123 yo yo.pdf

Introduction to
Pharmaceutical
Chemical Analysis

Introduction to
Pharmaceutical
Chemical Analysis
STEEN HANSEN
STIG PEDERSEN-BJERGAARD
KNUT RASMUSSEN

This edition first published 2012
Ó 2012 John Wiley & Sons Ltd.
Registered office
John Wiley & Sons Ltd, The Atrium, Southern Gate, Chichester, West Sussex, PO19 8SQ, United Kingdom
For details of our global editorial offices, for customer services and for information about how to apply for permission to reuse
the copyright material in this book please see our website at www.wiley.com.
The right of the author to be identified as the author of this work has been asserted in accordance with the Copyright,
Designs and Patents Act 1988.
All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form
or by any means, electronic, mechanical, photocopying, recording or otherwise, except as permitted by the UK Copyright,
Designs and Patents Act 1988, without the prior permission of the publisher.
Wiley also publishes its books in a variety of electronic formats. Some content that appears in print may not be available
in electronic books.
Designations used by companies to distinguish their products are often claimed as trademarks. All brand names and product
names used in this book are trade names, service marks, trademarks or registered trademarks of their respective owners.
The publisher is not associated with any product or vendor mentioned in this book. This publication is designed to provide
accurate and authoritative information in regard to the subject matter covered. It is sold on the understanding that the
publisher is not engaged in rendering professional services. If professional advice or other expert assistance is required,
the services of a competent professional should be sought.
The publisher and the author make no representations or warranties with respect to the accuracy or completeness of the
contents of this work and specifically disclaim all warranties, including without limitation any implied warranties of fitness for a
particular purpose. This work is sold with the understanding that the publisher is not engaged in rendering professional services.
The advice and strategies contained herein may not be suitable for every situation. In view of ongoing research, equipment
modifications, changes in governmental regulations, and the constant flow of information relating to the use of experimental
reagents, equipment, and devices, the reader is urged to review and evaluate the information provided in the package
insert or instructions for each chemical, piece of equipment, reagent, or device for, among other things, any changes in the
instructions or indication of usage and for added warnings and precautions. The fact that an organization or Website is referred
to in this work as a citation and/or a potential source of further information does not mean that the author or the publisher
endorses the information the organization or Website may provide or recommendations it may make. Further, readers should be
aware that Internet Websites listed in this work may have changed or disappeared between when this work was written and
when it is read. Nowarranty may be created or extended by anypromotional statements for this work. Neither the publisher nor the
author shall be liable for any damages arising herefrom.
Library of Congress Cataloging-in-Publication Data
Hansen, Steen, 1947–
Chemical analysis in pharmaceutical sciences / Steen Hansen, Stig
Pedersen-Bjergaard, Knut Rasmussen.
p. ; cm.
Includes bibliographical references and index.
ISBN 978-0-470-66121-5 (cloth) – ISBN 978-0-470-66122-2 (pbk.)
1. Drugs–Analysis. 2. Pharmaceutical chemistry. I. Pedersen-Bjergaard,
Stig. II. Rasmussen, Knut. III. Title.
[DNLM: 1. Pharmaceutical Preparations–analysis. 2. Chemistry,
Pharmaceutical. 3. Drug Compounding–standards. QV 55]
RS189.H277 2012
615.109–dc23
2011030362
A catalogue record for this book is available from the British Library.
Print ISBN (Hardback): 9780470661215
Print ISBN (Paperback): 9780470661222
ePDF ISBN: 9781119953609
oBook ISBN: 9781119953647
ePub ISBN: 9781119954330
Mobi ISBN: 9781119954347
Set in 10/12pt Times Roman by Thomson Digital, Noida, India

Table of Contents
Preface xv
1 Introduction to Pharmaceutical Analysis 1
1.1 Applications and Definitions 1
1.2 The Life of Medicines 4
1.3 The Quality of Medical Products 8
1.4 Summary 11
2 International Pharmacopoeias, Regulations and Guidelines 13
2.1 Overview of Legislation 13
2.2 Legislation and Regulations for Industrial Production 14
2.3 Life Time of Drugs and Drug Substances 17
2.4 Pharmacopoeias 18
2.5 International Harmonization 19
2.5.1 International Conference on Harmonization 20
2.5.2 Pharmacopoeial Discussion Group 20
2.6 Legislation and Regulations for Pharmacy Production 20
2.7 Summary 21
3 Fundamental Chemical Properties, Buffers and pH 23
3.1 pH and pKa 23
3.2 Partition 25
3.3 Stereochemistry 28
3.4 Stability Testing 29
3.5 Summary 30
4 Fundamentals of Pharmaceutical Analysis 33
4.1 What is a Pharmaceutical (Chemical) Analysis? 33
4.2 How to Specify Quantities and Concentrations? 35
4.3 Basic Laboratory Equipment 37
4.3.1 The Analytical Balance 37
4.3.2 Pipettes 41
4.3.3 Volumetric Flasks 44
4.3.4 Burettes 47

4.4 How to Make Solutions and Dilutions 47
4.5 Calibration of Analytical Methods 49
4.6 Errors, Accuracy, and Precision 50
4.6.1 Systematic and Random Errors 50
4.6.2 Accuracy and Precision 51
4.7 Statistics 52
4.7.1 Mean Value and Standard Deviation 52
4.7.2 Confidence Intervals 54
4.7.3 Comparison of Means with a t-Test 55
4.7.4 Q-Test to Reject Outliers 56
4.7.5 Linear Regression with the Method of Least Squares 57
4.7.6 How to Present an Analytical Result 58
4.8 Some Words and Concepts 62
4.8.1 Analysis and Determination 62
4.8.2 Sample Replicates and Measuring Replicates 62
4.8.3 Interference 62
4.8.4 Blind Samples 62
5 Titrimetric Methods 65
5.1 Introduction 65
5.2 Acid–Base Titrations 72
5.3 Acid–Base Titrations in Non-Aqueous Media 75
5.4 Redox Titrations 78
5.5 Other Principles of Titration 81
5.6 Summary 82
6 Introduction to Spectroscopic Methods 83
6.1 Electromagnetic Radiation 83
6.2 Molecules and Electromagnetic Radiation 85
6.3 Atoms and Electromagnetic Radiation 86
6.4 Summary 88
7 UV Spectrophotometry 89
7.1 Principle of Quantitative Determination 89
7.2 Principle of Identification 94
7.3 Which Substances Have Strong UV Absorbance? 95
7.4 Instrumentation 95
7.5 Practical Work and Method Development 99
7.6 Areas of Usage and Performance 101
7.7 System Testing 101
7.8 Summary 102
8 IR Spectrophotometry 103
8.1 IR Spectrophotometry 103
8.3 Scope 109
vi Table of Contents

8.4 Instrument Calibration 109
8.5 NIR Spectrophotometry 110
8.6 Applications 112
8.7 Summary 114
9 Atomic Spectrometry 115
9.1 Atomic Absorption Spectrometry 115
9.3 Applications and Performance 121
9.4 Practical Work and Method Development 122
9.5 Atomic Emission Spectrometry 123
9.7 Summary 124
10 Chromatography 127
10.1 General Principles 127
10.2 Retention 131
10.3 Column Efficiency 133
10.4 Selectivity 135
10.5 Peak Symmetry 136
10.6 Resolution 138
10.7 Chromatographic Techniques 140
10.8 Summary 140
11 Chromatographic Separation Principles 141
11.1 General Introduction 141
11.2 Normal Phase Chromatography 142
11.2.1 Silica 142
11.2.2 Interactions 143
11.2.3 Order of Elution 144
11.2.4 Other Stationary Phases 145
11.2.5 Mobile Phases 146
11.2.6 Summary of Normal Phase Chromatography 147
11.3 Reversed Phase Chromatography 148
11.3.1 Stationary Phases 148
11.3.2 Retention Mechanisms 150
11.3.3 Mobile Phases 152
11.3.4 Ion-Pair Chromatography 155
11.3.5 Summary of Reversed Phase Chromatography 155
11.4 Hydrophilic Interaction Chromatography 156
11.5 Chiral Separations 156
11.6 Size Exclusion Chromatography 158
11.6.1 Principle 158
11.6.2 Summary of SEC 160
11.7 Ion Exchange Chromatography 160
Table of Contents vii

12 Thin-Layer Chromatography 163
12.1 Introduction 163
12.2 Apparatus 164
12.3 TLC Plates 166
12.4 Stationary Phases 166
12.5 Mobile Phases 167
12.6 Chromatographic Development 168
12.7 Detection 169
12.8 Applications of TLC 169
12.9 Quantitative Analysis and Instrumentation 170
12.10 Summary 171
13 High Performance Liquid Chromatography 173
13.2 The Chromatographic Separation Process 175
13.3 The Column 177
13.4 Pumps 180
13.5 Detectors 182
13.5.1 UV detector 182
13.5.2 Fluorescence Detector 184
13.5.3 Electrochemical Detector 186
13.5.4 Refractive Index, Evaporative Light Scattering
and Corona Discharge Detectors 186
13.5.5 Combination of Detectors 187
13.6 Injectors 187
13.7 Mobile Phases 188
13.8 Solvents for Sample Preparation 189
13.9 Reporting the Results 189
13.10 Summary 190
14 Gas Chromatography 191
14.2 Apparatus 192
14.3 Temperature 193
14.4 Carrier Gas 195
14.5 Stationary Phases 196
14.6 Selectivity in GC 197
14.7 Columns 198
14.7.1 Capillary Columns 198
14.7.2 Packed Columns 199
14.8 Injection Systems 200
14.8.1 Injection Systems for Capillary Columns 200
14.8.2 Injection Systems for Packed Columns 202
14.9 Detectors 203
14.9.1 Flame Ionization Detector 203
14.9.2 Nitrogen–Phosphorus Detector 203
viii Table of Contents

14.9.3 Thermal Conductivity Detector 204
14.9.4 Electron Capture Detector 204
14.9.5 Mass Spectrometry Detector 206
14.10 Derivatization 206
14.10.1 Silylation 206
14.10.2 Alkylation 207
14.10.3 Acylation 207
14.11 The Uses of GC 208
14.12 More Advanced GC techniques 209
14.13 Summary 209
15 Capillary Electrophoresis 211
15.1 Principle and Theory 211
15.2 Electroosmotic Flow 213
15.4 The Capillary 217
15.5 Sample Introduction 218
15.6 Capillary Zone Electrophoresis; an Example 221
15.7 Micellar Electrokinetic Chromatography 222
15.8 Chiral Separations 224
15.9 Coated Capillaries 225
15.10 Non-Aqueous CE 229
15.11 Summary 229
16 Mass Spectrometry 231
16.2 Basic Theory 233
16.3 Electron Ionization 236
16.4 Identification using Electron Ionization Spectra 237
16.5 Characterization of Totally Unknowns using Electron
Ionization Spectra 239
16.6 Chemical Ionization 244
16.7 Electrospray Ionization 246
16.8 Atmospheric Pressure Chemical Ionization 247
16.9 High-Resolution Mass Spectrometry 248
16.11 Chromatography Coupled with Mass Spectrometry 253
16.12 Quantitative GC-MS and LC-MS 256
16.13 Areas of Usage and Performance 257
16.14 Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry 257
16.15 Inductively Coupled Plasma Mass Spectrometry 258
16.16 Summary 259
17 Miscellaneous Chemical Techniques 261
17.1 Potentiometric Determination of Ions using Ion-Selective Electrodes 261
17.2 Paper Chromatography 263
Table of Contents ix

