environmental conditions Statistics and reproducibility Secondary infection (contaminant)

effect rating, an endpoint of 3+-4+ is sought. In animals in which a lethal infection is used as parameter, an approximate 90% lethal dose is used. Virus concentrations that are too high usually overwhelm the antiviral effect of a test material, whereas viral levels that are too low often yield unsatisfactory end points, and consequently the test results are often suspect and require repeating. The route of viral inoculation into animals should also be considered and should attempt to mimic the infection seen in the human while proceeding at a rate that is not too rapid for an antiviral substance to have an effect. Host Selection In vitro antiviral systems ideally utilize a cell that has sufficient susceptibility to the infecting virus that a discernible infection can be produced. Investigators usually seek readily observable cytopathic effect as this infection manifestation, although a medium color change due to pH alteration or specific immunofluorescence has also been used with some success. It must be recognized, however, that the antiviral activity of some test substances is quite dependent on the cell line used. The broad-spectrum antiviral ribavirin, for example, has been shown to exert a strong herpesvirus inhibition in tests run in human carcinoma cells, rabbit kidney cells, human skin fibroblast cells, and chick embryo cells, whereas in monkey kidney and human embryonic lung fibroblast cells no activity is seen against the same virus [ 9] • Against other viruses, such as parainfluenza or cytomegalo virus, however, strong inhibitory effects are seen in the latter cells. No valid explanation for this cell-dependent antiviral effect is yet known, but such data strongly support the need for multiple cells used in in vitro antiviral experiments. The growth stage of the cell, that is, whether the cell is in a resting state or is actively metabolizing, will also influence the antiviral test

438

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results. Actively metabolizing cells are much more sensitive to the toxic effects of test substances, and hence possible virus inhibitory activity may be masked by cell alteration when tests are run in such cells. In vivo antiviral experiments may be highly dependent on the age, sex, species, and strain of animal employed in such studies. Older animals are often more resistant to the virus challenge than are younger animals, and the younger animals usually are less able to tolerate the administration of the substance being tested. Fighting tendencies of the male, especially as seen in the mouse, often influence the results of the experiment. The susceptibility to the challenge virus will vary markedly according to the species and strain of animal used and may influence the infection parameter to be chosen. Unless large supplies of test material are available to the investigator, however, attempts are usually made to use small animals, which would require relatively lesser quantities of that test substance. Many antiviral researchers tend to use designated specific pathogen-free animals to avoid the problem of animals that have a latent infection of some indigenous virus or are carrying specific antibodies to certain virus that often occur naturally in some animal colonies. Characteristics and Use of the Test Substance The quantity of the test antibiotic available for antiviral testing will seriously influence the kind and quantity of antiviral testing being planned: researchers have tended to use those systems requiring the minimum quantity of test substance. This usually in valves the use of disposable 96 well microplates for in vitro experiments [ 10) and mice for initial in vivo experiments. Often, in initial antiviral experiments run on a new test substance, the !ability of the substance is unknown. It is preferred, therefore, to avoid heating, undue exposure to light, excessive agitation of solutions containing the material being evaluated, and metal contact. In vitro antiviral experiments require the use of aqueous media to maintain the cells used in the study. Such media dictate the need for at least a partial aqueous solubility of the test substance if activity is to be discerned. Certain solvents, such as dimethylsulfoxide, may be used sparingly to assist in achieving a satisfactory degree of solution, although care should be taken in using such solvents to be assured they are not themselves virus inhibitory or cytotoxic in the cell system used. Antiviral studies run in animals must attempt to take into account such factors as the route by which the test antibiotic is to be administered, the vehicle to be used, especially for topical treatment of cutaneous infections, and the estimated rate of metabolic breakdown of the test substance. The selection of the route of administration will vary somewhat, depending on the philosophy of the investigator; some groups desire only to know if a substance will be efficacious by a clinically acceptable route, such as oral. Others prefer to known initially if the substance has any in vivo antiviral activity whatsoever and will then use a route allowing the greatest amount of test substance to reach the target infected area. The intraperitoneal and less practical intravenous routes are usually best for the latter, if the target area is not reached by topical applications. Some investigators have found the direct, target-organ, injection route to be efficacious, especially for initial therapy of neurotropic infections [ 11) . Later studies may then be designed to determine if routes more acceptable for human use will also allow a positive antiviral effect to be exerted.

Antiviral Activity

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The vehicle used for topical applications must allow the material to remain on infected tissues, penetrate interstitial spaces, and release the test antibiotic to allow its penetration into the cell. We have found the type of vehicle used to markedly influence the antiviral effectiveness of ribavirin [ 12, 13]. No ideal vehicle is known, since each test substance will vary in regard to its solubility, for example. Metabolic breakdown of the test substance can be a primary cause for the in vivo failure of a material that exhibits a high degree of antiviral activity in vitro. An example of this is the nucleoside analog cytarabine (1-13-Darabinofuranosylcytosine), which has significant in vitro activity against DNAcontaining viruses [ 14] yet has failed to exert acceptably positive effects against those viruses when administered by standard parental routes in animal systems [ 15]. This compound has a marked metabolic instability due to deamination to the inactive uracil arabinoside [ 16-18] . It is significant that target-organ treatment of herpesvirus infections with this compound yielded positive results [ 11] . Evaluation Parameters

Later portions of this chapter will deal in detail with the evaluation parameter to be used in antiviral experiments in vitro and in vivo. Certainly, the selection of the parameter is of primary importance to any antiviral study. Such parameters should be readily discernible, measurable, and relate, whenever possible, to the human disease, if control of the human infection is to be the ultimate target of the antiviral program. A properly equipped laboratory used in antiviral studies should include a camera and lighting setup to record photographically those lesions, organ enlargements, and other visible abnormalities that may be used as parameters for in vivo antiviral evaluation. Plaques, syncytia, and other manifestations of viral cytopathic effect seen in vitro may also be photographed. Such photographs provide a permanent record of important experiments and also allow the investigator to more carefully review the experiment at a later time. Timing Considerations

The sensitivity of the antiviral test can be influenced considerably by the various timing considerations indicated in Table 2. In studies run in vitro, care should be taken to use a standard time to evaluate cytopathic changes. If those changes occur earlier than expected, they usually indicate the viral inoculum was too high, which, as pointed out earlier, tends to overwhelm any antiviral effects exerted by the substance being tested. Similarly, if the viral cytopathic effects develop later than the expected time, an insufficient viral inoculum is usually responsible and the test may therefore be overly sensitive. For such viruses as herpes, rhino, or parainfluenza, we generally employ a 72 hr incubation period after virus exposure if viral cytopathic effects as determined microscopically are used. These same considerations of too early or too late manifestations of infection apply to animal experiments as well. Although toxicity is not the main point of concern here, as will be pointed out subsequently, the proper antiviral experiment will include concurrently run toxicity controls. Such toxicity observations in animals should be for a sufficiently long period to allow for the appearance of delayed toxicity. Usually a 21-30 day holding period is satisfactory, unless treatments are prolonged.

440

Sidwell

The selection of a time for initiation of therapy again will be dependent on the philosophy of the researcher. Some groups desire to use only treatment schedules that will clearly demonstrate a therapeutic (as compared to a prophylactic) effect of the test substance. In such circumstances treatment is usually delayed until some manifestation of the infection can be seen. Others desirous of first determining if their test material has any degree of antiviral effect may elect to begin therapy earlier, often even before virus exposure, which would significantly increase the sensitivity of the experiment. The frequency and duration of therapy again may vary, but in initial experiments multiple daily treatments continued through the peak of the infection will usually increase the efficacy of the test material. Potential immunomodulators being considered as antiviral agents would probably need to be used by different treatment schedules dictated by the time of immune modulation expected. Knowledge of the pharmacokinetics of the substance being evaluated would aid greatly in selection of the best treatment schedule. Controls

Adequate controls are mandatory for the results of an antiviral experiment to be accurately evaluated. Toxicity controls (uninfected, but treated concomitantly with infected) will indicate if the treatments used are themselves visibly harmful to the host. Such controls should be run at each treatment concentration. Virus controls of two types are needed. The first, and most important, is a placebo-treated host infected at the same time as those that will be treated with test substance, the placebo being the same vehicle as that in which the test substance is administered. A second virus control that may be used is an infected host that receives no treatment. The latter will illustrate whether placebo treatment alone influences the usual disease development. A normal control is an uninfected, untreated host held in the same environment to serve as a standard against which the infected hosts can be compared • The normal control is particularly helpful in ascertaining that the original condition of the host and the environment are acceptable. Finally, if available, a known positive antiviral drug should be run in an infected host in parallel with all the controls just mentioned. Such a control will demonstrate whether the test was at the correct level of sensitivity and will provide an important standard against which the activity of the test material can be compared. Ideally, the known positive drug will be one that is now accepted clinically in one or more major countries. A listing of those drugs is seen in Table 3. Environmental Conditions

Antiviral experiments run using cell culture systems are highly dependent on temperature, pH, the media used, and the container in which the cells are established. With respect to media, one must recognize that the serum usually included as an additive may vary markedly in concentration of normal ingredients, such as endotoxin, hemoglobin, glucose, growth hormone, etc. [ 19]. *

*Detailed information on the influence of serum factors are described in the quarterly publication, Art to Science in Tissue Culture, available by request from HyClone Laboratories, Inc. , Logan, Utah 84321.

441

Antiviral Activity

Table 3 Representative Clinically Active Antiviral Drugsa Drug

Chemical name

Viral disease inhibitedb

Acyclovir

9( 2- Hydroxyethoxyrnethyl) guanine

Herpes eye, cutaneous, and genital infections

Amantadine HCl

1-Adamantanamine HCl

Influenza A

Cytarabine

1-(3-D-Arabinofuranosylcytosine

Herpes eye , cutaneous infections

ldoxuridinec

5-Iodo- 21-deoxyuridine

Herpes eye, cutaneous, and encephalitis infections

Methisazone

1-Methylisatin-3thiosemicarbazone

Smallpox , severe vaccinia

RibavirinC

1- (3- D-ribofuranosy11, 2-4-triazole-3carboxamide

Hepatitis, influenza A and B , respiratory syncytial disease , measles

Rimantadine HCl

a.-Methyl-1-adaman tanamine HCl

Influenza A

Trifluorothymidinec

5-Trifluoromethyl- 2'deoxyuridine

Herpes eye infections

VidarabineC

9-(3-D-arabinofuranosyladenine

Herpes eye, encephalitis infections

aNot included are immunomodulators (interferon inducers, cellular immunity enhancers and others) or virus-inactivating substances. bniseases for which approval has been given to treat. CA microbiological process is available for complete or partial synthesis of these drugs.

