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Analytical and Chromatographic Techniques in Radiopharmaceutical Chemistry
Edited by Donald M. Wieland Michael C. Tobes Thomas J. Mangner
With 115 Figures
Spri nger -Veri ag New York Berlin Heidelberg Tokyo
Donald M. Wieland Division of Nuclear Medicine
Department of Internal Medicine
The University of Michigan
Ann Arbor, MI48109
USA
Thomas J. Mangner Division of Nuclear Medicine
Department of Internal Medicine The University of Michigan Ann Arbor, MI48109 USA
Michael C. Tobes Division of Nuclear Medicine
Department of Internal Medicine
The University of Michigan
Ann Arbor, MI48109
USA
Library of Congress Cataloging in Publication Data Main entry under title: Analytical and chromatographic techniques in radiopharmaceutical chemistry.
Updated and expanded versions of presentations at a symposium held June 4, 1984, in Los Angeles, Calif., under the sponsorship of the Radiopharmaceutical Science Council of the Society of Nuclear Medicine.
Includes bibliographies and index. 1. Radiopharmaceuticals--Analysis-Congresses.
2. Thin layer chromatography-Congresses. 3. Liquid chromatography-Congresses. 4. Chemistry, AnalyticCongresses. I. Wieland, Donald M. II. Tobes, Michael C. III. Mangner, Thomas J. IV. Society of Nuclear Medicine (1953- ). Radiopharmaceutical Science Council. [DNLM: 1. Chemistry, Analytical-methodscongresses. 2. Chromatography, High Pressure Liquidcongresses. 3. Chromatography, Thin Layer--congresses. 4. Radiochemistry-methods--congresses. QD 605 A532] RS190.R34A53 1985 615.84 85-14707
© 1986 by Springer-Verlag New York Inc.
Softcover reprint of the hardcover 1 st edition 1986
All rights reserved. No part of this book may be translated or reproduced in any form without written permission from Springer-Verlag, 175 Fifth Avenue, New York, New York 10010, U.S.A. The use of general descriptive names, trade names, trademarks, etc., in this publication, even if the former are not especially identified, is not to be taken as a sign that such names, as understood by the Trade Marks and Merchandise Marks Act, may accordingly be used
freely by anyone.
Typeset by BiComp, Inc., York, Pennsylvania.
987 6 5 4 3 2
ISBN-13: 978-1-4612-9331-6 e-ISBN-13: 978-1-4612-4854-5 DOl: 1 0.1 007/978-1-4612-4854-5
Foreword
In 1906, Michael T. Sweet first developed the chromatographic method by using an adsorbant to separate pigments. Since that time, the technological advances in TLC and HPLC have brought about new definitions of purity in parallel with the advances. Radiopharmaceutical chemistry is especially dependent on the chromatographic technique because of the relatively small amount of material in most radiopharmaceuticals-often so small that the usual physical methods of analytical chemistry cannot be used. As a result, this collection of papers represents the key to successful radiopharmaceutical development by setting the standard for the present-day definition of radiochemical purity.
William C. Eckelman, Ph.D. Diagnostics Associate Director The Squibb Institute for Medical Research New Brunswick, New Jersey
Preface
The chapters herein are updated and expanded versions of presentations that the authors made at a symposium held on June 4, 1984 in Los Angeles, California under the sponsorship of the Radiopharmaceutical Science Council of the Society of Nuclear Medicine. All manuscripts were refereed.
The intent of the symposium organizers was to enlist participants who work on a day-to-day basis with the analytical and chromatographic techniques to be discussed at the symposium. We feel confident that this distillation of hands-on experience will be of value to graduate students as well as experienced researchers in radiopharmaceutical chemistry and related fields which use radiotracer methodology.
The short history of radiopharmaceutical chemistry has been marked by vivid examples of the value of conscientious use of analytical and chromatographic techniques. Nearly a decade ago, radio-TLC revealed the presence of a radioactive "impurity" in preparations of the adrenal cortex imaging agent I-131-19-iodocholesterol. Identification of this "impurity" showed in fact that it was the active agent I-131-6J3-iodomethyl-19-norcholesterol. The recent renaissance in Tc-99m radiopharmaceuticals, especially the cationic heart agents, has been accompanied by successful application of reverse phase radio-HPLC to purity analyses of these agents. Application of similar radio-HPLC techniques to clinical mainstays such as Tc-99m bone agents, many of which were first synthesized in the "HPLC-Iess" days of the early 1970s, has revealed that certain of these agents are complex mixtures. Just as radio-HPLC was being embraced as the definitive technique for purity confirmation, a 1984 report using fluorine nuclear magnetic resonance revealed that F-18-2-fluorodeoxyglucose, a radiotracer of major importance in determining regional glucose metabolism by positron emission tomography, was in certain cases contaminated with varying amounts of its isomer, F-18-2-fluorodeoxy-
vi i i Preface
mannose. To date no radio-HPLC system has been reported that distinguishes these fluoro isomers.