17.3 Supercritical Fluid Chromatography 264
17.4 Gel Electrophoresis 265
17.5 Iso-Electric Focusing 267
17.6 Nuclear Magnetic Resonance Spectrometry 268
17.7 Raman Spectrometry 270
18 Sample Preparation 273
18.1 Why is Sample Preparation Required? 273
18.2 Main Strategies 274
18.3 Recovery and Enrichment 276
18.4 Protein Precipitation 278
18.5 Liquid–Liquid Extraction 279
18.5.1 Fundamentals 279
18.5.2 A Closer Look at the Theory 279
18.5.3 Extraction Solvents 282
18.5.4 Calculation of Recovery 283
18.5.5 Multiple Extractions 285
18.5.6 LLE with Back-Extraction 286
18.6 Solid–Liquid Extraction 287
18.7 Solid Phase Extraction 287
18.7.1 Fundamentals 287
18.7.2 The SPE Column 288
18.7.3 Conditioning 289
18.7.4 Equipment 290
18.7.5 Reversed-Phase SPE 290
18.7.6 Secondary Interactions 292
18.7.7 Ion Exchange SPE 293
18.7.8 Mixed-Mode SPE 295
18.7.9 Normal-Phase SPE 297
18.8 Summary 298
19 Analytical Chemical Characteristics of Selected Drug Substances 299
19.1 Amitriptyline and Mianserin 299
19.2 Morphine and Codeine 301
19.3 Ibuprofen and Naproxen 302
19.4 Furosemide 304
19.5 Paracetamol (Acetaminophen) 306
19.6 Neutral Drugs 307
20 Quantification and Quality of Analytical Data 309
20.1 Peak Height and Peak Area 309
20.2 Calibration Methods 310
20.2.1 External Standard Method 310
20.2.2 Internal Standard Method 313
20.2.3 Standard Addition 314
20.2.4 Normalization 314
x Table of Contents

20.3 Validation 314
20.3.1 Analytical Procedure 317
20.3.2 Accuracy 317
20.3.3 Precision 318
20.3.4 Specificity 320
20.3.5 Detection Limit 320
20.3.6 Quantification Limit 321
20.3.7 Linearity and Range 321
20.3.8 Robustness 323
20.3.9 Test Methods in the European Pharmacopeia 325
20.4 System Suitability 325
20.4.1 Adjustment of Chromatographic Conditions 326
21 Chemical Analysis of Drug Substances 327
21.1 What is a Pharmaceutical Raw Material, how is it Produced
and why must it be Controlled? 327
21.2 The Pharmacopoeias – the Basis for Control of Pharmaceutical
Raw Materials 330
21.3 Which Contaminants are Found in Raw Materials,
What are the Requirements in a Maximum Content and Why? 337
21.3.1 Well Defined Chemical Compounds 339
21.3.2 Mixtures of Organic Compounds 343
21.4 How to Check the Identity of Pharmaceutical Raw Materials 344
21.4.1 Overview of the Identification Procedures 344
21.4.2 Techniques used for the Identification of Well Defined
Chemical Compounds 344
21.4.2.1 Infrared Absorption Spectrophotometry 344
21.4.2.2 Ultraviolet and Visible Absorption
Spectrophotometry 347
21.4.2.3 Thin-Layer Chromatography 351
21.4.2.4 Melting Point 352
21.4.2.5 Polarimetry 353
21.4.2.6 High Performance Liquid Chromatography 356
21.4.2.7 Chloride and Sulfate Identification 359
21.5 How to Test for Impurities in Pharmaceutical Raw Materials 359
21.5.1 Main Purity Tests for Well Defined Chemical Compounds 359
21.5.1.1 Appearance of Solution 361
21.5.1.2 Absorbance 364
21.5.1.3 Acidity/Alkalinity 365
21.5.1.4 Optical Rotation 365
21.5.1.5 Related Substances 366
21.5.1.6 Solvent Residues 372
21.5.1.7 Foreign Anions 372
21.5.1.8 Cationic Impurities 376
21.5.1.9 Loss on Drying 378
21.5.1.10 Determination of Water 379
Table of Contents xi

21.5.2 Purity Tests for Raw Materials of the Type
of Mixtures of Organic Compounds 382
21.5.2.1 Oxidizing Substances 383
21.5.2.2 Acid Value 383
21.5.2.3 Hydroxyl Value 384
21.5.2.4 Iodine Value 384
21.5.2.5 Peroxide Value 385
21.5.2.6 Saponification Value 385
21.5.2.7 Unsaponifiable Matter 386
21.5.2.8 Other Tests 386
21.5.3 Identification of the Raw Materials of the Type of Mixtures
of Organic Compounds 388
21.6 How to Determine the Purity of Pharmaceutical Raw Materials 389
21.6.1 Acid–Base Titration in Aqueous Environment 389
21.6.2 Acid–Base Titration in a Non-Aqueous Environment 393
21.6.3 Redox Titrations 396
21.6.4 High Performance Liquid Chromatography 396
21.6.5 UV spectrophotometry 401
21.7 How to Control Compounds for Which no Pharmacopoeia
Monograph Exists 402
21.8 How are Ph.Eur. and USP Updated? 402
22 Chemical Analysis of Final Pharmaceutical Products 405
22.1 Quality Control of Final Pharmaceutical Products 405
22.2 Monographs and Chemical Testing 406
22.3 Identification of the Active Pharmaceutical Ingredient 412
22.4 Assay of the Active Pharmaceutical Ingredient 427
22.5 Chemical Tests for Final Pharmaceutical Products 446
22.5.1 Test for Related Substances 446
22.5.2 Uniformity of Content 449
22.5.3 Dissolution 451
23 Analysis of Drugs in Biological Fluids 453
23.1.1 Drug Development 453
23.1.2 Therapeutic Drug Monitoring 455
23.1.3 Forensic and Toxicological Analysis 456
23.1.4 Doping Control Analysis 457
23.2 The Biological Matrix 458
23.3 Bioanalytical Methods 460
23.3.1 Sampling 460
23.3.2 Sample Preparation 461
23.3.3 Protein Precipitation 462
23.3.4 Liquid–Liquid Extraction 463
23.3.5 Solid-Phase Extraction 463
23.3.6 Separation 464
xii Table of Contents

23.3.7 Detection 464
23.3.8 Calibration and Quantification 465
23.4 Examples 466
23.4.1 Sample Preparation 466
23.4.1.1 Sample Preparation Procedure by LLE 466
23.4.1.2 Comments to the Procedure 466
23.4.1.3 Sample Preparation Procedure by LLE
and Back Extraction 467
23.4.1.5 Sample Preparation Procedure by SPE 467
23.4.1.7 Sample Preparation Procedure by Protein
Precipitation 468
23.4.2 Quantitative Determination 468
23.4.2.1 Quantitative Determination of Amitriptyline
in Serum by LC-MS 468
23.4.2.3 Determination of Valproic Acid in Serum
by GC-MS 471
23.4.3 Identification 472
23.4.3.1 Sample Preparation Procedure for Unknown
Screening by Mixed Mode Cation Exchange 472
23.4.3.3 GC-MS Procedure for Unknown Screening 473
23.4.3.5 LC-MS-MS Procedure for Unknown Screening 475
Index 477
Table of Contents xiii

Preface
This textbook, entitled “Introduction to Pharmaceutical Chemical Analysis”, is the first
textbook giving a systematic introduction to the chemical analysis of pharmaceutical
raw materials, finished pharmaceutical products, and drugs in biological fluids, as
carried out in the pharmaceutical laboratories worldwide. In addition to this, the
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. The textbook is primarily intended for the pharmacy student,
to teach the requirements in “analytical chemistry” for the 5-year pharmacy curriculum,
but the textbook is also intended for analytical chemists moving into the field of
pharmaceutical analysis.
The field of pharmaceutical analysis is very broad and challenging to define and limit, and
thereforewe have made priority to some major areas offocus. First, the textbook has a major
focus on low-molecular-weight drug substances. This “low-molecular” focus was selected
to limit the size of the book, but also because we have a clear ambition of linking all the
discussions of the different chemical techniques and methods to the chemical properties of
the drug substances. We feel this is very important for a good understanding, and this
understanding is much easier to obtain for low-molecular drug substances than for
macromolecules. Thus, although macromolecules, like peptides and proteins, are also
used as drugs, they are not discussed in this textbook.
Second, this textbook has a major focus on pharmaceutical routine applications,
including how drug substances are analyzed as raw materials prior to pharmaceutical
production, how they are analyzed in finished pharmaceutical products, and how they are
analyzed in patient samples following administration. This “routine” focus was also
selected to limit the size of the book. Thus, applications of pharmaceutical analysis during
development of new drugs and during pharmaceutical research have not been discussed.
However, many of these applications are similar to the routine applications in terms of
fundamental understanding, and as long as the readers understand the routine applications,
they also have the best fundament to understand the more advanced applications.
Third, the textbook has a major focus on classical analytical techniques such as titration,
chromatography, electrophoresis, and spectroscopy. This “classical” focus was a natural
consequence of the “low-molecular” and “routine” focuses discussed above. Additionally,
we feel that discussing the most important techniques comprehensively is much more
valuable for the reader than mentioning all the techniques involved in pharmaceutical
analysis. In future revisions however, we may include more new analytical techniques as
they are gradually included as official methods in the international pharmacopoeias.

