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HPLC in Pharmacy

and Biochemistry

Edited by
Assistent Professor Dr. Magda Sarsiinova, D. Sc. Dr.-Ing. Oldfich Hanc,
D. Sc. Dr. nat. Sci. Bohumil Kakac, C. Sc.

With contributions by
Oldfich Hanc, Jan Hrivnak, Bohumil Kakac, Vladimir Miller, Zdenek Pefina,
Jan Rernen, Magda Sarsiinova, Juraj Tolgyessy

Huthig Buch Verlag Heidelberg


Contents

Preface v
The Development of Chromatographic Methods 1

General Part

1. Principles of Chromatographic Methods (M. Sarsiinova) 5


1.1 Comparison of HPLC with Other Types of Chromatography 7
1.1.1 HPLC and Liquid Column Chromatography (LC) 8
1.1.2 HPLC and GC 8
1.1.3 HPLC and Thin Layer Chromatography (TLC) 8

Theoretical Part

2. Principles of HPLC (M. Sarsiinova) 11


2.1. Physical Relations of Chromatography (Z. Pefina) 13
2.1.1 Elementary Idea 13
2.1.2 Simulation of the Chromatographic Phenomena 14
2.1.3 The Concept of the Theoretical Plate 15
2.2 Separation Mechanisms and Physico-Chemical Forces
(M. Sarsunova) 17

Practical Part

3. Working Techniques (V. Miller) 25


3.1 The Liquid Chromatograph 25
3.2 Chromatographic Columns: Their Preparation and Filling 27
3.2.1 Selection of the Stationary Phase 27
3.2.2 Chromatographic Columns and Packings 28
3.2.2.1 Columns for Adsorption and Partition LC 29
3.2.2.2 Columns for Chromatography with a Polar Chemically Bonded
Phase 29
3.2.2.3 Columns for Reverse Phase Chromatography 31
viii Contents

3.2.2.4 Columns for Ion-Exchange Chromatography 34


3.2.2.5 Columns for Gel Chromatography 34
3.2.3 Efficiency of Chromatographic Columns 37
3.2.4 Column Packing Procedure 38
3.3 Selection of the Mobile Phase (J. Hrivnak) 39
3.3.1 Transport of the Mobile Phase 43
3.3.2 Gradient Elution 43
3.4 Injection of Samples (J. Remeii, J. Hrivnak) 45
3.5 Detectors (B. Kakac, V. Miller) 47
3.5.1 Spectrophotometric Detectors 48
3.5.2 Fluorescence Detectors 49
3.5.3 Refractometric, Electrochemical and MS Detectors 50
3.6 Qualitative Analysis (V. Miller) 52
3.7 Quantitative Analysis 53
3.8 Preparative Liquid Chromatography 56
3.9 High Performance Liquid Radiochromatography (HPLRC)
(J. Tolgyessy) 59
3.9.1 Measurement of Radioactivity 59
3.9.2 Working Technique and Equipment 62
3.9.2.1 Discontinuous Analysis 63
3.9.2.2 Continuous Analysis 64
3.9.3 Application of HPLRC 70

Applications Part

4. Applications of HPLC in Pharmaceutical and Toxicological


Analysis (M. Sarsunova) 73
4.1 Drugs Acting upon the Central Nervous System (CNS) 73
4.1.1 Analeptics 73
4.1.2 Antipyretics with Analgesic Activity 78
4.1.2.1 Antirheumatics and Antiphlogistics 85
4.1.3 Antiepileptics 87
4.1.4 Hypnotics and Sedatives 90
4.1.5 Psychotropic Drugs (B. Kakac) 94
4.2 Chemotherapeutics that Act upon the Peripheral and Autonomic
Nervous Systems (M. Sarsunova) 108
4.2.1 Local Anesthetics 108
4.2.2 Myorelaxants 109
4.2.3 Adrenergic Drugs 110
4.2.4 Cholinergics, Cholinolytics, and Spasmolytics Ill
Contents