Such serum components have the potential to influence cell growth and viral replication and hence the results of antiviral experiments. Some serum companies now furnish biochemical content reports on each lot of serum purchased. Serum enzymes may also degrade some test compounds [20}. Care should be taken to standardize all these environmental factors as the in vitro antiviral study is run. Experiments run in laboratory animals are also influenced by environmental factors. Particularly temperature, accidental deprivation of food or water, erratic light changes, abnormal odors and sound, and trauma of shipping will all affect an animal's response to virus and therapy. Quarantine procedures as recommended in the U.S. Animal Welfare Act of 1970 [ 211 should be followed to avoid shipping trauma effects. Statistics and Reproducibility A concept used by most chemotherapists in evaluating the relative efficacy of a potential chemotherapeutic agent is the therapeutic index (TI). This is

442

Sidwell

defined as the maximum tolerated dose (MTD) divided by the minimum effective or inhibitory dose (MIC). To provide adequate data for such a calculation, it is necessary to use sufficient dosage levels of test substance to bridge the range between toxicity and total lack of antiviral effect and to use sufficient numbers of cell culture cups or animals at each dosage level to allow areasonable estimation of both MTD and MIC. In cell culture systems the MIC is often used as an expression of drug efficacy, but it is obvious that a substance with a relatively strong cytotoxicity will also have an MIC that is quite low. Thus MIC data alone, without taking into account MTD, will often be misleading. A virus rating (VR) system has been used by us and others [10,22,23] to provide a numerical score for in vitro antiviral activity. The VR calculation takes into account virus concentration and visible cytotoxicity. The antiviral investigator is well advised to consult with a statistician regarding the statistical design of antiviral experiments, especially experiments using animals. Reasonably simple statistical tests, such as chi-square analysis, Fisher exact test, and Student's t test, are commonly used. The results of any study should be readily reproducible, which can only be determined by repeating at least the essential portions of the study. Secondary (Contaminating) Infections A serious concern in running in vitro or in vivo antiviral studies is the problem of the secondary or contaminating infection. In cell culture studies, such contaminates will often sufficiently destroy or interfere with the cell system to totally obscure any virus infection. This problem can be alleviated to a degree by addition of bacterial and fungal-inhibitory antibiotics to the medium. In our in vitro antiviral studies, we do not add antibiotics to our cells prior to actually running the experiment because we would prefer to know our cells are contaminated and discard them. A more subtle problem, however, is the presence of contaminating mycoplasma that adversely affects cell growth, interferes with virus infections in the cell, and also may enzymatically alter the test compound [ 2]. Several laboratories offer a mycoplasmadetecting service at a nominal fee; many researchers routinely use this service to be assured their cell systems are free of this serious contaminant. Laboratory animals are often exposed to a variety of agents that can be enzootic in animal colonies. The commonly occurring viral infections of laboratory mice and rats are summarized well by Crispens [ 24]. In addition to causing production losses in the colony, these infections can interfere with the desired infection to be induced and can seriously alter the normal immunological response of the animal. Many researchers, therefore, use only animals from commercial suppliers that continually monitor their colonies for the presence of many of these infections and designate their animals as "specific pathogen-free." Secondary bacterial infections that may interfere with certain viral infections, such as influenza virus-induced pneumonia, can often be controlled by employment of a broad-spectrum antibacterial antibiotic in the drinking water. IN VITRO ANTIVIRAL EVALUATION PROCEDURES

The initial or primary screening tool for antiviral studies is the cell culture test. Such tests utilize living cells susceptible to the targeted virus. The cells are cultivated in a defined synthetic medium, usually supplemented by

Antiviral Activity

443

some type of serum (such as fetal bovine, calf, or horse), a buffer, and sometimes bacterial- and fungal-inhibiting antibiotics. In most antiviral tests, the cells are allowed to divide to become a thinly confluent monolayer, at which time the medium is changed and new medium containing a pretitered concentration of virus is added to the cells, usually shortly following, at the same time as, or shortly before addition of a test substance to the cells. The virus infection will usually subsequently alter the cell, causing a visibly discernible cytopathic effect. Such effects can be manifested as alteration in cell shape, size, in the actual destruction of the cell, or in the cell detaching from the monolayer. Most of these effects are readily seen microscopically; major cell destruction can be seen as plaques visible to the naked eye , particularly if an agar overlay is added to the cells to isolate the infection. pH alteration usually occurs in such virally infected cells, resulting in a color change in the medium due to an indicator dye incorporated into the medium. More subtle viral infections can be demonstrated by specific immunofluorescence procedures [ 25) . We have used this latter technique successfully for antiviral studies with rota virus [ 26, 27]. Using these basic virological procedures, a number of tests can be applied for ascertaining antiviral activity. Inhibition of microscopically determined viral cytopathic effect [ 10, 23) , plaque inhibition [ 28, 29) , virus titer reduction [ 23, 30) , and inhibition of uptake of radiolabeled thymidine or uridine into viral nucleic acid [31) have all been used. Several variations of the plaque reduction test have been described. These include the use of drugimpregnated paper disks [29,32,33), agar blocks [34), and gradient plate techniques [ 35) • Certain leukemia viruses replicate in tissue culture but do not produce cytopathology, making antiviral studies with them difficult. A technique to accomplish this has been an indirect method, wherein a rat tumor cell (XC cell) is placed in contact with mouse embryo (ME) cells infected with leukemia virus, and syncytium formation results [ 36) . Ultraviolet irradiation of the viral-infected ME cells followed by addition of XC cells results in definitive plaque formation [ 37) ; this plaque assay method has been used successfully in vitro for the study of antiviral compounds [ 38, 39). In studies we have performed with bovine leukemia virus [ 40) , inhibition of viral syncytia formation induced directly in a feline cell line (F-81) was used. Inhibition of internal and external viral polypeptide antigen as measured by complement fixation was also used as a parameter for evaluation against the bovine leukemia virus in the same report. As discussed earlier, it is imperative that adequate toxicity controls be included with in vitro antiviral experiments. In initial tests, examination of the treated but uninfected cell monolayer for cell abnormalities is often used. As follow-up tests are performed, however, additional measurements of cytotoxicity should be considered. These include actual counts of viable cells, trypan blue dye exclusion [30], neutral red dye uptake [41), and determination of total cell protein [ 42) • Much emphasis has recently been placed on more sensitive methods for measurement of cytotoxicity, with increasing use reported of the radiolabeled metabolic precursors [26,27,42a,43-45]. In these methods, the lowest concentration (MIC) of the test substance that inhibits uptake and incorporation of the precursor into cells is compared with the in vitro MIC against viruses. A "selective index" (SI) has been utilized by Drach and Shipman [46] as a means of expressing this difference in effect. The SI is calculated using 50% inhibitory concentrations (ID50) with type 1 herpesvirus as target with the following formula:

444

Sidwell

SI

= log

m 50 concentration for DNA synthesis in uninfected cells ID 50 concentration for viral DNA synthesis in virus-infected cells

The SI is positive if viral DNA synthesis is inhibited preferentially and negative if uninfected cellular DNA synthesis is more strongly inhibited. In our studies with reoviruses, rota viruses, and blue tongue viruses, we used as MIC that concentration of test substance inhibiting uptake or incorporation by at least 20% and inhibiting viral cytopathic effect or immunofluorescencestained cells virus infected by at least 10% [ 27, 45, 47]. Although some investigators have used rapidly dividing cells in performing their isotope uptake studies, we prefer using confluent, resting cell monolayers, since the antiviral experiments are also run in the resting cells and more accurate comparisons of specificity of effect can be determined. Certain viruses, a significant example is the Epstein- Barr virus, do not cause acceptable cytopathic effects in cells, and other means have and to be used to study in vitro antiviral effects against such agents. Among these procedures of determining inhibition of viral replication are measurement of DNA replication using labeled viral DNA, viral genome numbers are determined by viral complementary RNA-DNA hybridization, expression of viral antigen by indirect immunofluorescence, and viral DNA polymerase activity [ 48-50]. These methods have been used successfully for demonstrating the EpsteinBarr virus-inhibitory effects of acyclovir [ 48-50]. Despite all the best efforts to standardize the in vitro test and to demonstrate specific antiviral effects in those tests, such tests will not always accurately predict antiviral activity that may occur in animal systems. In addition to the factors discussed earlier, which must be considered in the design of the in vitro antiviral experiment, other factors not controllable in the cell culture systems may also affect the correlation between in vitro and in vivo results. Metabolizing enzymes in the animal not found in the cell system may either degrade the test substance to an inactive form or activate an otherwise inactive material. The ability of the animal to absorb the test compound and to distribute adequate quantities to the infected organ is of major importance. The aqueous insolubility of the material will limit its in vitro antiviral activity, yet in the animal itself such material may be absorbed. The animal's immune system may play a positive role in aiding in the control of the viral infection; many antiviral substances, particularly those that act by temporarily inhibiting viral replication, require some eventual assistance of the immune system in rendering a maximal antiviral effect. Test substances that may act as immunomodulators in exerting an antiviral effect will usually not be found active in an in vitro system; when the investigator suspects that such materials have this mechanism of action, it would be preferable to go directly to the in vivo evaluative system. For most new test materials, however, it is most advantageous to utilize the in vitro system in initial antiviral studies. In our experience, the correlations found between in vitro and in vivo activity far exceed the few incidences when such correlations do not occur. Example It is appropriate to present an example of an in vitro antiviral experiment

to illustrate the various concepts discussed in this chapter. The following is essentially the procedure we use when a new test substance is submitted

Antiviral Activity

445

to us for antiviral testing. For simplicity, we will assume the material is to be evaluated against type 2 herpesvirus. A typical in vitro evaluation will utilize seven concentrations of test substance, beginning with a maximum level of 1000 µg/ml, and varying by onehalf log dilutions through 1 µg/ml. Since our material is to be diluted 1: 2 with virus when placed on cells, we begin with 2000 µg/ml. Let us assume our material is somewhat insoluble in aqueous solutions. The medium to be employed is Eagle's minimum essential medium (MEM) supplemented with 5% fetal bovine serum, 0.18% NaHC03, and 50 µg/ml gentamicin. Since the test substance did not readily go into solution, the mixture is vortexed using a standard vortex mixer (American Scientific Products, McGaw Park, Illinois) for 1 min. The material is then sonified using a Branson ultrasonic cleaner for an additional 10 min. In our example, some particulate material is still visible, so this is centrifuged out using low-speed centrifugation for 5 min. The supernatant is then used for our antiviral test. Included with the test will be a previously prepared known active substance; vidarabine (9-S-Darabinofuranosyladenine) is typically used for our herpesvirus testing at an initial concentration of 2000 µg/ml. The test is performed in sterile disposable plastic microplates containing 96 flat-bottomed circular cups in which a monolayer of continuously passaged African green monkey kidney (MA-104) cells was established 18 hr previously using MEM supplemented with 10% fetal bovine serum devoid of antibiotic, To ensure the panel remains in a sterile, airtight condition, the plate is wrapped in a standard plastic wrap (Saran, Dow Chemical, Midland, Michigan) which is removed when the test is performed. To maintain a sterile environment during the test, we temporarily cover the plate with sterile paper toweling cut to fit the plate. To the appropriate panel cups, 0.1 ml of test compound is added, which is then followed within a 10 min period with 0.1 ml of virus (we utilize a well-recognized virus strain, E194 of type 2 herpes virus in this case) also diluted in cell culture medium. The plate is then resealed with plastic wrap, incubated for 3 days, and examined microscopically for cytopathic effect ( CPE). Included in each test panel are toxicity controls (containing virus and medium only), and cell controls (containing medium only). A layout of a typical panel in which three compounds are evaluated against one virus is shown in Figure 1. All additions of virus, medium, and test compound are done with standard 1 or 2 ml pipettes. Viral CPE is determined with the 40x magnification of an inverted microscope, graded on a standard scale of 0 (normal cells) to 4 (virtually complete destruction of the cell layer). Reduction of CPE resulting from exposure to test compound is evaluated using the VR method discussed earlier. To determine the VR , the sum of the values assigned to the CPE of each cup of the treated, infected cells (T) at each drug level is subtracted from the sum value of the CPE in an equal number of virus control (C) cups. If the test compound is slightly toxic at one or more levels, the total C-T value of those levels is divided by 2. The C-T total of all drug levels is then divided by 10 times the number of test cups used per drug level; in this example this number is 3. The results of the antiviral test with our test compound, which we shall label merely as "substance A," are seen in Table 4. Included on the table are the appropriate VR calculations. It can be seen that substance A was reasonably inhibitory to the virus, with a VR determined of >1.2 and an MIC of 21

8.5

9.1

12.8

9.5

10.8*

11.9

0.2

3.1

2.7

1.2**

2.4

1. 7**

1.1**

Mean vaginal lesion, days 3-8

Infected, treateda Mean surv time (days)

Table 6 Example of an In Vivo Type 2 Herpesvirus Antiviral Experiment

0

4.1

3.8

1.4**

3.8

2.1*

1. 5**

Vaginal virus titer (log)

)..