This textbook first gives a short introduction to the field of pharmacy, to the field of
pharmaceutical analysis, and to the regulations and guidelines relevant for the field
(Chapters 1 and 2). This is an important motivation for the reader, but is also a basis for
understanding the “landscape” of pharmaceutical analysis. Then, the textbook gives a short
chemistry course to make sure that the reader is at an appropriate level in terms of chemical
understanding (Chapter 3). This is very important, as we try to link every discussion later in
the book to chemical structures. The third part of the book (Chapters 4–20) describes all the
analytical techniques (tools). In this part of the textbook, we basically fill up the tool box to
be used in the final part. In the latter (Chapters 21–23), we describe how the different tools
(analytical techniques) are used for the analysis of pharmaceutical raw materials, for the
analysis of finished pharmaceutical products, and for the analysis of patient samples. Unlike
many other textbooks, we have no student problems. However, we have replaced the student
problems with many real examples, and for each example, we have given priority to
fundamental understanding of the chemistry and to calculations. Thus, in this textbook, you
learn how to calculate the concentration of a certain drug in your sample based on the
number displayed on your analytical instrument. Welcome to the challenging world of
pharmaceutical analysis!
Copenhagen/Oslo, April 2011
Steen Honor
e Hansen Knut Einar Rasmussen Stig Pedersen-Bjergaard
University of Copenhagen University of Oslo University of Oslo
University of Copenhagen
xvi Preface

1
Introduction to Pharmaceutical Analysis
This chapter briefly reviews the life of medical products and the manufacture of medical
products according to international regulations and guidelines. Based on this review the
major areas and usage of pharmaceutical analysis are identified.
1.1 Applications and Definitions
The European Pharmacopeia defines a medical product as:
(a) Any substance or combination of substances presented as having properties for
treating or preventing disease in human beings and/or animals; or (b) any substance
or combination of substances that may be used in or administered to human beings
and/or animals with a view either to restoring, correcting or modifying physiological
functions by exerting a pharmacological, immunological or metabolic action, or to
making a medical diagnosis.
A medical product contains a substance that is pharmacologically active and that
substance is called the active ingredient (AI) or active pharmaceutical ingredient (API)
defined as follows:
Any substance intended to be used in the manufacture of a medicinal product and
that, when so used, becomes an active ingredient of the medicinal product. Such
substances are intended to furnish a pharmacological activity or other direct effect
in the diagnosis, cure, mitigation, treatment or prevention of disease, or to affect the
structure and function of the body.
Introduction to Pharmaceutical Chemical Analysis, First Edition. Steen Honoré Hansen, Stig Pedersen-Bjergaard
and Knut Rasmussen.
Ó 2012 John Wiley Sons, Ltd. Published 2012 by John Wiley Sons, Ltd.

An herbal medical product is:
A medicinal product, exclusively containing as active ingredients one or more herbal
drugs or one or more herbal drug preparations, or one or more such herbal drugs in
combination with one or more such herbal drug preparation.
Drug substances are administered very rare as the pure active substance. Typically the
active substance and excipients (auxiliary substances) are combined into dosage forms to
produce the final medical product. An excipient is:
Any constituent of a medicinal product that is not an active substance.
Adjuvants, stabilizers, antimicrobial preservatives, diluents, antioxidants, for example,
are excipients.
The dosage form can be, for example, a tablet or a capsule or syrup to be administered
orally, injections that are for parenteral administration into the body, or ointments for
topical administration. Figure 1.1 shows typical dosage forms.
Formulation is the process in which different chemical substances, including the active
ingredient and excipients are combined to produce a final medical product. It involves
developing a preparation of the drug that is both stable and acceptable to the patient. For
orally taken drugs this usually involves incorporating the drug and excipients in a solid
Tablets
Capsules
Syrup for oral
administration
Solution for injection
Figure 1.1 Different dosage forms
2 Introduction to Pharmaceutical Chemical Analysis

dosage form such as a tablet or a capsule or a liquid dosage forms such as a syrup. The main
function of excipients is summarized as follows:
. Ensure that the preparation has a shape and size that is easy to use for the patient;
. Ensure that the active substance is optimally adsorbed in the patient;
. Ensure that the preparation has an acceptable shelf life;
. Ensure that the preparation does not have an unpleasant taste or odor;
. Ensure easy production.
There is a wide spectrum of different excipients, which varies widely from preparation
to preparation. To illustrate this, Table 1.1 shows the excipients of a tablet and syrup
which both contain paracetamol as the active ingredient. Paracetamol is both an analgesic
(an ¼ no, algesis ¼ pain) and a antipyretic (anti ¼ against, pyretos ¼ fever), which means
that it is used against pain and fever.
The tablets, which in this example, have a total weight of 285 mg contains 250 mg of
paracetamol (active ingredient), while the remaining 35 mg is made up of excipients.
The excipients are a disintegrating agent, a lubricant, a glidant and a binder. Binders,
lubricating and gliding agents are added to facilitate manufacture. A disintegrating agent
ensures rapid disintegration of the tablet in the stomach.
Paracetamol syrup, which contains 24 mg/ml of paracetamol, is composed mainly of
water. In addition, it is added sweetening and flavoring agents for better taste. Antimicrobial
preservatives and antioxidants are added to prevent bacterial growth and chemical degra-
dation. In addition agents that increase the viscosity and stabilizes the pH are added.
Medical products may be divided into over the counter drugs (OTC), which may be
sold directly to the consumer in pharmacies and supermarkets without restrictions, and
Table 1.1 Excipients of a paracetamol tablet and a paracetamol syrup
Content Amount (mg) Function
Tablet (weight 285 mg)
Paracetamol 250 Active ingredient
Hydroxypropyl cellulose Binder
Maize starch Disintegrant
Talcum Glidant
Magnesium stearate Lubricant
Syrup (volume 1 ml)
Paracetamol 24 Active ingredient
Sorbitol Sweetener
Glycerol Sweetener
Polyvinylpyrrolidone Thickening agent
Saccharine sodium salt Sweetener
Methylparabene Preservative
Ethylparabene Preservative
Propylparabene Preservative
Sodium metabisulfite Antioxidant
Citric acid pH regulator
Sodium citrate pH regulator
Strawberry aroma Flavoring agent
Water Solvent
Introduction to Pharmaceutical Analysis 3

prescription only medicine (POM) that must be prescribed by a licensed practitioner.
Medical products are predominantly produced by the pharmaceutical companies, only in
rare occasions are pharmaceutical products produced in hospitals and in pharmacies.
New products are often patented to give the developer exclusive right to produce them.
Those that are not patented or with expired patents, are called generic drugs since they can
be produced by other companies without restrictions or licenses from the patent holder.
According to the European Federation of Pharmaceutical Industries and Associations
(EFPIA), the pharmaceutical industry in Europe employed some 630 000 people, including
110 000 in research and development, in 2009. The trade surplus was Euro 55 200 million,
and Euro 26 000 million was spent on pharmaceutical research and development. The
retail value of the pharmaceutical market was Euro 215 000 million, which is just under
30% of the world market.
1.2 The Life of Medicines
Figure 1.2 outlines a typical industrial production of a pharmaceutical product.
Production starts by ordering the current active ingredient and the necessary starting
materials. In some cases, the company produces some of the ingredients, but most commonly
they are produced elsewhere by various industrial raw material suppliers. The raw materials
arrive in relatively large quantities (1–500 kg) and are typically packed in cardboard drums
or in plastic containers. Figure 1.3 shows an example of a received batch of raw material in
the photo gallery from a manufacturing facility.
Arrival of starting and packaging materials
Manufacturing
Sampling of starting materials
Filling
Labelling
Packaging
Documentation
and
Control
Documentation and control of finshed product
and product release
Figure 1.2 Illustration of the manufacturing of a medical product

Figure 1.4 shows an outline of some areas found in a manufacturing facility.
Upon arrival the raw materials are registered in the manufacturer’s documentation
system, tagged with internal labels and stored in a separate area of the warehouse or in
a separate room where they are in quarantine until they are released for production. Samples
of the raw materials are collected and analyzed to ensure that the raw materials are of
Figure 1.3 Photo gallery of a manufacturing facility: arrival of raw materials, weighing,
sampling, tablet pressing, filling, labelling, and packaging. Reproduced with permission from
Fagbokforlaget

satisfactory quality. This is the first of several important areas where pharmaceutical
analysis are vital. We focus further on this in Chapter 21. If the results are in accordancewith
the specifications of the manufacturer the raw materials are labeled as released materials,
and transferred to the production facility. Production starts with weighing or measuring
the active ingredient and excipients in appropriate amounts for the subsequent production
(see Figure 1.3). Then, the raw materials are transferred to the manufacturing machinery.
Manufacture of tablets uses several types of equipment such as machinery for granulation,
drying and tablet pressing (see Figure 1.3). The manufacture of liquid dosage forms is
carried out in large tanks, while the production of ointments and creams are carried in
large pots with agitator and heating. When the product leaves the production site samples
Figure 1.3 (Continued)

Figure 1.3 (Continued)

for a comprehensive finished product control is collected. A number of analytical tests
are made, and this is another important field of pharmaceutical analysis which is discussed
in detail in Chapter 22. The products are in quarantine until the results of the testing show
compliance with specifications. The released product is filled in appropriate containers
(filling; see Figure 1.3), the containers are marked with labels (labeling; see Figure 1.3) and
the containers are packed in cardboard boxes (packaging; see Figure 1.3). Assessment of the
finished product embrace all relevant factors, including production conditions, results of in-
process testing, a review of manufacturing (including packaging), documentation, compli-
ance with Finished Product Specifications and examination of the final finished pack.
As shown above, the industrial production of pharmaceuticals is a comprehensive process
that takes place over many different steps. Typically, production is a batch process, which
means that the products are made in limited batches. Each time a batch is produced a new
manufacturing process is started from the beginning with new starting materials. Between
each production of a given product, the equipment is often used for the production of other
products. Consequently the production facility must be cleaned thoroughly between each
batch to prevent the material from an earlier production contaminating other products
(cross contamination).
After leaving the manufacturer the products are sent to pharmaceutical wholesalers,
which provide for their further distribution to pharmacies, hospitals or other retailers
where they becomes available to the patients. Medicines have a broad scope of usage, and
are used against many types of illness and pain in various parts of the body.
At the start of medication, it is common to follow a standard treatment, but it is well
known that different patients may exhibit large variations in response. In such cases it is
important to adjust the dosage. One example is the treatment of hypertension. The dosage
may be reduced when the blood pressure is too low and the dosage may be increased
when the blood pressure is too high. For other types of treatment, such as depression,
psychosis and epilepsy, the measurement of effect is difficult; and in those cases therapeutic
drug monitoring (TDM) is advised. In TDM blood samples are collected and analyzed
to ensure that the drug level is appropriate. The analysis of drugs in biological fluids is
called bioanalysis. In addition to TDM, bioanalysis is crucial in drug development
programs, in forensic and toxicological analysis and in doping control testing in sports.
Bioanalysis is a third major area of pharmaceutical analysis, which is discussed in
Chapter 23.
1.3 The Quality of Medical Products
The purchaser of food and drink normally discovers that a product is associated with a
significant quality problem if has either an abnormal taste, unusual smell or a look that
seems abnormal. Medicines are, however, special. For example, there is no way patients can
decide whether a tablet contains the active ingredient, whether it is the correct dosage,
or whether any contaminants or degradation products are present. The patient is not in a
position to recognize that a medicine is incorrect or defective. The patient literally takes
medicines entirely on trust and is at the end of a chain of implicit trust which extends
back through administering, dispensing, prescribing and distributing, right back to those

