Kevin D. Altria Analysis of Pharmaceuticals by Capillary Electrophoresis
CH ROMAIOGRAPH IA Edited by Kevin D. Altria, Glaxo Wellcome R&D, UK CE Series There are currently a number of general textbooks covering Capillary Electrophoresis where information on a range of applications and techniques can be found. Readers who are interested in a specific area of CE struggle to find truly comprehensive treatments of their areas of interest. The CHROMATOGRAPHIA CE series has been established to allow comprehensive books to be produced covering individual topics. The books are written by well known authors in their specialist application areas and cover CE topics such as DNA analysis, analysis of pharmaceuticals, chiral separations, MECC, carbohydrate analysis, biomedical applications and troubleshooting ince. Volume 1: C. Heller (Ed.), Analysis of Nucleic Acids by Capillary Electrophoresis Volume 2: K. D. Altria, Analysis of Pharmaceuticals by Capillary Electrophoresis
Kevin D. Altria Analysis of Pharmaceuticals by Capillary Electrophoresis II vleweg
All rights reserved Friedr. Vieweg & Sohn Vedagsgesellschaft mbh, BraunschweiglWiesbaden, 1998 Softcover reprint of the hardcover 1st edition 1998 Vieweg is a subsidiary company of Bertelsmann Professional Information. No part of this publication may be produced, stored in a retrieval system or transmitted, mechanical, photocopying or otherwise, without prior permission of the copyright holder. http://www.vieweg.de Produced by Lengericher Handelsdruckerei, Lengerich ISBN-13: 978-3-322-85013-3 DOl: 10.1007/978-3-322-85011-9 e-isbn-13: 978-3-322-85011-9
v Preface During the 1980's the analysis of pharmaceuticals was dominated by the use of High Performance Liquid Chromatography (HPLC). Other separative techniques such as Gas Chromatography and Thin Layer Chromatography offered alternatives but their quantitative capabilities and/or solute range could not approach that of HPLC. The majority of pharmaceuticals are ionic and it would be reasonable to assume that electrophoresis may be useful in the analysis of pharmaceuticals. However, the electrophoretic instruments available in the 1980's were labour intensive and employed post-separation detection procedures. During the late 1980's and early 1990's extensive research was conducted into the possibilities of conducting electrophoretic separations in capillaries. This approach allowed on-line detection and could be performed on fully automated equipment. This research led to the advent of modern day capillary electrophoresis (CE) instruments which offer similar performance and automation levels to that of HPLC. Research was also focused on developing applications for CE and particular attention was paid to applications within the pharmaceutical analysis area. These applications proved that CE could be applied to a wide range of drug types including water insoluble and neutral compounds. The ability to achieve efficient chiral separations of drugs also increased the popularity of the technique. CE with indirect UV detection has become established as a simple and effective alternative to ion-exchange chromatography for the determination of small inorganic or organic ions. Routine CE methods are now used in many industrial pharmaceutical companies and applications include determination of related impurities, main component assay, chiral separations and drug residue determinations. Research into CE continues to broaden the application range in drug analysis. In particular current research is focused in the area of nonaqueous solvent systems and the