""' ~

'
21.0

5.3

5.2

7.9

6.7*

8.1*

8.1

5.6

Mean surv time (days)

0

2.9

3.1

1.1*

2.5

1.6*

1. 2**

1. 9

Mean lung consol score

0

21.2

20.9

1.6**

16.8

5.8**

1.1**

8.1

Mean lung HA titer

Infected, treatedb

aDifference between initial weight and weight following final treatment of toxicity controls. b*P < 0.05; **P < 0.01. CLungs in which HA titers of 1: 2 or less were demonstrated. dsterile water.

Normal controls

Untreated, infected

5/5

2.8

1/20

50

Ribavirin

5/5

5/10*

-

37.5

Substance B

2.7

5}5

8/10**

3/10

Surv/ total

3/20

75

Substance B

1. 9

-0.7

Host wt changea (g)

5/5

4/5

Surv/ total

-

150

Substance B

Placebo-treated, d infected

300

Dosage (mg/kg per day)

Substance B

Treatment group

---

Toxicity controls

Table 10 Example of an In Vivo Influenza Virus Antiviral Experiment

5/5

2/20

3/20

8/10**

3}10

7/10**

8/10**

5/10

Lung HA negc/total

464

Sidwell

Table 11 Parainfluenza Virus Animal Models Virus type

Animal/injection site

1

Mouse /intranasal

Pneumonia-associated mortality; lung consolidation; virus recovery from nasal washings, lungs

225 226

1

Ferret /intranasal

Nasal discharge; virus recovery from nasal wash, respiratory tissues

227

1

Rat /intranasal

Nasal discharge, virus recovery from nasal wash , respiratory tissues

228

1,2

Hamster /intranasal

Nasal discharge; virus recovery from nasal wash , respiratory tissues

229

1

Pig /intranasal

Pneumonia; virus recovery from nasal wash, respiratory tissues

230 231

2

Vervet monkey I intranasal

Nasal discharge; virus recovery fron nasal wash

232

2

Baboon /intranasal

Nasal discharge; virus recovery from nasal wash

233

3

Hamster /intranasal

Virus recovery from nasal wash; mild lung consolidation

234 235

3

Patas monkey I intranasal

Fever

236

Evaluation parameter

Reference

bronchitis [217-219]. They also may induce pneumonia and common coldlike symptoms in adults [220,221]. Type 2 parainfluenza is associated with acute croup in young children [ 222] and may also cause common coldlike symptoms in adults [ 223] • Type 4 parinfluenza virus antibodies have been found widely in human populations [ 224] , but the role of this virus in human disease has yet to be established. These viruses , then , appear also to be among those considered as targets for antiviral studies, although no clinically active antiviral drugs l:J.ave yet been demonstrated. Animal models for the parainfluenza viruses are indicated in Table 11. Type 1 parainfluenza virus causes the most pronounced and severe infections, particularly in mice. The type 2 virus is known to induce discernible infections only in monkeys and baboons, although the respiratory infections induced quite closely resemble those seen in humans. The type 3 parainfluenza virus does not readily infect mice, but an intranasal instillation of the virus to hamsters results in an infection predominantly of the upper respiratory tract characterized by nasal discharge containing a high titer of virus, histologic changes in the respiratory tract, and a strong serum antibody response.

Antiviral Activity

465

Soret [ 234] effectively used this model, which appears quite human related in the disease manifestations, to evaluate antiviral drugs. We modified the model to expose the hamsters to a small-particle aerosol type 3 parainfluenza virus and found the animal would then develop a moderate lung consolidation [ 235] • No animal models are known for the study of the type 4 parainfluenza virus; guinea pigs do develop serum antibody to the virus upon viral challenge, the inhibition of which has been used as a chemotherapy model for other viruses [ 98] •

Respiratory Syncytial Virus The respiratory syncytial virus is a major cause of bronchiolitis and pneumonia in infants and children [ 23 7, 238] • The viral disease is also reported to occur in adults [ 239] , and the virus infection has been implicated as a cause of death in infants with congenital heart disease [ 240] • The viral disease occurs most frequently in infants 2-3 months old, requiring immunization to be administered by the time the infant is 1 month old [ 216] • Effective vaccines for this virus are still in experimental stages [216]. One antiviral drug, ribavirin, has recently been shown to be active against the human disease when administered by small-particle aerosol [ 241, 242] • The respiratory syncytial virus has been reported to infect mice, hamsters, cotton rats, and ferrets, with virus readily recoverable from respiratory tissues, but no clinical signs of disease are manifested [ 243- 246] • The cotton rat model, wherein the anesthetized animal is infected by intranasal instillation of virus, has been used by Hruska et al. [ 247] to evaluate the efficacy of ribavirin. The compound reduced the amount of recoverable virus in nasal turbinates and lung tissues. Primates reportedly also are somewhat susceptible to respiratory syncytial virus infection , with respiratory disease demonstrable [ 248- 250] • Mice inoculated intracerebrally with one strain of this virus, adapted by over 30 passages through mice, will develop CNS signs and die of infection [251]. Such an infection is less applicable to the respiratory diseases usually associated with this virus. Arenaviridae The Arenaviridae family contains the American hemorrhagic fever viruses and the Lassa fever virus, both very serious human pathogens at present restricted to rather limited geographic areas. The American hemorrhagic fever viruses include Junin (located in Argentina) , Machupo and Latino (Bolivia), Anapari (Brazil), Tamaimi (Florida), Pichinde (Colombia), Parana (Paraguay), and Tacaribe (Trinidad) [240]. These have been more broadly characterized as Bolivian and Argentine hemorrhagic fevers by Rosen [ 172] • Lassa fever, first recognized in 1969 in Nigeria, is an often fatal hemorrhagic fever disease of western Africa [ 172, 252] • The importance of both American hemorrhagic fever and Lassa fever lies in the ease in which either could be transmitted anywhere in the world and potentially be of epidemic significance world wide. No vaccines are available. The Junin virus causes humanlike hemorrhagic disease in guinea pigs, hamsters, and mice [ 253, 254]. Paralysis can be induced in infant mice by Pichinde and Tacaribe viruses [ 255] • With adaptation, the Pichinde virus can also induce fatal infections in hamsters and guinea pigs [ 256] • The Machupo virus will induce chronic infections in hamsters [257] and will induce fatal infections in marmosets [258] and rhesus monkeys [259]. The

466

Sidwell

Lassa fever virus will induce fatal infections in guinea pigs [256] and in monkeys [ 260] • Most of these viruses are highly restricted in their transport and experimental use, with P-4 containment often required. As a result , few facilities are available for studies with these diseases. The U. S . Army Medical Research Institute of Infectious Diseases at Fort Detrick (Frederick, Maryland) has such appropriately equipped laboratories, and in vivo viral chemotherapy studies have been reported with Pichinde, Machupo, and Lassa fever viruses [256,261]. Rhabdoviridae

Two viruses classified in the Rhabdoviridae family should be considered possible targets for antiviral drugs. One is the Marburg virus, which is endemic in Africa, and the other is the widely disseminated rabies virus. The Marburg virus causes Marburg disease, a highly fatal hemorrhagic disease with transmission and global impact potential similar to those of the hemorrhagic fever viruses of the Arenaviridae family discussed previously [ 172]. The rabies virus disease continues to be a frightening, fatal disease, usually transmitted by animal bite, that is especially of concern in many developing nations [ 262] . The Marburg virus will induce fatal infections in infant mice [263], guinea pigs, and monkeys [ 264] . No chemotherapy studies are known to have been run on this virus, which, like the viruses of the Arenaviridae family, is highly restricted in its experimental use. The rabies virus will infect essentially all laboratory animals, usually producing a paralytic fatal disease when administered intracerebrally, intramuscularly, or intranasally [ 265-267]. The viral infection often occurs after a considerable incubation time, especially if the animal is infected intramuscularly in one of its extremities. Such a slowly developing CNS infection has allowed investigators to determine the influence of antiviral compounds, as well as immunostimulatory substances, on the progress of disease, with reasonable assurance of extrapolation to the human situation. Most antiviral studies have been run in mice [268-272] and in rabbits [272,273]. Togaviridae

Viruses of primary importance to this chapter that are included in the Togaviridae family are those of Alphavirus and Flavivirus genera, also known as arboviruses group A and B. The arboviruses are the causal agents of encephalitis, an often fatal disease that can occur in epidemic proportions throughout the world. In the United States, the case rate has been determined to be as high as 1. 8 in 100, 000 population [ 274]. The Alpha viruses are the 20 mosquito-transmitted group A arboviruses described by Casals and Brown [ 275]. Most studied of these are eastern, western, and Venezuelan equine encephalomyelitis, Sindbis virus, and Chikungungya virus. The Flaviviruses comprise more than 30 group B arboviruses [ 276] , the more recognized being St. Louis encephalitis, West Nile, Japanese B encephalitis, Murray Valley encephalitis, yellow fever, dengue, and tick-borne encephalitis viruses. Most of these viruses are associated with particular geographic areas where they are often a major public health problem. A number of effective vaccines are available for most of these viral diseases, but the lack of widespread use of those vaccines often hampers their effectiveness. No clinically effective antiviral drugs are available for any of these encepahlitis diseases .

