PHARMACEUTICALDRUG ANALYSIS

Methodology-Theory-lnstrumentationPharmaceutical Assays-Cognate Assays

Ashutosh KarProfessor, School of Pharmacy, Addis Ababa University

Addis Ababa (Ethiopia)Formerly Dean and Chairman, Faculty of Pharmaceutical Sciences

Guru lambheshwar University, Hisar (INDIA)

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Contents

Preface to the Second Edition (uii)

Preface to the First Edition (we)

PART I: GENERAL ASPECTS

1. PHARMACEUTICAL CHEMICALS : PURITY AND MANAGEMENT 31.1. Introduction 31.2 Purity 4

1.2.1. Broad-Based Highest Attainable Standard 41.2.2. Biological Response Vs. Chemical Purity 51.2.3. Official Standards vis-a-vis Manufacturing Standards 5

1.3. Management 61.3.1. Description of the Drug or Finished Product 61.3.2. Sampling Procedures and Errors 61.3.3. Bioavailability 91.3.4. Identification Tests 101.3.5. Physical Constants 111.3.6. Limit Tests vis-a-vis Quantitative Determinations 171.3.7. Various Types of Tests for Quantitative Determinations 171.3.8. Limit Tests for Metallic Impurities 251.3.9. Limit Test's for Acid Radical Impurities 301.3.10. Limit Tests for Non-Metallic Impurities 37

2. THEORY AND TECHNIQUE OF QUANTITATIVE ANALYSIS 41

2.1. Introduction 412.2. Volumetric Analysis 42

2.2.1. Theory 422.2.2. Definitions 422.2.3. Volumetric Apparatus 442.2.4. General Considerations 512.2.5. Technique of Volumetric Analysis 52

2.3. Gravimetric Analysis 532.4. Biomedical Analytical Chemistry 53

2.4.1. Colorimetric Assays 532.4.2. Enzymatic Assays 582.4.3. Radioimmuno Assays (RIAS) 63

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3. ERRORS IN PHARMACEUTICAL ANALYSIS AND STATISTICAL 71VALIDATION

3A. Errors in Pharmaceutical Analysis 713A.1. Introduction 713A.2. Classification of Errors 72

3A.2.1. Determinate (Systematic) Errors 723A.2.2. Indeterminate (Random) Errors 733A.2.3. Accuracy 743A.2.4. Precision 743A.2.5. Minimising Systematic Errors 75

3B. Statistical Validation 773B.1. Introduction 773B.2. Statistical Validation 77

3B.2.1. Statistical Treatment of Finite Samples 773B.2.2. Distribution of Random Numbers 793B.2.3. Significant Figures 803B.2.4. Comparision of Results 813B.2.5. Method of Least Squares 833B.2.6. Recommendations for Criteria of Rejecting an Observation 853B.2.7. Sampling Statistics 87

PART II : CHEMICAL METHODSA. Titrixnetric Methods: Acidimetry and Alkalimetry

4. AQUEOUS TITRATIONS 95

4.1. Introduction 954.1.1. Lowry and Bronsted's Theory of Acids and Bases 954.1.2. Lewis's Theory 974.1.3. Usanovich Theory 974.1.4. Lux-Flood Concept 98

4.2. Theory of Acidimetry 984.2.1. Direction Titration Method 984.2.2. Residual Titration Method 98

4.3. Assay of Drugs 984.3.1. Direction Titration Method 994.3.2. Residual Titration Method 100

4.4. Theory of Alkalimetry 1014.4.1. Direction Tirration Method (DTM) 1024.4.2. Residual Titration Methods (RTM) 103

5. NON-AQUEOUS TITRATIONS 106

5.1. Introduction 1065.2. Theory 107

5.2.1. Solvents 107

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5.3. Methodology 1085.3.1. Preparation of 0.1 N Perchloric Acid 1085.3.2. Standardization of 0.1 N Perchloric Acid 1095.3.3. Choice of Indicators 1105.3.4. Effect of Temperature on Assays 110

5.4. Assay by Non-Aqueous Titrations 1105.4.1. Acidimetry in Non-Aqueous Titrations I l l5.4.2. Alkalimetry in Non-Aqueous Titrations 117

