Introduction to pharmaceutical chemical analysis

This textbook is the first to present a systematic introduction to chemical analysis of pharmaceutical raw materials, finished pharmaceutical products, and of drugs in biological fluids, which are carried out in pharmaceutical laboratories worldwide.

In addition, this textbook teaches the fundamentals of all the major analytical techniques used in the pharmaceutical laboratory, and teaches the international pharmacopoeias and guidelines of importance for the field. It is primarily intended for the pharmacy student, to teach the requirements in "analytical chemistry" for the 5 years pharmacy curriculum, but the textbook is also intended for analytical chemists moving into the field of pharmaceutical analysis.

Addresses the basic concepts, then establishes the foundations for the common analytical methods that are currently used in the quantitative and qualitative chemical analysis of pharmaceutical drugs Provides an understanding of common analytical techniques used in all areas of pharmaceutical development Suitable for a foundation course in chemical and pharmaceutical sciences Aimed at undergraduate students of degrees in Pharmaceutical Science/Chemistry Analytical Science/Chemistry, Forensic analysis Includes many illustrative examples

Author Notes

Steen Hansen is professor in analytical chemistry at the Faculty of Pharmaceutical Sciences at Copenhagen University (Denmark), and has extensive experience in the teaching of analytical chemistry for pharmacy students. His main research interests are in basic separation sciences (primarily HPLC and CE), in particular on separation mechanisms, but also extend into hyphenated techniques using NMR and ICP-MS besides conventional detection techniques. He has authored and/or coauthored more than 240 papers in international peer-review journals. For 20 years, he was the President of the Scandinavian Section of The Chromatographic Discussion Group until this was renamed the Separation Sciences Foundation of which he is President.

Knut Einar Rasmussen is professor in pharmacy at the School of Pharmacy at University of Oslo (Norway) and has similarly long experience in teaching of drug analysis for Pharmacy students. He has published extensively and his main research areas are focused on the development of innovative state-of-the-art miniaturized methods in separation science combined with detection and structure determination of drugs, naturally occurring biomolecules/biomarkers and environmental biomarkers.

Stig Pedersen-Bjergaard is professor at at the School of Pharmacy, University of Oslo (Oslo, Norway) as well as in 2008, being appointed as full Professor (part time) at Department of Pharmaceutics and Analytical Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen (Copenhagen, Denmark). SPB has specialized in analytical micro extraction technologies, on development and applications of artificial liquid membranes, and on electrokinetic separation methods. SPB has published more than 90 papers in international journals, given more than 65 presentations at international symposia, authored 6 patent applications, and is a member of the Editorial board for Analytica Chimica Acta and International Journal of Analytical Chemistry .

