- 041, STUDIES IN ANALYTICAL CHEMISTRY 5 QUANTITATIVE ANALYSIS OF STEROIDS S. G5ROG Head of Department, Chemical Works G. Richter Ltd., Honorary Professor of Pharmacy, Semmelweis University Medical School, Budapest, Hungary UNIVERSITATST BIBL1OTHEK ELSEVIER SCIENTIFIC PUBLISHING COMPANY Amsterdam Oxford New York 1983
CONTENTS Preface 11 1. INTRODUCTION 13 1.1 Problems and tendencies in contemporary steroid analysis 13 1.2 Literature sources on analysis of steroids 15 1.3 Scope and limitations 17 References 18 2. SEX HORMONES AND RELATED MATERIALS 19 2.1 Fundamentals of the chemistry of sex hormones 19 2.2 Methods of analysis of sex hormones 2*4 2.2.1 Ultraviolet spectrophotometric methods based on natural absorption 24 a,f)-unsaturated ketones 24 3,5-Dienes 28 Phenolic ring A 29 2.2.2 Ultraviolet spectrophotometric and colorimetric methods based on chemical reactions 32 Determination of keto groups 32 Phenolic ring A 40 Miscellaneous 44 2.2.3 Fluorimetry 45 Methods based on native fluorescence 45 Methods based on acid-induced fluorescence 47 Miscellaneous fluorimetric methods 51 2.2.4 Infrared spectrophotometry 53 2.2.5 NMR spectrometry 55 2.2.6 Polarography 56 2.2.7 Titrimetric methods 60 2.2.8 Gravimetric methods 64 2.2.9 Column chromatography 64 2.2.10 Thin-layer chromatography 68 Separation and detection problems 68 Quantitative analysis using spot elution 71 In situ methods of quantitation 72 2.2.11 Gas chromatography 75 Packed columns 75 Capillary columns 78
Detectors 82 Problems of derivatization 84 Choice of derivates 86 Preparation of samples for quantitative analysis 94 Practical applications 95 2.2.12 High-performance liquid chromatography 99 The chromatographic system 100 Detectors 108 Derivatization methods 110 Practical applications 115 2.2.13 Radioimmunoassay and other radioisotope methods 116 Radioactive tracer methods 116 Double isotope derivative method 117 Competitive protein binding method 118 Radioimmunoassay 119 2.2.14 Enzymatic methods 128 Colorimetric and fiuorimetric methods 128 Radioenzymatic methods 130 Enzyme immunoassay 130 2.2.15 Mass spectrometry and related techniques 134 General aspects 134 Mass fragmentography 139 2.2.16 Miscellaneous methods 142 2.3 Problems in the analysis of sex hormones 143 2.3.1 Purity control of bulk hormones 143 2.3.2 Determination of sex hormones in pharmaceutical preparations 144 2.3.3 Determination of sex hormones and their metabolites in biological samples 148 General aspects 148 Extraction procedures 149 Chromatographic separation and purification of extracts 150.Cleavage of conjugates 151 Spectroscopic methods of quantitation. 152 Chromatographic methods of quantitation 153 Radioanalytical methods of quantitation 154 Some schemes of complex analytical procedures 155 2.3.4. Determination of sex hormones and anabolic agents in foods 159 References 161 3. CORTICOSTEROIDS 184 3.1 Fundamentals of the chemistry of corticosteroids 184 3.2 Methods of analysis of corticosteroids 187 3.2.1 Ultraviolet spectrophotometric and colorimetric methods based on chemical reactions 187 The tetrazolium method 187 The Porter-Silber method 189 Other methods 191 3.2.2 Fluorimetry 195 3.2.3 Polarography 197 3.2.4 Column chromatography 199 3.2.5 Paper and thin-layer chromatography 201 Paper chromatography 201
TLC separation and detection problems 202 Quantitative analysis 202 3.2.6 Gas chromatography 205 The thermal instability of the 17-side-chain 205 Derivatization methods 206 3.2.7 High-performance liquid chromatography 211 3.2.8 Radioimmunoassay and other radioisotope methods 221 Competitive protein binding method 222 Radioimmunoassay 223 312.9 Enzymatic methods 227 Photometric and fluorimetric methods 227 Enzyme immunoassay 227 3.2.10 Mass spectrometry and related techniques 228 3.2.11 Miscellaneous methods 230 3.3 Problems in the analysis of corticosteroids 231 3.3.1 Purity control of bulk corticosteroids 231 3.3.2 Determination of corticosteroids in pharmaceutical preparations 232 3.3.3 Determination of corticosteroids and their metabolites in biological samples 234 Preparation of samples for quantitative analysis 234 Quantitative determination 235 3.3.4 Determination of corticosteroids in foods 237 References 237 4. CHOLESTEROL AND RELATED STEROLS 247 4.1 Fundamentals of the chemistry of sterols 247 4.2 Methods for analysis of sterols 249 4.2.1 Spectropnoiometric and colorimetric methods 249 4.2.2 Fluorimetry 252 4.2.3 Digitonide precipitation methods 253 4.2.4 Electroanalytical methods 255 4.2.5 Column chromatography 256 4.2.6 Thin-layer chromatography 257 Separation and detection problems 257 Quantitative analysis 259 4.2.7 Gas chromatography, 261 Derivatization 261 Separation problems 264 Quantitative analysis 265 4.2.8 High-performance liquid chromatography 267 4.2.9 Enzymatic methods 268 General aspects 268 Colorimetric methods 270 Fluorimetric methods, 272 Electroanalytical methods 272 4.2.10 Mass spectrometry and related techniques 275 4.2.11 Miscellaneous methods. 