Stability of Drugs)

STABILITY OF DRUGS
AND DRUG PRODUCTS
Iqbal Ahmad, T.I.

M.Sc. (KU), Ph.D. (London), D.Sc. (KU)
Royal Society Fellow
Professor, Department of Pharmaceutical Chemistry
Former Professor and Chairman
Department of Pharmaceutical Chemistry
Faculty of Pharmacy, University of Karachi
Muhammad Ali Sheraz

B. Pharm. M. Phil., Ph.D. (BMU)
Associate Professor and
Chairman, Department of Pharmacy Practice
Sofia Ahmed
B. Pharm. M. Phil., Ph.D. (BMU)
Associate Professor and
Chairperson, Department of Pharmaceutics
Faculty of Pharmaceutical Sciences

Baqai Medical University, Karachi
HIGHER EDUCATION COMMISSION

ISLAMABAD - PAKISTAN
1
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HEC – Cataloging in Publication (CIP Data):
Iqbal Ahmad, Dr.
Chemical and Photo stability of Drugs and Formulated Products
1. Drug Stability
616.86 - dc23 2016
ISBN: 978-969-417-189-0
First Edition: 2016
Copies Printed: 500
Published By: Higher Education Commission – Pakistan
Disclaimer: The publisher has used its best efforts for this publication through a rigorous system of
evaluation and quality standards, but does not assume, and hereby disclaims, any liability to any person
for any loss or damage caused by the errors or omissions in this publication, whether such errors or
emissions result from negligence, accident, or any other cause.
2
Dedicated to
Professor Dr. Syed Sabir Ali (Late)
Founder Dean,
Faculty of Pharmaceutical Sciences,
Baqai Medical University, Karachi
3
CONTENTS
LIST OF FIGURES xi
LIST OF TABLES xii
LIST OF ABBREVIATIONS xiii
FOREWORD xv
PREFACE xvi
ACKNOWLEDGMENTS xvii
ABOUT THE AUTHORS xviii
1 INTRODUCTION 1
1.1 DEFINITION OF STABILITY 1
1.2 TYPES OF STABILITY AND DOSAGE FORMS 1
1.3 FACTORS INFLUENCING STABILITY 2
1.3.1 External Factors 2
1.3.2 Internal Factors 2
1.4 IMPORTANT TYPES OF STABILITY 2
1.4.1 Chemical Stability 2
1.4.2 Photostability 3
1.4.3 Physical Stability 3
1.4.4 Solid State Stability 3
1.4.5 Microbiological Stability 4
1.5 ANALYTICAL METHODS 4
1.6 STABILITY EVALUATION 4
1.7 STABILITY TESTING 4
1.8 FORCED DEGRADATION STUDIES 5
1.9 STATISTICAL APPLICATIONS 5
1.10 ROLE OF PHARMACIST 5
1.11 LITERATURE ON DRUG STABILITY 6
1.12 CONTENTS OF MONOGRAPH 6
REFERENCES 7
2 CHEMICAL KINETICS 13
2.1 INTRODUCTION 13
2.2 BASIC KINETIC PRINCIPLES 13
2.2.1 Reaction Rate 13
2.2.2 Molecularity and Order of Reaction 14
2.2.2.1 Molecularity 14
2.2.2.2 Order 14
2.2.3 Half-Life and Shelf-Life of Drug 14
2.2.3.1 Half-life (t1/2) 14
2.2.3.2 Shelf-life (t90 or t95) 14
2.2.3.3 Expiration dating 14
2.3 KINETICS OF CHEMICAL REACTIONS 15
2.3.1 Zero-Order Reaction 15
2.3.2 Pseudo Zero-Order Reaction 15
2.3.3 First-Order Reaction 16
2.3.4 Pseudo First-Order Reaction 17
2.3.5 Second-Order Reaction 17
2.3.6 Determination of Reaction Order 18
2.3.6.1 Substitution method 18
2.3.6.2 Graphical method 18
4
2.3.6.3 Half-life method 18
2.4 COMPLEX CHEMICAL REACTIONS 19
2.4.1 Reversible First-Order Reaction 19
2.4.1.1 Example of calculation of equilibrium constant and rate constants for a
20
reversible first-order reaction
2.4.2 Parallel Reactions 21
2.4.2.1 Parallel reactions involving the formation of two products 21
2.4.2.2 Parallel reactions involving the formation of three products 22
2.4.3 Consecutive Reactions 23
2.4.4 Enzyme Catalysed Reactions 24
2.5 FACTORS AFFECTING DRUG DEGRADATION REACTIONS 25
2.5.1 Temperature 25
2.5.2 Q10 Values 26
2.5.2.1 Q ΔT calculation 27
2.5.2.2 Shelf-life calculation 27
2.5.3 Nonisothermal Prediction of Rate of Degradation 27
2.5.4 pH 28
2.5.5 Catalysis 28
2.5.5.1 Specific acid-base catalysis 28
2.5.5.2 General acid-base catalysis 30
2.5.6 Ionic Strength Effect (Primary Salt Effect) 31
2.5.7 Solvent 31
2.5.8 Oxygen 32
2.5.8.1 Oxidizable drugs 32
2.5.8.2 Oxidation reactions 33
2.5.9 Surfactant 34
2.5.10 Moisture 34
2.5.11 Problems 34
REFERENCES 37
3 CHEMICAL STABILITY 43
3.1 INTRODUCTION 43
3.2 STUDY OF THE CHEMICAL STABILITY OF A DRUG 43
3.3 CHEMICAL DEGRADATION REACTIONS 44
3.3.1 Hydrolysis 44
3.3.1.1 Hydrolysis of esters 44
3.3.1.2 Hydrolysis of amides 45
3.3.1.3 Hydrolysis by ring opening 46
3.3.2 Oxidation 48
3.3.3 Decarboxylation 50
3.3.4 Elimination 50
3.3.5 Isomerization 51
3.3.6 Dimerization 51
3.3.7 Epimerization 52
3.3.8 Dehydration 52
3.3.9 Dehydrogenation 53
3.3.10 Dehalogenation 53
3.4 CHEMICAL STABILITY/DEGRADATION STUDIES 53
3.4.1 Aqueous Solution 53
3.4.2 Pharmaceutical Preparations 54
REFERENCES 57
4 PHOTOSTABILITY 61
4.1 INTRODUCTION 61
4.2 PHOTOSTABILITY AND RELATED ASPECTS 61
4.2.1 Photostability 61
5
4.2.2 Effects of Photoinstability 61
4.2.2.1 Chemical and physical changes 61
4.2.2.2 Biological effects on administration 62
4.2.2.3 Light induced side effects through interaction with endogenous substances 62
4.2.3 Objectives of Photostability Studies 62
4.2.4 Industrial Awareness on Photostability 62
4.3 PHOTOCHEMISTRY 63
4.3.1 Basic Laws of Photochemistry 63
4.3.2 Stages of Photochemical Reactions 63
4.3.3 Role of Photochemistry in Photostability Studies 63
4.4 PHOTOCHEMICAL REACTIONS 63
4.4.1 Regions of UV, Visible and Sunlight Radiation 64
4.4.2 Important Chemical Functions for Photoreactivity in Organic Molecules 64
4.4.3 Photophysical Processes 64
4.5 PRIMARY PHOTOCHEMICAL REACTIONS 65
4.5.1 Flash Photolysis 65
4.5.2 Laser Flash Photolysis 66
4.5.3 Two-Laser Flash Photolysis 66
4.5.4 Time-Resolved Spectroscopy 67
4.5.5 Excited State Reactions 67
4.5.6 Photosensitized Reactions 68
4.5.6.1 Type I: Free radical mechanism 68
4.5.6.2 Type II: Mechanism involving singlet oxygen 68
4.6 PHOTODEGRADATION REACTIONS 69
4.6.1 Photooxidation Reactions 70
4.6.1.1 Photooxidation of benzaldehyde 70
4.6.1.2 Photooxidation of ascorbic acid 71
4.6.2 Photoreduction Reactions 71
4.6.2.1 Photoreduction of riboflavin 71
4.6.3 Photodealkylation Reactions 72
4.6.3.1 Photodealkylation of riboflavin 72
4.6.4 Photoaddition Reactions 72
4.6.4.1 Photoaddition of riboflavin 72
4.6.5 Photoaquation Reactions 72
4.6.5.1 Photoaquation of cyanocobalamin 72
4.6.6 Photodegradation of Moxifloxacin 73
4.6.6.1 Acid solution 73
4.6.6.2 Alkaline solution 73
4.6.7 Other Photodegradation Reactions 75
4.6.8 Photochemical Interactions 75
4.6.8.1 Interaction of riboflavin with ascorbic acid 75
4.6.8.2 Interaction of nicotinamide with ascorbic acid 75
4.6.8.3 Interaction of α-tocopherol with ascorbic acid 76
4.6.8.4 Interaction of nicotinamide with riboflavin 76
4.6.8.5 Interaction of ascorbic acid with cyanocobalamin 76
REFERENCES 78
5 PHYSICAL STABILITY 83
5.1 INTRODUCTION 83
5.2 ANALYTICAL TECHNIQUES IN THE STUDY OF PHYSICAL STATE 83
5.2.1 Thermal Methods 83
5.2.1.1 Thermogravimetric analysis (TGA) 83
5.2.1.2 Differential scanning calorimetry (DSC) 83
5.2.1.3 Differential thermal analysis (DTA) 83
5.2.1.4 Microcalorimetry 83
5.2.1.5 Isothermal calorimetry 84
6
5.2.1.6 Dilatometry 84
5.2.1.7 Hot-stage microscopy 84
5.2.2 Spectroscopic Methods 84
5.2.2.1 Vibrational spectroscopy 84
5.2.2.2 Fourier transform infrared (FTIR) spectroscopy 84
5.2.2.3 Diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) 84
5.2.2.4 Solid state nuclear magnetic resonance (SSNMR) spectroscopy 84
5.2.2.5 Dynamic light scattering (DLS) 84
5.2.2.6 X-ray powder diffraction (XRPD) 85
5.2.2.7 Single crystal X-ray diffraction (XRD) 85
5.2.3 Other Techniques 85
5.2.3.1 Polarized light microscopy 85
5.2.3.2 Particle electrophoresis 85
5.3 CHANGES IN PHYSICAL STABILITY 85
5.4 FACTORS AFFECTING PHYSICAL STABILITY 86
5.4.1 Internal Factors 86
5.4.2 External Factors 86
5.4.3 Amorphous State 86
5.4.4 Dosage Forms 87
5.4.4.1 Solid dispersions 87
5.4.4.2 Semi-solid dispersions 87
5.4.4.3 Creams 89
5.4.4.4 Liposomes 90
5.4.4.5 Proteins 90
5.4.5 Crystalline State 90
5.4.6 Polymorphism 91
5.4.6.1 Conformational polymorphism 92
5.4.6.2 Solvatomorphism 92
5.4.6.3 Packing polymorphism 92
5.4.6.4 Pseudopolymorphism 93
5.4.6.5 Forced polymorphism 93
5.4.7 Characterization of Polymorphs 93
5.4.8 Pharmaceutical Implications 95
REFERENCES 96
6 SOLID STATE STABILITY 101

6.1 INTRODUCTION 101
6.2 TOPOCHEMICAL REACTIONS 102
6.3 CHEMICAL DEGRADATION REACTIONS 103
6.3.1 Solvolysis 103
6.3.2 Oxidation 104
6.3.3 Deamidation 105
6.3.4 Pyrolysis 106
6.3.5 Photolysis 106
6.4 FACTORS AFFECTING STABILITY IN THE SOLID-STATE 107
6.4.1 Moisture 107
6.4.2 Temperature 107
6.5 DRUG INTERACTIONS 108
6.6 KINETICS OF SOLID STATE DEGRADATION 109
6.7 SOLID STATE STABILITY STUDIES 110
6.7.1 Structural Studies 110
6.7.2 Kinetic Studies 111
6.7.3 Effect of Excipients 114
6.7.4 Effect of Aging 114
REFERENCES 116
7
7 FORCED DRUG DEGRADATION 121
7.2 OBJECTIVES 122
7.3 FACTORS INVOLVED IN DEGRADATION 122
7.3.1 Degradation Conditions 122
7.3.2 Degradation Limits 122
7.3.3 Method of Analysis 123
7.4 FORCED DEGRADATION STUDIES IN THE DEVELOPMENT OF DRUGS 124
7.5 ANALYTICAL TECHNIQUES USED IN FORCED DEGRADATION
124
STUDIES
7.6 DRUG DEGRADATION STUDIES 124
REFERENCES 128
8 PACKAGING EFFECTS ON DRUG STABILITY 131