B. Redox Methods

6. PERMANGANATE, DICHROMATE AND CERIC SULPHATE 125TITRATION METHODS

6.1. Introduction 1256.2. Theory 1256.3. Assay Methods 126

6.3.1. Permanganate Methods 1266.3.2. Dichromate Methods 1306.3.3. Ceric Sulphate Titration Methods 133

7. IODIMETRIC AND IODOMETRIC TITRATIONS 137

7.1. Introduction 1377.2. Theory 1377.3. Assay Methods 138

7.3.1. Iodimetric Assays 1387.3.2. Iodometric Assays 144

C. Precipitation Methods

8. ARGENTOMETRIC METHODS 151

8.1. Introduction 1518.2. Theory 1528.3. Assay Methods 152

8.3.1. Direct Titration with Silver Nitrate 1538.3.2. Ammonium Thiocyanate-Silver Nitrate Titrations

(Volhard's Method) 155

D. Complexometric Methods

9. COMPLEXOMETRIC ANALYSIS 161

9.1. Introduction 1619.2. Theory 161

9.2.1. Effect of pH on Complexation 1639.2.2. Stability of Complexes 1639.2.3. Colouration of Complexes

9.2.4. Titrability of Polyvalent Metal Ions Employing Disodium Edetate 1649.2.5. Usage of pM Indicators in Complexometric Titrations 164

9.3. Assay Methods 1659.3.1. Direction Titration Methods 1669.3.2. Masking and Demasking Agents 1689.3.3. Residual Titration Methods 169

E. Gravimetric Methods

10. GRAVIMETRIC ANALYSIS 173

10.1. Introduction 17310.2. Theory 173

10.2.1. Law of Mass Action and Reversible Reactions 17410.2.2. Principle of Solubility Product 17510.2.3. Comnion Ion Effect 176

10.3. Assay Methods 17710.3.1. Substances Assayed Gravimetrically 17710.3.2. Substances Assayed After Conversion 181

F. Thermoanalytical Methods

11. THERMOANALYTICAL ANALYSIS 193

11.1. Introduction11.2. Thermogravimetric Analysis (TGA) , 194

11.2.1. Theory 19411.2.2. Instrumentation 19411.2.3. Methodology 195

11.3. Differential Thermal Analysis (DTA) 19811.3.1. Theory 19811.3.2. Instrumentation 19811.3.3. Methodology 19911.3.4. Applications 200

11.4. Thermometric Titrations (TT) 20011.4.1. Theory 20011.4.2. Instrumentation 20011.4.3. Methodology 20211.4.4. Applications ,...202

G. Miscellaneous Methods

12. DIAZOTIZATION (SODIUM NITRITE TITRATION) 207

12.1. Introduction 20712.2. Theory 20712.3. Assay Methods 208

12.3.1. Preparation of 0.1 M Sodium Nitrite Solution 2 0 8

12.3.2. Standardization of 0.1 M Sodium Nitrite Solution with Sulphanilamide... 208

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12.3.3. Calcium Aminosalicylate 20912.3.4. Isocarboxazid 20912.3.5. Phthalylsulphathiazole 21012.3.6. Cognate Assays 210

13. ESTIMATION OF PHENOLS AND RELATED COMPOUNDS 213

13.1. Introduction 21313.2. Theory 21413.3. Assay Methods 214

13.3.1. Titrations with 0.1 N Bromine 21413.3.2. Titrations with Potassium Bromate 21713.3.3. Titrations with Potassium Iodate 219

14. KARL FISCHER METHOD FOR DETERMINATION OF WATER 223

14.1. Introduction 22314.2. Theory 22314.3. Instrumentation 224

14.3.1. Automated Electrochemical Karl Fischer Analysis 22514.4. Applications of Karl Fischer Method for Determination of Water in

Pharmaceutical Analysis 22614.4.1. Prednisolone Sodium Phosphate 22614.4.2. Cognate Assays 226

15. TETRAZOLIUM ASSAY OF STEROIDS 228

15.1. Introduction 22815.2. Theory 22815.3. Assay of Pharmaceutical Substances 229

15.3.1. Hydrocortisone Acetate 22915.3.2. Cognate Assays 230

PART III : ELECTROCHEMICAL METHODS

16. POTENTIOMETRIC METHODS 233

16.1. Introduction 23316.2. Theory 233

16.2.1. General Considerations 23516.2.2. End-Point Determination 237

16.3. Instrumentation 23916.3.1. Electrodes 24016.3.2. Automatic Titrator (Preset End-Point Titrator) 249

16.4. Applications of Potentiometric Titrations in Pharmaceutical Analysis 25016.4.1. Cognate Assays 251

17. AMPEROMETRIC METHODS 253

17.1. Introduction 253

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17.2. Theory 25417.2.1 Titration Curves 25417.2.2. Corrections for the Volume Change 25617.2.3. Advantages of Amperometric Titrations 257

17.3. Instrumentation 25717.3.1. Amperometric Titrations with the Dropping Mercury Electrode 25717.3.2. Amperometric Titrations with a Rotating Platinum