responsible for manufacture of the product. It is therefore mandatory that the pharma-
ceutical industry maintains the highest standards of quality in the development, manufac-
ture and control of medical products.
Government heath authorities are naturally concerned about the quality of medicines,
and regulate the development, manufacture and marketing of medical products by a number
of laws and guidelines. These are discussed in Chapter 2. The regulations and guidelines
are to assure the safety, protection and well being of the consumer or patient. Two important
areas are:
. Marketing authorization of medical products;
. Manufacturing authorization of medical products.
Marketing authorization, also called a license, is required before any medicine can be used
to treat people. Only when the regulatory bodies are satisfied that the product works as it
should, and that it is acceptably safe, is it given a marketing authorization or product license.
The regulatory system also imposes rigorous standards on manufacturers. Manufacturing
authorization is required by all pharmaceutical manufacturers and ensures that only
authorized manufacturers manufacture all licensed products. Competent authorities regu-
larly inspect the activities of the manufacturers and annually collect samples of marketed
medicines for assessment of quality. National Medical Control Agencies have the power to
QC- Batch release
QC - Analytical
Validation
Shipping and Receiving
Regulatory Affairs
Facilities Office
Manufacturing
Packaging and Labelling
Product
Development
Laboratory
Quality
Assurance
Figure 1.4 Outline of some areas found in a manufacturing facility

withdraw a product from the market and to suspend production. These Agencies can also
prosecute a manufacturer if the law has been broken.
The holder of a manufacturing authorisation must manufacture medical products so as
to ensure that they are fit for their intended use. The products should comply with the
requirements of the marketing authorization and should not put patients at risk due to
inadequate safety, quality or efficacy. The attainment of quality is the responsibility of the
management, and it relies on a comprehensively designed and correctly implemented
system of Quality Assurance (QA) incorporating Good Manufacturing Practice (GMP) and
Quality Control (QC). The system should be fully documented. The basic concepts of QA,
GMP and QC are inter-related, as shown in Figure 1.5.
Quality Assurance is a wide-ranging concept, which covers all matters that influence
the quality of a product. It is the sum of all organized arrangements that are made to
ensure that medical products are of the quality required for their intended use. Quality
Assurance therefore incorporates GMP, which ensures that products are consistently
produced and controlled to the quality standards appropriate to their intended use and
as required by the Marketing Authorization. The basics of GMP are that all manufacturing
processes are clearly defined, systematically reviewed and shown to be capable of
consistently manufacturing products of the required quality. Quality Control is part of
GMP and is concerned with sampling, specifications and testing, and with the organization,
documentation and release procedures. Release procedures should ensure that the necessary
and relevant tests are actually carried out and that materials are not released for use, nor
QA
GMP
QC
Figure 1.5 Illustration of the QA/GMP/QC inter-relationship

products released for sale or supply, until their quality has been judged to be satisfactory.
The independence of the Quality Control Department from other departments is considered
fundamental. Quality Control is not confined to laboratory operations, but must be involved
in all decisions that may concern the quality of a product.
According to European regulations each batch of finished product must be certified by a
Qualified Person (QP) before being released for sale or supply.
Before certifying a batch the QP should ensure that at least the following requirements
have been met:
. The batch and its manufacture comply with the provisions of the marketing authorization.
. Manufacture has been carried out in accordance with GMP.
. The principal manufacturing and testing processes have been validated (validation is
defined as the documented act of demonstrating that processes will consistently lead to
the expected results).
. Any deviations or planned changes in production or quality control have been authorized
by the persons responsible in accordance with a defined system.
. All the necessary checks and tests have been performed.
. All necessary production and quality control documentation has been completed.
. The QP should in addition take into account any other factors of which he is aware
which are relevant for the quality of the batch.
Good documentation constitutes an essential part of the quality assurance system and
constitutes a vital part of batch release and certification by the QP. Clearly written documen-
tation and standard operating procedures (SOP) prevent errors from spoken communication
and permit tracing of batch history. The documentation include:
. Specifications that in detail describe the requirements that must be fulfilled prior to
quality evaluation;
. Manufacturing formulae, processing and packaging instructions;
. Procedures that give directions for performing operations such as cleaning, sampling
testing and equipment operation;
. Records providing a history of each batch or product.
The batch documentation shall be retained for at least one year after the expiry date of
the batches.
1.4 Summary
Government health authorities have regulated the development, manufacture and mar-
keting of medical products by a number of laws and guidelines to assure the safety,
protection and well being of the patient. Market authorization is required before any
medical product can be marketed and only authorized manufacturers can produce
authorized products. Authorized manufacture is based on a correctly implemented system
of Quality Assurance incorporating Good Manufacturing Practice and Quality Control.

2
International Pharmacopoeias,
Regulations and Guidelines
This chapter reviews a number of laws, guidelines and regulations important in the
pharmaceutical production and thus also for the subject pharmaceutical analysis. The
manufacture of drugs is international and therefore this chapter focuses on international
affairs. But also national laws, guidelines, and regulations are important for manufacturers
in order to fulfil the requirements of the relevant authorities. All these laws, guidelines and
regulations are subject to regular updates and the latest edition should therefore always
be consulted.
2.1 Overview of Legislation
At the end of Chapter 1 it was mentioned that drug manufacturers have to fulfil require-
ments given in a number of laws, guidelines and regulations that will ensure high quality
of pharmaceutical preparations. Production and the related control of medicines are
often governed by the following two laws on a national basis:
. Law on medicinal products (Medicines Act);
. Law on pharmacy (Pharmacy Act).
The Medicines Act regulates, among other things, the requirements for medicines, clinical
trials (the testing of drugs on humans), the manufacturing process, import, wholesale sales,
retail sales and the advertising of medicines. This chapter focuses on what is relevant
in connection to drug development and production conducted in the pharmaceutical
industry and in pharmacies. We first discuss what is applicable for Proprietary Medicinal
Products manufactured industrially, while medicinal products produced in pharmacies
(stock production, small-scale production) are treated at the end of this chapter.
and Knut Rasmussen.

2.2 Legislation and Regulations for Industrial Production
A key point in the Medicines Act is that all medicinal products to be sold in a country must
have a marketing authorization granted by the local authorities (Ministry of Health). In the
EU a common legislation is available if a pharmaceutical company wants to register its
products in more than one EU country. This legislation is managed by the European
Medicines Agency (EMA). In the United States the Food and Drug Administration (FDA) is
the regulatory authority for the control of drugs.
The drug manufacturer or the importer of the drug is responsible for filing the application
for marketing authorization. The Ministry of Health through the State Medicines Agency
is responsible for evaluating the application based on the stated quality, safety and efficacy
of the drug. The market authorization is granted for five years and may thereafter be
renewed. The marketing authorization may be withdrawn before the expiration by the
Ministry of Health if:
. The product is no longer considered to meet the requirements for quality, safety or efficacy.
. The product does not have the specific qualitative or quantitative composition.
. The provisions that apply to pharmaceutical preparations have not been observed.
This means that a drug may be withdrawn from the market if there are problems associated
with its use, or if the composition of the preparation does not comply with the marketing
authorization. The Medicines Act contains no detailed information about what an applica-
tion for marketing authorization shall contain, but some headlines are given in Table 2.1.
All analytical chemical control procedures and data for drug analysis that should be
performed to ensure the quality of the forthcoming product are to be described in Section 2
of the application for marketing authorization (chemical, pharmaceutical and biological
documentation). No further information about this is givenin the pharmaceutical regulation,
but the applicant has to refer to the EU directives and guidelines (for Europe) or to FDA
(for USA) where further specification of what should be included can be found.
Table 2.1 Requirements for the content of an application for marketing authorization
Point Description
1 Administrative information (name of applicant, name of manufacturer)
2 Name of the drug and its composition
3 Chemical, pharmaceutical, and biological documentation
4 Toxicological and pharmacological documentation
5 Clinical documentation
6 Expert reports
7 Proposals for advertisements
8 Proposed labeling preparations
9 Proposed leaflet
10 Proposals for prescription status
11 Documentation of the manufacturing authorization
12 Confirmation of fees paid to the Medicines Agency
13a
A copy of the marketing authorization for the drug in other EEA countries
14a
A copy of the advertisement and package leaflet approved in other EEA countries
a
Points 13 and 14 only apply within Europe.

Table 2.2 lists an excerpt of the type of information that must be included on chemical
evidence based on applicable EU directives.
As you can see from Table 2.2, detailed information has to be filed about the methods to be
used to control the raw materials before production, about the methods to be used during
production and the methods to be used for control of the finished product. In addition, the
application must contain the results from the test analysis of the different trial production
batches, with documentation that the methods used have been tested and found suitable for
use (validation). The entire control scheme based on drug analysis for the forthcoming
Table 2.2 Excerpts from the content requirements of the application for marketing
authorization for medicinal producton chemical evidence. Only the topicsmost relevant to drug
analysis are included
Topic Requirements
Control of active substance
(active pharmaceutical
ingredient; API)
Characteristics of the active substance and purity
requirements (specification)
Detailed description of the applicant’s chemical methods
for the confirmation of the identity of the active substance
for control of the purity
Detailed information on the active substance
Nomenclature of the active substance
Description of the active substance
Manufacturing method for the active substance
(chemical synthesis)
Quality control methods of the manufacturer of the
active substance
Known impurities in the active substance
Results of the chemical control of previously produced
batches of the active substance
Control of excipients Characteristics of the excipients and purity requirements
(specification)
Detailed description of the applicant’s methods for
confirmation of identity of the excipient
for control of the purity of the excipient
Information on excipients
Control of production
preparations
for control of production mixtures
Control of finished product Detailed description of the applicant’s chemical methods
for the confirmation of the identity and determination of
the content of the active substance in the preparation
for the confirmation of the identity of a dye (excipient)
for the determination of the levels of antimicrobial
additives and preservatives
Documentation (validation) of the suitability of all the
chemical methods
Results of the chemical control of previously produced
batches of the product
International Pharmacopoeias, Regulations and Guidelines 15

preparations should therefore be documented in the application for marketing approval,
and this, together with the rest of the information shown in Table 2.1 forms the basis for
the authorities evaluation of the product application. The requirements for toxicological,
pharmacological and clinical documentation are different when applying for new drugs
or for preparations containing known active substances, and especially in the former case,
applications for marketing approval require very comprehensive documentation. If a
pharmaceutical manufacturer later wish to make changes in product composition or
changes of control methods it must be approved by the Ministry of Health at the National
Medicines Agency.
The Medicines Act states that any medicinal products can only be market if granted a
marketing authorization, and the law also states that anyone who manufactures drugs must
have a manufacturing authorization from the National Medicines Agency, and that all
premises and laboratories shall be in accordance with good manufacturing practice (GMP).
The word “manufacturing” in the Medicines act refers to production, packaging, repacka-
ging, labeling, relabeling and release of drugs, as well as the necessary controls in connection
totheseactivities.Therefore analyticalchemicalworkisincludedinthe MedicinesAct andin
GMP guidelines. The Medicines Act gives only little detail on the areas of manufacturing
authorization and good manufacturing practice, and more information about this should be
found in, for example, regulations on the manufacture and import of drugs. This regulation
specifies that a manufacturing authorization applies to certain premises at the manufacturing
site and only for certain drugs or drug types. This means that a pharmaceutical manufacturer
producing several different products must have a marketing authorization for each product,
while in principle only one manufacturing authorization is needed. The consequence of this
regulation is that the pharmaceutical analysis performed in conjunction with quality control
of pharmaceutical production has to be performed by a manufacturer with manufacturing
authorization for pharmaceuticals; and pharmaceutical analysis, like the rest of the
manufacturing process, is subject to good manufacturing practice (GMP). Good Laboratory
Practice (GLP) is another set of rules that should be consulted, and FDA, OECD and EU all
have rules, directives and so on in this field.
In the directive “Regulation on the manufacture and import of drugs” only few details
on what GMP means are given, but in this case, refer to the EU Directive. GMP is a
comprehensive regulatory framework, and the main content is presented in Table 2.3.
Table 2.3 The main elements of the GMP/GLP regulations
Paragraph Description
1 Demands for a quality department and for quality control in all stages
of production
2 Requirements for staff
3 Requirements for premises and equipment
4 Documentation requirements
5 Requirements for production
6 Requirements for quality control
7 Requirements in connection with external contract work
8 Requirements for the withdrawal of products
9 Requirements for self-inspection