Preface	p. xv
1 Introduction to Pharmaceutical Analysis	p. 1
1.1 Applications and Definitions	p. 1
1.2 The Life of Medicines	p. 4
1.3 The Quality of Medical Products	p. 8
1.4 Summary	p. 11
2 International Pharmacopoeias, Regulations and Guidelines	p. 13
2.1 Overview of Legislation	p. 13
2.2 Legislation and Regulations for Industrial Production	p. 14
2.3 Life Time of Drugs and Drug Substances	p. 17
2.4 Pharmacopoeias	p. 18
2.5 International Harmonization	p. 19
2.5.1 International Conference on Harmonization	p. 20
2.5.2 Pharmacopoeial Discussion Group	p. 20
2.6 Legislation and Regulations for Pharmacy Production	p. 20
2.7 Summary	p. 21
3 Fundamental Chemical Properties, Buffers and pH	p. 23
3.1 pH and pKa	p. 23
3.2 Partition	p. 25
3.3 Stereochemistry	p. 28
3.4 Stability Testing	p. 29
3.5 Summary	p. 30
4 Fundamentals of Pharmaceutical Analysis	p. 33
4.1 What is a Pharmaceutical (Chemical) Analysis?	p. 33
4.2 How to Specify Quantities and Concentrations?	p. 35
4.3 Basic Laboratory Equipment	p. 37
4.3.1 The Analytical Balance	p. 37
4.3.2 Pipettes	p. 41
4.3.3 Volumetric Flasks	p. 44
4.3.4 Burettes	p. 47
4.4 How to Make Solutions and Dilutions	p. 47
4.5 Calibration of Analytical Methods	p. 49
4.6 Errors, Accuracy, and Precision	p. 50
4.6.1 Systematic and Random Errors	p. 50
4.6.2 Accuracy and Precision	p. 51
4.7 Statistics	p. 52
4.7.1 Mean Value and Standard Deviation	p. 52
4.7.2 Confidence Intervals	p. 54
4.7.3 Comparison of Means with a t-Test	p. 55
4.7.4 Q-Test to Reject Outliers	p. 56
4.7.5 Linear Regression with the Method of Least Squares	p. 57
4.7.6 How to Present an Analytical Result	p. 58
4.8 Some Words and Concepts	p. 62
4.8.1 Analysis and Determination	p. 62
4.8.2 Sample Replicates and Measuring Replicates	p. 62
4.8.3 Interference	p. 62
4.8.4 Blind Samples	p. 62
5 Titrimetric Methods	p. 65
5.1 Introduction	p. 65
5.2 Acid-Base Titrations	p. 72
5.3 Acid-Base Titrations in Non-Aqueous Media	p. 75
5.4 Redox Titrations	p. 78
5.5 Other Principles of Titration	p. 81
5.6 Summary	p. 82
6 Introduction to Spectroscopic Methods	p. 83
6.1 Electromagnetic Radiation	p. 83
6.2 Molecules and Electromagnetic Radiation	p. 85
6.3 Atoms and Electromagnetic Radiation	p. 86
6.4 Summary	p. 88
7 UV Spectrophotometry	p. 89
7.1 Principle of Quantitative Determination	p. 89
7.2 Principle of Identification	p. 94
7.3 Which Substances Have Strong UV Absorbance?	p. 95
7.4 Instrumentation	p. 95
7.5 Practical Work and Method Development	p. 99
7.6 Areas of Usage and Performance	p. 101
7.7 System Testing	p. 101
7.8 Summary	p. 102
8 IR Spectrophotometry	p. 103
8.1 IR Spectrophotometry	p. 103
8.2 Instrumentation	p. 106
8.3 Scope	p. 109
8.4 Instrument Calibration	p. 109
8.5 NIR Spectrophotometry	p. 110
8.6 Applications	p. 112
8.7 Summary	p. 114
9 Atomic Spectrometry	p. 115
9.1 Atomic Absorption Spectrometry	p. 115
9.2 Instrumentation	p. 118
9.3 Applications and Performance	p. 121
9.4 Practical Work and Method Development	p. 122
9.5 Atomic Emission Spectrometry	p. 123
9.6 Instrumentation	p. 124
9.7 Summary	p. 124
10 Chromatography	p. 127
10.1 General Principles	p. 127
10.2 Retention	p. 131
10.3 Column Efficiency	p. 133
10.4 Selectivity	p. 135
10.5 Peak Symmetry	p. 136
10.6 Resolution	p. 138
10.7 Chromatographic Techniques	p. 140
10.8 Summary	p. 140
11 Chromatographic Separation Principles	p. 141
11.1 General Introduction	p. 141
11.2 Normal Phase Chromatography	p. 142