4.2.5 Antagonists of Histamine H 2 Receptors 112


4.3 Drugs that Affect the Circulatory, Digestive, and Excretory
Systems 113
4.3.1 Diuretics 113
4.3.2 Oral Antidiabetics 115
4.3.3 Anticoagulants 117
4.3.4 Vasodilators and Antihypertensive Drugs 119
4.3.5 Antiarrhythmics 122
4.3.6 Antisclerotic and Antilipid Drugs 125
4.3.7 P-Adrenolytics 125
4.4 Antiinvasive Drugs 127
4.4.1 Disinfectants and Antiseptic Drugs 127
4.4.2 Sulfonamides 129
4.4.3 Antibiotics (B. Kakac) 132
4.4.4 Antibacterial, Antiparasitic, and Antiviral Drugs (M. Sarsunova) 150
4.4.5 Antituberculotics 153
4.4.6 Dermatologic Preparations 154
4.4.7 Antineoplastics 155
4.4.8 Other Drugs 162
4.4.8.1 Anthelmintics 162
4.4.8.2 Disulfiram 162
4.4.8.3 Antiemetics 163
4.4.8.4 Antifibrinolytics 163
4.4.8.5 Contraceptives 163
4.4.8.6 Antiparkinsonics 163
4.5 Natural Biocatalysts (B. Kakac) 164
4.5.1 Vitamins 164
4.5.1.1 Water Soluble Vitamins 164
4.5.1.2 Fat Soluble Vitamins 169
4.5.2 Thyrostatics (M. Sarsunova) 179
4.6 Drugs of Natural Origin 179
4.6.1 Alkaloids 179
4.6.1.1 Ergot Alkaloids (V. Miller) 179
4.6.1.2 Quinine Alkaloids (M. Sarsunova) 184
4.6.1.3 Pilocarpine and Related Compounds 185
4.6.1.4 Opium Alkaloids 186
4.6.1.5 Strychnine Alkaloids 189
4.6.1.6 Tropane Alkaloids 190
4.6.1.7 Ipecacuane Alkaloids 192
4.6.1.8 Other Alkaloids and Mixtures 193
4.6.2 Cardiac Glycosides (B. Kakac) 198
Contents

4.6.2.1 Digitalis Glycosides 198


4.6.2.2 Other Cardiac Glycosides 203
4.6.3 Prostaglandins 205
4.7 Auxiliary and Diagnostic Compounds (M. Sarsiinova) 210
4.7.1 Dyes 210
4.7.2 Auxiliary Compounds Used in the Pharmaceutical Industry 212
4.7.3 Antioxidants, Preservatives, and Antimicrobial Additives 213
4.7.4 X-Ray Diagnostics 217
5. Other Applications of HPLC in Drug Research (Z. Pefina) 218
5.1 Applications of Chromatographic Data to Structure-Activity
Relations 218
6. Applications of HPLC in Biochemistry and Clinical Analysis
(O. Hanc) 223
6.1 Biogenic Amines 227
6.1.1 Catecholamines 229
6.1.2 Serotonine, Precursors and Metabolites 235
6.2 Amino Acids 237
6.2.1 Thyronine Derivatives 241
6.3 Peptides and Proteins 243
6.3.1 Peptide Hormones 248
6.3.2 Enzymes and Enzymology 255
6.4 Saccharides and Their Derivatives 258
6.5 Nucleic Acids and Their Components 262
6.6 Lipids and Their Components 266
6.7 Steroids 269
6.7.1 Sterols 271
6.7.2 Bile Acids 273
6.7.3 Estrogens 276
6.7.4 Androgens 282
6.7.5 Gestagens 285
6.7.6 Corticoids 288
6.8 Natural Coloring Matters and Their Components 296
6.9 Plant Components 299
6.10 Components and Products of Microorganisms 309

Abbreviations 313

Subject Index 315