8.2 DEFINITIONS 131
8.3 TYPES 132
8.3.1 Primary Packaging Material 132
8.3.2 Secondary Packaging Material 132
8.4 FUNCTIONS 132
8.5 SELECTION 133
8.6 PACKAGING STUDIES 133
8.6.1 Solid Dosage Forms 133
8.6.2 Liquid Dosage Forms 134
8.7 STABILITY PREDICTION IN PACKAGED PRODUCTS 136
8.8 STABILITY TESTING 136
REFERENCES 137
9 STABILIZATION 139
9.2 PREVENTION OF DEGRADATION REACTIONS 139
9.2.1 Common Degradation Reactions 139
9.2.1.1 Hydrolysis 139
9.2.1.2 Oxidation 140
9.2.1.3 Photolysis 140
9.2.2 Prevention of Degradation Reactions Involving Steric Structural Variations 141
9.2.2.1 Cyclization 141
9.2.2.2 Dimerization 142
9.2.2.3 Epimerization 142
9.2.2.4 Racemization 143
9.2.2.5 Polymerization 143
9.3 METHODS OF STABILIZATION 143
9.3.1 Temperature Control 143
9.3.2 Cyclodextrin Complexation 144
9.3.3 Polymer Complexation 144
9.3.4 Use of Stabilizers 145
9.3.5 Liposomal Formulation 145
9.4 CHEMICAL AND PHOTOSTABILIZATION STUDIES 145
9.4.1 Chemical Stabilization 145
9.4.1.1 Amorphous drugs 145
9.4.1.2 Binary co-amorphous mixtures 146
9.4.1.3 Solid dosage forms 147
9.4.1.4 Liquid dosage forms 147
9.4.2 Photostabilization 149
9.4.2.1 Solid and semisolid dosage forms 149
9.4.2.2 Liquid dosage forms 150
8
REFERENCES 151
10 STABILITY OF HERBAL DRUGS AND PRODUCTS 157

10.2 DEFINITIONS 157
10.2.1 Herbal Drugs 158
10.2.2 Processed Herbal Drugs 158
10.2.3 Herbal Drug Preparations 158
10.2.4 Herbal Drug Extracts 158
10.3 QUALITY CONTROL METHODS 159
10.3.1 Herbal Products 159
10.3.2 Essential Oils 159
10.3.3 Herbal Extracts 159
10.4 FINGERPRINT ANALYSIS OF HERBAL DRUGS 160
10.5 STORAGE 165
10.6 PHOTOSENSITIVITY REACTIONS OF HERBS 165
10.7 STABILITY OF HERBAL DRUGS AND PRODUCTS 165
10.7.1 Photodegradation of Herbal Drugs 165
10.7.2 Chemical Degradation of Herbal Drugs 168
10.8 STABILITY/DEGRADATION STUDIES OF HERBAL DRUGS IN
169
FORMULATIONS
10.9 STABILITY TESTING OF HERBAL PRODUCTS 170
10.10 HERB-DRUG INTERACTIONS 171
REFERENCES 173
11 STABILITY-INDICATING ASSAY METHODS 179

11.2 DEFINITIONS 179
11.3 DEVELOPMENT OF ANALYTICAL METHODOLOGY FOR ASSAY OF A
179
DRUG COMPOUND
11.4 DEVELOPMENT OF ANALYTICAL METHODOLOGY FOR STABILITY-
180
INDICATING ASSAY OF A DRUG COMPOUND
11.5 STABILITY-INDICATING SPECTROMETRIC ASSAY METHODS 181
11.5.1 One-Component Assay 181
11.5.2 Multicomponent Assay 181
11.5.2.1 Two-component assay (additive absorbencies) 181
11.5.2.2 Three-component assay (additive absorbencies) 182
11.5.3 Advantages 183
11.5.4 Applications 183
11.6 STABILITY-INDICATING THIN-LAYER CHROMATOGRAPHIC (TLC) AND
HIGH-PERFORMANCE THIN-LAYER CHROMATOGRAPHIC (HP-TLC) 184
ASSAY METHODS
11.7 STABILITY-INDICATING HIGH-PERFORMANCE LIQUID
194
CHROMATOGRAPHIC (HPLC) ASSAY METHODS
11.7.1 Development of HPLC Stability-Indicating Assay Methods 194
11.7.2 Applications 194
11.7.2.1 Drug mixture 194
11.7.2.2 Stress testing/forced degradation studies 194
11.8 VALIDATION OF STABILITY-INDICATING ASSAY METHODS 194
11.8.1 Linearity 196
11.8.2 Range 197
11.8.3 Accuracy 197
11.8.4 Precision 198
11.8.4.1 Repeatability 199
11.8.4.2 Intermediate precision 199
11.8.4.3 Reproducibility 199
9
11.8.5 Specificity 200
11.8.6 Sensitivity 202
11.8.6.1 Limit of detection (LOD) 202
11.8.6.2 Limit of quantification (LOQ) 202
11.8.7 Robustness 202
REFERENCES 204
12 REGULATORY ASPECTS OF STABILITY TESTING 209

12.2 OBJECTIVES 210
12.2.1 The Development Phase 210
12.2.2 The Approval Phase 210
12.2.3 The Post-Approval Phase 210
12.3 DESIGN OF STABILITY STUDIES 211
12.3.1 Stress Testing 211
12.3.2 Selection of Batches 211
12.3.3 Container Closure System 211
12.3.4 Test Procedure and Test Criteria 212
12.3.5 Frequency of Testing 212
12.3.5.1 Long term or real-time studies 212
12.3.5.2 Accelerated studies 212
12.3.5.3 Intermediate studies 212
12.4 STORAGE CONDITIONS 213
12.4.1 General Case 213
12.4.2 Drug Substance or Drug Product Intended for Storage in a Refrigerator 214
12.4.3 Drug Substance or Drug Product Intended for Storage in a Freezer 215
12.4.4 Drug Products Packaged in Impermeable Containers 215
12.4.5 Drug Products Packaged in Semi-Permeable Containers 215
12.4.6 Drug Substance or Drug Product Intended for Storage Below -20 oC 216
12.5 PHOTOSTABILITY 216
12.5.1 Light Sources 216
12.5.2 Testing Criteria 217
12.5.3 Presentation of Samples 218
12.5.4 Post Exposure Sample Analysis 218
12.5.5 Recommendation for Handling and Packaging 218
12.6 EVALUATION OF TEST RESULTS 218
12.7 STABILITY REPORT 219
12.8 STATEMENTS AND LABELING 219
12.9 STABILITY COMMITMENT 219
12.10 ONGOING STABILITY STUDIES 220
12.11 IN-USE STABILITY TESTING 220
12.12 VARIATIONS 221
REFERENCES 223
INDEX 225
10
LIST OF FIGURES
2.1 Zero-order plot of A versus time. 15

2.2 First-order plot of log A versus time. 16
2.3 Second-order plot of 1/[A] versus time. 18
2.4 Rate–pH profile for photodegradation of moxifloxacin in aqueous solution. 29
2.5 Rate–pH profile for the photolysis of riboflavin in aqueous solution. 29
2.6 Rate–pH profile for the photolysis of cyanocobalamin in aqueous solution. 30
4.1 Photooxidation of benzaldehyde. 70
4.2 Chemical structures of riboflavin and photoproducts. ?
4.3 Proposed pathway for the photodegradation of MF in acid solution. 73
4.4 Proposed pathway for the photodegradation of MF in alkaline solution. 74
4.5 Proposed pathway for the photodegradation of MF in alkaline solution. 74
6.1 Solid crystalline state dimer of 4-aminosalicylic acid, a and c indicate 103
directions in the arrangement of crystals.
7.1 Stress conditions used for the degradation of drug substances and drug 123
products.
11.1 Chemical structure of riboflavin. 180
11.2 Hydrolysis of aspirin. 185
11.3 Hydrolysis of procaine HCl in alkaline solution. 186
11.4 Alkaline hydrolysis of riboflavin at pH 11.0. 187
11.5 Alkaline hydrolysis of formylmethylflavin at pH 11.0. 188
11.6 Thermolysis of aqueous soluiton of reserpine (pH 3.0) at 60°C. 189
11.7 Hydrolysis and photooxidation of sulfacetamide in aqueous solution. 190
11.8 Photolysis of riboflavin at pH 7.0. 191
11.9 Photoaddition reaction of riboflavin at pH 7.0. 192
11.10 Photolysis of ascorbic acid in the presence of riboflavin at pH 4.0. 193
11.11 Calibration curve of sulfacetamide sodium in aqueous solution, pH 7.0. 196
11.12 Overlay UV spectra of sulfacetamide sodium in aqueous solution, pH 7.0. 197
11.13 Illustration for accuracy and precision. 199
11.14 HPLC Chromatogram of carvedilol and its photodegradation products. 200
11.15 Absorption spectra of riboflavin (——), cyclodehydroriboflavin (…....), 201
formylmethylflavin (------) at pH 2.0 in KCl–HCl buffer.
12.1 Flow chart for photostability testing of drug products. 218
11
LIST OF TABLES
1.1 Types and criteria for acceptable levels of stability. 2