developing technique of capillary electrochromatography (CEC). In CEC the capillaries used in CE are packed with HPLC stationary phase material and a high voltage is used to achieve separations by combining electrokinetic and chromatographic processes. All of these application and development areas are covered in individual chapters within this book. CE is still a rapidly developing technique and new applications and developments appear on a weekly basis. This book broadly reflects the current state of the art for CE analysis of pharmaceuticals and contains several hundred references to specific applications and methods. The main purpose of the book is to present the application possibilities of CE but I hope the extensive use of tabulated application data should also make the book useful as a reference point for specific applications. London 1998 Kevin D Altria
VI Preface Acknowledgement I would like to extend my appreciation to Dr. Angelika Schulz from Vieweg Publishing who has kindly supported the creation of the Chromatographia CE Series throughout its continued development. My thanks are also extended once again to my family and friends who have helped me to retain? a little sanity during the preparation of this volume. In particular my gratitude is extended to my wife Fatima and my colleagues Dave Rudd and Simon Bryant. Contact details Adress: Phone: Fax: E-mail: Website: Kevin D Altria, Pharmaceutical Development, GlaxoWellcome R&D, Park Road, Ware, Herts. SGl2 ODP, UK ++44-1920-883616 ++44-1920-883873 KDA8029@ggr.co.uk http://dspace.dial.pipex.com/townlterracelho81ikda.htm
VII Contents Preface... V Contents... VII 1 Introduction to CE and the Use of CE in Pharmaceutical Analysis........ 1 1.1 Capillary Electrophoresis (CE) Theory and Background... 1 1.2 CE Instrumentation... 3 1.2.1 Capillaries... 3 1.2.2 Temperature control... 3 1.2.3 Sampl~ introduction... 3 1.2.4 Detectors... 3 1.3 Band Broadening Effects in CE... 4 1.3.1 Heat dissipation... 4 1.3.2 Electroendosmotic flow... 4 1.3.3 On-capillary detection... 5 1.3.4 Molecular diffusion... 6 1.3.5 Injection related broadening... 6 1.4 Separation Modes Available... 6 1.4.1 Free Solution Capillary Electrophoresis (FSCE)... 6 1.4.2 Micellar electrokinetic capillary chromatography (MECC)... 9 1.4.3 Capillary Gel Electrophoresis (CGE)... 11 1.4.4 Capillary Isoelectric Focusing (ClEF)... 13 1.4.5 Capillary electrochromatography (CEC)... 13 1.5 Application of CE to Specific Drug Classes... 14 1.6 The Role of CE in Pharmaceutical Analysis... 16 References... 18 2 Main Component Assay by CE... 21 2.1 Introduction... 21 2.2 Reported Applications... 21 2.2.1 Low ph... 23 2.2.2 High ph... 27 2.2.3 MECC... 30 2.4 Identity Confirmation Testing... 33 2.5 General Considerations in Quantitative Analysis... 33 2.5.1 Precision... 33 2.5.2 Accuracy... 38 2.5.3 Linearity... 39 2.6 Comparison of CE and HPLC for Drug Assay... 39 References... 40 3 Determination of Drug Related Impurities... 44 3.1 Introduction... 44 3.2 Separations Using Low ph Electrolytes... 49 3.3 High ph... 52