Antiviral Activity

467

A generalized statement can be made regarding essentially all the group A and B arboviruses, and that is that they will induce a fatal encephalitis in a variety of laboratory animals, but especially the mouse when injected intracerebrally. Younger mice are usually more susceptible than the adult animal. Peripheral exposure, including intranasal instillation, will often also produce a CNS-associated illness. With several of the viruses (such as yellow fever virus), some adaptation to the animal is required before reproducible infections occur. This adaptation is accomplished by successive passage of the virus through the brain of the animal [ 277]. Examples of chemotherapy experiments with these viruses include reports by Odelola [ 278] using West Nile virus in mice, Stephen et al. [ 256] using Chikungunya virus infections in monkeys and yellow fever virus and Venezuelan equine encephalitis virus infections of mice, Bauer and Sadler [ 279] with Semliki Forest, dengue, and less known arboviruses in mice, Mizuma et al. [280] using Japanese encephalitis in mice, Kramer et al. [ 281] using St. Louis encephalitis in mice, and Gresikova et al. [282] with tick-borne encephalitis in mice. Retroviridae

The viruses in the Retroviridae family that are of particular interest to this chapter are the type C oncoviruses, which are known to be oncogenic for avian, mammalian, reptilian, and piscine hosts. These viruses are especially interesting because they cause leukemias and solid tumors, which are often quite similar to certain human neoplasms, and the genomes of these viruses are part of the genetic complement of many, if not all, vertebrate species. Of significance have been the recent reports of the isolation of human retroviruses from patients with T-cell leukemias and lymphomas [5-7] and of the demonstration of antibodies to these viruses in the sera of such patients (see review, Reference 7). No animal model is known for the study of the human T-cell leukemia and lymphoma virus, but other retroviruses are known to cause leukemia, lymphoma, and other forms of cancer in a wide variety of animals. These include chickens and other fowl, induced by avian leukosis viruses (see review, Reference 283) ; rats [ 284- 289] , guinea pigs [ 290] , hamsters [ 291] , and primates (see review, reference 292), all induced by type C oncoviruses of the respective species and by Rous sarcoma virus [ 293, 294] ; cats, caused by feline leukemia virus [ 295, 296] , a feline fibrosarcomata virus [ 297, 298] , and R. D. 11 virus [ 299] ; dogs, the causative agents also feline leukemia virus [ 300] , feline fibrosarcomata virus [ 297, 298] , and Rous sarcoma virus [ 301, 302] ; and cattle, sheep, and goats, induced by bovine leukemia virus [303-305]. A laboratory animal that has proven to be of exceptional value as a model for study of type C oncoviruses has been the mouse. Sarcomas can readily be induced in these animals by several murine sarcoma viruses [306-309]. A number of murine leukemia viruses have also been studied in considerable depth, with the Gross (see review, Reference 310), Moloney [311], Graffi [312], and Friend [313] viruses the most widely described. Numerous antiviral studies have been reported with these various murine viruses. Parameters for evaluation have included inhibition of splenomegaly [314-318], increase in survivor numbers and mean survival time [319,320], decrease in histologic signs of disease [315], and reduction in infectious virus in plasma and spleen of infected mice [ 317, 318, 321]. Care should be taken in chemotherapy studies in which utilization is made of a parameter, such as inhibition of splenomegaly or tumor growth, to be sure that host

468

Sidwell

weight loss also is not occurring, since it has been shown [ 322, 323] that loss of host weight can bring about a concomitant reduction in tumor growth although does not appear to influence organ and plasma virus titers [ 323] . CONCLUSIONS

It should be quite apparent that there are no all-encompassing or standardized tests available for the evaluation of the antiviral activity of antibiotics. The reviewer is impressed, instead, by the multitude of tests available to researchers in this field for many of the clinically important virus diseases. It is hoped that the material provided here will enable the investigator to more knowledgeably select those test systems that will accurately predict the potential antiviral activity of test antibiotics. As a concluding thought, it should be pointed out that viruses cause serious, economically important diseases of other hosts as well as the human. The veterinary viral diseases are most significant, and many are uncontrollable except by such severe measures as highly restrictive quarantine and herd destruction. Plant diseases are even less controlled, except through quarantine, crop destruction, and the development of strains of plants that may be resistant to a particular virus disease. Researchers who develop substances that are significantly inhibitory to human viruses as seen by the evaluation procedures described here should also seriously consider extending their antiviral studies to include related veterinary and plant viruses. The procedures for such evaluations could be the subject of further reviews. REFERENCES

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

R. E. F. Matthews, Classification and Nomenclature of Viruses, Karger, Basel, 1979. R. W. Sidwell, in Chemotherapy of Infectious Disease (H. H. Gadebusch, ed.), CRC Press, Cleveland, 1976, p. 31. J. D. Snyder and M. H. Merson, Bull. WHO, 60:605 (1982). L. Rosen, in Human Diseases Caused by Viruses (H. Rothschild, F. Allison, Jr. , C. Howe, and C. F. Chapman, eds.) , Oxford University Press, New York, 1978, p. 135. B. J. Poiesz, F. W. Ruscetti, A. F. Gazdar, P. A. Bunn, J. D. Minna, and R. C. Gallo, Proc. Natl. Acad. Sci. (U.S.), 77:7415 (1980). B. J. Poiesz, F. W. Ruscetti, M. S. Reitz, V. S. Kalyanaraman, and R. c. Gallo, Nature, 294:268 (1981). M. Popovic, M. S. Reitz, M. G. Sarngadharan, M. Robert-Guroff, V. S. Kalyanaraman , Y. Nakao, I. Miyoshi, J. Minowada, M. Yoshida, Y. Ito, and R. C. Gallo, Nature, 300;63 (1982). C. McLaren, C. D. Sibrack, and D. W. Barry, Amer. J. Med., 73: 376 (1982). J. H. Huffman, R. W. Sidwell, G. P. Khare, J. T. Witkowski, L. B. Allen and R. K. Robins, Antimicrob. Agents Chemother., 3:235 (1973). R. W. Sidwell and J. H. Huffman, Appl. Micr·ob., 22:797 (1971). L. B. Allen and R. W. Sidwell, Antimicrob. Agents Chemother., 2: 229 (1972).

Antiviral Activity

12. 13. 14. 15. 16. 17. 18. 19. 20. 21.

22. 23. 24. 25. 26.

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G. A. Eddy, S. K. Scott, F. S. Wagner, and 0. M. Brand, Bull.

WHO, 52:517 (1975).

D. H. Walker, H. Walff, J. V. Lange, and F. A. Murphy, Bull.

WHO, 52:523 (1975).

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L. Gross, Oncogenic Viruses, 2nd ed., Pergamon Press, Oxford, 1970. J. B. Moloney, JNCI, 24:933 (1960). A. Graffi, Ann. N. Y. A cad. Sci., 68: 540 (1957). C. Friend, in Perspectives in Virology, Vol. 1, Wiley, New York, 1959' p. 262. K. Sugiura, Gann, 50:251 (1959). E. A. Mirand, N. Back, T. C. Prentice, J. L. Ambrus, and J. T. Grace, Jr., Proc. Soc. Exp. Biol. Med., 108:360 (1961). P. J. Dawson, A. H. Fieldsteel, and W. L. Bostick, Proc. Soc. Exp. Biol. Med., 119: 206 ( 1965). M. A. Chirigos, E. Luber, R. March, and H. Pettigrew, Cancer Chemother. Rep., 45:29 (1965). R. W. Sidwell, G. J. Dixon, S. M. Sellers, and F. M. Schabel, Jr., Cancer Chemother. Rep., 50:299 (1966). R. W. Sidwell, G. J. Dixon, and F. M. Schabel, Jr., in Progress in Antimicrobial and Anticancer Chemotherapy (H. Umezawa, ed.), University of Tokyo Press, Tokyo, 1970, p. 26.

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13 Fertilized Sea Urchin Eggs as a Model for Detecting Cell Division Inhibitors ROBERT S. JACOBS and LESLIE WILSON

University of California, Santa Barbara, California

Properties and Utility of Naturally Synchronous Cultured Echinoderm Embryos Description of the Sea Urchin Embryo Cell Cycle Assay Protocol Results Standard Drugs Marine Natural Products References

482 483 485 485 485 487 491

When we initiated our marine pharmacology program in 1977, we developed a battery of screening tests designed to detect unusual biological activity of purified marine natural products with the view that some of these agents, once identified, could serve as useful experimental probes or models for future drug development. We decided to adapt the fertilized sea urchin egg as a cell culture system and determine if this test model provided us with unusual f>harmacological results. Although not free of special drawbacks inherent in the system, the fertilized sea urchin egg has shown a significant degree of utility both as a.-i experimental model for investigating mechanisms of drug action and as a selective screen that appears relatively insensitive to several classes of antineoplastic agents. This degree of selectivity afforded us the opportunity to identify several new classes of cell division inhibitors from marine sources that thus far appear to be exerting their effects by novel mechanisms of action [ 1- 5] . Described here are details of this assay that we hope will prove helpful, as well as an overview of the results one can obtain. References refer the reader to published literature describing in more elaborate detail results on mechanisms of drug action obtained using this experimental model.

481

482

Jacobs and Wilson

PROPERTIES AND UTILITY OF NATURALLY SYNCHRONOUS CULTURED ECHINODERM EMBRYOS

The sea urchin embryo cell culture system employed in much of this work exhibits many advantages over conventional mammalian cell culture. Eggs fertilized at the same time undergo numerous highly synchronous divisions, exhibiting a degree of synchrony generally much better than that obtainable using the drug-induced synchronization or physical selection techniques required of mammalian cells. Moreover, sea urchin embryos retain this degree of division synchrony at least until completion of the third cleavage [ 6) and thus do not suffer appreciably from the phenomenon of synchrony decay that so plagues experiments with mammalian cells. This tight synchrony through multiple divisions also allows one to identify a drug-induced delay or decay in cell cycle progression with a precision virtually impossible using synchronized mammalian cells. There are also other, less obvious, benefits to be derived from the use of this cell culture system. First, the fertilized egg is a nontransformed cell of stable karyotype. This is usually not the case when using cultures of established mammalian cell lines [ 7) . Second, the use of cultured mammalian cells to identify cytotoxic compounds involves considerable time and expense (the latter arising both from the cost of the medium and from such culture equipment as filtration manifolds, incubators, compressed gas, and electronic cell counters). In contrast, sea urchin embryos require only seawater as a medium, can be incubated in a simple water bath, need not be cultured under rigorously sterile conditions, and exhibit a cell cycle time of 120 min or less, thus allowing cell cycle inhibition studies to be completed within just a few hours. Third, the large size of the sea urchin embryo (75-100 µmin diameter, depending upon the species used) makes it relatively easy to examine the assembly and development of the mitotic spindle apparatus in unfixed material using simple low- or medium-power light microscopy. This is a technically more difficult undertaking with the far smaller mammalian cells. Fourth, the presence of large amounts of maternal "masked" messenger RNA present in echinoderm embryos is thought to preclude transcriptional inhibition as a cytotoxic mechanism of action in early development [ 8) • The sea urchin embryo is thus believed to represent a naturally occurring pharmacologically selective bioassay system that conveniently allows the exclusion of an important cytotoxic process (transcriptional inhibition) as a mechanism responsible for the inhibition of cell division. To employ a mammalian cell system counterpart would require the production and/or selection of mutant lines resistant to each and every transcriptional inhibitor, obviously a timeconsuming, expensive, and laborious undertaking. Finally, the sea urchin embryo is a totipotent zygotic cell that can be routinely cultured well beyond the gastrula stage of development. This property allows the rapid identification and study of any drug-induced alterations in the differentiation sequence that may be manifest as changes in embryo morphology. It is of course impossible to exploit this capability using cultured mammalian somatic cells. In addition to its natural synchrony , the sea urchin embryo cell culture system thus possesses a number of unique and distinct advantages over con ventional mammalian cell culture. It is also true, however, that certain disadvantages exist due to differences between the cell cycles of invertebrate zygotes and mammalian cells. The sea urchin embryo exhibits