11.2.1 Silica	p. 142
11.2.2 Interactions	p. 143
11.2.3 Order of Elution	p. 144
11.2.4 Other Stationary Phases	p. 145
11.2.5 Mobile Phases	p. 146
11.2.6 Summary of Normal Phase Chromatography	p. 147
11.3 Reversed Phase Chromatography	p. 148
11.3.1 Stationary Phases	p. 148
11.3.2 Retention Mechanisms	p. 150
11.3.3 Mobile Phases	p. 152
11.3.4 Ion-Pair Chromatography	p. 155
11.3.5 Summary of Reversed Phase Chromatography	p. 155
11.4 Hydrophilic Interaction Chromatography	p. 156
11.5 Chiral Separations	p. 156
11.6 Size Exclusion Chromatography	p. 158
11.6.1 Principle	p. 158
11.6.2 Summary of SEC	p. 160
11.7 Ion Exchange Chromatography	p. 160
12 Thin-Layer Chromatography	p. 163
12.1 Introduction	p. 163
12.2 Apparatus	p. 164
12.3 TLC Plates	p. 166
12.4 Stationary Phases	p. 166
12.5 Mobile Phases	p. 167
12.6 Chromatographic Development	p. 168
12.7 Detection	p. 169
12.8 Applications of TLC	p. 169
12.9 Quantitative Analysis and Instrumentation	p. 170
12.10 Summary	p. 171
13 High Performance Liquid Chromatography	p. 173
13.1 Introduction	p. 173
13.2 The Chromatographic Separation Process	p. 175
13.3 The Column	p. 177
13.4 Pumps	p. 180
13.5 Detectors	p. 182
13.5.1 UV detector	p. 182
13.5.2 Fluorescence Detector	p. 184
13.5.3 Electrochemical Detector	p. 186
13.5.4 Refractive Index, Evaporative Light Scattering and Corona Discharge Detectors	p. 186
13.5.5 Combination of Detectors	p. 187
13.6 Injectors	p. 187
13.7 Mobile Phases	p. 188
13.8 Solvents for Sample Preparation	p. 189
13.9 Reporting the Results	p. 189
13.10 Summary	p. 190
14 Gas Chromatography	p. 191
14.1 Introduction	p. 191
14.2 Apparatus	p. 192
14.3 Temperature	p. 193
14.4 Carrier Gas	p. 195
14.5 Stationary Phases	p. 196
14.6 Selectivity in GC	p. 197
14.7 Columns	p. 198
14.7.1 Capillary Columns	p. 198
14.7.2 Packed Columns	p. 199
14.8 Injection Systems	p. 200
14.8.1 Injection Systems for Capillary Columns	p. 200
14.8.2 Injection Systems for Packed Columns	p. 202
14.9 Detectors	p. 203
14.9.1 Flame Ionization Detector	p. 203
14.9.2 Nitrogen-Phosphorus Detector	p. 203
14.9.3 Thermal Conductivity Detector	p. 204
14.9.4 Electron Capture Detector	p. 204
14.9.5 Mass Spectrometry Detector	p. 206
14.10 Derivatization	p. 206
14.10.1 Silylation	p. 206
14.10.2 Alkylation	p. 207
14.10.3 Acylation	p. 207
14.11 The Uses of GC	p. 208
14.12 More Advanced GC techniques	p. 209
14.13 Summary	p. 209
15 Capillary Electrophoresis	p. 211
15.1 Principle and Theory	p. 211
15.2 Electroosmotic Flow	p. 213
15.3 Instrumentation	p. 214
15.4 The Capillary	p. 217
15.5 Sample Introduction	p. 218
15.6 Capillary Zone Electrophoresis; an Example	p. 221
15.7 Micellar Electrokinetic Chromatography	p. 222
15.8 Chiral Separations	p. 224
15.9 Coated Capillaries	p. 225
15.10 Non-Aqueous CE	p. 229
15.11 Summary	p. 229
16 Mass Spectrometry	p. 231
16.1 Introduction	p. 231
16.2 Basic Theory	p. 233
16.3 Electron Ionization	p. 236
16.4 Identification using Electron Ionization Spectra	p. 237
16.5 Characterization of Totally Unknowns using Electron Ionization Spectra	p. 239
16.6 Chemical Ionization	p. 244
16.7 Electrospray Ionization	p. 246
16.8 Atmospheric Pressure Chemical Ionization	p. 247
16.9 High-Resolution Mass Spectrometry	p. 248
16.10 Instrumentation	p. 250
16.11 Chromatography Coupled with Mass Spectrometry	p. 253
16.12 Quantitative GC-MS and LC-MS	p. 256
16.13 Areas of Usage and Performance	p. 257
