This volume of the Handbook of Experimental Pharmacology (Concepts in Biochemical Pharmacology) will show that pharma­ cology has finally arrived as a true discipline in its own right, and is no longer the handmaiden of organic chemistry and physiology. Instead it is an amalgam of all the biological sciences including biochemistry, biophysical chemistry, physiology, pathology and clinical medicine. In the volumes that make up Concepts in Bio­ chemical Pharmacology we hope to convince Medical Schools what should now be obvious, that pharmacology is no longer that dull topic bridging the basic sciences with medicine, but is probably the most important subject in the medical curriculum. We are grateful for the advice of Dr. Byron Clark, Director of the Pharmacology-Toxicology Program at the National Institutes of Health whose support made possible much of the work described in this volume. Contents Section Four: Methods 01 Stooging the MetoholiBm 01 Drugs Subsection A. Assay 01 Drugs and Their M etoholites Chapter 22 : Basic Principles in Development of Methods for Drug Assay. B. B. BRODIE. With 2 Figures 1 1 A. Introduction . . . . . . . . . . B. Principles of Developing a Method. . . . . . 1 I. Section of Method of Assay . . . . . . 1 II. Choice of Solvent for Extraction of Drug 2 III. Adsorption of Drugs by Glass Surfaces . . . . . . . . . . . . . . 3 IV. Recoveries of Known Amounts of Compound from Biological Material. 4 V. Assessment of Sensitivity 5 VI. Assessment of Specificity 5 References. . . . . . . . . . .

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Section Four: Methods of Studying the Metabolism of Drugs.- Subsection A. Assay of Drugs and Their Metabolites.- 22: Basic Principles in Development of Methods for Drug Assay. With 2 Figures.- A. Introduction.- B. Principles of Developing a Method.- I. Section of Method of Assay.- II. Choice of Solvent for Extraction of Drug.- III. Adsorption of Drugs by Glass Surfaces.- IV. Recoveries of Known Amounts of Compound from Biological Material.- V. Assessment of Sensitivity.- VI. Assessment of Specificity.- References.- 23: Absorption Spectrophotometry.- A. Ultraviolet Absorption Spectrophotometry.- I. Chromophores.- 1. Chromophore Coupling with Electron Fusing.- 2. Chromophore Coupling by a Saturated Link.- 3. Chromophore Coupling by Conjugated Systems.- 4. Effect of Tautomerism.- II. Environmental Influences.- 1. Effects of Solvent.- 2. Effects of Ionic Strength.- III. Applications of Ultraviolet Spectrophotometry to Drug Assay.- 1. Assays Based on a Change in pH.- 2. Assays Based on a Change of Redox State.- 3. Assays Based on Production of UV Absorption by Chemical Reaction.- 4. Blank Correction by Differential Wavelength Readings.- 5. Estimation of Drug and Metabolite at Differential Wavelengths.- B. Visible Absorption Spectrophotometry.- I. General.- II. Application of Visible Spectrophotometry to Drug Assay.- 1. Assays Based on Changes in pH.- 2. Assays Based on Changes of Redox State.- 3. Production of Visible Absorption by Chemical Reaction.- 4. Other Factors Affecting Visible Absorption.- C. Infrared Absorption Spectrophotometry.- References.- 24: Fluorometry. With 1 Figure.- A. Introduction.- B. Principles.- C. Practical Considerations.- I. Concentration.- II. Background Fluorescence.- III. Surface Adsorption of Fluorophors.- IV. Scattered Light.- D. Luminescence Analysis.- I. Types of Fluoroscence Assay Procedures.- II. Phosphorescence Analysis.- III. Application of Fluorescence Analysis to Drug Metabolism Studies.- IV. The Application of Fluorescence Spectroscopy to Drug Binding Studies.- References.- 25: Radioactive Techniques: The Use of Labeled Drugs. With 2 Figures.- A. Introduction.- B. Synthesis of Labeled Compounds.- I. Chemical Synthesis Procedures.- 1. Procedures for 3H.- 2. Procedures for 14C.- II. Biosynthesis Procedures.- 1. In Vivo Procedures.- 2. In Vitro Procedures.- C. Stability of Labeled Compounds.- D. Measurement of Labeled Drugs.