Detecting Cell Division Inhibitors

483

a cell cycle considerably different from that of a somatic mammalian cell. There is no obvious somatic cell equivalent to several processes that occur in the sea urchin, for example, to the initiation of metabolic activity, structural reorganization, and developmental differentiation that occurs upon fertilization. The activation and clonal expansion of lymphocytes by specific antigen certainly bears some resemblance, but the degree of differentiation and metabolic activation is far more restricted in those cells relative to fertilized echinoderm eggs. In the case of typical epithelial or fibroblast cells cultured in vitro and stimulated to proliferate by release from high culture density, the difference is even greater. To provide the reader with some appreciation of the limitations to the use of sea urchin embryos as a model of the typical somatic mammalian cell, a brief review of the sea urchin embryo cell cycle is presented. DESCRIPTION OF THE SEA URCHIN EMBRYO CELL CYCLE

In sea urchin embryos (as in all echinoderm embryos), the fertilization "program" is initiated when the acrosomal process of the sperm penetrates the vitelline layer of the egg and fuses with the egg plasma membrane. Within approximately 3 sec [ 9], there follows an influx of both Na+ [ 10, 11] and ca2+ [ 12, 13], resulting in depolarization of the egg plasma membrane and establishment of the "fast block" to polyspermy [ 14, 15]. Within the next few seconds there follows a rise in intracellular ca2+ via mobilization of intracellular ca2+ stores [ 16-18] , which in turn activates the "early events" of fertilization. The first of these "early events" (occurring within 20 sec after sperm-egg fusion) is a Ca2+-mediated fusion of the cortical granules with the plasma membrane. This exocytotic process releases colloidal material and peroxidase en zymes into the perivitelline space [ 19, 20]. The enzymes released by the cortical granules then cause the vitelline layer to elevate the fertilization envelope (a now spherical membrane completely enclosing the zygote). This extracellular structure is subsequently "hardened" by ovoperoxidase-mediated phenolic coupling of tyrosyl residues present within the fertilization envelope proteins [ 21]. Insertion of the cortical granule membranes into the egg plasma membranes during exocytosis results in the formation of a mosaic membrane [ 22, 23] , a process temporally associated with a change in conductance of the membrane to Na+ and H+. Beginning at approximately 50-60 sec after fertilization, a Na+ /H+ facilitated transport system becomes activated, resulting in an exchange of extracellular Na+ for intracellular H+ and a subsequent cytoplasmic alkalinization [ 24-26]. At the same time, subcortical actin (which was present within the thousands of short microvilli covering the surface of the unfertilized egg) is reorganized as a result of the increasing intracellular pH and free ca2+ concentration [ 27]. As a consequence of this actin reorganization (assembly or sliding?), the short microvilli rapidly elongate and "engulf" the sperm [ 28]. Additionally, this microvillar elongation process may also be responsible for taking up excess membrane introduced by the cortical granules [ 29] . Immediately after this cortical granule exocytosis, there follows a burst of oxygen consumption. This increase in the rate of oxygen consumption can be quantitatively accounted for by the generation of hydrogen peroxide

Jacobs and Wilson

484

and is absent when an inhibitor of cortical granule discharge (procaine) is present at fertilization [ 30]. Warburg [ 31] believed this increase in the rate of 02 consumption to be a "respiratory burst," but it is now known to be calcium dependent, cyanide insensitive, and nonmitochrondrial. This phase of 02 consumption is thus not believed to be respiratory but is associated with the oxidation of naphthoquinones derived from pigment granules, the product of this oxidation being H202 [32]. In addition to initiating the cortical reaction, the intracellular mobilization of ca2+ also activates a calmodulin-dependent NAD kinase [33]. Within approximately 120-150 sec, the rising cytoplasmic alkalinization reaches a maximum (pH 7. 2 as opposed to pH 6. 6 in the unfertilized egg), which is then held constant for about 5 min. Although the pH gradually declines over the next 30 min, this degree of cytoplasmic alkalinization is sufficient to trigger the "late events" of fertilization. These events include: 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Development of the K+ conductance [34,35] Activation of Na+-dependent amino acid and nucleoside transport systems [36-38] Increase in the amount of translatable polyadenylated mRNA [39-42] Increase in the rate of translation [ 43- 451 Pronuclear centration and fusion of the egg and sperm pronuclei [46-48] Initiation and completion of the 81 period of DNA synthesis [49,50] Formation and disappearance of the "clear streak" structure of perinuclear microtubules [ 51, 52] Initiation of mitosis and assembly of the mitotic spindle apparatus [53-55] Formation of the "contractile ring" [56,57] Completion of mitosis coincident with the initiation of the 82 period of DNA synthesis [ 58, 59] and disassembly of both the mitotic spindle and the contractile ring [ 60]

The first cell cycle of the sea urchin embryo can thus be divided into two general periods: the "early events" of gamete fusion, egg membrane reorganization, and metabolic activation, followed by the "late events" of increased protein synthesis, syngamy, 81, 02, and mitosis (which partially overlaps 82). Although the events leading up through syngamy end at the beginning of the 81 period of DNA synthesis, this "pre-8 period" is not considered the equivalent of the 01 phase typically found in somatic mammalian cells. In fact, the sea urchin embryo is generally considered to be devoid of a 01 period, at least up until the blastula stage of development [61]. It is also important to note that the 02 period is quite short (approximately 10-15 min in Strongylocentrotus purpuratus sea urchin embryos) and represents a smaller fraction of the total cell cycle time than the 02 period of a typical mammalian cell. Following the completion of the "early events" (which are of course unique to the first cell cycle of embryonic development) , the sea urchin embryo cell cycle is thus seen to be highly abbreviated, essentially cycling from 8 to M to 8 with no 01 and a relatively short 02 phase. One drawback to the use of sea urchin embryos is apparent: agents that block cell division by acting in 01 will have no effect in this system. Another potential disadvantage to the use of the sea urchin embryo resides

Detecting Cell Division Inhibitors

485

in the fact that agents that inhibit egg cleavage by specifically inhibiting some pre-Sl event (events generally unique to zygotes) might have no effect on the growth and division of cultured somatic mammalian cells. ASSAY PROTOCOL

For all assays, fresh seawater is collected and filtered through Whatman 4 filter paper. Sea urchins, either S. purpuratus or Lytechinus pictus, are induced to spawn by injection of 0. 5 M KCl through the soft tissue of the oral surface into the coelomic cavity. Gametes emerge through the aboral surface. Sperm are collected using a Pasteur pipette and stored in a test tube on ice. Female urchins are placed atop a beaker of seawater, and eggs simply drop to the bottom of the beaker. Eggs are washed in three changes of seawater to remove the jelly coat, thus ensuring uniform fertilization, hand centrifuged in a graduated tube to determine the volume of packed eggs, and then added to an appropriate volume of seawater to obtain a 1% egg slurry. When eggs have been completely prepared for fertilization, 10 µl of drug solution is preloaded into duplicate test vials. Approximately 50 µl sperm are added to 25 ml seawater and gently stirred, and 1 ml of this solution is added to the egg slurry. Fertilization occurs within 60-90 sec. After 1 ml of fertilized egg slurry is added to each preloaded test vial, vials are placed in a shaking water bath (15°C for S. purpuratus and 19°C for L. pictus) and incubated until completion of first division in control embryos. A minimum of 300 embryos from each vial is observed under low-power microscope for completion of division and abnormalities (such as cell lysis). The percent of inhibition is the percentage of cells that are not divided at this time. RESULTS

Standard Drugs We have tested a number of antineoplastic agents in the sea urchin assay and compared them with effects seen in the Chinese hamster ovary cell culture (Table 1). As can be seen by inspection of the table, a wide spectrum of agents does not inhibit the first division cycle in the sea urchin. All these agents act specifically on such processes as DNA synthesis and transcription and are relatively cell cycle specific. Thus, the first cycle after fertilization cannot easily be interrupted by these cytotoxic agents, extending earlier work [ 8] and suggesting that in RNA transcription and possibly DNA synthesis is not required for cleavage during this initial replication period. This leaves protein synthesis, and protein function as potential sites of drug action. Thus the sea urchin has been found to be sensitive to cytochalasin B , colcemid, podophyllotoxin, vinblastine, and several new marine natural products recently reported in the literature that appear to act in part by inhibition of protein function [ 1] . The antifungal agents are another group of standard drugs in which the sea urchin egg appears sensitive. They are summarized in Table 2. Amphotericin B and nystatin are believed to act by binding to a sterol moiety, primarily ergosterol, present in the membrane of certain fungi. These polyene antibiotics, by virtue of their interaction with sterols, produce pores or channels in the membrane allowing leaking and loss of small molecules

82 94 88 67 67 73

100 100 100 100 100 100 100 0 0 100 100 100

Natural products Microtubu le assembly inhibitors

Antibiotic s

34

0 0 0 0

Hydroxyu rea, Guanazole Cytochala sin B , Daunomyc in , Actinomy cin D Colcemid, Podophyl lotoxin, Vinblastin e

88 37 89 0 0 0 0

0 0 0 0

Amethopt erin, 6-Mercap topurine, 5-Fluorou racil, Cytosine arabinosid e

Folic acid analogs, Purine analogs, Pyrimidin e analogs

Antimetab olites

Miscellane ous agents

54

44

80

0 0

0 0

Chloramb ucil, Mechloret hamine

Name

Nitrogen mustards

Type

CHO cells in culture

Alkylating agents

Class

S. purpuratu s

Echinoder m embryos L. pictus

% Inhibition

16.0 µg/ml Table 1 Antineopl astic Agents Tested for Inhibition of Cell Division at Concentr ation=

ol'>.

;::s

0

Ci;'

~

Q.

;::s

Q

Cl.l

O'

0

g

....

C)

Oo

487

Detecting Cell Division Inhibitors

Table 2 Antifungal and Antibiotic Agents Tested for Inhibition of Cleavage in the Echinoderm Assay (Concentration = 16 µg/ml)

% Inhibition by species Name

S. purpuratus

L. pictus

Antifungal agents Amphotericin B

100

100

Griseofulvin

100

100

Haloprogin

100

100

Nystatin

100

100

Pyrrolnitrin

100

100

5-Fluorocytosine

0

0

Miconazole

0

0

Tolnaftate

0

0

Penicillin G

0

0

Streptomycin

0

0

Chloramphenicol

0

0

Antibiotic agents

from intracellular pools. We are not sure if this is the mechanism of action of these drugs on the sea urchin. Similarly, haloprogin and pyrrolnitrin activity is not completely understood. 5-Fluorocytosine, which is converted to 5-fluorouracil, would not be expected to be active. Thus, the specificity of the fertilized sea urchin egg could be summarized as one in which DNA synthesis is not an absolute requirement for early cleavage. Drugs that block the first cleavage would be expected to act as steps affecting mRNA function forward to cytokinesis. Griseofulvin is a potent microtubule assembly inhibitor and would be expected to inhibit cell division by a mechanism similar to colchicine. The binding site for griseofulvin, however, is distinct from colchicine and the vinca alkaloids [ 62] •

Marine Natural Products We have investigated the effects of several hundred marine natural products in which the structure was known and sufficient material available for study. The sources of the compounds were a variety of algae, sponges, soft corals, and other forms of marine life. Bioassay-directed isolation was accomplished by our collaborators, Faulkner and Fenical (Scripps Institute of Oceanography) and Crews (University of California, Santa Cruz). The chemical structures of these substances were of great variety, varying from simple quinones to more complex carbon skeletons. A few of the types of structures encountered are shown in Figure 1. We subjected this set of compounds to a comparative study to determine if any of these substances pre-

wt17

wf16

(JO,...M)

(15J.1Ml

(>60J,1M)

wf5

wf37

wf21

(IOµM)

(10,..M)

(J.6,...M) ( 18,uM)

( 11 µM)

( 30 ,uM)

!'-./s

s~s).