VIII Contents 3.4 MECC... 54 3.5 Comparison with HPLC... 55 3.5.1 Sensitivity... 56 3.6 Peak: identity confirmation... 57 3.7 Applications... 59 3.7.1 Chemical purity testing of drug substance... 61 3.7.2 Chemical purity testing of formulated product...... 62 3.7.3 Stability testing... 65 3.7.4 Impurity profiling... 66 References... 66 4 Separation and Quantitation of Enantiomers... 70 4.1 Introduction... 70 4.2 Cyclodextrins... 70 4.2.1 Low ph electrolytes containing cyclodextrins... 77 4.2.2 Cyclodextrins at high ph... 77 4.2.3 Cyclodextrins in Non-aqueous CE... 79 4.3 Crown ethers... 79 4.3.1 Crown ethers in Non-aqueous CE... 79 4.4 SDS-CD MECC... 80 4.5 Carbohydrates... 80 4.6 Proteins... 80 4.7 Bile salt MECC... 81 4.8 Antibiotics... 81 4.9 Synthetic surfactants... 82 4.10 Method development... 83 4.10.1 Selector type... 83 4.10.2 Temperature... 85 4.10.3 Electrolyte selection... 85 4.10.4 MECC electrolyte optimisation... 86 4.11 Quantitative Applications... 86 4.11.1 Enantiomeric purity testing... 87 4.11.2 Reaction rate monitoring... 88 4.11.3 Formulation stability testing... 88 4.11.4 Clinical applications... 89 4.12 Method Validation... 89 4.12.1 Detection limits... 89 4.12.2 Precision... 90 4.12.3 Linearity... 90 4.12.4 Recovery... 91 4.12.5 Cross-validation... 91 4.12.6 Freedom from interference... 91 4.12.7 Selectivity... 92 4.12.8 Robustness evaluation... 92 4.12.9 Method transfer... 92 4.13 Conclusions... 93 Stop press update... 93 References... 94
IX 5 Determinations of Drug Counter-Ions and Ionic Impurities by CE............ I0l 5.1 Introduction... 101 5.2 Separation of inorganic anions... 103 5.2.1 Indirect detection of anions... 104 5.2.2 Direct detection of anions... 105 5.3 Metal Ion Determinations... 106 5.3.1 Determination of metal ion content by indirect UV detection... 106 5.3.2 Direct UV detection of metal ion complexes... 108 5.4 Quantitative Procedures... 109 5.5 Determination ofinorganic Anion Counter-Ion Levels in Basic Drugs... 109 5.6 Determination of Organic Acid Anion Counter-Ion Levels in Basic Drugs... 109 5.7 Quantitation of Metal Counter-Ion Levels in Acidic Drugs... 113 5.8 Determination of Ionic Contaminants in Drug Substance... 116 5.9 Benefits and Disadvantages of CE Methods in Stoichiometric Analysis... 118 5.9.1 Disadvantages... 118 5.9.2 Advantages... 118 References... 119 6 Trace Analysis and Residues Determination................. 121 6.1 Introduction... 121 6.2 Drug Residue Analysis... 121 6.2.1 Basic drugs residues... 123 6.2.2 Acidic drug residues... 126 6.3 Detergent Solution Residue Analysis... 127 6.3.1 Surfactant residues... 127 6.3.2 Metal ion residues from detergent solutions... 128 6.3.3 EDTA residues... 129 6.4 Drug Doping Levels... 130 6.5 Environmental Analysis... 130 6.6 Advantages and Disadvantages of the Use of CE in Residues Analysis... 130 6.6.1 Disadvantages... 130 6.6.2 Advantages... 130 References... 131 7 Pharmaceutical Raw Materials and Excipients Analysis... 133 7.1 Introduction... 133 7.2 Alcohols... 135 7.3 Carbohydrates... 136 7.4 Cyc10dextrins... 137 7.5 Dyes... 137 7.6 Fatty Acids... 137 7.7 Flavouring Agents... 138 7.8 Inorganic Anions... 139 7.9 Lecithins... 140 7.10 Metal Ions... 140 7.11 Organic Acids... 141 7.12 Polycarboxylic Acids... 142 7.13 Preservatives... 142
x Contents 7.14 Starting Materials... 144 7.15 Surfactants... 145 7.16 Water Purity... 148 References... 149 8 Analysis of dissolution test sample solutions... 153 8.1 Introduction... 153 8.2 Total Drug Content Release Testing... 154 8.3 Dissolution Profile Monitoring... 156 8.4 Monitoring of the Dissolution of Chiral Drugs... 157 8.5 Multi-Component Analysis... 159 8.6 Benefits and Disadvantages of Adopting CE for Dissolution Analysis... 159 References... 160 9 Determination of Vitamins by Capillary Electrophoresis... 162 9.1 Introduction... 162 9.2 Assay... 165 9.3 Related Impurities Determinations... 169 9.4 Clinical Determinations... 169 9.5 Identity Confirmation... 169 9.6 Microemulsion Electrokinetic Capillary Chromatography (MEEKC)... 170 9.7 Conclusions... 171 References... 