1.2 Types of pharmaceutical dosage forms. 2
2.1 Order of reactions, half-life and shelf-life equations. 19
2.2 Q10 factors for 10° interval and Ea values. 26
2.3 Rate–pH profiles for the degradation of drugs. 28
6.1 Moisture content of commonly used tablet excipients at 25°C on 107
storage at different relative humidities (RH).
6.2 A comparison of the apparent zero-order rate constants (k0) for the 108
degradation of various vitamin A derivatives at 50°C and their melting
points.
7.1 Widely used conditions for conducting forced degradation. 123
7.2 Application of analytical techniques in forced degradation studies. 124
8.1 Packaging preservation of product stability against environmental 132
factors.
8.2 t90 Values of drugs in plastic and glass containers. 135
9.1 Stabilization of drug substances in solid state/solid dosage forms. 148
9.2 Stabilization of drug substances in liquid dosage forms. 149
10.1 Analytical methods for the study of herbal drugs. 160
10.2 Sensitivity and storage of some herbal drugs and products. 161
10.3 Herbs causing skin sensitivity on exposure to sunlight. 165
10.4 Some light sensitive drugs and products. 165
10.5 Photodegradation of herbal drugs by sunlight. 166
10.6 Storage conditions for stability testing of drug substances. 171
10.7 Adverse effects of herbs and herbal products. 172
11.1 Analytical parameters for the validation of sulfacetamide sodium. 196
11.2 Accuracy and precision of sulfacetamide sodium by the UV 198
spectrometric method at 95% confidence interval.
11.3 Comparison of tolfenamic acid recovery by titrimetric, UV and FTIR 198
spectrometric methods.
12.1 Mean climatic conditions: calculated data and derived storage 210
conditions.
12.2 Stability protocol design using bracketing. 213
12.3 Stability protocol design using matrixing. 213
12.4 General case (drug substance or drug product). 214
12.5 Drug substance or drug product intended for storage in a refrigerator. 214
12.6 Drug substance or drug product intended for storage in a freezer. 215
12.7 Drug products packaged in semi-permeable containers. 215
12
LIST OF ABBREVIATIONS
Abbreviation Name
a Absorptivity
A Absorbance
AAS Atomic absorption spectrometry
ANN Artificial neural networks
Arg Arginine
ATR Attenuated total reflectance
BP British Pharmacopoeia
CD Circular dichroism
CDs Cyclodextrins
CE Capillary electrophoresis
CDRF Cyclodehydroriboflavin
CMF Carboxymethylflavin
C Concentration
Cp Heat capacity
CRH Critical relative humidity
COSY Correlation spectroscopy
CTAB Cetyltrimethylammonium bromide
Cys Cystine
2D NMR 2 dimensional nuclear magnetic resonance spectroscopy
DFT Density function theory
DLS Dynamic light scattering
DRIFTS Diffuse reflectance infrared Fourier transform spectroscopy
DSC Differential scanning calorimetry
DTA Differential thermal analysis
EMC Equilibrium moisture content
EP European Pharmacopoeia
F Flavin
FDA Federal Drug Authority
FMF Formylmethylflavin
FMO Front molecular orbital
fs Femto second
FTIR Fourier transform infrared
GC Gas chromatography
GC–MS Gas chromatography-mass spectrometry
GE Gel electrophoresis
GMP Good Manufacturing Practice
HOMO Highest occupied molecular orbital
HPLC High performance liquid chromatography
HP–TLC High performance thin-layer chromatography
HSQC Heteronuclear single quantum coherence spectroscopy
ICH International Conference on Harmonization
ISO International Organization for Standardization
IR Infrared
K Kelvin
klx–h Kilolux hour
LASER Light amplification by stimulated emission of radiation
LC Liquid chromatography
LDPE Low density polyethylene
13
LF Lumiflavin
LC–MS Liquid chromatography-mass spectrometry
LC–MS/TOF Liquid chromatography-mass spectrometry/time-of-flight
LOD Limit of detection
LOQ Limit of quantification
Lys Lysine
LUMO Lowest unoccupied molecular orbital
MAS–SSNMR Magic angle spinning solid state nuclear magnetic resonance
MC Methyl cellulose
MCC Microcrystalline cellulose
M/L Mole per litre
MEP Molecular electrostatic potential
Min–1 Per min
MS Mass spectrometry
MS/MS Tandem mass spectrometry
Ms–1 Mole per second
M–1 s–1 Per mole per second
NA Nicotinamide
NCE New chemical entity
NF Norfloxacin
NIR Near infrared
NMR Nuclear magnetic resonance
NSAIDs Nonsteroidal anti-inflammatory drugs
PDA Photodiode array
PEG Polyethylene glycol
PDI Polydispersity index
PLS Partial least squares
PSD Particle size distribution
ps Pico second
PVA Polyvinyl alcohol
PVP Polyvinyl pyrrolidone
Q–TOF–ESI–MS/MS Quadrupole time-of-light electrospray ionization tandem mass
spectrometry
RF riboflavin
RH Relative humidity
RP–HPLC Reversed phase high performance liquid chromatography
s–1 Per second
SER Serine
SSNMR Solid state nuclear magnetic resonance
SLN Solid lipid nanoparticles
S/N ratio Signal to noise ratio
Tg value Glass transition temperature
TGA Thermogravimetric analysis
TLC Thin-layer chromatography
Tm Melting point
Tmc Critical mobility temperature
Trp Tryptophan
UPLC Ultra performance liquid chromatography
USP United States Pharmacopeia
UV Ultraviolet
Vis Visible
WHO World Health Organization
W/m2 Watt per square meter
XRPD X-ray powder diffraction
XRD X-ray diffraction
14
FOREWORD
The stability of drugs and drug products is a subject of great importance for the assessment
of the quality, efficacy and safety of the products. The knowledge of various aspects of stability is
essential for the drug development process. Stability testing provides information about the factors
that affect the expiration dating of drug products.
The authors have wide experience of teaching and research in the field and their efforts to
present various aspects of the subject in the form of a monograph are commendable. No attempts
have been made to write monographs in specialized areas of pharmaceutical disciplines in this
country. This monograph meets the requirements of M.Phil. / Ph.D. courses in drug stability in
various universities and would be of great help to postgraduate students in finding the relevant
information in a unified source. An understanding of the advanced concepts and their applications
would assist in the development of different dosage forms.
The monograph is a valuable contribution to the existing literature in the field of drug
stability and would also be useful to teachers, pharmacists and R & D personnel in pharmaceutical
industries.
Prof. Dr. Zahida Baqai

MBBS, MRCOG, FRCOG, FICS, FCPS, Ph. D.
Vice Chancellor
Baqai Medical University
15
PREFACE
This monograph has been prepared to meet the requirements of

M. Phil./Ph. D. courses in drug stability and related fields taught in the Faculties of Pharmaceutical
Sciences in Pakistan. It covers a wide range of topics related to drug stability with chapters on
general introduction and those concerning chemical kinetics, chemical stability, photostability,
physical stability, solid-state stability, forced drug degradation, packaging effects on stability,
stabilization, stability of herbal drugs and products, stability-indicating assay methods and
regulatory aspects of stability testing. Each chapter provides a brief introduction to the topic,
definitions of the terms used, theoretical background, relevant literature and discussion of the
material. An understanding of various aspects of drug stability is essential for the development of
different dosage forms. A prominent feature of each topic is the inclusion of current and previous
research studies to apprise the students of the developments being made in the field to enable
them to design their own research projects in a specific area of the subject. The monograph would
be helpful to the teachers in providing a suitable background of various aspects of drug stability
and to workers engaged in quality control, stability testing and drug development in pharmaceutical
industries.
This monograph on drug stability is the only one of its kind prepared on the subject for
postgraduate students. The authors have made the best of efforts in the selection, compilation and
presentation of the subject material. However, any inadvertent errors and omissions are regretted.
The authors would be grateful for pointing out any errors or shortcomings in the text.
Iqbal Ahmad
Muhammad Ali Sheraz
Sofia Ahmed
August, 2016
16
ACKNOWLEDGMENT
The authors are very grateful to Professor Dr. Syed Fazal Hussain of the Faculty of
Pharmaceutical Sciences, Baqai Medical University, Karachi, Professor Dr. Anwar Ejaz Baig of the
Department of Pharmaceutics, Faculty of Pharmacy, Ziauddin Medical University, Karachi,
Professor Dr. Faiyaz H. M. Vaid of the Department of Pharmaceutical Chemistry, Faculty of
Pharmacy & Pharmaceutical Sciences, University of Karachi, Professor Dr. Usmanghani Khan,
Consultant, Herbion Pakistan (Pvt) Ltd, Karachi, Professor Dr. Iqbal Azhar, Dean, Faculty of
Pharmacy & Pharmaceutical Sciences, University of Karachi, and Dr. Saif-ur-Rehman Khattak,
Director, Central Drugs Laboratory Karachi, Drug Regulatory Authority of Pakistan, for their kind
help and valuable suggestions for the improvement of the monograph. The authors are very grateful
to Dr. Saif-ur-Rehman Khattak for contributing a chapter on regulatory aspects of drug stability.
They are also thankful to Mr. Zubair Anwar, Ph. D. scholar, for literature search and computer work.
One of the authors (I.A.) is highly appreciative of the patience and support of his wife, Shamim
Iqbal, during the preparation of this monograph.
The authors express their heartfelt gratitude to the Higher Education Commission,
Government of Pakistan, for the publication of this monograph.
17
ABOUT THE AUTHORS
Dr. Iqbal Ahmad is Professor of Pharmaceutical Chemistry and Director, Postgraduate Studies at
the Faculty of Pharmaceutical Sciences, Baqai Medical University, Karachi. He previously served
as Professor and Chairman Department of Pharmaceutical Chemistry at the Faculty of Pharmacy,
University of Karachi. He obtained a Ph. D. degree in Pharmaceutical Chemistry from the University
of London and conducted Postdoctoral research at North E Wales Institute of Higher Education,
UK and Department of Biochemistry, University of Arizona, USA. He has the privilege of working
with Professor Lord George Porter, Nobel Laureate, at Imperial College London on a Royal Society
Fellowship. He has vast experience of teaching and research extending over a period of 50 years
and has to his credit more than 200 publications including 2 books and 12 chapters. He has
supervised more than 60 students for M. Pharm., M. Phil., and Ph. D. degrees at the University of
Karachi and Baqai Medical University. He was awarded the D. Sc. degree in Pharmaceutical
Chemistry by the University of Karachi and Tamgha-e-Imtiaz by Government of Pakistan in 2014
for his outstanding academic and research contribution.
Dr. Muhammad Ali Sheraz is Associate Professor and Chairman of the Department of Pharmacy
Practice at the Faculty of Pharmaceutical Sciences, Baqai Medical University, Karachi. He obtained
a Ph. D. degree in Pharmaceutics from Baqai Medical University and conducted Postdoctoral
research at the University of Sheffield, UK on a fellowship awarded by Higher Education
Commission of Pakistan. He is a HEC approved supervisor for M. Phil., and Ph. D. studies. He has
published more than 60 research papers and has co-authored 6 chapters and a book published in
USA. He has so far supervised 5 students for M. Phil. degree. He is also the Editor in Chief of the
Baqai Journal of Health Sciences.
Dr. Sofia Ahmed is Associate Professor and Chairperson of the Department of Pharmaceutics at
the Faculty of Pharmaceutical Sciences, Baqai Medical University, Karachi. She obtained a Ph. D.,
degree in Pharmaceutics from Baqai Medical University and then conducted Postdoctoral research
at the University of Sheffield, UK on a fellowship awarded by Higher Education Commission of
Pakistan. She is a HEC approved supervisor for M. Phil., and Ph. D., studies and has published
more than 60 research papers. She has also co-authored 6 chapters and a book published in USA.
She has supervised 3 students for M. Phil. degree.
CHAPTER – 1
18
INTRODUCTION
The stability of drug substances and drug products is a subject of great interest to
pharmacists, drug manufacturers and regulatory agencies. Stability is a critical quality attribute, a
measure of good manufacturing practices (GMP) and an integral part of drug development
process. It is of fundamental importance among all the characteristics of a drug product since any
physical and chemical change with time may affect the quality, efficacy and safety of the product.
Stability is a regulatory requirement for the registration of drug products in most of the countries.
This is necessary to ensure that a safe and effective product is available to the patient throughout
its shelf-life.
Drug substances are susceptible to chemical, physical and microbiological degradation
under different conditions due to their sensitivity to environmental factors that may lead to a change
in the chemical structure or the physical state. This could have serious consequences on their
biological efficacy and safety. It is essential for the manufacturer to ensure the quality of the
product under the conditions to which it is exposed during manufacture, transportation and
storage.
Stability studies are necessary for the selection of suitable packaging materials and
storage conditions to avoid chemical and physical changes and interactions between the drug and
the excipients. Pharmaceutical products included in the pharmacopoeias must be stored under
specified conditions to maintain quality attributes during the shelf-life period. Preventive measures
are necessary for the storage of drug products under adverse climatic conditions (i.e. high
temperature and high humidity). Stability considerations are important in the development of
therapeutically effective dosage forms.
Stability studies are required for all finished products by the manufacturer including the
products that are reconstituted or diluted with saline solution or 5% dextrose solution before use.
The reconstituted or diluted solutions of a product also need to be subjected to stability assessment
over the recommended storage period. The compatibility of drugs in admixtures and the stability
of preservatives/stabilizers used should also be investigated. This would provide information on
drug interactions and the efficacy of preservatives/stabilizers during the shelf-lives of the products.
The cost of treating a new chemical entity (NCE) through the drug development process
involving discovery, toxicology, clinical development and commercialization ranges from $ 800
million to $ 1.2 billion. In the optimization of the drug development process, a deep understanding
of the key factors affecting the stability profile of the drug product and the execution of an effective
stability program are important in the commercialization of the product (Huynh-Ba, 2009).
1.1 DEFINITION OF STABILITY
Stability is considered as the period of time under specific storage conditions and in a
specific container-closure system that a product will retain within predefined limits all of its original
characteristics. The United States Pharmacopeia (USP, 2016) defines stability as the extent to
which a product retains, within specified limits, and throughout its period of storage and use (i.e.
its shelf-life), the same properties and characteristics that it possessed at the time of its
manufacture.
1.2 TYPES OF STABILITY AND DOSAGE FORMS
The different types of stability (i.e. chemical, physical, microbiological, therapeutic and
toxicological) and their criteria for acceptable levels (USP, 2012) are given in Table 1.1. The
stability of drug substances depends on the dosage forms of the product and their susceptibility to
environmental conditions. The various pharmaceutical dosage forms are described in Table 1.2.
These may undergo a chemical and/or physical change during manufacture, storage and use
affecting their stability.
Table 1.1 Types and criteria for acceptable levels of stability
19
Type of Conditions maintained throughout the shelf-life of the drug
stability product
Chemical Each active ingredient retains its chemical integrity and labeled
potency, within the specified limits.
Physical The original physical properties, including appearance, palatability,
uniformity, dissolution and suspendability, are retained.
Microbiological Sterility or resistance to microbial growth is retained according to
the specified requirements. Antimicrobial agents that are present
retain effectiveness within the specified limits.
Therapeutic The therapeutic effect remains unchanged.
Toxicological No significant increase in toxicity.
Table 1.2 Types of pharmaceutical dosage forms
Dosage form Phase Example