The historical lesson is clear-no single analytical technique should be relied on. Working at nanogram levels, radiopharmaceutical chemists have always had cause to be light sleepers. Hopefully, reading this book will keep them up all night.
Donald M. Wieland, Ph.D. Michael C. Tobes, Ph.D. Thomas J. Mangner, Ph.D.
Acknowledgments
The editors and the Radiophannaceutical Science Council would like to thank the speakers, panel participants, poster presentors, and commercial exhibitors who participated in the symposium. A special thanks to Joanna Fowler of Brookhaven National Laboratories for moderating the panel discussion and to Richard Chamberlain of the Society of Nuclear Medicine Central Office for organizational assistance. The editors are grateful to Linder Markham for secretarial assistance.
Research at the University of Michigan Medical Center in Radiophannaceutical Chemistry and Nuclear Medicine is supported by the Department of Internal Medicine and the following grants from the National Institutes of Health: Contract No. HL-27555, NCI Training Grant, Contract No. 5-T32-CA09015-09 and the U.S. Department of Energy, Contract No. DE-AC02-76EV02031.
Contents
THIN-LAYER CHROMATOGRAPHY
Instrumental Evaluation of Thin-Layer Chromatograms
Colin F. Poole, Hal T. Butler, Myra E. Coddens, and Sheila A. Schuette 3
Introduction 3 Sample Application 4 Mode Selection for Scanning Densitometry 10 Instrumentation for Scanning Densitometry 13 Instrument Parameters that Affect the Performance of Slit-Scanning Densitometers 16 Protocol for Measuring the Sensitivity of a Slit-Scanning Densitometer 18 Qualitative Sample Identification by HPTLC and Scanning Densitometry 23 Quantitation of Separated Components by Scanning Densitometry 28 Reagents Used to Enhance Fluorescence of Organic Compounds on Thin-Layer Plates 32 Radiochromatography on Thin-Layer Plates 33 Conclusions 35 References 35
2 Radioanalytical Techniques: ITLC, TLC, Mini-Columns, and Electrophoresis
Alan P. Carpenter, Jf.
Introduction 39 ITLC 40 Radio-TLC 45 Mini-Columns 56
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Electrophoresis 60 Conclusions 68 Future Considerations 68 References 68
3 Radio-Thin-Layer Chromatogram Imaging Systems-Performance and Design
Sheryl J. Hays
Introduction 71 Instrumentation 72 Resolution and Efficiency 73 Performance and Operation of the TLC Imaging Systems 74 Conclusion 75 References 77
4 Detection of Radiochromatograms and Electropherograms with PositionSensitive Wire Chambers
Heinz Filthuth
Introduction 79 The TLC Linear Analyzer 81 Data Acquisition System 83 Multiplate Detector 84 Performance of the Linear Analyzer 85 Quantitative Measurements 86 Experience with the Linear Analyzer 90 Determination of the Radiochemical Purity of Radiopharmaceuticals 98 Summary 98 References 99
HIGH PRESSURE LIQUID CHROMATOGRAPHY
5 Components for the Design of a Radio-HPLC System
Adrian D. Nunn and Alan R. Fritzberg
Introduction 103 Columns and Packings 104 Components of a Radio-HPLC System 108 Conclusion 122 References 123
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Contents xiii
6 Overall Radio-HPLC Design
Chester A. Mathis, Reese M. Jones, and Joseph H. Chasko
Introduction 125 Components of the Basic System 128 A More Complex Radio-HPLC System 130 Additional Considerations 137 A Specialized HPLC System 140 Summary 147 References 147
7 Quantitation of Radiolabeled Molecules Separated by High Pressure Liquid Chromatography
Michael J. Kessler
Introduction 149 Detection of HPLC-Separated Radioactive Compounds 150 Detector Design 154 Considerations in Use of Detector 158 Applications 163 Conclusion 167 References 167