Microelectrode 25817.3.3. Amperometric Titrations with Twin-Polarized Microelectrodes

(Biamperometric Titrations or Dead-Stop-End-Point Method) 26017.4. Applications of Amperometric Titrations in Pharmaceutical Substances 261

17.4.1. Procainamide Hydrochloride 26117.4.2. Cognate Assay 26117.4.3. Assay of Nickel with Dimethylglyoxime 26117.4.4. Assay of Lead with Potassium Dichromate Solution 261

PART IV : OPTICAL METHODS

18. REFRACTOMETRY 265

18.1. Introduction 26518.2. Theory 26618.3. Instrumentation 26818.4. Determination of Refractive Index of Pharmaceutical Substances 27018.5. Applications of Refractivity 271

19. POLARIMETRY 274

19.1. Introduction 27419.2. Theory 27519.3. Instrumentation 27719.4. Determination of Optical Activity of Pharmaceutical Substances 278

19.4.1. Determination of Optical Rotation of Pharmaceutical Substances 27819.4.2. Determination of Specific Optical Rotation of Pharmaceutical

Substances 279

20. NEPHELOMETRY AND TURBIDIMETRY 283

20.1. Introduction , 28320.2. Theory ; 28420.3. Instruments for Nephelometry and Turbidimetry 284

20.3.1. Instruments for Nephelometry 2 8 4

20.3.2. Instruments for Turbidimetry 28720.4. Assay of Pharmaceutical Substances 2 8 7

20.4.1. Turbidimetric Assay 28720.4.2. Nephelometric Assay 289

21. ULTRAVIOLET AND ABSORPTION METHODS 2 9 3

21.1. Introduction 293

(xix )

21.2. Theory 29321.2.1. Electromagnetic Spectrum 29321.2.2. Schematic Representation of Electromagnetic Spectrum 29421.2.3. Molar Absorptivity 29521.2.4. Laws of Photometry 29621.2.5. Spectral Presentation 29621.2.6. Structural Features 29721.2.7. Absorption of Radiant Energy by Molecules 29821.2.8. Factors Influencing Absorption of Radiant Energy 301

21.3. Instrumentation 30321.3.1. Single Beam Spectrophotometer 30321.3.2. Double Beam Spectrophotometer 304

21.4. Assay Methods 30621.4.1. Methodology 30621.4.2. Spectrophotometers 30621.4.3. Preparation of Sample 30721.4.4. Measurement of Extinction (E) 30721.4.5. Examples 30721.4.6. UV-Absorption Characteristics of Some Official Pharmaceutical

Substances 311

22. INFRARED SPECTROPHOTOMETRY 314

22.1. Introduction 31422.1.1. Group Frequency Region 31522.1.2. Fingerprint Region 315

22.2. Theory 31522.2.1. Molecular Vibrations 31522.2.2. Factors Influencing Vibrational Frequencies 318

22.3. Instrumentation 32322.3.1. Single Monochromator Infrared Spectrophotometers 32422.3.2. Double-Monochromator Infrared Spectrophotometer 326

22.4. Applications of IR-Spectroscopy in Pharmaceutical Assays 33022.4.1. Applications of IR-Spectroscopy in the Analysis of Pharmaceutical

Substances 33022.4.2. Applications of IR-Spectroscopy in the Analysis of Pharmaceutical

Dosage Forms 33122.5. Applications of IR-Spectroscopy in Analytical Chemistry 333

22.5.1. Determination of cis-trans Isomer Ratio in Clomiphene Citrate 33322.5.2. To Distinguish and Characterize the pri-, sec- and tert-amine Salts

from One Another 33322.5.3. IR-Spectroscopy in the Study of Complex Formations 33422.5.4. IR-Spectroscopy in Quantitative Reaction Sequence Study 33422.5.5. IR-Spectroscopy in the Identification of Functional Groups 33422.5.6. IR-Spectroscopy : Identification by Fingerprinting 33522.5.7. Interpretation of an IR-Spectrum 335

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23. NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY 339

23.1. Introduction 33923.1.1. The NMR Phenomenon 34023.1.2. Informations Provided by TC-NMR (Proton-NMR) 341

23.2. Theory 34223.2.1. Orientations of Magnetic Nucleus Under Bo 34323.2.2. Precessional Frequency (v) 34323.2.3. Saturation of the Signal 34423.2.4. Absorption Positions in NMR-spectrum 34423.2.5. Chemical Shift 34423.2.6. Spin-Spin Interactions 34523.2.7. 3H-NMR (Tritium NMR-Spectroscopy) 34723.2.8. 13C-NMR-Spectroscopy 34823.2.9. 2D-NMR (Two Dimensional Correlation Spectroscopy or Two