- E. Specific Procedures for the Analysis of Labeled Compounds.- F. Use of Labeled Compounds to Study Drug Metabolism.- 6. Discussion of Basic Principles.- References.- 26: Radioactive Techniques: Radioactive Isotope Derivatives of Nonlabeled Drugs.- I. Estimation of Primary or Secondary Amines.- II. Estimation of Tertiary Amines.- III. Estimation of Drugs by Chelation.- IV. Estimation of Steroids as Radioactive Derivatives.- V. Enzymatic Production of Labeled Derivatives.- VI. Potential Applications of Radiolabeled Derivatizing Reagents.- References.- 27: Gas Chromatography. With 18 Figures.- I. Introduction.- II. Theory of Gas Chromatography.- III. Columns.- IV. Detectors.- V. Techniques in Gas Chromatography.- VI. Analytical Applications of Gas Chromatography in Pharmacology.- References.- 28: Enzymatic Assays in Pharmacology. With 4 Figures.- I. Introduction.- II. Enzymatic Assays with Pharmacological Agents as Substrates.- III. Enzymatic Assays with Drugs as Inhibitors.- IV. Competitive Protein Binding Analysis.- V. Conclusion.- References.- 29: Bioassay. With 2 Figures.- A. General Considerations.- I. Specificity.- II. Accuracy.- III. Automation.- B. Individual Assays.- I. Assays Used on Grounds of Sensitivity or Specificity.- II. Assays Used for Confirmation of Biological Activity (Steroids).- III. Assays Used because Chemical Methods are not yet Available.- IV. Proteins.- C. Conclusion.- References.- 30: Immunoassay. With 2 Figures.- References.- Subsection B. Isolation and Identification of Drug Metabolites.- 31: Paper, Column and Thin Layer Chromatography, Counter-Current Distribution and Electrophoresis..- A. Introduction.- B. Extraction from Tissues.- C. Counter-Current Distribution.- I. Theory.- II. Prediction of Separability.- III. Choice of Solvent Pairs for Counter-Current Distribution.- D. Chromatography.- I. The Properties of Adsorbents Used in Column Chromatography.- II. Liquid-Liquid Partition Chromatography.- III. Paper and Thin Layer Chromatography.- 1. Introduction.- 2. Paper Chromatography.- 3. Thin Layer Chromatography.- E. Electrophoresis.- References.- 32: Isotope Dilution Analysis. With 4 Figures.- A. Introduction.- B. Isotope Dilution Analysis.- 1. Direct Methods.- 2. Inverse Methods.- C. Modifications of Isotope Dilution Analysis.- D. Some Analytic Techniques Used in Conjunction with Radioassay Methods.- 1. Conversion of Samples to a Uniform Physical State.- 2. Addition and Purity of Carrier Material.- 3. Isolation of Drugs and Drug Metabolites.- 4. Purification of Isolated Material.- 5. Concentration and Radioactivity Measurements.- E. Applications of Isotopic Dilution Techniques.- 1. Examples of Direct Methods.- a) Pentobarbital.- b) Tolbutamide.- 2. Examples of Indirect Methods.- a) Warfarin.- b) Probenecid.- c) Fluroxene.- d) Folic Acid Antagonists.- e) Cyclophosphamide.- f) Thalidomide.- g) Tartrazine.- h) Benzene and Cyclohexane.- References.- 33: Gas Chromatography-Mass Spectrometry. With 8 Figures.- A. Introduction.- B. Operation of the Combined Gas Chromatograph-Mass Spectrometer.- C. Mass Spectral Fragmentation Processes.- D. Applications of Mass Spectrometry to the Problem of Identifying Drug Metabolites.- 1. Ethanol.- 2. Aspirin.- 3. Phenacetin.- 4. Tremorine.- 5. Siduron.- 6. Naphthalene.- 7. Probenecid.- 8. Brief Accounts of Mass Spectral Studies of Miscellaneous Compounds of Interest to Pharmacologists and Toxicologists.- a) Limonene.- b) Phytanic Acid.- c) Isoprene.- d) Acetylcholine.- e) The "Pink Spot" in Urine from Schizophrenics.- f) Bee Scent.- g) Trans-stilbene.- h) Pteridines.- i) Tetracycline.- j) Polychlorinated Biphenyls (PCB's).- k) DDT.- E. Forensic Applications.- References.- 34: The Application of Various Spectroscopies to the Identification of Drug Metabolites. With 5 Figures.- A. Introduction.- B. Sample Requirements and Isolation Procedures.- C. Mass Spectrometry.- D. Nuclear Magnetic Resonance Spectroscopy.- E. Infrared Spectroscopy.- F. Ultraviolet Spectroscopy.- G. Optical Rotatory Dispersion and C…