Solid One or more than one solid Tablets, capsules, lozenges, pills,
granules, powders, suppositories
Semisolid One or two liquid and one solid Ointments, gels, pastes
Liquid Liquid Solutions, parenterals, syrups,
elixirs, drops, gargles
Emulsion Liquid/liquid or liquid/solid Creams
Inhaler Solid/gas or Liquid/gas Aerosols
1.3 FACTORS INFLUENCING STABILITY

Several factors are involved in altering the chemical and physical characteristics of drug
substances and drug products. These factors may influence the stability of different dosage forms
during manufacture and storage and can be described as:
1.3.1 External Factors
These include temperature, light, moisture, oxygen, carbon dioxide and microbial
contaminants.
1.3.2 Internal Factors
These include pH, solvent, medium polarity, buffer species, ionic strength, particle size,
metal contaminants, and drug-drug, drug-excipients and drug-container interactions.
The external factors can be controlled by using suitable packaging materials and
appropriate storage conditions. The effect of internal factors can be minimized by the selection of
optimum formulation conditions to achieve an acceptable level of stability. The shelf-life of the
packaged product can then be determined under the recommended storage conditions.
1.4 IMPORTANT TYPES OF STABILITY
1.4.1 Chemical Stability
The chemical reactions undergone by drug substances in liquid dosage forms and
affecting the stability of a product include hydrolysis (e.g. esters, amide, imides), oxidation (e.g.
ascorbic acid, epinephrine, vitamin A), epimerization (e.g. tetracyclines, moxalactam, etoposide),
isomerization (e.g. cytarabine, amphotericin B, cyclosporine A), decarboxlyation (e.g. 4-
aminosalicylic acid, etodolac), dehydration (e.g. glucose, erythromycin, prostaglandin E 1 and E2)
and others.
20
The screening of degradation products for their potential toxicity is part of the safety
evaluation program. Computer-assisted technologies are now being used for the prediction of
toxicological behavior of pharmaceutical degradation products (Jamrogliewicz, 2016).
1.4.2 Photostability
The photostability of drug substances and drug products is an important factor in the
assessment of the overall stability of solid and liquid dosage forms. A large number of
pharmacopoeial drugs are sensitive to light and their formulated products may be degraded during
manufacture, storage and administration. This could result in the loss of potency, change in
efficacy and adverse biological effects. Knowledge of the photochemical behavior of drugs under
stipulated light exposure conditions could provide guidance for handling, packaging and labeling
of the products. The use of suitable packaging material can provide protection to the products from
photodegradation. Opaque and amber colored containers are suitable for light protection in the UV
and visible region. The important photodegradation reactions of drugs include photooxidation (e.g.
ascorbic acid), photoreduction (e.g. riboflavin), photoaquation (e.g. cyanocobalamin),
photocyclization (e.g. meclofenamic acid), photodealkylation (e.g. chloroquine),
photodecarboxylation (e.g. amino acids), photoisomerization (e.g. aztreonam), photodimerization
(e.g. primaquine), photo-induced hydrolysis (e.g. sulfacetamide), and photo-induced ring cleavage
(e.g. norfloxacin).
1.4.3 Physical Stability
The physical stability of drug products takes into consideration the physical changes
occurring in the products. These changes depend on the physical properties of the drugs such as
melting point, particle size, polymorphic behavior, texture and morphology.
The physical stability of liquid dosage forms is affected by changes in appearance,
alteration in viscosity, discoloration, precipitation, polymorph formation (low solubility), drug
adsorption (container surface) and microbial growth.
The changes in the physical stability of solid dosage forms involve polymorphic transition,
solvation and desolvation, salt and salt exchange, amorphization and reversion to crystalline form
and moisture adsorption. These changes may lead to the physical destabilization of the product.
1.4.4 Solid State Stability
The solid state stability deals with the physical and chemical transformations occurring in
the solid state (or solid dosage forms) under the influence of factors such as moisture and
temperature or during storage with time (such as polymorphic transitions). The physical changes
in the solid state have been discussed by Santos (1999) and involve:
 Particle size growth and surface area changes.
 Precipitation from solution at refrigerated temperatures.
 Degree of hydration.
 Deliquescence or softening.
 Crystallization of amorphous material.
 Solid state transitions.
The chemical degradation of drugs in the solid state and in the solid dosage forms occurs
in the presence of moisture and at high temperature. It involves reactions such as solvolysis (e.g.
acetylsalicylic acid), oxidation (e.g. ascorbic acid), decarboxylation (e.g. carbenicillin sodium),
deamidation (e.g. peptides), pyrolysis (e.g. fluconazole) and photolysis (e.g. furosemide). The solid
state degradation of drugs is affected by properties such as melting point, crystalline state and
hygroscopic character of the drug.
1.4.5 Microbiological Stability
21
The microbiological stability of drug products is essential for the efficacy and safety of the
products. The sterility or resistance to microbial growth should be maintained throughout the shelf-
life period. The efficacy of the preservative should remain unaltered within the specified limit. The
multidose aqueous preparations contain a preservative to protect against spoilage during use. The
preservative has no influence on the vulnerability of the product to contamination (i.e. the access
of organisms into it that largely depends on the container design). However, a good design
minimizes the level of organisms introduced during use and operates in harmony with an effective
preservative to protect the consumer (Hodges, 1999). A pathogen-contaminated product can result
in severe consequences both for the consumer and the manufacturer, and, therefore, adequate
preservative activity is vital for the product. For regulatory approval, it is necessary to show
adequate preservative performance at the time of manufacturing as well as later during the shelf-
life period. The requirements for the biological assessment of preservative activity have been
discussed by Hodges (1999).
1.5 ANALYTICAL METHODS
Many regulatory agencies require an assessment of the individual and the total limits of
degradation products in the specifications of the drug products. This can be achieved by the
application of a stability-indicating assay method, such as high-performance liquid
chromatography (HPLC), for the determination of the intact drug as well as its degradation
products. The method should be validated to ensure the desired specificity for a particular system.
It can also be applied to assess the stability of drug products manufactured in several dosage
forms with variable strengths and stored in different packaging. Aubry et al. (2009) have discussed
the development of stability-indicating assay methods.
1.6 STABILITY EVALUATION
The purpose of stability studies is to establish, based on testing a minimum of three
batches of the drug substance and evaluating the stability information (including, as appropriate,
results of the physical, chemical, biological and microbiological tests), a re-test period applicable
to all future batches of the drug substance manufactured under similar circumstances. The degree
of variability of individual batches affects the confidence that a future production batch will remain
within specification throughout the assigned re-test period (ICH Guideline, 2003).
The design of the stability studies of drug products should be based on the evaluation of
all factors that may cause a physical, chemical and/or biological change during the recommended
storage period. It should include the assay of the drug and degradation products, and
measurement of change in pH, color, appearance, etc. for liquid dosage forms; and hardness,
dissolution, moisture content, etc. for solid dosage forms and any other tests depending on the
dosage form.
An understanding of the stimuli causing the degradation of drugs and the mode of their
degradation is helpful in the evaluation of the stability of drug products. The various stimuli that
lead to the degradation of a drug include oxygen, temperature, humidity and light. The pH of the
medium, buffer content, metal contaminants, etc. also plays a part in the degradation process. The
evaluation of the stability of drugs enables the development of safe and effective dosage forms,
selection of suitable packagings, establishment of appropriate storage conditions and assignment
of shelf-lives.
1.7 STABILITY TESTING
Stability testing is an integral part of drug development process and is an essential
requirement for the registration of drug products. The ICH (2003), WHO (2009) and FDA (1998,
2014) have provided guidelines for the stability testing of new drug substances and products which
involve long term, intermediate and accelerated stability studies. The purpose of stability testing is
to provide evidence on how the quality of a drug substance or drug product varies with time under
the influence of a variety of environmental factors such as temperature, humidity, and light, and to
establish a reset period for the drug substance or a shelf-life for the drug product and
recommended storage conditions (ICH, 2003). Similar ICH guideline is also available for
22
photostability testing of new drug substances and products. The photostability testing should be
an integral part of stress testing and should be conducted on at least one primary batch of drug
product, if appropriate. The purpose of photostability testing is to evaluate the intrinsic
photostability characteristics of new drug substances and products to demonstrate that the light
exposure does not result in an unacceptable change. The standard conditions for photostability
testing are described in ICH Q1B guideline (ICH, 1996).
1.8 FORCED DEGRADATION STUDIES
Forced degradations studies of new drug substances and drug excipients involve a
degradation process at conditions that are more severe than those of the accelerated or stress
conditions. These studies are required for the establishment of the degradation pathways,
characterization of the degradation products, determination of intrinsic stability of drug substances,
elucidation of the mechanism of degradation reactions, and development of stability-indicating
assay methods. Pharmaceutical industry conducts forced degradation studies on drugs during the
preformulation stage to select appropriate active ingredients and excipients, to characterize
degradation products, to assess compatibility of ingredients and to conduct formulation
development. Different aspects of the forced degradation of pharmaceuticals have been reviewed
by Reynolds (2004).
1.9 STATISTICAL APPLICATIONS
Statistics plays an important role in the stability studies of drug products (Carstensen et
al, 1992; Helboe, 1992; Lin et al., 1993; Chow and Liu, 1995; Chen et al., 1997). Statistical
methods have been proposed for the design and analysis of stability studies (Nordbrock, 1992;
Carstensen et al., 1992; Fairweather et al., 1995; Chen et al., 1997) and for testing and
classification of stability data with multiple factors (Chow and Shao, 1989, 1990; Chen et al., 1995;
Golden et al., 1996; Ahn et al., 1997).
Statistical treatment of stability data provides information on the effect of batch-to-batch
variations, dosage unit to dosage unit variations, small scale-production scale process variations,
packaging variations and strength variations on the expiration dating. The ICH (1994) guideline for
industry on stability testing of new drug substances and products has recommended the use of
bracketing and matrixing as an experimental design for testing the stability data to obtain expiration
dating of the products. Bracketing involves the design of a stability schedule such that only
samples on the extremes of certain design factor, e.g. strength, package size, are tested at all time
points as in a full design. The design assumes that the stability of any intermediate level is
represented by the stability of extremes tested. Matrixing involves the design of a stability schedule
such that a selected subset of the total number of possible samples for all factor combination is
tested at a specific time point. At a subsequent time point, another subset of samples for all factor
combinations is tested. The design assumes that stability of each subset of samples tested
represents the stability of all samples at a given time point (ICH, 2003).
1.10 ROLE OF PHARMACIST
The pharmacist has to play an important role in ensuring the quality, efficacy and safety
of the products dispensed under his supervision. He should be aware of the factors involved in the
destabilization of drugs under adverse climatic conditions and evolve a strategy to overcome them.
He should ensure that the products meet the acceptable criteria of stability under the prescribed
storage conditions during their shelf-life period. It is the time period during which a drug product is
expected to remain within the approved shelf-life specifications, provided that it is stored under the
conditions defined on the container label. It is also referred to as expiration dating period (ICH,
2003).
1.11 LITERATURE ON DRUG STABILITY
Extensive literature on various aspects of drug stability is available. Some of the important
sources are as follows.
23
Books
Windheuser (1970), Connors et al. (1986), Rubinstein (1989), Albini and Fasani (1998),
Mazzo (1999), Carstensen and Rhodes (2000), Yoshioka and Stella (2000), Tonnesen (2004),
Baertschi (2005), Piechocki and Thoma (2007), Huynh-Ba (2009), Trissel (2009), Grimm et al.
(2011), Loftsson (2014).
Chapters
Schwartz and Nelson (1966), Ho (1972), Simonelli and Dresback (1972), Lintner (1973),
Hashmi (1973), Lachman et al. (1986), Racz (1989), Lim et al. (1993), Grimm (2000), Matthews
(2000), Valvani (2000), Pugh (2002), Tonnesen (2002), Hawely and Van Arendonk (2002),
Guillory and Poust (2002), Ghosh (2005), Fasani and Albini (2005), Ahmad and Vaid (2006),
Florence and Attwood (2006), O’Donnell and Bokser (2006), Singh (2006), Jackson and Lowey
(2010), Sinko (2011), Govindarjan (2014).
Reviews
Macek (1960), Garrett (1967), Maudling and Zoglio (1970) Tingstad and Dudzinski (1973),
Allen (1974), Carstensen (1974), Madsen et al. (1974), Zoglio et al. (1975), Amirjahed (1977),
Mollica et al. (1978), DeRitter (1982), Carstensen and Rhodes (1984), Ahmad (1985), Sugden
(1985), Greenhill and McLelland (1990), Wessels et al. (1997), Singh (1999), Singh and Bakshi
(2000a,b), Tonnesen (2001), Boreen et al. (2003), Glass et al. (2004), Waterman and Adami
(2005), Phalekar et al. (2008), Panda et al. (2013), Bajaj et al. (2012), Baertschi et al. (2013, 2015),
Vinodi et al. (2015), Ahmad et al. (2016).
Pharmacopoeias
British Pharmacopoeia (2016), United States Pharmacopeia (2016), European
Pharmacopoeia (2015), and other pharmacopoeias.
Regulatory Aspects
Food and Drug Administration Guidelines (FDA) (1987, 1998), International Conference
on Harmonization (ICH) Guidelines (1996, 1997, 2002, 2003), World Health Organization (WHO)
Guideline (2009).
1.12 CONTENTS OF MONOGRAPH
This monograph presents an overall view of different aspects of drug stability to cover the
course contents for M. Phil. /Ph. D. program in different disciplines of pharmaceutical sciences.
Chapters 2, 3, 4, 5 and 6 are devoted to chemical kinetics, chemical stability, photostability,
physical stability and solid state stability. Chapters 7, 8, and 9 deal with forced drug degradation,
packaging effects on stability and stabilization. The last three chapters 10, 11 and 12 cover stability
of herbal drugs and products, stability-indicating assay methods and regulatory aspects of stability
testing.
24
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Loftsson T. Drug Stability for Pharmaceutical Scientists, Elsevier, Amsterdam, The Netherlands,
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Maudling HV, Zoglio MA. Flexible nonisothermal stability studies. J Pharm Sci. 1970;59:333–337.
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29
30
CHAPTER – 2
CHEMICAL KINETICS
2.1 INTRODUCTION
Chemical kinetics deals with the quantitative study of the rates of chemical reactions and
the factors that influence them. The study of the stability of drug products involves the evaluation
of the kinetics of chemical degradation reactions of drugs in dosage forms. This is necessary to
predict the shelf-life of the product under defined storage conditions. The drug substances possess
diverse chemical structures and may follow one or more than one modes of degradation with
different orders of reaction under various conditions. The most commonly occurring degradation
reactions of drugs include oxidation, hydrolysis and photolysis. These reactions may occur during
manufacturing, storage and use of drug products. The practicing pharmacist should be aware of
the effects of these processes on the potency loss and/or toxicity development in the product to
ensure the safety of the patient.
An accurate assessment of the potency loss of a drug in a product can be made by the
application of a stability-indicating assay method that is also capable of determining the
degradants and related compounds. The assay data are then subjected to kinetic treatment to
determine the shelf-life of the product and to establish the expiration dating. A change in
formulation parameters to improve the stability of the product may require a reconsideration of the
packaging and storage conditions. This would again need an establishment of the reset period or
shelf-life under the proposed storage conditions.
The determination of the rates of degradation reactions undergone by drug substances
and the prediction of shelf-lives and expiration dates of drug products requires a sound knowledge
of the fundamental principles of chemical kinetics. The kinetic parameters could be useful in the
elucidation of the mechanisms of degradation reactions and thus enable to adopt appropriate
measures for the stabilization of the product. Several excellent accounts of the subject with
applications to the degradation kinetics of drug substances and drug products are presented in
books (Lintner, 1973; Lachman and DeLuca, 1986; Carstensen, 2000; Pugh, 2002; Ghosh, 2005;
Guillory and Poust, 2002; Wigent, 2006; Florence and Attwood, 2006; Sinko, 2011), monographs
(Windheuser, 1970; Connors et al., 1986; Laidler, 1987; Carstensen and Rhodes, 2000; Yoshioka
and Stella, 2000; Loftsson, 2014) and reviews (Macek, 1960; Garrett, 1967; Mollica, 1978;
Connors, 1981; DeRitter, 1982, Waterman and Adami, 2005; Ahmad et al., 2016a). This chapter
deals with a brief description of the fundamental principles of chemical kinetics, their application
to drug stability and the factors affecting the stability of pharmaceuticals.
2.2 BASIC KINETIC PRINCIPLES
2.2.1 Reaction Rate
The rate of a reaction is defined as the change in concentration of a reactant or products
per unit time. Consider a reaction in which two reactants, A and B, react to yield products, C and
D.
aA + bB cC + dD (2.1)
where a, b, c, d represent the number of molecules, A, B the reactants and C, D the products. The
rate of this reaction can be expressed as:
1 d[A] 1 d[B] 1 d[C] 1 d[D]
Rate = – =– = = (2.2)
a dt b dt c dt d dt
31
where d[A], d[B], d[C] and d[D] each is an infinitesimal change in the concentration of A, B, C and
D during the infinitesimal time dt. A decrease in concentration is denoted by a minus sign.
The relationship between the rate of reaction and the concentration of reactants is given
by eq. (2.3)
Rate = k [A]a [B]b (2.3)
where k is the rate constant.
If the concentration is expressed in M/L, the rate constant is expressed as moles per unit
time, for example, Ms–1 or M–1s–1.
The rate of a reaction is directly proportional to the concentration of the reactants, each
concentration being raised to a certain power, usually equal to the number of molecules, a and b,
of the reactants, A and B, respectively.
2.2.2 Molecularity and Order of Reaction
2.2.2.1 Molecularity
Molecularity is defined as the number of molecules taking part in a chemical reaction. A
reaction in which only one reactant molecule in involved is called a Unimolecular reaction, if two
reactant molecules are involved, it is called a bimolecular reaction, and if three reactant molecules
are involved, it is called a termolecular reaction.
2.2.2.2 Order
The order of a reaction is defined as the sum of exponents of the concentrations of
reactants involved in the rate equation. It can also be defined with respect to a single reactant. For
example in eq. (2.3), the reaction order with respect to A is a and with respect to B is b. If a + b =
1, it is a first-order reaction, if a + b = 2, it is a second-order reaction and if a + b = 3, it is a third–
order reaction.
The molecularity and the order are the same for a simple one–step reaction.
2.2.3 Half-Life and Shelf-Life of Drug
2.2.3.1 Half-life (t1/2)
The half-life (t1/2) of a reaction is defined as the time required for the drug concentration to
decrease to 50% of its initial concentration. The value of t1/2 is a function of the rate constant of
the reaction.
2.2.3.2 Shelf-life (t90 or t95)
The shelf-life (t90 or t95) of a product is defined as the time required for the product after
manufacture to decrease to the minimum acceptable level of the label claim (i.e. 90% or 95%). It
is also a function of the reaction rate constant.
2.2.3.3 Expiration dating
A drug product should comply with the official standards of identity, strength, quality and
purity during the expiration dating period. This period can be defined as “the time interval that a
drug product is expected to remain within an approved shelf-life specification, provided that it is
stored according to label storage conditions and that it is in the original container closure system”.
The expiry/expiration date is the actual date period on the container/label of a drug product
designating the time during which a batch of a drug product is expected to remain within the
approved shelf-life specifications if stored under defined conditions and after which it must not be
used (Hawely and Van Arendonk, 2002).
The expiration dating of drug products manufactured in a particular country is to be
determined according to the storage conditions prevailing in the climatic zone of that country. In
Pakistan this applies to the conditions prescribed for Climate Zone IVa (hot and humid).
32
2.3 Kinetics of Chemical Reactions
2.3.1 Zero-Order Reaction
In a zero-order reaction, the rate of disappearance of a reactant, A, remains constant and
is independent of concentration as shown by eq. (2.4):
–
d[A] = k0 (2.4)
dt
The integrated form of eq. (2.4) is
A = A0 – k0t (2.5)
where
A is the amount of A0 remaining at time t,
A0 is the initial concentration of A, and
k0 is the zero-order rate constant.
The rate constant, k0, of a zero-order reactions can be determined from the slope of a
linear plot of A versus t (Fig. 2.1)
Fig. 2.1 Zero-order plot of A versus time.