A brief discussion of the main headlines of the rules without going into further detail shall
be given.
Paragraphs 1, 2, 3, 4, 6 and 9 in Table 2.3 are of particular relevance for pharmaceutical
analysis relatedtoproduction.Paragraph 1oftheGMPregulations requiresallmanufacturers
of drugs to have an overall system of quality assurance throughout the company. Manu-
facturers are also required to employ enough competent persons with the necessary
theoretical and practical skills (paragraph 2). The manufacturer is required to have an
organization chart, where each employee’s duties and responsibilities are defined, so that
there is no uncertainty about the duties and responsibilities. Under item 3, specified detailed
requirements for premises and equipment used during the manufacture of pharmaceuticals
are given. This ensures that the final products are of high quality and at the same time the
working environment of the staff is in accordance with official requirements. The latter is
important since drug production involves handling highly potent compounds. These rules
also apply to premises and personnel involved in drug analysis. Documentation is essential
throughout the manufacturing process including chemical analysis and quality control
(paragraph 4). Written documentation shall ensure that no misunderstandings occur during
manufacture. All information related to the production and control of individual production
sections shall be easily recovered for example at release of production batches, at inspections
and in the case of withdrawal of production batches. The requirement for documentation
applies to all departments in a manufacturing company, and is thus also an important part of
everyday life for the staff that performs drug analysis. Paragraph 6 of the GMP regulations
requires all companies that manufacture drugs to have a quality control (QC) department.
Furthermore, the company should have a quality assurance (QA) organization. This should
be sufficiently staffed with competent personnel and shall be independent of the other
departments in the company controlling the different steps in the manufacturing process.
BasedondocumentationfromthevariousstepsinmanufacturingQA hasthe responsibilityto
release products (discussed in Chapter 1). QA is thus also responsible for the drug analyses
performed (raw material control, production control, product control) and to interpret the
results from these. Manufacturers are under Clause 9 ordered to perform self-inspection,
which means that staff in the company at regular intervals must inspect the various
departments and ensure that they work as intended according to GMP.
The regulations on the manufacture and import of drugs oblige the manufacturer to follow
the manufacturing operations that are approved for marketing authorization. This means that
raw material control (Chapter 21), production control and product control (Chapter 22) must
alwaysbeperformedforeachproductionbatchofthemethodsthatareapprovedformarketing
authorization.Inaddition,theregulationsrequirethateachmanufacturer appointsatleastone
qualifiedpersonwhoisapprovedbytheNationalMedicinesAgency.Onlyqualifiedpersonnel
can assess the results from raw material control, production control and product control, and
together with the other documentation (see Chapter 1) release the production lots.
2.3 Life Time of Drugs and Drug Substances
Although some drugs (e.g., tablets) often have a stability that may last for many years
there is a maximum life time for all drugs and drug substances of five years. This is to

avoid any discussion on how long a time drugs and drug substances may be stored. However,
drug substances should always comply with the monographs in the pharmacopoeias or
similar standards.
Drug preparations in general should at the time of production at the most deviate 5%
from the declared content. On the basis of stability testing a larger deviation of the lower
limit of up to 10% within the life time of the drug is accepted. The life time is also named the
shelf-life of the drug.
2.4 Pharmacopoeias
Standards for finished pharmaceutical products and for raw materials used for such products
have been given in pharmacopoeias for many years. It all started in the seventeenth century
and many countries have their own pharmacopoeia. Pharmacopoeia Nordica 63 was the
first pharmacopoeia to be authorized in more than one country (the Nordic countries).
Now the European Pharmacopoeia (Ph.Eur.) – first published in 1969 – is authorized in
36 European countries (2010) and also the United States Pharmacopoeia is used in a
number of other countries.
The standards (also called monographs) in the pharmacopoeias are the official require-
ments for raw materials and finished products. Standards should be compiled as minimum
requirements and shall ensure that medicines are generally of high quality. In order to obtain
marketing approval, it is important that the manufacturer’s own requirements for the
product (see Table 2.2), which are called specifications, meet the government standards.
When using the monographs in the pharmacopoeias it is important also to be aware of the
provisions and requirements given in some of the general texts in the pharmacopoeias (e.g.,
test for residual solvents in not specified in each individual monograph but is a general
requirement for all raw materials). In addition, general monographs on dosage forms are
given in the Ph.Eur. The main contents of Ph.Eur. are given in Table 2.4.
Besides the European Pharmacopoeia (Figure 2.1) most European countries have a
national supplement with some local provisions. The British Pharmacopoeia includes
monographs on finished products similar to what can be found in USP.
A large number of standards for drug substances which are not still under patent are
included in Ph.Eur. and USP, and in these standards requirements for identity and purity
are given. Besides these requirements the pharmacopoeias also provide detailed procedures
for how to perform the identification and control of purity for each drug substance. In
the application for the marketing authorization, it is usual that the manufacturer makes
Table 2.4 Main content of the European Pharmacopoeia (Ph.Eur.)
Item Description
1 Official purity requirements for raw materials (standards)
2 Official methods for control of raw materials
3 Official requirements for pharmaceutical formulations (dosage forms)
4 Official requirements for finished preparations (e.g., vaccines)

reference to this information for the documentation of the chemical raw materials
(see Table 2.2). In case of production the producer has to prove that the requirements
for identity and purity are met, and normally he will refer to the methods given in
the pharmacopoeia.
There are also many active pharmaceutical ingredients (APIs) that is patent protected
by a single pharmaceutical company. Since there is normally only one company who has
the right to produce such a drug substance it is not necessary to describe it in the
pharmacopoeia, but the requirements (specifications) and procedures used by that company,
and approved by the Ministry of Health, are generally similar to those found in similar
monographs in the pharmacopoeias. As the patent rights of pharmaceutical raw materials
run out, they are normally included in the pharmacopoeias if they still are in use as
marketed medicines.
2.5 International Harmonization
Due to globalization and expansion in international trade there is a growing need to
develop global quality standards for medicines. As standards are a vital instrument for
registration, market surveillance and the free movement and trade of medicines among
as many countries as possible, harmonization among the world’s three major pharma-
copoeias, the European Pharmacopoeia (Ph.Eur.), the Japanese Pharmacopoeia (JP)
and the United States Pharmacopoeia (USP), is an important task. This harmonization
process is now well under way in between the three pharmacopoeias but there is still long
way to go before the standardization of medicines becomes truely international.
Figure 2.1 The European Pharmacopoeia (Ph.Eur.) 7th edn, 2011

2.5.1 International Conference on Harmonization
In 1990 a trilateral program, the International Conference on Harmonization (ICH), for the
harmonization of testing of medicines among the European Union, the United States and
Japan was set up. This program aims to reduce the overall cost of pharmaceutical research
and development worldwide by avoiding the duplication of work such as the preparation of
dossiers and studies, thus reducing the time required for innovative medicines to become
available. This conference takes places twice a year with the location of meetings rotating
among Europe, Japan and the United States. ICH has also published a number of guidelines
which have become important document especially for the pharmaceutical industry. The
most important ICH guidelines for analytical chemists are presented in Table 2.5.
2.5.2 Pharmacopoeial Discussion Group
Further work on harmonization between the three pharmacopoeias is carried out by the
Pharmacopoeial Discussion Group (PDG). The PDG considers proposals made by national
associations of manufacturers of pharmaceutical products and excipients in order to select
general methods of analysis and excipient monographs for addition to its work program. To
promote these exchanges and synergy, since 2001 the PDG has organized, upon request,
hearings for representatives of the pharmaceutical and excipient industries. Harmonizations
of monographs on active pharmaceutical ingredients have now also been initiated.
2.6 Legislation and Regulations for Pharmacy Production
So far we have focused on industrially produced drugs, but since pharmaceutical prepara-
tions are also produced on a small scale in many pharmacies, the regulations for these will
briefly be discussed. For drugs made in pharmacies The Pharmacy Act applies as the overall
legislation. The Pharmacy Act states that the Ministry of Health can provide manufacturing
authorization to the pharmacy. This means that, like the pharmaceutical industry, the
pharmacy must also have a manufacturing authorization in order to produce drugs. The
Pharmacies Act further stipulates that the manufacture shall be in accordance with good
manufacturing practice (GMP). Further reading of the regulations on the manufacture of
medicines in pharmacies given by the Ministry of Health explain that the raw materials and
finished drugs shall meet the requirements of the Ph.Eur. Normally the pharmacy buys raw
Table 2.5 ICH guidelines of important relevance for the pharmaceutical analysis
Reference Description
Q1A(R2) Stability testing of new drug substances and products
Q2(R1) Validation of analytical procedures: text and methodology
Q2B Validation of analytical procedures: methodology [in Q2(R1)]
Q3A(R2) Impurities in new drug substances
Q3B(R2) Impurities in new drug products
Q3C(R4) Impurities: guideline for residual solvents

materials which have been tested for identity and purity according to Ph.Eur. or another
official standard by the supplier, and therefore the pharmacy only has to secure identity.
Smal-scale preparation of products having no marketing authorization does not require a
final testing for the content by drug analysis. The pharmacy is obliged to otherwise ensure
that the quality is consistent with the Ph.Eur., and this is executed by working according to
GMP. If the pharmacy produces preparations on a larger scale, which is called the stock
production, the pharmacy is required to perform final testing of the content.
2.7 Summary
Pharmaceutical research related to quality control of pharmaceutical production (raw
material control, production control, product control) is intended to verify that the current
specifications are met. The area of drug substances, drug development and drug production
is heavily controlled by a number of laws, regulations and guidelines. All chemical methods
for control of a preparation shall be documented in the marketing authorization. The
government standards are given in pharmacopoeias, and manufacturers are obliged to
follow these standards or to prepare their own specifications of similar or even better quality.
Drug manufacturers should have a manufacturing authorization, and production and
inspection shall be performed in accordance with good manufacturing practice (GMP).