16.14 Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry	p. 257
16.15 Inductively Coupled Plasma Mass Spectrometry	p. 258
16.16 Summary	p. 259
17 Miscellaneous Chemical Techniques	p. 261
17.1 Potentiometric Determination of Ions using Ion-Selective Electrodes	p. 261
17.2 Paper Chromatography	p. 263
17.3 Supercritical Fluid Chromatography	p. 264
17.4 Gel Electrophoresis	p. 265
17.5 Iso-Electric Focusing	p. 267
17.6 Nuclear Magnetic Resonance Spectrometry	p. 268
17.7 Raman Spectrometry	p. 270
18 Sample Preparation	p. 273
18.1 Why is Sample Preparation Required?	p. 273
18.2 Main Strategies	p. 274
18.3 Recovery and Enrichment	p. 276
18.4 Protein Precipitation	p. 278
18.5 Liquid-Liquid Extraction	p. 279
18.5.1 Fundamentals	p. 279
18.5.2 A Closer Look at the Theory	p. 279
18.5.3 Extraction Solvents	p. 282
18.5.4 Calculation of Recovery	p. 283
18.5.5 Multiple Extractions	p. 285
18.5.6 LLE with Back-Extraction	p. 286
18.6 Solid-Liquid Extraction	p. 287
18.7 Solid Phase Extraction	p. 287
18.7.1 Fundamentals	p. 287
18.7.2 The SPE Column	p. 288
18.7.3 Conditioning	p. 289
18.7.4 Equipment	p. 290
18.7.5 Reversed-Phase SPE	p. 290
18.7.6 Secondary Interactions	p. 292
18.7.7 Ion Exchange SPE	p. 293
18.7.8 Mixed-Mode SPE	p. 295
18.7.9 Normal-Phase SPE	p. 297
18.8 Summary	p. 298
19 Analytical Chemical Characteristics of Selected Drug Substances	p. 299
19.1 Amitriptyline and Mianserin	p. 299
19.2 Morphine and Codeine	p. 301
19.3 Ibuprofen and Naproxen	p. 302
19.4 Furosemide	p. 304
19.5 Paracetamol (Acetaminophen)	p. 306
19.6 Neutral Drugs	p. 307
20 Quantification and Quality of Analytical Data	p. 309
20.1 Peak Height and Peak Area	p. 309
20.2 Calibration Methods	p. 310
20.2.1 External Standard Method	p. 310
20.2.2 Internal Standard Method	p. 313
20.2.3 Standard Addition	p. 314
20.2.4 Normalization	p. 314
20.3 Validation	p. 314
20.3.1 Analytical Procedure	p. 317
20.3.2 Accuracy	p. 317
20.3.3 Precision	p. 318
20.3.4 Specificity	p. 320
20.3.5 Detection Limit	p. 320
20.3.6 Quantification Limit	p. 321
20.3.7 Linearity and Range	p. 321
20.3.8 Robustness	p. 323
20.3.9 Test Methods in the European Pharmacopeia	p. 325
20.4 System Suitability	p. 325
20.4.1 Adjustment of Chromatographic Conditions	p. 326
21 Chemical Analysis of Drug Substances	p. 327
21.1 What is a Pharmaceutical Raw Material, how is it Produced and why must it be Controlled?	p. 327
21.2 The Pharmacopoeias - the Basis for Control of Pharmaceutical Raw Materials	p. 330
21.3 Which Contaminants are Found in Raw Materials, What are the Requirements in a Maximum Content and Why?	p. 337
21.3.1 Well Defined Chemical Compounds	p. 339
21.3.2 Mixtures of Organic Compounds	p. 343
21.4 How to Check the Identity of Pharmaceutical Raw Materials	p. 344
21.4.1 Overview of the Identification Procedures	p. 344
21.4.2 Techniques used for the Identification of Well Defined Chemical Compounds	p. 344
21.4.2.1 Infrared Absorption Spectrophotometry	p. 344
21.4.2.2 Ultraviolet and Visible Absorption Spectrophotometry	p. 347
21.4.2.3 Thin-Layer Chromatography	p. 351
21.4.2.4 Melting Point	p. 352
21.4.2.5 Polarimetry	p. 353
21.4.2.6 High Performance Liquid Chromatography	p. 356
21.4.2.7 Chloride and Sulfate Identification	p. 359
21.5 How to Test for Impurities in Pharmaceutical Raw Materials	p. 359
21.5.1 Main Purity Tests for Well Defined Chemical Compounds	p. 359
21.5.1.1 Appearance of Solution	p. 361