The half-life (t1/2) of a zero-order reaction is directly proportional to the initial concentration
of the reactant, and is inversely proportional to the rate constant (Table 2.1).
Example: Fading of color of dyes (Garrett and Carper, 1955).
2.3.2 Pseudo Zero-Order Reaction
In certain pharmaceutical systems such as suspensions the drug is degraded by a first-
order reaction (Section 2.3.3). However, the drug present in the solid form in the suspension
dissolves in the aqueous phase and thus a constant concentration of the drug is maintained in the
solution. In this case although the reaction is first-order with respect to the drug, it follows zero-
order reaction.
The rate of the reaction can be expressed as:
33
–
d[A] = k1 [A] = k0 (2.6)
dt
where
k1 is the first-order rate constant,
A is the concentration of dissolved drug, and
k0 is the zero-order rate constant (The rate constant, k0, is determined as mentioned in
section 2.3.1).
Examples:
1. Hydrolysis of aspirin in aqueous suspensions (Blaug and Wesolowski, 1959).
2. Hydrolysis of amoxicillin in aqueous suspensions (Zia et al., 1977).
2.3.3 First-Order Reaction
In a first-order reaction, the rate of disappearance of the reactant, A, is proportional to the
concentration of A at time t as given by eq. (2.7)
–
d[A] = k1 A (2.7)
dt
The integrated form of eq. (2.7) can be expressed by eq. (2.8)
ln A – ln A0 = –k1t (2.8)
or
ln A = ln A0 – k1t (2.9)
Rearranging and converting logarithms to the base 10
k1 2.303 A0
log (2.10)
= t A
In a first-order reaction there is an exponential decrease in the concentration of the
reactant A with time.
The rate constant, k1, for a first-order reaction can be obtained from the slope of a linear
plot of log A versus t (Fig. 2.2).
Fig. 2.2 First-order plot of log A versus time.
34
The t1/2 of first-order reaction is independent of the initial concentration of the reactant and
depends on the rate constant (Table 2.1). First-order reactions are the most widely occurring
reactions in the degradation of drugs in formulated products.
Examples:
1. Hydrolysis of aspirin (Edwards, 1950).
2. Oxidation of ascorbic acid solutions (Blaug and Hajratwala, 1972).
3. Photolysis of riboflavin in aqueous solutions (Ahmad et al., 2004a).
2.3.4 Pseudo First-order Reaction
A chemical reaction in which one of the reactants is present in large excess, shows an order that
is different from that of the actual order. This type of reaction is called pseudo first-order reaction.
Consider a reaction
A+B Products
This is a second-order reaction and the rate is given by eq. (2.11)
Rate = k [A] [B] (2.11)
Since [B] is present in large excess, its concentration is considered constant during the course of
the reactions and eq. (2.11) can be expressed as
Rate = k′ [A] (2.12)
where
k′ = k [B]
Thus the reaction is first-order with a rate constant k′ and is termed as an apparent or pseudo first-
order reaction.
This applies to the hydrolysis of drugs in aqueous solution in which the amount of water is
in large excess and does not alter during the course of reaction.
Example: Hydrolysis of ethyl acetate (Pugh, 2002).
2.3.5 Second-order Reaction
In a second-order reaction two molecules react to yield the products
A+B Products
The rate of the reaction is proportional to the concentration of the two reactants.
–dA dB
= = k2 [A] [B] (2.13)
dt dt
where
k2 is the second-order rate constant, and
A and B are the concentrations of the two reactants.
In a simple case, if [A] = [B], each having the same molar concentration or two [A]
molecules react, the rate of the reaction can be expressed as:
–dA
= k2 [A]2 (2.14)
dt
The integrated form of eq. (2.14) is:
35
1 1
– = k2t (2.15)
[A] [A0]
or
1 1
= + k2t (2.16)
[A] [A0]
The rate constant, k2, for a second-order reaction can be determined from the slope of a
linear plot of 1 / [A] versus t (Fig. 2.3).
The t1/2 of a second-order reaction is inversely proportional to the initial concentration of
the reactant and the rate constant (Table 2.1).
Examples:
1. Hydrolysis of esters in alkaline solution (Pugh, 2002).
2. Thermolysis of formylmethylflavin in acid solution (Ahmad and Vaid, 2008).
3. Photolysis of formylmethylflavin in organic solvents (Ahmad et al., 2006).
Fig. 2.3. Second-order plot of 1/[A] versus time.