171 10 Overview of Application of CE to determine drugs in biofluids... 173 10.1 Introduction... 173 10.2 Sample Pretreatment Procedures... 173 10.3 Direct Sample Injection... 177 10.4 Sample Matrix Effects... 179 10.5 Sensitivity Enhancement... 179 10.6 Quantitative Precision... 181 10.7 Applications... 181 10.8 Chiral Clinical Applications... 185 10.9 Non-Aqueous CE and CEC Clinical Applications... 186 References... 188 11 Method Validation................... 193 11.1 Introduction... 193 11.2 Specific method validation aspects... 193 11.2.1 Specificity (selectivity)... 193 11.2.2 Linearity... 195 11.2.3 Sensitivity... 196 11.2.4 Accuracy/recovery... 196 11.2.5 Injection Repeatability... 197 11.2.6 Method Repeatability... 199 11.2.7 Method Robustness... 200 11.2.8 Cross-Validation... 200 11.2.9 Solution Stability... 200 11.3 Response factors... 201 11.4 Peak Homogeneity... 202
XI 11.5 Method Transfer... 203 11.6 System Suitability... 203 11.7 Conclusions... 203 References... 204 12 Capillary Electrochromatography... 206 12.1 Introduction... 206 12.2 CEC Instrumentation... 206 12.3 CEC Operation... 208 12.4 Detection Options in CEC... 210 12.5 Analytical Performance of CEC.... 211 12.6 Applications... 213 12.6.1 Pharmaceutical applications... 213 12.7 Chiral CEC... 215 12.8 Benefits and Disadvantages of CEC Compared to CE and HPLC... 216 12.8.1 Advantages ofcec... 216 12.8.2 Current disadvantages of CEC... 218 12.9 Conclusions... 219 Stop press update... 219 References... 220 13 Use of non-aqueous electrolytes in pharmaceutical analysis... 223 13.1 Introduction... 223 13.2 Basic Drugs... 224 13.3 Acidic Drugs... 227 13.4 ImpuritylMetabolites Determinations... 227 13.5 Chiral Separations... 227 13.6 Excipients and Raw Materials... 230 13.7 Main Component Assay... 230 13.8 Inorganic Ions and Small Organic Ions... 232 13.9 Practicalities of Routine NACE Operation... 233 13.10 Comparison of the Advantages ofnace and Aqueous CE... 234 References... 234 14 The Use of Chemometrics and Experimental Designs in CE Method Development and Robustness Testing... 236 14.1 Introduction... 236 14.1.1 Full and Fractional Factorial designs... 238 14.1.2 Central Composite and Overlapping Resolution Mapping Designs... 238 14.1.3 Simplex optimisation... 238 14.2 MECC Method Development... 239 14.3 FSCE Method Development... 240 14.4 Chiral CE Method Development...... 242 14.5 CEC Method Development...... 243 14.6 Robustness Testing... 244 14.7 Peak Identification... 247 14.8 Selection of Experimental Design... 248 References... 248
XII Contents 15 Forensic Applications of CE...................... 251 15.1 Introduction... 251 15.2 Identity Confirmation... 251 15.3 Assay... 251 15.4 Clinical Applications... 254 15.5 Purity Determination... 256 15.6 Inorganic Ions... 258 15.7 Non-Aqueous CE... 259 15.8 Chiral... 260 References... 261 16 Determination of Radioactive Compounds by CE................. 263 16.1 Introduction... 263 16.2 Radioactivity Detectors... 264 16.3 Radiopharmaceutical Purity and Assay Determinations... 266 References... 267 17 Miscellaneous Pharmaceutical Analysis Related Areas of CE... 269 17.1 Drug Diet Determinations... 269 17.2 Regulatory Aspects... 269 17.3 Biopharmaceuticals... 271 17.3.1 Proteins... 271 17.3.2 Peptides... 273 17.3.3 Oligonucleotides... 274 17.4 Combinatorial Libraries... 274 17.5 Physicochemical Property Determinations Using CE... 275 17.5.1 Binding constants... 275 17.5.2 Dissociation constants... 276 17.5.3 Partition coefficients... 276 17.5.4 Isoelectric point determinations... 277 References... 277 Subject Index... 280