Dimensional Cosy Spectrum) 34923.3. Interpretation of a NMR-Spectrum 350

23.3.1. Chemical Shift (5) (Relative to Reference Compound, Usually Me4Si) 35123.3.2. Relative Peak Area 35123.3.3. Multiplicity of the Signal 35123.3.4. Coupling Constant (J) 351

23.4. Instrumentation 35223.5. Applications of NMR-Spectroscopy in Pharmaceutical Analysis 353

23.5.1. Identification Testing 35323.5.2. Assay of Drugs 353

24. EMISSION SPECTROSCOPY 357

24.1. Introduction 35724.2. Theory 35724.3. Instrumentation 361

24.3.1. Excitation Sources 36124.3.2. Electrodes 36324.3.3. Sample Handling 36424.3.4. Monochromators 36424-3.5. Detectors 36524.3.6. Spectrographs 366

24.4. Applications of Emission Spectroscopy 368

25. FLAME SPECTROSCOPY 370

25.1. Introduction 37025.2. Theory 37125.3. Instrumentation , 372

25.3.1. Simple Flame Photometer 37225.3.2. Internal Standard Flame Photometer 373

25.4. Applications of Flame Emission Spectroscopy in Pharmaceutical Analysis 37525.4.1. Assay of Sodium, Potassium and Calcium in Blood Serum and Water 375

i

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25.4.2. Assay of Barium, Potassium and Sodium in Calcium Acetate 37525.4.3. Cognate Assays 376

26. ATOMIC ABSORPTION SPECTROSCOPY 378

26.1. Introduction 37826.2. Theory 379

26.2.1. Merits of AAS Over FES ;. 37926.2.2. Demerits of AAS 380

26.3. Instrumentation 38026.3.1. Single-Beam Atomic Absorption Spectrophotometer 38026.3.2. Double-Beam Atomic Absorption Spectrophotometer 382

26.4. Important Aspects of Atomic Absorption Spectroscopy 38326.4.1. Analytical Techniques 38326.4.2. Detection Limit and Sensitivity 38426.4.3. Interferences 385

26.5. Application of Atomic Absorption Spectroscopy in Pharmaceutical Analysis 38726.5.1. Assay of Total Zinc in Insulin Zinc Suspension 38726.5.2. Assay of Palladium in Carbenicillin Sodium 38726.5.3. Cognate Assays 388

PART V : ASSAY METHODS BASED ON SEPARATION TECHNIEQUES

27. LIQUID-LIQUID EXTRACTION 393

27.1. Introduction 39327.2. Theory 394

27.2.1. Error Due to the Volume Change 39527.2.2. Effectiveness of an Extraction 396

27.3. Factors Influencing Solvent Extraction 39727.3.1. Effect of Temperature and Inert Solutes 39727.3.2. Effect of pH on Extraction 39827.3.3. Effect of Ion-Pair Formation 39927.3.4. Effect of Synergistic Extraction 400

27.4. Emulsion Problem Encountered in Extractions 40127.5. Assay Methods Based on Liquid-Liquid Extraction 403

27.5.1. Determination of Copper (I) as the Neo-Cuproin Complex 40327.5.2. Determination of Iron (III) as the 8-Hydroxy Quinolate Complex

[Iron (III) Oxinate] 40327.5.3. Determination of Lead (I) by the Dithizone Method 40427.5.4. Determination of Molybdenum (VI) by the Thiocyanate Method 40527.5.5. Determinations of Nickel (ID 406

28. THIN LAYER CHROMATOGRAPHY (TLC) 409

28.1. Introduction 40928.2. Theory 410

28.2.1. Versatility of TLC over Paper and Column Chromatography 410

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28.3. Experimental Techniques of TLC 41128.3.1. Preparation of Thin Layers on Plates 41128.3.2. Choice of Adsorbents 41328.3.3. Choice of Solvent System in TLC 41528.3.4. Activation of Adsorbent 41728.3.5. Purification of Silica Gel-G Layers 41728.3.6. Spotting of the Components 41828.3.7. Development of Thin Layers 41828.3.8. Special Techniques in TLC 41928.3.9. Chemical Reactions on TLC Plates 42228.3.10. Combination of TLC with other Techniques 42328.3.11. Detection of Components 42328.3.12. Evaluation of the Chromatogram 424

28.4. Applications of TLC in Pharmaceutical Analysis 42528.4.1. Presence of Specific Substances as Impurities in Drug Substances 42528.4.2. Related Substances Present in Official Drugs 42728.4.3. Foreign Alkaloids Present in Alkaloidal Drugs 42828.4.4. Foreign Steroids Present in Steroidal Drugs 42828.4.5. Ninhydrin Positive Substances Present in Official Amino Acids 429