2.3.6 Determination of Reaction Order
The order of a reaction can be determined by the following methods.
2.3.6.1 Substitution method
The concentration data obtained on the degradation of a drug at various time intervals during a
kinetic study are substituted in the integrated forms of the equations for zero- , first- and second-
order reactions and the values of the rate constant, k, are calculated. The reaction is of the order
for which the rate equation gives a constant value of k within the experimental error limits.
2.3.6.2 Graphical method
The graphical method of the determination of the order of a reaction involves the plotting of
concentration or a function of concentration data for the reactant A versus t. A linear plot of A
versus t represents a zero-order reaction, a linear plot of log A versus t represents a first-order
reaction and a linear plot of 1/[A] versus t represents a second-order reaction.
2.3.6.3 Half-life method
The half-life period (t1/2) of a reaction is expressed as:
36
1
[A]n– (2.17)
1
where
n is the order of reaction. For a second-order reaction it is assumed that A = B.
For a reaction carried out at two different initial concentrations, A1, A2, the t1½ and t2 ½ are
given by the relation:
t1½ A2 n–1
= (2.18)
t2 ½ A1
Rearranging and converting to the log form gives

log [t1½ / t2 ½]
n= +1 (2.19)
log [A2/A1]
The t1/2 values are determined from plots of A versus t at two different concentrations and
finding the values of t at A1/2 and A2/2. The substitution of the values of t1/2 and the initial
concentrations in eq. (2.19) gives the order of reaction.
Table 2.1 Order of reaction, half-life and shelf-life equations
Integrated rate
Units of
Order equation Half-life (t1/2) Shelf-life (t90)
k
(linear form)
conc.
time–1
Zero A = A0–k0t 0.5 A0/ k0 = A0/ 2k0 0.1 A0/ k0
(e.g. Ms–
1)
time–1 2.303 A0
log A = log A0– 2.303 log A0 log
First (e.g. s–1) k1 0.90A0
k1t / 2.303 k1 0.50A0
conc–1.
1/ A = 1/ A0 + time–1
Second 1 / A0 k2 –
k2t (e.g. M–
1s–1)
2.4 Complex Chemical Reactions

The degradation reactions of many drugs may not follow zero–, first– or second-order
kinetics. These reactions may include more than one–step with the same or different pathways
and could be complex involving reversible, parallel or consecutive reactions.
2.4.1 Reversible First-Order Reaction
A reversible first-order reaction may be represented as:
k
A B
k'
Where k and kˊ are the first-order rate constants for the forward and reversible reactions,
respectively.
If the initial concentration of A is a units and that at time t is (a–x) units, the concentration
of B at time t is x units.
37
The net rate of reaction at time t is expressed as:
dx
= k (a–x) – kˊx (2.20)
dt
At equilibrium
dx
= 0 (2.21)
dt
and
k (a–xe) = kˊxe (2.22)
where xe is the value of x at equilibrium

therefore,
k xe
=K (2.23)
k' a–xe
where K is the equilibrium constant of the reaction and can be calculated from the analytical data
(concentration) as a function of time. Substituting the value of kˊ obtained from eq. (2.22) into eq.
(2.20) gives:
dx kxe
= k (a–xe) – (a–xe)
dt xe
ka
= (xe –x) (2.24)
xe
Integrating eq. (2.24) between the limits of t = 0 and t = t, and x = 0 and x = x gives:
kat xe
=
xe– (2.25)
xe ln
x
It is seen from eq. (2.22) that ka/xe = k + kˊ and substitution of this value is eq. (2.25) gives:
xe
(k + kˊ)t = ln xe–
x
and
2.303 xe
t= (k + log
xe– x
kˊ)
2.303 2.303
t= (k + log xe– (k + log (xe– x) (2.26)
kˊ) kˊ)
A graph of t versus log (xe– x) gives a straight line of slope – 2.303 / (k + kˊ), which can be
used to calculate the values of k and kˊ using the values of equilibrium constant (K) for the reactions
(Griffiths and Thomas, 1963). In view of the complexity of reversible reactions the solution to a
problem is presented.
2.4.1.1 Example of calculation of equilibrium constant and rate constants for a reversible
first-order reaction (Griffiths and Thomas, 1963)
38
Problem
The acid catalyzed conversion of a hydroxyl acid into lactone has been carried out in 0.1
M HCl solution at 20°C. The initial concentration of the acid was 18.20 units and the concentration
of the lactone as a function of time was:
Time (min) 0 20 35 50 65 80 100 ∞
Lactone conc. (units) 0 2.40 3.65 4.91 6.09 7.10 8.05 13.30
Calculation
A graph of t versus log (xe– x) gives a straight line with a slope
Form the –2.303

= –240
(k + kˊ)
and k + kˊ = 9.60 × 10–3 min–1 (2.27)
experimental data xe = 13.30 and a = 18.20
xe 13.30
and the equilibrium constant K = = = 2.71
a – xe 4.90
since
k
= K = 2.71 and k = 2.71 k'
k'
substituting the value of k in eq. (2.27) gives:
3.71 kˊ = 9.60 × 10–3 min–1
kˊ = 2.59 × 10–3 min–1 (first-order rate constant for the forward reaction).
k = 7.01 × 10–3 min–1 (first-order rate constant for the reversible reaction).
and
K = 2.71 (equilibrium constant for the reaction).
Example:
Hydrolysis of triazolam in aqueous solution (Konishi et al., 1982).
2.4.2 Parallel Reactions
Many drugs degrade simultaneously by two or more pathways. The major reaction
pathway depends on the experimental conditions.
2.4.2.1 Parallel reactions involving the formation of two products
Consider the degradation of a molecule A into products, B and C, by parallel first-order
reactions.
where
k1 and k2 are the rate constants for the formation of the products, B and C, respectively.
The rate of the reactions can be expressed as:
39
–
d[A] = k1A + k2A = (k1+ k2) [A] = kobs (2.28)
dt
where kobs is the overall rate constant and is the sum of the rate constants, k1 and k2, for the
individual reactions. Using the concentration of the products, B and C, the values of the two rate
constants can be determined.
k1 [B]
= (2.29)
k2 [C]
kobs = k1 (1 + [C] / [B]) = k2 (1 + [C] / [B]) (2.30)
Examples:
1. Simultaneous photolysis and photoaddition reaction of riboflavin in aqueous solutions
(Ahmad et al., 2004b).
2. Effect of divalent anions on photodegradation kinetics and pathways of riboflavin in
aqueous solution (Ahmad et al., 2010).
2.4.2.2 Parallel reactions involving the formation of three products
Consider the degradation of a molecule A into products, B, C and D, involving parallel first-
order reactions.
The method of calculation involved in the determination of the first-order rate constants,
k1, k2 and k3, for these reactions has been reported (Frost and Pearson, 1964; Ahmad et al.,
2016b).
Considering A, B, C and D to represent the corresponding concentrations during the
reactions and A0 as the initial concentration, the overall rate of the reaction can be expressed as:
–dA
= k1A + k2A + k3A = (k1 + k2 + k3) A (2.31)
dt
= kobs A
kobs = k1 + k2 + k3
and
(A0)
ln = kobs t (2.32)
(A)
or
A = A0 e–kt (2.33)
The reaction is simple first-order as far as the loss of A is concerned.
dB
= k1A = k1 A0 e–kt
dt
and
–k1
B= A0 e–kt + constant
kobs
40
or
B = B0 + (k1 A0/k) (1 – e–kt)
C = C0 + (k2 A0/k) (1 – e–kt) (2.34)
D = D0 + (k3 A0/k) (1 – e–kt)
If
B0 = C0 = D0 the equations simplify and
C/B = k2/k1
and
D/B = k3/k1 (2.35)
or
B: C: D = k1: k2 : k3
The product concentrations occur in constant ratio to each other. These are independent
of the time and the initial concentration of the reactant and can be used for the calculation of the
three rate constants.
Examples:
1. Liquid-phase pyrolysis of α-pinene (Fuguitt and Hawkins, 1947).
2. Photodegradation reactions of riboflavin in aqueous solution(Ahmad et al., 2016b).
2.4.3 Consecutive Reactions
The simple form of a consecutive reaction can be expressed as:
k1 k2
A B C (2.36)
Where k1 and k2 are the first-order rate constants for the degradation of A to B, an intermediate,
leading to the formation of C as the final product.
The rate of degradation of A is given by the eq. (2.37)
–d[A]
= k1 [A] (2.37)
dt
The rate of change of [B] is expressed by the eq. (2.38)
–d[B]
= k1 [A] – k2 [B] (2.38)
dt
and the rate of formation of [C] by eq. (2.39)
–d[C]
= k2 [B] (2.39)
dt
The integrated form of eq. (2.37) is
[A] = [A0] e–k1t (2.40)
A combination of eq. (2.38) and eq. (2.39) gives
–d[B]
= k1 [A0] e–k1t – k2 [B] (2.41)
dt
[k2 – k1]
[B] = (e–k1t – e–k2t) (2.42)
k1[A0]
Since
[A0] = [A] + [B] + [C] (2.43)
41
[C] = [A0] – [A] – [B] (2.44)
or
[C] 1
[A0] 1+ k2 e–k1t – e–k2t) (2.45)
= [k1 + k2]
Using the Eqs. (2.40), (2.42) and (2.45), the values of the rate constants, k1 and k2 and the
concentration of the final product C can be obtained.
Example:
Effect of borate buffer on the photolysis of riboflavin in aqueous solution (Ahmad et al., 2008).
2.4.4 Enzyme Catalyzed Reactions
Enzyme catalyzed reactions occur in biological system and proceed as follows.
1. Formation of a complex between the enzymes (E) and the substrate (S).
k
E+S ES (2.46)
k'
2. Breakdown of the complex to form the products (P) and regeneration of the enzyme.
k'' (2.47)
ES P+E
These reactions can be described by the application of Michaelis-Menton equation.
Consider a fraction of enzyme molecules (α) that is involved in the formation of the complex. The
rate of complex formation (eq. (2.46), forward reaction) would be proportional to the concentration
of the free enzyme, (1–α) [E]0 and also to the concentration of the substrate.
where [E]0 is the total concentration of the enzyme.
Therefore
v = k (1–α) [E]0 [S] (2.48)
The rate of the reverse reaction is proportional to the concentration of complex (α).
Therefore
v' = k' α [E]0
At equilibrium
v = v'
and
k (1–α) [E]0 [S] = k' α [E]0
Therefore
α k
(1– = [S] (2.49)
k'
α)
Since k / k' = K, equilibrium constant for the reaction (eq. (2.46)), eq. (2.49) can be
expressed as:
α k
(1– = [S] (2.49)
k'
α)
K [S]
α= (2.50)
1 + K [S]
42
Assuming that the reaction (eq. (2.49)) is quite slow for the equilibrium (eq. (2.48)) to be
undisturbed, the rate of reactions. v˶, being proportional to the concentration of the complex, would
be:
v˶ = k˶ α [E]0
k˶ K [S]
= [E]0
1 + K [S]
k˶ K [S]
= [E]0 (2.51)
Km + [S]
where Km = 1/K and is called Michaelis constant. It is the dissociation constant of the enzyme-
substrate complex.
Eq. (2.51) may be rearranged as:
v˶ (Km + [S]) – k˶ [S] [E]0
Therefore
v˶ k˶ [E]0 v˶
= – (2.52)
[S] Km Km
A plot of v˶ / [S] versus v˶ should be a straight line of slope – 1/ Km. The intercept on the vt
axis is v˶ [E]0, the rate when α = 1. It indicates the maximum rate when in the presence of a high
concentration of the substrate, the enzyme is completely in the complex form. Under these
conditions the rate of the reaction if proportional to the concentration of the complex, is
independent of substrate concentration and attains a limiting value (Griffiths and Thomas, 1963).
2.5 FACTORS AFFECTING DRUG DEGRADATION REACTIONS
In the evaluation of the stability of drug substance and drug products, it is necessary to
consider the factors that affect the rate of degradation under various conditions and hence the
shelf-life of the product. This information could be useful in achieving the stabilization of the
product.
2.5.1 Temperature
Collision between molecules initiates a chemical reaction and higher the number of
collision per unit time, higher is the rate of reaction. The number of collisions increases with an
increase in temperature and hence the rate of reaction. A two to three times increase in the rate
of many reactions with a 10 degree increase in temperature has been observed. The energy of
activation (Ea) is the minimum amount of energy required for a reaction to occur. In drug products
an increase in temperature T leads to an increase in degradation. The relationship between T and
the rate constant k for the degradation of a drug is given by the Arrhenius equation.
k = Ae–Ea /RT (2.53)
or ln k = ln A – Ea / RT (2.54)
where
k is the reaction rate constant of any order
A is the frequency factor
Ea is the activation energy
R is the gas constant (8.314 J mol–1K–1), and
T is the absolute temperature in K.
43
A plot of ln k versus the reciprocal of T would be linear with a slope equal to –Ea/R and
an intercept on the vertical axis equal to ln A. This plot can be used to determine the rate constant
for the degradation of the drug at any temperature (e.g. 298 K) and hence the product shelf-life at
room temperature (25°C).
The value Ea can also be calculated by determining k at two temperatures, T1 and T2 using
the equation
k2 Ea (T2 – T1)
log = (2.55)
k1 2.303R T1 T2
Eq. (2.55) can be applied to the determination of the rate constant at one temperature,
using the values of Ea and the rate constant at another temperature.
A study of the thermal degradation 7,8-dimethyl-10-formylmethylisoalloxazine, a riboflavin
analog, in acid solution at 40–60°C has been conducted. The values of activation energy (Ea) and
the frequency factor (A) for the reaction have been determined as 15.0 kcal/mol (62.8 kJ/mol) and
2.43×1010 s–1, respectively (Ahmad and Vaid, 2008).
2.5.2 Q10 Values
Connors et al. (1986) introduced the Q10 method to determine the shelf-lives of drugs
stored at different temperatures. The method can be used to estimate the effect of 10°C rise in
temperature on the degradation of drugs. The Q10 is defined as “the factor by which the rate
constants increase with a 10°C increase in temperature” and is expressed as:
Q10 k(T1 + 10)