Jf2J

Br

o~I

wf13

Cl

HO~I

wf6

Figure 1 Chemical structures of active cytotoxins in the fertilized sea urchin egg. Numbers in parentheses are ED 50 values.

0

HO

HO

wf15

""'

Q

;::s

0

fj)'

~

Q..

;::s

Q

Cll

O'

0

C'l

~

Clo Clo

Detecting Cell Division Inhibitors

489

vented microtubule assembly in vitro as a first step in exploring their mechanism and also to verify the specificity of the sea urchin. Halogenated Sesquiterpenes from Laurencia obtusa and Laurencia elata

The first compounds we found active against division of sea urchin embryos were elatol (WF-6) and elatone (WF-13). Elatol was isolated as a metabolite of the red algae L. obtusa and L. elata [63]. Elatone is the oxidation product of elatol. These halogenated sesquiterpenes have been extensively studied in our laboratory [64,65] and served as important models for the development of our in vitro methods. Figure 2 demonstrates a time of addition study carried out with elatol. As can be seen, if addition of the drug is delayed until 30 min past fertilization, there occurs a regression of effect to the point that, when added late in the cell cycle (>90 min), cell division is relatively unimpeded. In addition to these studies, we have found that both elatol and elatone inhibit the incorporation of thymidine into DNA during the first cleavage of s. purpuratus embryos. In microtubule assembly studies, we found elatone to inhibit the assembly of beef brain microtubules, whereas elatol produced no significant effect on assembly.

DRUG CONTACT TIME (MIN) 180

150

120

90

60

30

0

30

60

90

120

150

180 MIN

100 90

z

80

0

70

CID

60

J:

z

so

~

40

....

30 20 10 0

0

TIME Of ELATOL ADDITION AFTER FERTILIZATION

Figure 2 Percentage inhibition of first cleavage versus the time of addition of elatol (WF-6; 16 µg/ml) to fertilized sea urchin eggs. The first cleavage in controls occurs at approximately 120 min after fertilization.

490

Jacobs and Wilson

Table 3 Natural Products Tested for Inhibition of Cleavage in the Sea Urchin Assay and Brain Microtubule Assembly (MTA)

Name

Source

ED100 Sea urchin (µg/ml)

% Inhibition MTA

16

100

Pseudopterogorgia rigida

8

100

WF-17

Pseudopterogorgia rigida

16

100

WF-21

Stypopodium zonale

8

100

JF-23

Chondria californica

32

95

WF-5

Ircinia variabilis

16

10

WF-6

Laurencia obtusa

16

0

WF-15

Pseudopterogorgia rigida

16

0

JF-37

Pseudop lexaura

16

0

WF-13

Laurencia obtusa

WF-16

Sesquiterpenoids from the Gorgonian Coral Pseudopterogorgia rigida Three compounds from P. rigida, curcuphenol (WF-15), curcuquinone (WF-16), and curcuhydroquinone (WF-17), have been isolated, purified, and found active against Staphylococcus aureus and the marine pathogen V. anguillarum [ 66] . The corals were collected at Belize. In the sea urchin assay and microtubule assembly assay (Table 3) curcuphenol was found inactive whereas curcuquinone and curcuhydroquinone were found active. Stypoldione, an Ichthyotoxin from the Alga Stypopodium zonale

This compound (WF-21) was isolated and purified as the active component of the brown alga s. zonale that was shown toxic to reef-dwelling fish [67]. It is the most potent inhibitor of cell division we have identified thus far (Figure 1). Equivalent concentrations also prevented microtubule assembly in vitro (Table 3). It is interesting to note that the dihydroxy analog strypotriol is reportedly more toxic to fish than stypoldione [ 68] , yet in our sea urchin assay, the former compound proved inactive. A Sesterterpene from the Sponge

Ircinia variabilis

Variabilin (WF-5) was isolated and purified on the basis of its potent antibiotic activity against s. aureus [69]. Ircinia variabilis is widely distributed in the Gulf of California [ 70] • Variabilin was found to inhibit cell division in sea urchins. The potency of this compound is roughly equivalent to that of curcuquinone (Figure 1) but unlike the above compounds did not inhibit microtubule assembly.

Detecting Cell Division Inhibitors

491

A Cyclic Polysulfide from the Red Alga Chondria californica

A group of cyclic polysulfides from C. californica obtained from the Pacific at Isla San Jose, Mexico, were isolated and purified when crude fractions were found to have antibiotic activity against V. anguillarium [ 71] . One of these compounds (JF- 23) was shown to have weak inhibiting activity in both the cell division assay (Figure 1) and the microtubule assembly assay (Table 3). These types of structures are relatively rare in nature, and the compound was included in this study primarily because of its novel structure. Cembranolide from Gorgonians of the Pseudoplexaura Genus

Crassin acetate (WF-37) has been previously found to be active in several anticancer screens [ 64] • This compound was isolated from several species of Pseudoplexaura obtained from the Caribbean. In addition to antineoplastic activity, it has been shown to be antibiotic [65] and ichthyotoxic [66], In our laboratory, we found it to inhibit cell division in sea urchin embryos but not inhibit microtubule assembly (Table 1) • One can see that there is an apparent relationship between inhibition of the first cleavage and a somewhat high incidence of compounds that inhibit polymerization of microtubules. We have been unsuccessful so far in proving beyond doubt that these drugs act only by inhibiting assembly of microtubules. The most potent compounds studied thus far are elatone [ 2] and stypoldione [ 4] , which show that the activity of these compounds falls off rapidly if added close to the period of mitosis, but the cells do not accumulate in metaphase. With stypoldione in particular the chromosomes appear in some studies to be condensed and in early prophase. Thus, other mechanisms could be affected by this compound. Most pertinent here is that, for investigations of mechanism of drug action, the sea urchin model simplifies the number of possibilities by eliminating the antimetabolite class of agents that inhibit DNA synthesis and mRNA synthesis as a complex set of molecular events that need to be considered.

ACKNOWLEDGMENTS The authors are indebted to Ms. Elise Clason and Ms. Joan Jacobs for assistance in preparing this chapter and to Dr. Steven White for his helpful comments. The research reviewed here was sponsored by the National Oceanographic and Atmospheric Administration under grant NOAA-04-8-MOI189 and by State Resources Agency Project R/MP-21.

REFERENCES 1. 2. 3. 4. 5.

R. S. Jacobs, S. White, and L. Wilson, Fed. Proc., 40:26 (1981). S. White and R. S. Jacobs, Mol. Pharmacol., 20:614 (1981). T. O'Brien, R. S. Jacobs, and L. Wilson, Mol. Pharmacol., 24:493 ( 1983). S. J. White and R. S. Jacobs, Mol. Pharmacol., 24: 500 ( 1983). T. O'Brien, S. White, R. S. Jacobs, G. B. Boder, and L. Wilson, Hydrobiologia, 116/117: 141 (1984).

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6. 7. 8. 9. 10. 11. 12. 13; 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44.

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K. Okasaki, in The Sea Urchin Embryo (G. Czihak, ed.), SpringerVerlag, Berlin, 1975, pp. 177-232. P. M. Kraemer, D. F. Petersen, and M. A. Van Dilla, Science, 174: 714 ( 1971). P. R. Gross, L. I. Malkin, and W. A. Moyer, Proc. Natl. Acad. Sci. USA, 51:407 (1964). T. Schmidt, C. Patton, andD. Epel, Dev. Biol., 90:284 (1982). L. A. Jaffe, Nature, 261:68 (1976). J. D. Johnson, D. Epel, and M. Paul, Nature, 262:661 (1976). M. Paul and R. N. Johnston, J. Exp. Zool., 203:143 (1978). E. L. Chambers and J. de Armendi, Exp. Cell Res., 122:208 (1979). L. A. Jafee, Nature, 261 (1976). L. A. Jaffe and K. R. Robinson, Dev. Biol., 62:215 (1978). E. L. Chambers, B. C. Pressman, and B. Rose, Biochem. Biophys. Res. Commun., 60: 128 ( 1974). R. A. Steinhardt and D. Epel, Proc. Natl. Acad. Sci. USA, 71:1915 (1974). T. Schmidt, C. Patton, and D. Epel, Dev. Biol., 90:284 (1982). D. Epel and J. D. Johnson, in Biogenesis and Turnover of Membrane Macromolecules (J. S. Cook, ed.), Raven Press, New York, 1975, pp. 105-120. D. Epel, Sci. Amer., 237:129 (1977). G. Hall, Cell, 15: 343 ( 1978). D. Epel and J. D. Johnson, in Bio genesis and Turnover of Membrane Macromolecules (J. S. Cook, ed.), Raven Press, New York, 1975, pp. 105-120. E. M. Eddy and B. M. Shapiro, J. Cell Biol., 71:35 (1976). J. D. Johnson, D. Epel, and M. Paul, Nature, 262:661 (1978). L. A. Jaffe, Nature, 261:68 (1976). s. S. Shen and R. A. Steinhardt, Nature, 272:253 (1978). D. A. Begg, L. I. Rehbun, and H. Hyatt, J. Cell Biol., 93:24 (1982). D. Epel, Sci. Amer., 237:129 (1977). T. E. Schroeder, Dev. Biol., 70:306 (1979). C. Foerder, S. J. Klebanoff, and B. M. Shapiro, Proc. Natl. Acad. Sci. USA, 75:3183 (1978). O. Warburg, Hoppe-Seyler's Z. Physiol. Chem., 57: 1 ( 1908). G. Perry and D. Epel, Exp. Cell Res., 134:65 (1981). D. Epel, C. Patton, R. W. Wallace, and W. Y. Cheung, Cell, 23:543 (1981). R. A. Steinhardt, L. Ludin, and D. Mazia, Proc. Natl. Acad. Sci. USA, 68:2435 (1971). S.S. Shen and R. A. Steinhardt, Exp. Cell. Res., 125:55 (1980). D. Epel, Exp. Cell Res., 72:74 (1972). J. Piatigorsky and A. H. Whitely, Biochim. Biophys. Acta, 108: 404 ( 1965). J.M. MitchisonandJ. E. Cummins, J. Cell. Biol., 1:35 (1966). L. H. Kedes and P.R. Gross, J. Mol. Biol., 42, 559 (1969). L. H. Kedes and P. R. Gross, Nature, 223:1335 (1969). L. H. Ke des, P. R. Gross, G. Gognetti, and A. L. Hunter, J. Mol. Biol. , 45: 337 ( 1969). R. A. Raff, Cell Differ., 11:305 (1982). M. M. Winkler and R. A. Steinhardt, Dev. Biol., 84:432 (1981). R. A. Raff, J. W. Brandis, C. J. Huffman, A. L. Koch, and D. E. Leister, Dev. Biol. , 86: 265 ( 1981).