3
Fundamental Chemical Properties,
Buffers and pH
Chemical analysis is an important part of the quality assessment of drugs. A deeper
understanding of the analytical method requires knowledge about both the analytical
technique and the chemical properties of the analytes. Therefore a basic knowledge of a
number of physicochemical properties of molecules is needed to be able to understand and
further develop analytical chemical methods. For example, knowledge of spectroscopic
principles and techniques is needed when the choice of the detection technique for a
given separation method is made; and knowledge of pH and pKa values is important for
the design of many sample preparation techniques. Spectroscopic techniques are treated
in separate chapters, and this chapter discusses some important physicochemical and
chemical properties of drug substances and focuses on how to utilize the information in an
analytical chemical context.
3.1 pH and pKa
pH is an expression for the acidity or alkalinity of an aqueous solution and the pH
concept is extremely important, having great influence on living organisms as well as in
analytical chemistry.
Water can react with itself to form a hydronium ion and a hydroxide ion:
2H2O a H3Oþ
þ OH
ð3:1Þ
This is called autoprotolysis as the water acts as an acid as well as a base. The autoprotolysis
constant is:
Kw ¼
½H3Oþ
½OH

½H2O2
¼ 1014
ð3:2Þ
and Knut Rasmussen.

indicating that only a very small amount of water is ionized. The concentration of the two
ions in pure water is therefore 107
M of each ion.
pH is defined as the negative logarithm to the activity, aH
þ
, or the concentration of the
hydrogen ions (equivalent to the hydronium ions), [Hþ
].
pH ¼ logðaHþ
Þ logð½Hþ
Þ ð3:3Þ
Strong acidsand strong bases are fully ionized in dilute aqueous solution and the activity and
their concentrations of [Hþ
] therefore can be considered to be identical.
Weak acids and weak bases are not completely ionized in aqueous solution and are
therefore in equilibrium with the unionized acid or base. When we ignore the weak
autoprotolysis of water we get the following general equation for a weak acid:
HA a Hþ
þ A
) Ka ¼
½Hþ
½A

½HA
ð3:4Þ
When acid in form of Hþ
is added to such a system, the Hþ
will partly be removed and form
HA, and ifa base is added itwill partly be neutralized byHþ
and more HAwill dissociate. pH
will thus be maintained in the solution. A system like this is called a buffer system and the
purpose of a buffer is to maintain the pH in the solution.
As:
pKa ¼ logKa ð3:5Þ
it is obvious that the highest buffer capacity is achieved at a pH value close to the pKa
value of the buffer substance. This brings us to a most useful equation called the
Henderson–Hasselbalch equation:
pH ¼ pKaþ log
½A

½HA
ð3:6Þ
At pH ¼ pKa equal concentrations of the acid and corresponding base are present. If the ratio
between HA/A
becomes 9 : 1 (only 10% base) pH decreases by one unit; and if the ratio
becomes 99 : 1 (1% base) the pH value decreases by two units.
Equivalent estimations can be performed when increasing the base content.
It is convenient to have an knowledge of pKa values of a number of functional groups
as presented in Table 3.1.
Table 3.1 Typical pKa values of functional groups
Functional group pKa Comment (depending on
chemical structure)
R-COOH, carboxylic acid 4–5 Can be lower (more acidic)a
R-NH2; R1,R2,NH and R1,R2,R3,N,
aliphatic amines
8–10 Can be lower (less basic)a
Ar-OH, phenols 8–10 Can be lower (more acidic)a
R-OH, alcohols 14 Can be considered as neutral
substances
R-SO2OH, sulfonic acid Approx. 1
a
This depends on other groups in the molecule.

The pKa value for bases refers to the protonated form of the bases. However, the basicity
of bases may also be expressed equivalent to the pKa of acids. In that case the term pKb is
used and:
pKaþ pKb ¼ 14 ð3:7Þ
3.2 Partition
Thepartitioningofsubstancesbetweenimmisciblephases(gas–liquid,gas–solid,liquid–liquid
or liquid–solid) is of major importance for a number of analytical chemical techniques.
Sample molecules introduced into the two-phase system will be exposed to a number of
interactions (diffusion, collisions, dipole–dipole interactions, hydrogen bonding, electro-
static interactions, etc., as illustrated in Table 3.2) in the two phases. The interactions taking
place are dependent on the physical and chemical nature of the analytes as well as of the
mobile and stationary phases and may result in different partition of analytes between the
two phases.
The partition between phases is also influenced by pH, and thus a thorough knowledge
of the pH concept including pKa as well as the distribution constant will ease the develop-
ment of analytical methods (e.g., chromatographic methods). The distribution constant is
dependent on the nature of the two phases as well as the temperature. If we want to alter
the partition between the two phases, we must change one of these variables. The
equilibrium distribution for a substance A is given by the partition ratio, also called the
distribution constant, KD:
KD ¼
½Aorg
½Aaq
ð3:8Þ
where [A]org is the concentration of compound in the organic phase and [A]aq is the
concentration of compound in the water phase.
The distribution constant is a constant relating to a specific molecular species, but often the
moleculescanbepresentasdifferentspecies,forexample,bydissociatingintheaqueousphase:
HAþ H2O a A
þ H3Oþ
ð3:9Þ
Table 3.2 Energy in bonds or of intermolecular forces
Type of bond or
intermolecular force
Example of interacting
molecules
Energy
kJ mol1
kcal mol1
Covalent RH2C–CH2R 400–1200 100–300
Ionic R4Nþ
OOC-R 200–800 50–200
Hydrogen bond H3COH.....HOH 20–50 5–12
Dipole–dipole C6H5Cl/H3CCN 12–40 3–10
Dipole–induced dipole H3CCN/C6H6 10–25 2–6
Dispersion/van der Waal C6H6/C6H14 5–20 1–5
Fundamental Chemical Properties, Buffers and pH 25

or dimerizing in the organic phase:
2HA a ðHAÞ2 ð3:10Þ
These equilibria areveryfast,anditistherefore appropriate to lookat the totaldistributionof all
the species of a compound between the two phases:
DC ¼
½HAorgþ ½A
orgþ ½ðHAÞ2org
½HAaqþ ½A
aqþ ½ðHAÞ2aq
¼
½HAtotalorg
½HAtotalaq
ð3:11Þ
The concentration distribution ratio, DC, between the two phases can also be expressed as the
mass distribution ratio, Dm, by multiplying the concentrations with the matching phase
volumes:
Dm ¼
½HAtotalorg Vorg
½HAtotalaq Vaq
¼
½ðamount of HAÞtotalorg
½ðamount of HAÞtotalaq

½ðamount of HAÞtotalstat
½ðamount of HAÞtotalmob
ð3:12Þ
Where Vorg and Vaq refer the volumes of organic and water phases, respectively. The
subscript terms stat and mob refer to the stationary phase and the mobile phase used
in chromatography.
The fundamentals of partition are outlined in Chapter 18. The greater the partition coeffi-
cient the higher the affinity towards an organic phase will be. In the case of distribution to a
solid phase the partition can be governed by other characteristics than partition coefficients.
The partitioning of analytes in a system where one phase is a gas necessitates that the
analytes can enter the gas phase.
Discussions on the extraction and partition of compounds therefore most often refer to
liquid/liquid systems. Partition ratios are estimated using distribution between n-octanol
and water. If the compound can be ionized, the ionized form will have a much stronger
affinity towards the aqueous phase as water molecules will solvate the ions. The distri-
bution of an ionizable compound therefore very much depends on the pH of the aqueous
phase. From the Henderson–Hasselbalch equation given above the following equations
can be derived:
For acids : Dapp ¼
DC
1þ 10pH pKa
ð3:13Þ
For bases : Dapp ¼
DC
1þ 10pKa pH ð3:14Þ
If the distribution ratio, DC, and the pKa value are known for a compound, the apparent
distribution ratio, Dapp, at a given pH can be calculated.
Parameters such as the partition ratios in octanol/water are available as the so-called
log P values, and the distribution ratio of compounds between octanol and water at different
pH values in the water phase is tabulated as log D values. Computer programs can also be
used for estimation of pKa values, log P values, log D values and solubility of compounds.
The actual values of each parameter canvary when consulting different literature references,
and this is most often due to differences in the methods used for analysis. This is particularly
true for log P, log D and solubility data. In an analytical chemical context such parameters
should primarily be used as a guide.

Liquid/liquid extraction is often used in sample preparation. It is therefore of interest to
determine the fraction of analytes extracted under given conditions. This is given by the
general formula:
En ¼ 1
1
1þ DC
V2
V1

2
4
3
5
n
ð3:15Þ
where En is the extracted fraction, DC is the distribution ratio between the two phases V2
and V1 and n is the number of extractions. V1 is the phase that originally contains the
analyte and V2 is the phase to which the analytes is extracted.
Example 3.1: Ibuprofen (Figure 3.1) has a log P value of 3.72 and a pKa value of 4.43. If
10 ml of a sample solution of ibuprofen at pH 6.0 is to be extracted to 30 ml of an organic
solution, how much will be extracted?
A log P value of 3.72 corresponds to a KD of 5248. At pH 6.0 the apparent distribution
ratio will be 5248/1 þ 37 ¼ 138 (using Equation 3.13). Calculating the extracted fraction
gives 0.9997 or 99.97%. Doing the same extraction at pH 7.0 results in an extraction of
97.7%. Try to do this calculation yourself.
Example 3.2: Salicylic acid has a log P of 2.0 (corresponding to a KD of 100) and
pKa values of 3.0 and 13.7. Performing similar calculations as in the above example,
it can be shown that only 19% of the salicylic acid is extracted into 30 ml of organic
phase at pH 6.0. Lowering the pH to 5.0 gives an apparent Dc of about 1 and thus an
extraction of 75%.
A question could be whether multiple extractions using the same total amount of
organic solvent would improve the extraction yield. Consider using three extractions of
only10 ml each of organic solvent. Calculations using Equation 3.15 show that the total
extraction in the combined 30 ml will be 87.5% compared to the 75% obtained in only
one single 30-ml extraction. Multiple extractions are more efficient, but in the case of
salicylic acid it is necessary to perform the extraction at a lower pH value if a quantitative
extraction is needed.
Similar extraction calculations can be performed for bases using Equations 6.14 and 6.15.
It is obvious that quantitative extractions from an aqueous solution into an organic
phase are more easily achieved if extraction is performed when the analytes are not ionized.
Thus extraction of carboxylic acid should take place at low pH (pKa 2 or 3 pH units) and
bases at high pH (pKa þ 2 or 3 pH units).
COOH
OH O
OH
pKa = 3.0
pKa = 13.7
pKa = 4.43
log P = 2.0
log P = 3.72
Salicylic acid Ibuprofen
Figure 3.1 Chemical structures of ibuprofen and salicylic acid with log P and pKa values