(2.56)
= k T1
It is related to the activation energy, Ea

Ea 1 1
Q10 = exp – T+ – (2.57)
R T
10
Thus, Q10 is directly proportional to Ea and is inversely proportional to temperature. Using
eq. (2.57) the Q10 value can be calculated from the known value of Ea.
According to Connors et al. (1986), it is assumed that Ea is constant and would be the
same for any interval of temperature (for example, 20–30°C). The Ea values for drug degradation
reactions are usually in the range of 12–24 kcal/mole. The values of Ea corresponding to three
values of Q10 are given in Table 2.2.
Table 2.2 Q10 factors for 10° interval and Ea values.
Q10 (20–30°C) Ea (kcal/mol) kJ/mol
2.0 12.2 50.8
3.0 19.4 80.8
4.0 24.5 102.1
The values of Q10 = 2, 3, or 4 represent low, average and high estimates of Q 10 when Ea
is unknown and show that the rate of degradation of the majority of drugs increases by a factor of
two to four for a 10o increase in temperature in the range of 20–30°C.
For a given change in temperature, ΔT = T2 – T1, Q ΔT can be calculated as:
Q ΔT k (T – ΔT)
= Q10 (ΔT/10) (2.58)
= kT
If the shelf-life at one temperature T1 (t90 (T1)) is known, the shelf-life at a second
temperature can be calculated as:
t90 (T1) = a/k (T1) (2.59)
44
where a is a constant depending on the order of reaction.
Since
T2 = T1 + ΔT
t90 (T2) = a / k (T1 + ΔT) (2.60)
and combining this with eq. (2.58)
t90 (T2) = a / k T1 Q10 (ΔT/10) (2.61)
Since
t90 (T1) = a / k(T1)
t90 (T2) = t90 (T) / Q10 (ΔT/10) (2.62)
2.5.2.1 QΔT calculation
1. Calculate the factors by which rate constants may change for (a) a 20 to 40°C temperature
change and (b) a 20 to 0°C temperature change.
Solution:
Apply eq. (2.58)
(a) Q + 20 = Q1020/10
= 4.0, 9.0, 16.0 for Q10 = 2, 3, 4, respectively.
The values indicate that the rate increases between 4-fold and 16-fold, probably with an
average increase of about 6-fold.
(b) When ΔT = –20
Q –20 = Q10–20/10
= 1/4, 1/9, 1/16 for Q10 = 2, 3, 4, respectively.
The above values show that the rate decreases to between 1/4 and 1/16 of the initial rate.
2.5.2.2 Shelf-life calculation
The shelf-life of a reconstituted product is 100 h on storage in a refrigerator (5°C). What is
the shelf-life if the product is stored at room temperature (25°C)?
Solution:
Apply eq. (2.62)
t90 (25) = 100/2 (25–5)/10 = 25 h
2.5.3 Nonisothermal Prediction of Rate of Degradation
The evaluation of the stability of drugs can also be carried out by nonisothermal kinetics
(Hadjiioannou et al., 1993). The degradation rates are obtained by conducting an experiment in
which the temperature is programmed to change at a predetermined rate. The temperature and
time are related as:
1/T = 1/T0 + αt (2.63)
where
T0 is the initial temperature and α is a reciprocal rate constant.
The Arrhenius eq. (2.55) for time 0 and time t can be expressed as:
Ea (T2 – T1)
log kt = log k0 + (2.64)
2.303R T1 T2
45
Substitution of eq. (2.63) after rearrangement of eq. (2.64) gives
Ea
log kt = log k0 + (αt) (2.65)
2.303R
As temperature is a function of time t, kt is determined by a change in a range of
temperature. The slope of the line for eq. (2.65) is –Eaα / 2.303 and the intercept at time zero is
log k0. Using the values of k0 and Ea, and substitution of these values into the Arrhenius equation
(eq. (2.64)) would give the value of the rate constant at room temperature. The method of
programmed temperature is used for the prediction of shelf-lives of drug products.
2.5.4 pH
The pH of a solution has great influence on the rate of hydrolytic degradation reactions of
drugs in liquid dosage forms. Several studies have been conducted to evaluate the effect of pH on
the stability of drugs (Connors et al., 1986) and to determine the optimum pH range for the
stabilization of the product. The influence of pH on the hydrolysis of drugs is due to the catalytic
effect of H+ and OH– ions (specific acid-base catalysis) or different cationic and anionic buffer
species (general acid-base catalysis).
The effect of pH on the rate of degradation of a drug can be expressed in terms of rate–
pH profiles. These profiles can be used to determine the pH of maximum stability (pH max) of the
drug in a liquid dosage form. The different types of rate–pH profiles for the degradation of drugs
are reported in Table 2.3 (Connors et al., 1986).
Table 2.3 Rate–pH profiles for the degradation of drugs.

Type of profile Interpretation Relationships
V-shaped Specific acid and base catalysis pHmin = ½ pKw + ½ log kH+/ kOH–
Sigmoid curve One ionizable group affecting the pHinft = pKa (for k vs pH plot)
rate
Bell–shaped Two ionizable groups affecting pHmax = ½ (pK1 + pK2)
curve the rate
2.5.5 Catalysis
2.5.5.1 Specific acid–base catalysis
The degradation rate constant, kobs, for a specific acid–base catalyzed reaction involving H+ and
OH– ions can be expressed as:
kobs = k0 + kH+ [H+] + kOH [OH– ]
–
(2.66)
where
k0 is the rate constant of the uncatalyzed reaction,
kH+ is the rate constant for the specific acid–catalyzed reaction, and
kOH is the rate constant for the specific base–catalyzed reaction.
–
The specific acid-base catalyzed reactions are second-order reactions. However, at fixed
pH, where H+ and OH– ions are constant, the reaction apparently follows first-order kinetics.
A plot of kobs versus pH of the solution (rate–pH profile) for the specific acid-base catalyzed
photodegradation of the fluoroquinolone, moxifloxacin, is shown in Fig. 2.4 (Ahmad et al., 2014a).
The values of rate constants in the alkaline range are nearly twice compared to those determined
in the acid range indicating that OH– ions exert a greater catalytic effect on the reaction than that
of the H+ ions. The kobs has a minimum value at pH 7.5 at which the drug is most stable.
46
70.0
60.0
kobs × 104 (min-1)

50.0
40.0
30.0
20.0
10.0
0.0
0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0
pH
Fig. 2.4 Rate–pH profile for photodegradation of moxifloxacin in aqueous solution.
The ionization of a drug in aqueous solution may have considerable effect on the rate of
degradation of a drug. For example, riboflavin (vitamin B2) is a dipolar molecule with pKa values of
1.7 and 10.2. In the acid pH range, the photolysis of protonated riboflavin is catalyzed by H + ion
and in the alkaline pH range the anionic form of the molecule is subjected to degradation by OH –
ion catalysis. Riboflavin shows a bell-shaped log k–pH profile to exhibit the variations in the rate
as a function of pH (Fig. 2.5) (Ahmad et al., 2004a).
36.0
32.0
28.0
kobs × 102 (min-1)
24.0
20.0
16.0
12.0
8.0
4.0
0.0
0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0
pH
Fig. 2.5 Rate–pH profile for the photolysis of riboflavin in aqueous solution.
Cyanocobalamin (vitamin B12) undergoes photolysis by zero-order kinetics in aqueous
solution. It has a pKa values 3.5 and its protonated form is degraded faster than the neutral form
which is stable. Thus a pH range of 6–7 is most suitable for the stability of cyanocobalamin in
vitamin preparations (Fig. 2.6) (Ahmad et al., 1992). Such rate–pH profiles are necessary to
determine the pH range for the optimum stability of drugs in liquid dosage forms.
47
4.20
3.60
kobs × 107 (min-1)