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G. Schatten, Dev. Biol., 86: 426 (1981). H. Schatten, T. Bestor, and G. Schatten, Eur. J, Cell Biol., 22: 356a ( 1980). 47. G. Schatten and H. Schatten, Exp. Cell. Res., 135:311 (1981). 48. A. M. Zimmerman and S. Zimmerman, J, Cell. Biol., 34:483 (1967). 49. R. T. Hinegardner, B • Rao, and D. E. Feldman , Exp. Cell Res. , 36:53 (1964). 50. H. Schatten, S. Schatten, C. Petzeit, and D. Mazia, Eur. J. Cell Biol., 27: 74 ( 1982). 51. G. Schatten, Dev. Biol., 86:426 (1981). 52. P. Harris, M. Osborn, and K. Weber, J, Cell. Biol., 84:668 (1980). 53. G. Sluder, J. Cell. Biol., 80: 674 (1979). 54. G. Schatten, Dev. Biol., 86:426 (1981). 55. P. Harris, M. Osborn, and K. Weber, J. Cell. Biol. , 84: 668 ( 1980). 56. T. E. Schroeder, J. Cell. Biol., 53: 419 ( 1972). 57. T. E. Schroeder, Proc. Natl. Acad. Sci. USA, 70:1688 (1973). 58. R. T. Hinegardner, B. Rao, and D. E. Feldman, Exp. Cell Res., 36: 53 (1964). 59 • .M, Von Ledebur-Villiger, Exp. Cell Res., 72:285 (1972). 60. T. E. Schroeder, in Molecules and Cell Movement (S. Inoue and R. E. Stephens, eds.), Raven Press, New York, 1975, pp. 334-352. 61. M. Von Ledebur-Villiger, in The Sea Urchin Embryo (G. Czihak, ed.), Springer-Verlag, New York, 1975, pp. 539-545. 62. L. Wilson, Biochemistry, 9: 4999 (1970). 63. J. J, Sims, G. H. Y. Lin, and R. M. Wing, Tetrahedron Lett., 39: 3487 (1974). 64. S. White and R. S. Jacobs, Pharmacologist, 21: 170 (1979). 65. s. White, R. Tanalski, W. Fenical, and R. S. Jacobs, Pharmacologist, 20:219 (1978). 66. F. J. McEnroe and W. Fenical, Tetrahedron Lett., 34: 1661 (1978). 67. W. H. Gerwick, W. Fenical, N. Fritsch, and J. Clardy, Tetrahedron Lett., 145 ( 1979). 68. S. J. Wratten and D. J. Faulkner, J. Org. Chem. , 41: 2464 (1976). 69. A. J. Weinheimer and J. A. Matson, Lloydia, 38: 378 (1975). 70. L. S. Ciereszko, D. N. Sifford, and A. J, Weinheimer, An. N. Y. Acad. Sci., 90: 917 ( 1960). 71. C. F. Asnes and L. S. Wilson, Anal. Biochem., 98:64 (1979).

14 Critical Appraisal of Animal Models for Antibiotic Toxicity PATRICIA D. WILLIAMS and GIRARD H. HOTTENDORF Bristol-Myers Company, Syracuse, New York

Types of Antibiotic Toxicity Nephrotoxicity Oto toxicity Hypersensitivity Hematologic Toxicity Hepatotoxicity Isolated or Rare Toxicities Animals as Human Surrogates Advantages of Animal Models Criteria for Animal Model Selection Concerns for Animal Models Critical Appraisal Nephrotoxicity Ototoxicity Hypersensitivity Hematologic Toxicity Isolated or Rare Toxicities Future Directions of Antibiotic Toxicity Testing Animal Model Selection In Vitro Testing Procedures Conclusions References

496 496 498 498 499 499 499 500 500 501 505 505 505 512 515 516 516 517 517 517 518 518

Antibiotics represent a group of chemical substances that kill or suppress the growth of microorganisms. By definition such substances are themselves produced entirely or in part by various species of microorganisms (bacteria, fungi, or Actinomycetes) [ l]. Not surprisingly, they differ significantly in chemical and physical properties, as well as in antimicrobial spectrum and mechanisms of action. This heterogeneity is also reflected in the diversity of toxic reactions produced clinically by antibiotics. With roughly 30% of all 495

496

Williams and Hottendorf

hospitalized patients receiving antimicrobial chemotherapy, the spectrum of toxicities produced by these compounds is a significant factor in the clinical management of microbial disease [ 1] • As the number of antibiotics being developed grows, the toxicologist is challenged to predict not only the toxic liability of these new compounds in humans, but also to rank such potentials relative to other currently marketed antibiotics. Obviously, the central issue of this challenge involves the reliance of the toxicologist upon animal experimentation to predict human risk. This chapter involves an appraisal of animal models for testing antibiotic toxicity. This appraisal will include a review of existing animal models utilized to study specific target organ toxicities of antibiotics, as well as the factors involved in selecting an appropriate toxicity model, The future direction of antibiotic toxicity testing will also be discussed. TYPES OF ANTIBIOTIC TOXICITY It is beyond the scope and intent of this chapter to discuss all the possible

adverse or toxic reactions due to antibiotics. Such detailed information is available in several published works [ 2, 3] . With emphasis on the use of animal models for the evaluation of antibiotic toxicity, this chapter will concentrate upon the most commonly encountered toxicities associated with these chemotherapeutic agents. Nephrotoxicity Antibiotics are not only the principal cause of drug-induced nephrotoxicity, but the kidney is the major site of toxicity due to these compounds [ 4] . The kidney is particularly susceptible to toxicity due to such drugs as antibiotics, for a number of reasons [ 5] • First, because the kidney is often the sole source of elimination for antibiotics, it is necessarily exposed to the entire body burden of potentially toxic compounds. Second, even though the kidneys constitute less than 1% of the body weight, they receive 25% of the resting cardiac output. Thus, relative to other organs, large fractions of circulating antibiotics are delivered to the kidneys. Third, the functional ability of the kidney to extract substances from the blood or tubular lumen for the purpose of secretion or reabsorption allows for accumulation of substances in the renal parenchyma. For example , renal transport systems that function to secrete or reabsorb endogenous organic ions (either bases or acids) may be utilized to transport antibiotics, often organic ions themselves, into renal tubular cells. Finally, another factor contributing to the kidney's susceptibility to toxic reactions lies in its ability to concentrate substances along the nephron. As the result of salt and water reabsorption, luminal contents become progressively more concentrated, exposing renal cells to very high concentrations of antibiotics. The compromise in renal function due to antibiotics is typically manifested by an abrupt decline in kidney function referred to clinically as acute renal failure [ 6] • Fortunately, the nephrotoxicity due to most antibiotics appears to be clinically reversible [ 7, 8] . However, though clinical evidence of renal impairment may disappear with cessation of drug administration, scarring and permanent loss of tissue with functional compensation by the surviving nephrons may be occurring [ 9, 10] •

Critical Appraisal of Animal Models

497

A minoglycosides

Aminoglycosides represent an important class of antibiotics used clinically to treat gram-negative infections, often life-threatening in nature. This class includes such compounds as neomycin, gentamicin, tobramycin, amikacin, kanamycin, and netilmicin. Structurally, they consist of amino sugars linked to another moiety by a glycoside bond. Aminoglycoside-induced nephrotoxicity is well-documented in the human, with incidences ranging from 2 to 36% of patients receiving the drug [11-15]. In fact, aminoglycosides are the leading cause of antibiotic-induced acute renal failure [ 16]. Proximal tubule necrosis is a consistent feature of aminoglycoside nephrotoxicity in humans and experimental animals, primarily involving the pars convoluta of the proximal nephron [17-19].

Cephalosporins

Cephalosporin antibiotics possess activity against gram-positive as well as gram-negative organisms. Such compounds as cephaloridine, cephalothin, cefazolin, cephapirin, cephalexin, ceforanide, and cefamandole belong to this class of f3-lactam antibiotics. Though some cephalosporins are widely perceived as possessing nephrotoxic potential in humans [20], the documentation of such effects is not only very limited but is somewhat unconvincing due to the presence of other potential causes of renal dysfunction in those reports [ 21, 22]. Like the aminoglycosides, the nephrotoxicity of cephalosporins in experimental animals is also localized in the proximal tubule of the nephron [ 23, 24] . Like its relatives the penicillins, cephalosporins have been associated with hypersensitivity reactions resulting in acute interstitial nephritis [25,26]. Penicillins

The penicillins represent an important class of antibiotics with notable activity versus pneumococcal and streptococcal infections in humans. The incidence of nephrotoxicity induced by penicillin and its derivatives is a relatively rare but well-documented phenomenon [27-31]. Nephrotoxicity has been most commonly reported with semisynthetic penicillins, such as methicillin, ampicillin, nafcillin, and carbenicillin [ 25, 32-34]. Unlike the antibiotic-induced nephrotoxicities previously described, the mechanism underlying the nephrotoxicity induced by penicillins is thought to involve a hypersensitivity reaction rather than direct tubular injury [ 26] • The pattern of renal injury involves acute interstitial nephritis and glomerular damage accompanied by fever , rash, and eosinophilia. Tetracyclines

Tetracyclines are broad-spectrum antibiotics with notable activity against rickettsi.ae and Mycoplasma pneumonia. The use of outdated tetracyclines has been associated with a Fanconi-like syndrome characterized by proximal tubular damage resulting in proteinuria, glycosuria, aminoaciduria, hypercalciuria, hyperphosphaturia, and uricosura [35-37]. This effect is related to several degradation products of tetracycline (epi-, epianhydro-, and anhydrotetracycline). Tetracyclines may also lead to progressive azotemia due to their antianabolic effect [ 26] •

498

Williams and Hottendorf Miscellaneous Antimicrobial and Antifungal Agents

Because of their well-documented nephrotoxicity in humans, the polymixin antibiotics, namely, polymixin B and colistimethate (polymixin E) , have been removed from extensive clinical use, being reserved for treatment of gramnegative infections resistant to penicillins and aminoglycosides [ 1, 26, 39] . Polymixins cause acute necrosis in the proximal tubule, and nephrotoxicity is commonly observed at clinical doses resulting in proteinuria, cylinduria, hematuria, and urinary casts progressing to a decline in glomerular filtration rate. Other antimicrobial agents exhibiting nephrotoxic potential in humans include the polypeptide antibiotic, bacitracin, and the glycopeptide antibiotic, vancomycin [1,21,26]. Nephrotoxicity due to the polyene antifungal agent amphotericin B is an expected result of chemotherapy, with over 80% of the patients receiving the drug developing renal impairment [ 1, 39]. Distal tubular damage is the major feature of amphotericin nephrotoxicity associated with renal tubular acidosis and hypokalemia. Renal vasoconstriction accompained by glomerular and proximal tubular damage has also been observed [26]. Ototoxicity Like the kidney, the ear is equipped with specialized tissues that function to transport water, electrolytes, and other endogenous substances across cellular membranes. Coupled with known antigenic similarities between the ear and the kidney [ 40] , it is not surprising that agents that produce toxicity in one organ may also affect the other.. A number of antibiotics have been implicated as causing impairment of hearing and/or equilibrium associated with inner ear damage [ 41]. Of the commonly administered antibiotics, the aminoglycosides are most frequently associated with ototoxic reactions [ 21]. The prevalence of ototoxicity in humans has been reported to range from 2 to 20% [ 42] • The aminoglycosides vary in their propensity to cause vestibular (equilibrium) or cochlear (hearing) damage to the ear. Gentamicin, along with streptomycin and tobramycin, predominantly affects the vestibular apparatus; neomycin, kanamycin, and amikacin chiefly cause hearing loss [ 43]. If detected early, ototoxicity may be reversible, but with continued drug administration damage is often permanent. Other antibiotics less frequently associated with human ototoxicity include the tetracyclines, polymixin B , vancomycin, and erythromycin [ 43] • Hypersensitivity Virtually all antibiotics possess allergenic potential, producing such reactions as rash, fever, serum sickness, and anaphylaxis [ 44] • However, the various antibiotics differ markedly in their potential to produce hypersensitivity, with the penicillins the most common cause of drug allergy in humans [ 21]. Although the frequency of drug-induced allergic reactions is generally very low, the overall incidence of penicillin-induced hypersensitivity is estimated to be as high as 10% [ 1] . Because of the structural similarity between penicillins and cephalosporins, hypersensitivity reactions are also fairly common with ceppalosporins, and cross-reactivity of allergic reactions may occur following administration of a compound of the other class. As previously discussed, indirect drug toxicities secondary to hypersensitivity may be produced (such as acute interstitial nephritis or hematologic disorders).