3.3 Stereochemistry
The pharmacological activity of drug substances is very dependent on their physico-
chemical behavior, often discussed as their ADME properties (adsorption, distribution,
metabolism, excretion). The above-mentioned physicochemical parameters are of vital
importance, but also the stereochemistry of the drug substances is of importance as this
will affect the ADME properties. Describing the stereochemistry of a drug substance is
to visualize the spatial orientation of its components in space. Biological systems including
the human body contain large biomolecules which are constructed from building blocks
with unique stereochemistry. Biological systems are therefore able to distinguish between
isomers which only differ in their spatial configuration. Such isomers may therefore also
have different biological effects.
Figure 3.2 shows how isomers can be divided into several groups. Constitutional isomers
are of course different compounds with different chemical structures.
The diastereomers grouped under the stereoisomers contain compounds where the
isomers have different physicochemical characteristics with different melting points,
partition ratios and so on. These isomers are therefore easy to separate in chromatographic
systems. Cis–trans isomers belong to this group and a number of drug substances can be
found in this group. Two examples are given in Figure 3.3.
Isomerization at the double bond is often mediated by light and the compound should
therefore be protected from light exposure.
Enantiomers constitute a special group of stereoisomers. The two enantiomers that
constitute a pair contain a chiral center and are mirror images of each other. A chiral center
is an atom connected to three (S and P atoms) or four (N and C atoms) different ligands.
The most abundant chiral center is where a carbon atom is connected to four different
groups, but also nitrogen, phosphor and sulfur can be chiral centers. A compound containing
one or more chiral centers is able to rotate plane-polarized light either left or right. This is
Figure 3.2 Classification of isomers

denoted ( ) or (þ), respectively. However, this is not an unambiguous way to describe the
configuration of the chiral center as the direction and size of the rotation is dependent on
the solvent used for sample preparation. In older literature the terms d (dexter) and l (laevo)
were used to denote (þ) and ( ), respectively, but also the small capital letters D and L
have been used where reference was made to the configuration of glyceraldehyde. To give
an unambiguous description of the configuration of the chiral center the R/S nomenclature
has to be used. This nomenclature gives the absolute configuration of the position of groups
connected to the chiral atom and this nomenclature should always be used.
A pair of enantiomers has besides the rotation of the plane-polarized light identical
physicochemical characteristics. However, when they enter a chiral environment (e.g., a
biological system) they may behave differently. A large number of drug substances are
chiral, and for enantiomers it is often observed that one enantiomer has the beneficial
pharmacological effect while the other is either inactive or even gives rise to unwanted side
effects. It is therefore important to be able to control the purity of an enantiomeric drug
substance for content of the unwanted enantiomer.
Measuring the rotation of the plane-polarized light is one way to do this, but the technique
is notsensitive and a low percentage of theimpurity cannotbe detected. A chiral environment
is needed in order to separate the two enantiomers. This can be done by chromatography or
electrophoresis by introducing chirality into the system. This is described in Section 11.5.
3.4 Stability Testing
Drug substances and drug products should be stable or only degrade to a small extend during
their lifetime. It is therefore necessary to perform stability testing to obtain knowledge of
possible degradation processes and in this way establish the shelf-life of products. The
international ICH guidelines as well as guidelines from FDA in the United States describe
how to perform such studies. Many drug substances are fairly stable and under proper
storage are stable for at least five years, which is the normal authorized lifetime of a drug
substance or a drug. Also a number of dry drug formulations are very stable, but when it
comes to liquid preparations long time stability cannot be expected. Some drug substances
are susceptible to hydrolysis and/or oxidation which more readily take place in solution.
Reaction kinetics is used to calculate the shelf-life of pharmaceutical products. Depending
HO
OH
OH
S
N
Cl
N
OH
Clopenthixol Resveratrol
Figure 3.3 The chemical structure of cis-clopenthixol and trans-resveratrol

on the chemical reaction taking place the reaction kinetics can be divided into zero-, first-,
second-, or third-order reactions.
In zero-order reactions the reaction rate is independent of the analyte concentration. A
rate constant of 0.02 mmol/h corresponds to a degradation of 0.02 mmol/h and 0.048 mmol/
day. A solution containing 1 mol of a drug substance in this case degrades to 90% of its
original concentration (10 mmol of total degradation) within about 208 days. This gives the
product a shelf life of 0.5 year.
In most cases reaction kinetics are considered to be of first order or are approximated to a
first-order reaction, also denoted pseudo first order. In this case the reaction rate is dependent
ontheanalyteconcentration[A],andtheunitoftherateconstant,k,istime1
(e.g.,h1
ors1
):

d½A
dt
¼ k A
½ ð3:16Þ
The degradation process by time can be described as:
dx
dt
¼ kða xÞ a
ðx
0
dx
ða xÞ
¼
ðt
0
kdt ð3:17Þ
By rearrangement and integration and also taking the amount of degraded product, x,
into consideration the following equation can be derived:
t ¼
1
k
ln
a
a x
ð3:18Þ
And from this the half life of the substance can be calculated:
t0:5 ¼
1
k
ln
a
a 1
2 a
¼
1
k
ln 2 ð3:19Þ
The hydrolysis of aspirin (acetylsalicylic acid) is considered as a pseudo first-order reaction
and at pH 7.4 and 25
C the rate constant is about 1.4 102
h1
. Using Equation 3.19 gives a
half life of 49h and a 10% degradation takes place within 7.5 h.
It is therefore not possible to store liquid preparations of aspirin as the shelf life would be
only a few hours. Due to the relative fast degradation it is also important to consider the
stability of the prepared sample when performing analysis of aspirin tablets. In order not to
bias the obtained quantitative analysis data, the extracted tablet solution should be analyzed
as quickly as possible. Degradation to 0.5 or 1.0% takes place within 0.36 or 0.7 h,
corresponding to about 20 or 40 min, respectively. Longer storage of the sample solution
increases the bias of the analytical data.
3.5 Summary
The examples show that many drug substances have poor water solubility, and that
substances with high log P values are less water soluble than compounds with a lower
log P. The solubility is often better in organic solvents. Many organic bases are available
as hydrochlorides, sulfates or phosphates which generally are soluble in water. Water

solubility may be increased up to several thousand times if the pH is changed in order to
ionize functional groups.
In general, substances with high log P values are easily extracted from aqueous solutions
using an organic solvent. The higher the log P, the more effective will be the extraction.
Extraction may be performed in one or several steps. Substances with medium log P values
should generally be extracted using multiple extractions in order to have a high recovery.
The water phase must have a pH which suppresses ionization. Substances with high log P
values will also be retarded well in reversed phase chromatography.

4
Fundamentals of Pharmaceutical
Analysis
This chapterdiscusses the basics of pharmaceutical analysis, including the different types of
calculations related to pharmaceutical analysis. The chapter also includes a review of
simple laboratory equipment, how to make solutions and dilutions, how to calibrate
analytical methods, and how to use simple statistics on the analytical data. The chapter
concludes with a list of important terms and concepts in pharmaceutical analysis. It is
important that you read carefully through this chapter before proceeding to the subsequent
chapters. Important terms should be learned.
4.1 What is a Pharmaceutical (Chemical) Analysis?
A pharmaceutical analysis is intended to either identify or quantify one or more
substances in a given sample of pharmaceutical interest. In pharmaceutical analysis,
the substance or substances of interest are normally active pharmaceutical compounds,
pharmaceutical excipients, contaminants, or drug metabolites. A substance to be identi-
fied or quantified is called the analyte. The samples in pharmaceutical analysis are
typically pharmaceutical raw materials, finished pharmaceutical products, or biological
samples like human blood or urine containing one or more drug substances. The samples
consist of one or several analytes, and a sample matrix which is the rest of the sample.
Identification is intended to confirm the identity of the analytes. Identification can also be
referred to as qualitative analysis. A quantitative analysis is intended to measure the exact
concentration or the exact amount of the analyte in a given sample. A quantitative analysis
is also termed as a determination. As an example, paracetamol tablets containing 500 mg
paracetamol per tablet have to be controlled prior to release from production. This is
accomplished by pharmaceutical analysis. Paracetamol is the analyte, whereas the rest of
Introduction to Pharmaceutical Chemical Analysis, First Edition. Steen Hansen, Stig Pedersen-Bjergaard
and Knut Rasmussen.
2012 John Wiley Sons, Ltd. Published 2012 by John Wiley Sons, Ltd.

the tablet, consisting of different pharmaceutical excipients, is the sample matrix.
Identification of paracetamol in the tablets is performed to make sure that the
tablets contain the correct active pharmaceutical ingredient, whereas a quantitative
analysis is performed to measure the content of paracetamol and to check that this result
is exactly or close to 500 mg per tablet. In the latter case, a determination of paracetamol
is performed.
Procedures for pharmaceutical analysis are often complicated and consist of several
steps, as illustrated in Figure 4.1.
First, a sampling is performed, where the required number of samples are taken. During
sampling, it is essential that samples are taken in a representative manner, to give a correct
picture of the case under investigation. Exemplified with the 500-mg paracetamol tablets
discussed above, finished tablets have to be sampled in a systematic way during the entire
time scale of production to give an average of the total production. Sampling is beyond the
scope of this book and is not discussed further here. However, sampling is subject to much
focus in the laboratories involved in pharmaceutical analysis. Often, the samples must be
stored until further analysis. Sample storage is also avery important point, and the reason for
this is to avoid compositional changes of the sample during storage. If compositional
changes occur, the final analytical result will not reflect the original composition of the
sample. To protect samples, they are frequently stored at low temperature and protected
from light, as in a refrigerator or a freezer. This is especially important for liquid samples
where degradation chemistry likely occurs. For the example with 500-mg paracetamol
tablets, sample storage is not a critical issue as the tablets are relatively stable at room
temperature. Sample storage is also beyond the scope of this book and is not discussed
further here.
After sampling and storage, samples are normally pretreated in some way, and this is
called sample preparation. Sample preparation can be very simple or quite complicated,
depending on the sample. Sample preparation is discussed in detail later in this book.
Sample preparation ensures: (i) the sample can be analyzed in a subsequent step, (ii) the
sample is compatible with the final analytical method or instrument, (iii) the analyte is
present in a sufficient amount to be detected, and (iv) substances in the sample matrix that
can cause problems or interferences are removed. In the example with 500-mg paraceta-
mol tablets, the sample preparation normally includes pulverization of the tablets,
dissolution of the tablets, and filtration of material that has not dissolved. Finally, the
analytical measurement is performed, where the analyte or analytes are identified and
quantified. In this textbook, focus will principally be directed on analytical measurements
by titration, spectroscopy, chromatography, and electrophoresis, as discussed in subse-
quent chapters. In the example with 500-mg paracetamol tablets, spectroscopy is typically
used to identify paracetamol and to measure the quantity of paracetamol in the tablets.
After the analysis, the measurements are processed; the results are calculated and
presented in an analytical report.
Sampling Sample storage Sample preparation Analytical measurement
Figure 4.1 Different steps in a typical procedure in pharmaceutical analysis