29. GAS LIQUID CHROMATOGRAPHY (GLC) 431

29.1. Introduction 43129.2. Theory 432

29.2.1. Plate Theory 43229.2.2. Rate Theory 43329.2.3. Random Walk and Nonequilibrium Theory 433

29.3. Instrumentation 43529.3.1. Carrier Gas Pressure Regulator and Flow Meter 43629.3.2. Sample Injection System 43629.3.3. Separation Column 43729.3.4. Thermal Compartment 43729.3.5. Detectors 43729.3.6 Recording of Signal Current 44129.3.7. Integrator 441

29.4. Working Techniques for Quantitative Analysis 44229.4.1. Area Normalization 44329.4.2. Internal Standard Method 44429.4.3. Comparison Method 445

29.5. Applications of GLC in Pharmaceutical Analysis 44529.5.1. Assay of Drugs 44629.5.2. Determination of Specific Organic Compounds as Impurities in

Official Pharmaceutical Substances 44729.5.3. Determination of Related Substances in Official Drugs 44829.5.4. Determination of Water in a Drug 44929.5.5. Determination of Chloroform in Colchicine by Head-Space Gas

Chromatography :..449

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30. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) 452

30.1. Introduction 45230.2. Theory 45330.3. Instrumentation 455

30.3.1. Solvent Reservoir and Degassing System 45630.3.2. Pressure, Flow and Temperature 45630.3.3 Pumps and Sample Injection System 45730.3.4. Columns 45930.3.5. Detectors 46130.3.6. Strip Chart Recorder 46530.3.7. Data Handling Device and Microprocessor Control 466

30.4. Derivatization 46630.4.1. Pre-Column Off-Line Derivatization 46630.4.2. Post-Column On-Line Derivatization 46730.4.3. Reagents for Derivatization 467

30.5. Applications of HPLC in Pharmaceutical Analysis 46830.5.1. Isolation of Natural Pharmaceutically Active Compounds 46830.5.2. Control of Microbiological Processes 46830.5.3. Assay of Cephalosporins 46930.5.4. Assay of Frusemide 47030.5.5. Assay of Theophylline 47130.5.6. Assay of Corticosteroids 47230.5.7. Assay of Dichlorphenamide 47330.5.8. Assay of Human Insulin 47330.5.9. Cognate Assays 473

31. SIZE EXCLUSION CHROMATOGRAPHY 476

31.1. Introduction 47631.2. Theory 477

31.2.1. Distribution Coefficient (KJ 47831.2.2. Performance 47831.2.3. Materials 478

31.3. Apparatus 47931.3.1. Application of Sample 47931.3.2. Detection and Recording 479

31.4. Applications of Size Exclusion Chromatography in Pharmaceutical Analysis 48031.4.1. Determination of Relative Component Composition 48031.4.2. Determination of Molecular Weight 48031.4.3. Corticotrophin : For Impurities of Higher Molecular Weights 48031.4.4. Insulin : For Proteins of Higher Molecular Weight 48131.4.5. Human Insulin : For Proteins of Higher Molecular Weight 48131.4.6. Plasma Protein Solution : For Polymers and Aggregates 481

(xxiv)

PART VI : MISCELLANEOUS METHODS

32. RADIOIMMUNOASSAY 485

32.1. Introduction 48532.2. Theory 4 8 6

32.2.1. Hapten Determinants and Purity : The Key to ImmunologicalSpecificity 487

32.2.2. Importance of Antigenic Determinants 48732.2.3. Analysis by Competitive Antibody Binding or Isotopically

Labelled Compounds 48832.3. Instrumentation 491

32.3.1. Centrifuge 49132.3.2. Radioactive Counters 491

32.4. Methodology of the Assay 49232.5. Applications of Radioimmunoassay (RIA) in Pharmaceutical Analysis 492

32.5.1. Radioimmunoassay of Morphine 49332.5.2. Radioimmunoassay of Hydromorphone and Hydrocodone in Human

Plasma 49432.5.3. Radioimmunoassay of Clonazepam 49532.5.4. Radioimmunoassay of Flurazepam in Human Plasma 49632.5.5. Radioimmunoassay of Chlordiazepoxide in Plasma 49732.5.6. Radioimmunoassay of Barbiturates 49932.5.7. Radioimmunoassay of Flunisolide in Human Plasma 500

32.6. Novel Applications of RIA-Techniques 50232.6.1. Combined RIA-Techniques Isotope Dilution 50232.6.2. Stereospecificity 503

INDEX 510