Critical Appraisal of Animal Models

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Hematologic Toxicity Many antibiotics have been reported to cause blood disorders in humans, such as anemia, leukopenia, thrombocytopenia, and coagulation defects [41,45,46]. Although the majority of these reactions are secondary to drug hypersensitivity, several antibiotics cause hematologic toxicity by interacting or interfering directly with blood elements. Coagulation defects (bleeding) have been reported following high-dose therapy with penicillins and cephalosporins [ 47- 52]. The mechanism of this effect is thought to involve inhibition of platelet aggregation as well as decreased conversion of fibrinogen to fibrin and increased antithrombin III activity [ 43]. More recently, several cephalosporins, cefoperazone, cefamandole, and moxalactam have been associated with another bleeding disorder referred to as hypoprothrombinemia [ 53-55]. Prolonged bleeding times relative to deficient prothrombin synthesis characterize this disorder [ 56]. A common structural feature of the cephalosporins thought to be associated with this effect is the methyltetrazolethiol substituent on the R3 position [ 57] . Chloramphenicol is the leading cause of drug-induced aplastic anemia in humans [1,21,58]. This anemia is the result of destruction of erythroid precursors in bone marrow. The bone marrow aplasia is irreversible and is associated with fatal pancytopenia. Although the incidence of this toxicity is low (1 in 30, 000), the fatality rate approaches 100%. Though the mechanism of this toxicity is unknown, it has been suggested that it represents a form of hypersensitivity reaction. Hepatotoxicity Several antimicrobial agents have been implicated in causing liver damage in humans. The tetracyclines, namely chlorotetracycline, oxytetracycline, and tetracycline, have been reported to cause liver damage following administration of high doses orally or intravenously [ 59-61] • Clinically, the hepatic injury is manifested by hyperbilirubinemia and elevated serum glutamic oxalactic transaminase and alkaline phosphatase and may be accompanied by nausea, fever, and jaundice. Histologically, the lesion is characterized by fine droplet-type fatty metamorphosis with little necrosis [ 62]. The lauryl sulfate salt of erythromycin propionate, erythromycin estolate, may also produce hepatotoxicity in humans [ 21, 63, 64] • The hepatotoxicity is apparently specific for this ester since other forms of erythromycin have not been associated with this effect. Liver damage is characterized by jaundice, abnormal liver function tests, and eosinophilia. It is thought that this damage presents intrahepatic cholestasis resulting from sensitization. Consideration of hypersensitivity in the etiology of this hepatotoxicity is suggested by the low incidence, lack of relationship to the dose administered, frequent association with repeated exposure to the drug, and the presence of eosinophilia. Amphotericin B may also occasionally cause hepatic injury and failure, causing direct hepatocellular degeneration and necrosis [ 21, 65]. Isolated or Rare Toxicities As previously mentioned, the spectrum of specific toxicities reportedly due to individual antibiotics is virtually limitless. However, a few additional specific toxicities are worthy of mention due to their clinical significance.

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Disulfiramlike Activity

Disulfiram (Antabuse) is a drug used in the treatment of chronic alcoholism [ 1]. By altering the intermediary metabolism of alcohol, disulfiram causes an elevation in blood acetaldehyde levels following the ingestion of alcohol. This is accompanied by a number of unpleasant effects, such as flushing, respiratory difficulty, nausea, vomiting, sweating, hypotension, and blurred vision. The elevation in blood acetaldehyde is due to inhibition of liver aldehyde dehydrogenase. Several third-generation cephalosporin antibiotics of recent clinical use appear to mimic disulfiram activity, causing these effects in conjunction with ingestion of alcohol [ 66-68]. Cefoperazone, cefamandole, and moxalactam have been reported to cause disulfiramlike effects in humans, presumably due to the methyltetrazolethiol side chain common to all three antibiotics [ 69] • N eurotoxicity

High doses of penicillins may be associated with neurotoxic reactions in humans [ 70-72]. Cerebral toxicity is most frequently observed and is manifested as a seizure disorder. This disorder is characterized by decreased consciousness, myoclonic jerking, and grand mal seizures and is typically associated with massive doses of parenterally administered drug. Neuromuscular Blockade

Several major groups of antibiotics are capable of producing neuromuscular blockage in humans-the aminoglycosides, polymixins, and tetracyclines [ 43]. However, the rare occurrence of neuromuscular blockade and resultant respiratory paralysis in humans is almost always associated with the concomitant administration of neuromuscular depressants commonly employed in anesthesia or with other antibiotics capable of similar toxicity [ 73]. ANIMALS AS HUMAN SURROGATES

The toxicologist, challenged to assess human risk associated with antibiotics prior to their administration in humans, must rely on tests in nonhuman subjects. However, the inherent uncertainties in risk assessments performed in animals can be reduced by selecting animal models based upon appropriate criteria. Advantages of Animal Models

Because of the number of factors influencing toxic reactions to antibiotics, which are controllable in animal studies but vary considerably in the patient population, the accurate evaluation of an antibiotic's toxic potential may be more attainable by using animal models [ 10, 74]. The advantages of animal models are summarized in Table 1. One very obvious advantage is the availability of untreated control groups in animal experimentation, enabling meaningful determinations of druginduced effects against "biological background." In human studies, untreated control subjects are ethically unobtainable since biologically equivalent controls would necessitate withholding chemotherapy in infected patients. Second, large numbers of subjects can be employed in animal studies, whereas

Critical Appraisal of Animal Models

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Table 1 Advantages of Toxicity Comparisons in Animals Disease-free, uncompromised subjects Subjects unexposed to previous or concomitant toxins Comparisons with untreated control groups Ability to compare multiple dose levels Histopathologic evaluation of tissue sections Large experimental numbers

in human studies relatively smaller numbers of patients typify the experimental design. Accordingly, valid statistical analyses and confidence in experimental findings can be enhanced by the larger numbers available in animal studies. Another important advantage in using animal subjects is their state of health. Patients receiving antibiotics represent a spectrum of age, physical condition, and disease states, which can greatly influence the development of toxicity [ 26] • Such factors are also subject to considerable patient-to-patient variation. In addition to underlying disease states or physical condition, the previous or concurrent use of other potentially toxic drugs further obscures the evaluation of drugs in humans. The result is that it is often impossible to discern the precise role of the antibiotic in the development of human toxicity. On the other hand, a uniform, naive, and relatively disease-free population of animals can be tested. Last, and perhaps most important, is the capability to perform sensitive analyses, such as microscopic examinations on target tissues from animal subjects. Varying degrees of cellular necrosis and altered function precede as well as accompany changes measured by clinical parameters of organ function. Sensitivity in terms of the ability to detect and quantitate toxicity necessitates evaluations of subclinical toxicities, such as organ histopathology [75]. Also in animal experimentation, doseresponse data can be generated by multiple dose level protocols. Such data are critical for comparative analyses and are rarely available in human studies [ 75]. Criteria for Animal Model Selection

The criteria for selecting appropriate animal models for toxicologic study are based upon the many anatomic, physiologic , pharmacokinetic , and metabolic factors that determine toxic responses. Through an appreciation and understanding of these factors and how they interrelate to effect toxic reactions in humans, meaningful criteria can be identified and applied. The following section involves a discussion of criteria pertinent to animal model selection. Target Organ Toxicity and Characteristics

Model selection based upon target organ considerations applies to situations in which a specific toxic potential of an antibiotic relative to related compounds is of interest. The animal species selected should ( 1) possess target organ characteristics (anatomy and physiology) comparable to those of humans (2) exhibit drug-induced toxicity in the organ or system of interest, and ( 3)

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present morphological and functional characteristics of the toxic lesion in the human. In regard to target organ characteristics, it is fortunate that a remarkable similarity exists in the anatomy and physiology of mammalian species. This similarity provides the foundation for risk assessment as well as for all facets of applied biological research. However, exceptions and extremes with respect to this common scheme exist, and as illustrated here, these functional and morphological differences must be considered in model selection. Dose and Sensitivity

The prevailing practice of utilizing doses many times greater than the hurr.an clinical dose in animal toxicity testing has been questioned [ 42, 76, 77] • The relevance of the administration of high doses in animals to the lower clinical doses given to humans is not obvious if the higher doses invoke secondary metabolic or elimination pathways or exceed other physiologic thresholds. The use of high multiples of the clinical dose in animal comparisons overpredicted the human safety of netilmicin, for example [ 78] • High doses might also produce toxicities that are unrelated to the clinical toxicity. The acute lethalities produced by large single doses of aminoglycosides are the result of respiratory depression and do not reflect the oto- and nephrotoxicities that are of clinical importance. The rabbit has been generally accepted as the most sensitive species to the nephrotoxicity of cephaloridine [ 23, 24] • However, when the sensitivity of the rhesus monkey to cephaloridine nephrotoxicity was directly compared with that of the rabbit utilizing light microscopic examination of renal tissue, the rhesus monkey proved to be more sensitive [ 79] • Thus, sensitivity in the animal model is not only a reflection of species sensitivity but is directly related to the sensitivity of the methods used to evaluate toxicity. Renal function tests, including blood urea nitrogen and serum creatinine [ 80, 81] , endogenous creatinine and inulin clearance [ 82, 83] , and quantitation of urinary enzymes [84-86], are inaccurate and nonspecific. Light microscopsy is an accurate and sensitive method of assessing nephrotoxicity and can reveal aminoglycoside nephrotoxicity in animals at doses within the therapeutic range [17,75]. Pharmacokinetics and Metabolism

The principles of pharmacokinetics and metabolism are the foundation of any interspecies comparisons of drug efficacy and toxicity. Drug absorption, distribution, metabolism, and excretion (ADME) determine the qualitative and quantitative aspects of exposure. Selecting an animal species for toxicologic evaluation with a pattern of drug exposure congruent with humans lends a great deal of confidence and validity to the use of animal data. The criteria to be compared for model selection involve quantitative masurements for drug absorption (fractional absorption fa and absorption rate constant ka) , volume of distribution vd, and elimination (biological half-life tt and elimination rate constant ke). Figure 1 provides a graphic display of these processes. However, additional quantitative and qualitative information must be integrated into these measurements to make them meaningful. For example, binding of drugs to plasma proteins effects a number of relevant processes and measurements. Volume of distribution measurements based upon free drug in plasma can be overestimated if the quantity of protein-bound drug is not taken into account. The fraction of plasma protein-bound drug also limits the amount of

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