4.2 How to Specify Quantities and Concentrations?
In most cases, a quantitative pharmaceutical analysis is performed when the analyte is
present in a solution. A solution is a homogeneous mixture of two or more substances. A
minor species in a solution is called a solute and the analyte is an example of a solute. The
major species in a solution is the solvent. The amount of analyte (solute) is normally
expressed as the concentration. Concentration means the amount of solute per volume unit
of solution. In some contexts,the term molarity is used to expressthe concentration, which is
abbreviated M. The molarity of a certain solute is defined as follows:
Molarity ¼ number of moles of solute per liter of solution ð4:1Þ
An example of how to calculate the molarity during the preparation of a solution with a
known concentration of solute is given in Box 4.1.
When the concentrations are low, it is impractical to use M. In such cases, either
millimolar (mM), micromolar (mM), or nanomolar (nM) are used as defined in the following
way:
1M ¼ 103
mM ¼ 106
mm ¼ 109
nM ð4:2Þ
An example of how to convert from M to mM is shown in Box 4.2.
As an alternative to molarity, it is common to use concentrations expressed as mass per
volume unit. Either milligrams per milliliter (mg/ml), micrograms per milliliter (mg/ml),
nanograms per milliliter (ng/ml), or picograms per milliliter (pg/ml) can be used, which are
defined as follows:
1 g=ml ¼ 103
mg=ml ¼ 106
mg=ml ¼ 109
ng=ml ¼ 1012
pg=ml ð4:3Þ
An example of how to convert from mg/ml to mg/ml is shown in Box 4.3, and an example of
how to convert from mg/ml to M is shown in Box 4.4.
Box 4.1 Calculation of molarity
0.100 g paracetamol (molar mass ¼ 151.2 g/mol) is dissolved in water and the total
volume is adjusted to 500.0 ml. The molarity of paracetamol is calculated to:
0:100 g=151:2 g=mol
0:5000 l
¼ 1:32 103
M
Box 4.2 Conversion from M to mM
One sample has a content of 1.62 105
M paracetamol. This corresponds to the
following concentration in mM:
1:62 105
M 106
mM=M ¼ 16:2 mM
Fundamentals of Pharmaceutical Analysis 35

Concentrations can also be expressed as a percentage. The following definitions are used:
% weight ¼ %ðw=wÞ ¼
mass of solute
mass of solution
100% ð4:4Þ
% volume ¼ %ðv=vÞ ¼
volume of solute
volume of solution
100% ð4:5Þ
% weight=volume ¼ %ðw=vÞ ¼
mass of solute
volume of solution
100% ð4:6Þ
When expressing concentration as a percentage, it is highly important to specify whether %
weight, % volume, or % weight/volume has been used. % Weight is used to express the
concentration of solids or liquids either in solid samples or liquid samples. % Volume,
however, is used to express the concentration of liquids in liquid samples (or gases in
gaseous samples). % Weight/volume is used to specify the concentration of solids or liquids
in a solution. Boxes 4.5 and 4.6 show examples of how to calculate concentrations using the
terms % weight and % weight/volume, respectively.
At very low concentrations, it may be convenient to use the terms parts per million (ppm)
or parts per billion (ppb) instead of % weight. These are defined as follows:
Box 4.3 Conversion from mg/ml to mg/ml
One sample has a content of 0.0125 mg/ml paracetamol. This corresponds to the
following concentrations in mg/ml:
0:0125 mg=ml 103
mg=mg ¼ 12:5 mg=ml
Box 4.5 Calculation of % weight
5.01 g paracetamol is dissolved in 200.0 ml of ethanol, and the total weight of the
solution is determined to 162.81 g. The concentration of paracetamol in % weight is:
% ðw=wÞ ¼
5:01 g
162:81 g
100% ¼ 3:08% ðw=wÞ
Box 4.4 Conversion from mg/ml to M
One sample has a content of 12.5 mg/ml of paracetamol (Molar mass ¼ 151.2 g/mol).
This corresponds to the following concentration in M:
12:5 mg=ml ¼ 12:5 g=l )
12:5 g=l
151:2 g=mol
¼ 8:27 102
M

ppm ¼
mass of solute
mass of sample
106
ð4:7Þ
ppb ¼
mass of solute
mass of sample
109
ð4:8Þ
Box 4.7 shows an example of how to calculate concentration in terms of ppm.
For the analysis of pharmaceutical raw materials and preparations, the analyte concen-
trations are relatively high, and normally mg/ml is used to express concentrations. However,
for the analysis of drugs in biological samples, the analyte concentrations are normally
much lower, and here it is customary to express the concentrations in ng/ml or nM. A drug
concentration of 1 ng/ml, which is normal for several drugs in biological samples, is an
extremely low concentration, as visualized in Box 4.8. Box 4.9 shows an example how to
convert concentrations from ng/ml to nM.
4.3 Basic Laboratory Equipment
4.3.1 The Analytical Balance
Quantitative pharmaceutical analysis is always based on accurate weighing of the substance
or sample to be analyzed. Such weighing should be performed with an analytical balance to
get as high accuracy as possible. The analytical balance is therefore a fundamental
instrument in the pharmaceutical laboratory. Many laboratories have laboratory balances
available, but these do not provide sufficiently high accuracy for analytical purposes. An
analytical balance is shown in Figure 4.2. Analytical balances are equipped with a digital
display with direct recording of the mass. The most common analytical balances have a
weighing capacity up to 100 or 200 g. The sensitivity of the balance is defined as the smallest
increment of mass that can be measured, and the most common analytical balances have
Box 4.6 Calculation of % weight volume
5.01 g paracetamol is dissolved in ethanol and the volume of the solution adjusted to
200.0 ml. The concentration of paracetamol in % weight volume is:
% ðw=vÞ ¼
5:01 g
200:0 ml
100% ¼ 2:51% ðw=vÞ
Box 4.7 Calculation of ppm
0.106 g ethanol dissolved in 999.911 g water. The concentration of ethanol in ppm is:
ppm ¼
0:106 g
0:106 g þ 999:911 g
106
¼ 106 ppm

sensitivities in the range 0.01–0.1 mg. The analytical balance should be located on a heavy
table, such as a marble slab, to minimize vibrations. This is important to ensure stable
readings. Analytical balances have adjustable feet and a bubble meter that allow you to keep
the balance level.
Box 4.8 Illustration of typical drug concentration in human blood
A patient sample contains 1.0 ng/ml of fluphenazine. To visualize how little substance
this is per volume unit, one can imagine that a sugar cube is dissolved in water. How
much water is required to give a concentration of 1 ng/ml?
The weight of a sugar cube is about 2.3 g. The following amounts of water (V) must be
used to give a concentration of 1.0 ng/ml:
1:0 ng=ml ¼ 1:0 109
g=ml ¼ 1:0 106
g=l ¼ 2:3 g=V
V ¼
2:3 g
1:0 106
g=l
¼ 2:3 106
l
2.3 106
l equals about the amount of water in a swimming pool which is 2.5 m deep,
20 m in width and has a length of 50 m. A drug concentration of 1 ng/ml is equivalent to
the concentration you get if you solve a sugar cube in a large swimming pool! As you will
learn in this book, such low concentrations can be measured with our analytical
techniques!
With a concentration of 1.0 ng/ml, how many molecules are present per ml? Assume
the molar mass is 300 g/mol. In 1.0 ml, 1.0 ng drug is present, which is equal to
1.0 109
g, and which corresponds to the following number of moles:
n ¼
1:0 109
g
300 g=mol
¼ 3:3 1012
mol
This number of moles corresponds to the following number of molecules, by multipli-
cation by Avogadro’s number:
Number of molecules ¼ 3:3 1012
mol 6:022 1023
molecules=mol
¼ 2:2 1012
molecules
As you can see, although dealing with very low concentrations, there are still a lot of
molecules!
Box 4.9 Conversion from ng/ml to nM
A patient sample was found to contain 1.0 ng/ml fluphenazine. This substance has a
molar mass of 437.58 g/mol. The concentration is converted to nM in the following way:
1:0 ng=ml ¼ 1:0 mg=l ¼ 1:0 106
g=l )
1:0 106
g=l
437:58 g=mol
¼ 2:3 109
M ¼ 2:3 nM

A normal weighing procedure includes the following steps:
. Place an empty weighing vessel on the weighing pan.
. Reset the reading of the balance (tare to 0.0000 or 0.00000 g).
. Fill the substance or sample to be weighed into the weighing vessel.
. Record the mass on the digital display.
. Clean the balance after use.
Calibration is done regularly to ensure high accuracy. Analytical balances calibrate
themselves by placing a standard mass on the pan. It is important never to place drug
substances or samplesdirectly on the pan, as thiswill contaminate the system. Therefore, the
substances or the sample to be weighed should always be placed in a weighing vessel placed
on the pan. First, place an empty vessel on the pan. Use a paper towel or tissue to handle the
Figure 4.2 Photo of an analytical balance

vessel, because fingerprints will change its mass. Let the balance stabilize for a few seconds,
then reset the digital display to show 0.0000(0) g. Make sure that the weighing vessel is
centered on the pan and that the glass doors protecting the pan are closed to protect it from
drafts. Then place the substance or the sample to be weighed in the weighing vessel, make
sure that the weighing vessel is still centered, close the doors, and wait for the balance to
stabilize before the mass is recorded on the digital display. If some substance or sample is
spilled inside the balance, this should be removed immediately.
Like with many other instruments, errors can occur during operation of the analytical
balance. One source of weighing error occurs if the density of what you are weighing is
different from the density of the standard mass used for calibration. This is because every
time you place an item in the balance, it displaces an equivalent amount of air that also has a
weight. If the difference in density is largebetween the object to beweighed and the standard
mass used for calibration, the weighing error (% relative error) will be relatively large as
shown in Figure 4.3.
To correct for differences in density, the buoyancy equation can be used:
m2 ¼
m1 1 dair
dcw

1 dair
d
ð4:9Þ
where m1 is the measured weight, m2 is the corrected weight, dair is the density of air, dcw is
the density of standard mass used for calibration (typically 8.0 g/ml), and d is the density of
the object considered. Equation (4.9) is not frequently used in the pharmaceutical
laboratory, but it is important to be aware of the principle.
Density of object (g/l)
2 4 6 8 10 12 14 16 18
0.00
-0.10
-0.20
-0.30
% Relative error
Figure 4.3 Weighing error (% relative error) due to the difference in density between the object
to be weighed and the standard mass used for calibration. In this case, the analytical balance was
calibrated with a standard mass of density 8.0 g/l

123 yo yo.pdf

Recommended

Recommended

More Related Content

Similar to 123 yo yo.pdf

Similar to 123 yo yo.pdf (20)

Recently uploaded

Recently uploaded (20)

123 yo yo.pdf