8 Flow Detector Designs: Build Your Own or Buy?
Richard D. Hichwa
Introduction 171 Requirements 171 Types of Flow Cells 175 Data Acquisition and Analysis Considerations 177 Conclusions 178 References 179
APPLICA liONS
9 Radio-HPLC: Application to Organics and Metal Chelate Chemistry
Alan R. Fritzberg and Adrian D. Nunn
Introduction 183 Stability Under HPLC Conditions 188 Tc-N2S2 Chelates 188
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Use of HPLC in Stereochemical Studies of Tc and Re Penicillamine Complexes 200
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HPLC in the Development and Analysis of HIDAs 201 Phosphines 204 Phosphonates 205 Conclusions 206 References 207
10 Concepts and Techniques Used in Metabolic Tracer Studies
Jorge R. Barrio, Randy E. Keen, Diane C. Chugani, Gerald Bida, Nagichettiar Satyamurthy, and Michael E. Phelps 213
Introduction 213 Requirements of Analytical Techniques for Metabolic Studies 214 Applications in Tracer Kinetic Models 221 Conclusions 227 References 227
11. Development of No-Carrier-Added Radiopharmaceuticals with the Aid of Radio-HPLC
D. Scott Wilbur
Introduction 233 Radio-HPLC 234 Scaling Radiolabeling Reactions to nca Levels 236 Identification of the nca Radiolabeled Compound 237 Examples of nca Radiolabeling Experiments 238 Conclusions 245 References 248
12 From Cyclotron to Patient via HPLC
Michael R. Kilbourn, Michael J. Welch, Carmen S. Dence, and Keith R. Lechner
Introduction 251 HPLC Systems 251 Columns 253 Solvent Systems 255 Dedicated vs General Instruments 256 Detectors 257 Pre-Columns and Filters 258 Is HPLC Truly Necessary? 258 Conclusions 259 References 259
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13 Potential Artifacts in the Chromatography of Radiophannaceuticals
Thomas J. Mangner
Introduction 261 Choice of Analytical Methods 262 Radiochromatography 266 Potential Artifacts/Inconsistencies in Radiochromatography 270 Illustrative Examples 274 Summary 276 References 276
14 HPLC of Radiolabeled Antibodies
261
Donald J. Hnatowich 279
Introduction 279 Theory 280 An HPLC System for the Analysis of Radiolabeled Antibodies 283 Applications 285 Discussion 290 Conclusions 291 References 292
Index 295
Contributors
Jorge R. Barrio, Division of Nuclear Medicine and Biophysics, UCLA School of Medicine, Los Angeles, CA 90024, USA
Gerald Bida, Division of Nuclear Medicine and Biophysics, UCLA School of Medicine, Los Angeles, CA 90024, USA
Hal T. Butler, Department of Chemistry, Wayne State University, Detroit, MI 48202, USA
Alan P. Carpenter, Jr., Division of Analytical Chemistry, Radiopharmaceutical Research, Dupont/New England Nuclear Corporation, North Billerica, MA 01845, USA
Joseph H. Chasko, Donner Laboratory, Lawrence Berkeley Laboratory, University of California, Berkeley, CA 94720, USA
Diane C. Chugani, Division of Nuclear Medicine and Biophysics, UCLA School of Medicine, Los Angeles, CA 90024, USA
Myra E. Coddens, Department of Chemistry, Wayne State University, Detroit, MI 48202, USA
Carmen S. Dence, Division of Radiation Sciences, The Edward Mallinckrodt In-stitute of Radiology, Washington University, St. Louis, MO 63110, USA
Heinz Filthuth, Laboratorium Prof. Dr. Berthold, D-7547 Wildbad 1, West Germany
Alan R. Fritzberg, NeoRx Corporation, Seattle, WA 98119, USA
Sheryl J. Hays, Warner-Lambert Company, Pharmaceutical Research Division, Ann Arbor, MI 48105, USA
xviii Contributors
Richard D. Hichwa, Division of Nuclear Medicine, University of Michigan Medical Center, Ann Arbor, MI 48109, USA
Donald J. Hnatowich, Department of Nuclear Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA
Reese M. Jones, Donner Laboratory, Lawrence Berkeley Laboratory, University of California, Berkeley, CA 94720, USA
Randy E. Keen, Division of Nuclear Medicine and Biophysics, UCLA School of Medicine, Los Angeles, CA 90024, USA
Michael J. Kessler, Radiomatic Instruments & Chemical Co., Tampa, FL 33611, USA
Michael R. Kilbourn, Division of Radiation Sciences, The Edward Mallinckrodt Institute of Radiology, Washington University, St. Louis, MO 63110, USA
Keith R. Lechner, Division of Radiation Sciences, The Edward Mallinckrodt Institute of Radiology, Washington University, St. Louis, MO 63110, USA
Chester A. Mathis, Donner Laboratory, Lawrence Berkeley Laboratory, University of California, Berkeley, CA 94720, USA
Thomas J. Mangner, Division of Nuclear Medicine, University of Michigan Medical Center, Ann Arbor, MI 48109, USA
Adrian D. Nunn, Division of Radiopharmaceutical Research and Development, The Squibb Institute for Medical Research, New Brunswick, NJ 08903, USA
Michael E. Phelps, Division of Nuclear Medicine and Biophysics, UCLA School of Medicine, Los Angeles, CA 90024, USA
Colin F. Poole, Department of Chemistry, Wayne State University, Detroit, MI 48202, USA
Nagichettiar Satyamurthy, Division of Nuclear Medicine and Biophysics, UCLA School of Medicine, Los Angeles, CA 90024, USA
Sheila A. Schuette, Department of Chemistry, Wayne State University, Detroit, MI 48202, USA
Michael J. Welch, Division of Radiation Sciences, The Edward Mallinckrodt Institute of Radiology, Washington University, St. Louis, MO 63110, USA
D. Scott Wilbur, NeoRx Corporation, Seattle, WA 98119, USA