21.5.1.2 Absorbance	p. 364
21.5.1.3 Acidity/Alkalinity	p. 365
21.5.1.4 Optical Rotation	p. 365
21.5.1.5 Related Substances	p. 366
21.5.1.6 Solvent Residues	p. 372
21.5.1.7 Foreign Anions	p. 372
21.5.1.8 Cationic Impurities	p. 376
21.5.1.9 Loss on Drying	p. 378
21.5.1.10 Determination of Water	p. 379
21.5.2 Purity Tests for Raw Materials of the Type of Mixtures of Organic Compounds	p. 382
21.5.2.1 Oxidizing Substances	p. 383
21.5.2.2 Acid Value	p. 383
21.5.2.3 Hydroxyl Value	p. 384
21.5.2.4 Iodine Value	p. 384
21.5.2.5 Peroxide Value	p. 385
21.5.2.6 Saponification Value	p. 385
21.5.2.7 Unsaponifiable Matter	p. 386
21.5.2.8 Other Tests	p. 386
21.5.3 Identification of the Raw Materials of the Type of Mixtures of Organic Compounds	p. 388
21.6 How to Determine the Purity of Pharmaceutical Raw Materials	p. 389
21.6.1 Acid-Base Titration in Aqueous Environment	p. 389
21.6.2 Acid-Base Titration in a Non-Aqueous Environment	p. 393
21.6.3 Redox Titrations	p. 396
21.6.4 High Performance Liquid Chromatography	p. 396
21.6.5 UV spectrophotometry	p. 401
21.7 How to Control Compounds for Which no Pharmacopoeia Monograph Exists	p. 402
21.8 How are Ph.Eur. and USP Updated?	p. 402
22 Chemical Analysis of Final Pharmaceutical Products	p. 405
22.1 Quality Control of Final Pharmaceutical Products	p. 405
22.2 Monographs and Chemical Testing	p. 406
22.3 Identification of the Active Pharmaceutical Ingredient	p. 412
22.4 Assay of the Active Pharmaceutical Ingredient	p. 427
22.5 Chemical Tests for Final Pharmaceutical Products	p. 446
22.5.1 Test for Related Substances	p. 446
22.5.2 Uniformity of Content	p. 449
22.5.3 Dissolution	p. 451
23 Analysis of Drugs in Biological Fluids	p. 453
23.1 Introduction	p. 453
23.1.1 Drug Development	p. 453
23.1.2 Therapeutic Drug Monitoring	p. 455
23.1.3 Forensic and Toxicological Analysis	p. 456
23.1.4 Doping Control Analysis	p. 457
23.2 The Biological Matrix	p. 458
23.3 Bioanalytical Methods	p. 460
23.3.1 Sampling	p. 460
23.3.2 Sample Preparation	p. 461
23.3.3 Protein Precipitation	p. 462
23.3.4 Liquid-Liquid Extraction	p. 463
23.3.5 Solid-Phase Extraction	p. 463
23.3.6 Separation	p. 464
23.3.7 Detection	p. 464
23.3.8 Calibration and Quantification	p. 465
23.4 Examples	p. 466
23.4.1 Sample Preparation	p. 466
23.4.1.1 Sample Preparation Procedure by LLE	p. 466
23.4.1.2 Comments to the Procedure	p. 466
23.4.1.3 Sample Preparation Procedure by LLE and Back Extraction	p. 467
23.4.1.4 Comments to the Procedure	p. 467
23.4.1.5 Sample Preparation Procedure by SPE	p. 467
23.4.1.6 Comments to the Procedure	p. 468
23.4.1.7 Sample Preparation Procedure by Protein Precipitation	p. 468
23.4.1.8 Comments to the Procedure	p. 468
23.4.2 Quantitative Determination	p. 468
23.4.2.1 Quantitative Determination of Amitriptyline in Serum by LC-MS	p. 468
23.4.2.2 Comments to the Procedure	p. 469
23.4.2.3 Determination of Valproic Acid in Serum by GC-MS	p. 471
23.4.2.4 Comments to the Procedure	p. 471
23.4.3 Identification	p. 472
23.4.3.1 Sample Preparation Procedure for Unknown Screening by Mixed Mode Cation Exchange	p. 472
23.4.3.2 Comments to the Procedure	p. 472
23.4.3.3 GC-MS Procedure for Unknown Screening	p. 473
23.4.3.4 Comments to the Procedure	p. 473
23.4.3.5 LC-MS-MS Procedure for Unknown Screening	p. 475
23.4.3.6 Comments to the Procedure	p. 475
Index	p. 477

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