276 4.3 Problems in the analysis of sterols 277 4.3.1 Determination of cholesterol in blood serum and other biological samples 277 4.3.2 Determination of cholesterol in the raw materials of its production (wool. wax, spinal cord) 280
4.3.3 Determination of sterols in plants 281 4.3.4 Determination of sterols in foods 281 References 282 5. VITAMINS D AND RELATED MATERIALS 290 5.1 Fundamentals of the chemistry of vitamins D 290 5.2 Methods of vitamin D analysis 292 5.2.1 Ultraviolet spectrophotometric methods " 292 5.2.2 Colorimetric methods 294 5.2.3 Infrared spectrophotometry 296 5.2.4 NMR spectrometry 296 5.2.5 Polarography 297 5.2.6 Column chromatography 298 5.2.7 Thin-layer chromatography 300 5.2.8 Gas chromatography 305 5.2.9 High-performance liquid chromatography 311 5.2.10 Competitive protein binding method and other radioisotope techniques 320 5.2.11 Mass spectrometry and related techniques 324 5.2.12 Miscellaneous methods 325 5.3 Problems in the analysis of vitamins D 325 5.3.1 Purity control of crystalline vitamins D 325 5.3.2 Analysis of vitamin D resins and other irradiation mixtures 326 5.3.3 Determination of vitamins D in pharmaceutical preparations 327 5.3.4 Determination of vitamins D in foods 330 5.3.5 Determination of vitamins D and their metabolites in blood 331 References 322 6. BILE ACIDS 339 6.1 Fundamentals of the chemistry of bile acids 339 6.2 Methods of analysis of bile acids 341 6.2.1 Colorimetric methods 341 6.2.2 Fluorimetry 342 6.2.3 Column chromatography 343 6.2.4 Thin-layer chromatography " 344 Separation of bile acids and conjugates 345 TLC fractionation of bile acids 346 Quantitative analysis using spot elution 346 In situ methods of quantitation 347 6.2.5 Gas chromatography 347 6.2.6 High-performance liquid chromatography 354 6.2.7 Enzymatic methods 356 6.2.8 Mass spectrometry and related techniques 359 6.2.9 Radioimmunoassay 361 6.2.10 Miscellaneous methods 362 6.3 Problems in the analysis of bile acids 363 6.3.1 Determination of bile acids and conjugates in biological samples 363 6.3.2 Analysis of bulk bile acids and pharmaceutical preparations made therefrom 365 References 366
7. CARDIAC GLYCOSIDES 372 7.1 Fundamentals of the chemistry of cardiac glycosides 372 7.2 Methods of analysis of cardiac glycosides 374 7.2.1 Ultraviolet spectrophotometry 374 7.2.2 Colorimetric method" 375 7.2.3 Fluorimetry 380 7.2.4 Column chromatography 382 7.2.5 Thin-layer chromatography 382 Separation and detection problems 384 Quantitative analysis using spot elution 384 In situ methods of quantitation 386 7.2.6 Gas chromatography 388 7.2.7 High-performance liquid chromatography 393 7.2.8 Radioimmunoassay and enzyme immunoassay 393 Radioimmunoassay 396 Enzyme immunoassay 396 7.2.9 Miscellaneous methods 397 7.3 Problems in the analysis of cardiac glycosides 397 7.3.1 Purity control in isolated cardiac glycosides 399 7.3.2 Determination of cardiac glycosides in pharmaceutical preparations 400 7.3.3 Determination of cardiac glycosides in plants 402 7.3.4 Determination of cardiac glycosides and their metabolites in body fluids 403 References 8. DIOSGENIN AND RELATED SAPOGENINS 408 8.1 Fundamentals of the chemistry of sapogenins 408 8.2 Methods of analysis of sapogenins 409 8.2.1 Colorimetric methods 409 8.2.2 Infrared spectrophotometry 412 8.2.3 Thin-layer chromatography 412 8.2.4 Column and high-performance column chromatography 414 8.2.5 Gas chromatography 415 8.2.6 Miscellaneous methods 417 8.3 Problems in the analysis of sapogenins 418 8.3.1 Purity control of bulk sapogenins 418 8.3.2 Determination of sapogenins in plants 418 References 420 9. MISCELLANEOUS STEROIDS 422 9.1 Ecdysones 422 9.2 Solasodine and related alkaloids 426 9.3 Pancuronium bromide and related quaternary ammonium compounds 429 References 431 Subject index 435