3.00
2.40
1.80
1.20
0.60
0.00
0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0
pH
Fig. 2.6 Rate–pH profile for the photolysis of cyanocobalamin in aqueous solution.
2.5.5.2 General acid-base catalysis
Buffers are normally used to maintain the desired pH of liquid dosage forms. However, the
buffer species may act as proton donors (e.g., H 2PO4–) or proton acceptors (e.g., HPO42–) and thus
catalyze the degradation of drugs present in the formulation. It is, therefore, necessary to choose
a buffer system that has no or minimum effect on the stability of the drugs. General acid–base
catalysis refers to the catalysis of drug degradation that is carried out by the acidic or basic species
other than the H+ or OH– ion. In buffer catalyzed reaction, the activation energy is lowered which
leads to a greater number of collisions of molecules to accelerate the rate of reaction.
The kobs for a general acid–base catalyzed reaction involving the monoanion or the dianion
or both anions of H3PO4 in the degradation of a drug can be written as:
kobs = k0+kH+[H+]+kH2O+kOH–[OH–]+kH2PO4–[H2PO4–]+kHPO42–[HPO42–] (2.67)
= k0 + kˊ [B]T
where
k0, kH+ and kOH– are the rate constants as described in eq. (2.67),
kH2PO4– is the rate constant for the reaction catalyzed by H2PO4– ions,
kHPO42– is the rate constant for the reaction catalyzed by HPO42– ions,
kˊ is the overall rate constant for the reaction catalyzed by all the species, and
BT is the total buffer concentration.
The values of kH2PO4– and kHPO42– can be calculated by the method described by Florence
and Attwood (2006).
A plot of kobs versus BT gives an intercept of k0 and a slope of kˊ. The values of buffer
catalyzed rate constants can be determined by rearrangement of eq. (2.67) in a linear form
(kobs – [BT – [H2PO4–
kˊ kH2PO4– [H2PO4–] +
k0) = ] (2.68)
= kHPO42–
BT BT BT
Another plot of kˊ versus the fraction of acid buffer component [H2PO4–/BT] will give an
intercept at [H2PO4–/BT] = 0 which is equal to kHPO42–. The value kˊ at [H2PO4–/BT] = 1 gives the rate
constant, kH2PO4–. Thus the catalytic effect of the individual buffer species on the rate of degradation
of a drug can be evaluated. The phosphate, acetate and carbonate buffer catalyzed
photodegradation reactions of riboflavin have been reported (Ahmad et al., 2005, 2011, 2014b).
48
2.5.6 Ionic Strength Effect (Primary Salt Effect)
The rate of a drug degradation reaction involving two ionic species, A and B, with charges,
ZA and ZB, respectively, is affected by other ionic species such as electrolytes (e.g. NaCl) present
in the solution. The effect of ionic strength on the rate of a reaction can be expressed as:
log k = log k0 + 2 AZAZB√µ (2.69)
where
k0 is the rate constant in an infinitely dilution solution (µ = 0),
A is a constant for a given solvent and temperature (e.g. 0.5 for water at 25°C), and µ is the
ionic strength of the solution.
A plot of log k versus √µ gives a straight line with a slope of 1.02ZAZB (for water at 25°C)
and an intercept of log k0. An increase in the ionic strength would decrease the rate of reaction
between oppositely charged ions and increase in the rate of reaction between similarly charged
ions. In the case of barbituric acid the degradation in alkaline solution occurs by the attack of OH–
ions on the monoanion species of the molecule. An increase in the ionic strength of the solution
leads to an increase in the rate of degradation (Garrett et al., 1971). If one of the reactants is a
neutral molecule, ZA ZB = 0. In this case the rate constant should be independent of the ionic
strength in dilute solutions.
Eq. (2.69) can be applied to the reactions conducted at an ionic strength less than 0.01. If
the ionic strength is higher than this value (i.e., up to 0.1), a modified form of eq. (2.69) can be
used for drug degradation reactions.
log k = log k0 = 2 QZAZB √µ/1 + √µ (2.70)
Studies of the effect of ionic strength on the degradation rate of benzyl penicillin
(Carstensen, 1970), carbencillin (Zia et al., 1974), betamethasone-17 valerate (Khattak et al.,
2012) and riboflavin (Ahmad et al., 2016b) have been reported.
2.5.7 Solvent
Solvents can influence the rates of degradation of drugs in liquid dosage forms since these
may contain mixtures of water and organic solvents such as ethanol, propylene glycol and glycerin.
The organic solvents are often used to increase the solubility of drugs and in certain cases to
enhance the stability of the formulations. The addition of organic solvents may alter characteristics
such as the dielectric constant (a measure of solvent polarity) and the viscosity of the medium.
Variations in the dielectric constant of a solvent can results in a change in the free energy, ΔG,
and hence the rates of the reaction. Reactions between ions and dipoles are facilitated by the
solvents of high dielectric constant. These involve the formation of polar intermediate states and
thus proceed with an enhanced rate of reaction.
The effect of solvent dielectric constant on the rate of a reaction can be expressed by eq.
(2.71).
KZAZB
log kobs = log kε = ∞ – (2.71)
ε
where
kobs is the rate constant of the reaction,
kε = ∞ is the rate constant of the reaction in a solvent of infinite dielectric constant,
K is a constant for a given reaction at a given temperature,
ε is the dielectric constant of the reaction, and
ZAZB are the charges on A and B ions.
49
The rate constant, k, increases with an increase in the dielectric constant for ions of same
charge and decreases for ions of different charge.
A plot of log kobs versus 1/ε should be linear with a positive slope for reactant ions of
opposite sign and a negative slope for reactant ions of like signs.
A decrease in dielectric constant results in a decrease in the rates of anion-dipole
reactions and an increase in the rates of cation-dipole reactions. For example, the rate constant
for the hydrolysis of chloramphenicol in water-propylene glycol mixtures has been found to
increase with a decrease in the dielectric constant due to H 3O+ ion-dipole reaction in the presence
of perchloric acid (Marcus and Teraszka, 1959).
Several studies have been conducted to evaluate the effect of solvents on the kinetics of
degradation of drugs including riboflavin (Ahmad et al., 2015a), norfloxacin (Ahmad et al., 2015b)
levofloxacin (Ahmad et al., 2013), moxifloxacin (Ahmad et al., 2014a), β-lactams antibiotics (Hou
and Poole, 1969), aspirin (Bakar and Niazi, 1983) and indomethacin (Ghanem et al., 1979).
2.5.8 Oxygen
2.5.8.1 Oxidizable drugs
Many drugs are susceptible to oxidation and undergo degradation in solid or liquid dosage
forms in the presence of oxygen during processing or storage. Examples of these drugs include
5-aminosalicylic acid, ascorbic acid, captopril, cholecalciferol, epinephrine, hydroquinine,
fumagillin, hydrocortisone, ergocalciferol, 6-mercaptopurine, methyldopa, morphine,
phenylbutuzone, prednisolone, promethazine, spironolactone, sulpyrine, tetrazepam, vitamin A
and sulfides (Yoshioka and Stella, 2000; Connors et al., 1986). Antibiotics, steroids, vitamins, fats
and oils also undergo oxidative degradation by molecular oxygen. Molecular oxygen in the ground
state exists as a diradical or in the triplet state (3O2). It can be excited by UV light to form the singlet
state (1O2).
hv (2.72)
triplet state singlet state

Singlet oxygen is a strong oxidizing agent and is more reactive than triplet oxygen. It is
involved in many photosensitized oxidation reactions (Smith et al., 1973) .
50
Example:
Oxidation of ascorbic acid (Blaug and Hajratwala, 1972).
Protection from oxygen can be achieved by storing the drug products in an anaerobic
atmosphere, by purging the solution with nitrogen, addition of antioxidants and removal of metal
ions that initiate catalytic reaction. An oxidizable drug may be stabilized by the use of a compound
of lower reduction potential, Eo, than the drug. The oxidative degradation of a drug may be
minimized by adjusting the pH of the medium to a value where a reversible redox process may
occur. Solid dosage forms, e.g. tablets can be protected from oxygen by film coating and suitable
packaging.
2.5.8.2 Oxidation reactions
The majority of drugs exist in the reduced state and are, thus, susceptible to oxidation.
The absorption of UV and visible light may lead to photodegradation. The chemical and
photooxidation reactions involve one electron change in the molecule. The oxidation-reduction
reactions occur simultaneously and involve transfer of electrons. For example, the oxidation of iron
can be expressed by eq. (2.73):
Fe2+ Fe3+ + e– (2.73)
In organic compounds, the oxidation state of carbon atom is given by the number of bonds
between carbon and oxygen. The oxidation state of carbon compounds increases with the number
of these bonds. Consider the oxidation of methane
CH4 CH3OH CH2O HCOOH CO2 (2.74)
The oxidation of hydroquinone to quinone in aqueous solution involves the reaction of the
ionized form of the molecule depending on the pH of the solution (Connors et al., 1986).
-
OH O
O O
OH
O2
+ 2H +H2O2 + H2O (2.75)
-
OH O O O
The mechanism of oxidation of sodium sulfite (an antioxidants) in the presence of a metal
ion (M+) catalyst involves several steps and is described by Connors et al. (1986) as follows.
SO32– + M+ SO3–● + M● (2.76)

SO3–● + O2 SO–5● (2.77)
SO–5● + HSO3– HSO5– + SO3–● (pH ≤ 7) (2.78)
SO–5● + SO3 2– SO52– + SO3–● (pH ≤ 7) (2.79)
SO3 2– + HSO5– HSO4– + SO42– (pH ≤ 7) (2.80)
SO3 2– + SO5 2– 2SO42– (pH ≤ 7) (2.81)
SO3–● + SO–5● 2–
S2O6 + O2 (2.82)
SO–5● + inhibitor nonreactive products (2.83)
where
eq. (2.76) is the initial step of the reaction, eqs. (2.77)–(2.79) are the propagation steps,
eqs. (2.80) and (2.81) are the oxidation steps giving the ultimate oxidation product, SO 42–,
and eqs. (2.82) and (2.83) are the termination steps.
The pH dependence of the reaction is due to the amount of fractions of SO32– and HSO3–
ions present at a particular pH.
51
2.5.9 Surfactant
Surfactants are compounds that are capable of lowering the surface tension or interfacial
tension between the two liquids or between a liquid and a solid. Surfactants may act as detergents,
wetting agents, emulsifiers, foaming agents and dispersants. They may inhibit the rates of
degradation reactions and thus improve the stability of drugs. Several studies have been carried
out to evaluate the effect of surfactants on the stability of drugs in pharmaceutical systems. Some
of these studies are presented as follows:
An early study of the effect of surfactants on the rate of hydrolysis of esters using
benzocaine has been conducted. It has been found that the rate of hydrolysis of benzocaine in
alkali-stable nonionic surfactants varies with the concentration of the surfactant. The hydrolysis
takes place both in the micelle and in the aqueous phase. Anioinc and cationic surfactants stabilize
the drug to base catalysis, with an eighteen-fold increase in half-life in 5% lauryl sulfate solution
(Reigelman, 1960).
The effect of surfactant micelles on the aqueous stability of β-lactam antibiotics has been
studied by determining the apparent binding constants of the micellar-antibiotic complex as a
function of solution pH and ionic strength using dynamic dialysis method. The interaction of these
antibiotics in the nonionic and anionic micelles of polyoxyethylene-23-lauryl ether and sodium
lauryl sulfate showed large differences in the binding constants of undissociated and ionized
species of pencillins. Acid degradation of pencillins is protected in micellar solutions of the above
two surfactants (Tsuji et al., 1982). The forced degradation of aqueous paliperidone solutions
under photolytic stress conditions on exposure to sunlight for 72 h has shown major degradation
by HPLC in the presence of cationic and nonionic surfactants at concentration exceeding critical
micellar concentration (CMC) (Marothu et al., 2015).
The solid lipid nanoparticles (SLN) have been found to undergo enzymatic degradation by
pancreatic lipase at different rates in the presence of surfactants. The degradation of SLN depends
on the length of fatty acid chains in the glycerides and the surfactant used for the production of
SLN. It has been found that longer the fatty acid chain, the slower the degradation. The surfactant
accelerates (e.g. cholic acid sodium salt) or hinders (e.g. Poloxamer 407, a hydrophilic non-ionic
surfactant) the degradation of SLN due to steric factors (Olbrich and Muller, 1999).
The emulsion stability of surface active (e.g. phenobarbital) and non surface active (e.g.
benzocaine) drugs in triphasic systems in the presence of the ionic surfactant
cetyltrimethylammonium bromide (CTAB) and the nonionic surfactant Brij 97 (polyoxyethylene 10
oleoyl ether) has been studied by droplet size analysis using photon correlation spectroscopy. The
droplet size of CTAB–stabilized emulsion system has been found to be bigger than that of the Brij
97-stabilized system because of the relatively small dense interfacial packing of the cationic
surfactant. CTAB forms a complex with the drugs that increases the stability of the emulsion
(Chidambaram and Burgess, 2000).
2.5.10 Moisture
Moisture present in the surroundings may be adsorbed on the surface of solid drugs or
solid formulations and cause dissolution of the active ingredient. This may affect the drugs
susceptible to hydrolytic degradation, for example, aspirin, an ester, and sulfacetamide, an amide.
The hygroscopic content of the solid dosage forms may be detrimental in promoting hydrolytic
reactions.
Moisture may play the role of a catalyst is drug degradation reactions. Water may
participate as a reactant in degradation processes such as hydrolysis, isomerization or other
bimolecular reactions. In these reactions the rate of degradation of the drug is a function of the
concentration of water, H+ ions, or OH– ions and may be expressed as:
–
d[A] = kH+ [H+] [A] + kH2O [H2O] [A] + kOH– [OH+] [A] (2.84)
dt
52
Examples of effect of moisture on the kinetics of degradation of drugs include ascorbic
acid (Yamamoto and Kawai, 1959), thiamine salts (Yamamoto and Inazu, 1959a), aspirin
(Yamamoto and Inazu, 1959b), ranitidine HCl (Teraoka et al., 1993) and vitamin A (Carstensen et
al., 1966).
Moisture can change the physical characteristics of tablets, such as disintegration and
hardness and, thus may facilitate the degradation of active ingredients (Ahmad and Shaikh, 1994a,
1994b). Relationships between moisture content and % degradation of a drug (Kornblum and
Sciarrone, 1964) and moisture uptakes of tablets a function of storage time (Ahmad and Shaikh,
2003) have been reported.
2.5.11 Problems
Zero-Order Reactions
1. The degradation of a dye in liquid preparations follows zero-order kinetics at 25°C. The rate
of the reaction is 7.3×10–7 absorbance units per min.
Calculate
a) The half-life of a preparation with an initial absorbance of 0.240 at 450 nm.
b) The predicted life of the preparation at 25°C. When the absorbance of the solution is 0.100.
Answer
a) 114 days
b) 133 days
2. The first-order rate constant, k1, for the degradation of a drug at pH 5.0 is 2×10–7 s–1. The
solubility of the drug is 1 g/100 ml. For a suspension of the drug containing 2.5 g/100 ml,
calculate
a) Zero-order rate constant, k0.
b) Shelf-life in solution (zero-order dependent).
Answer
a) k0 = 2.20×10–7 g dL s–1
b) t90 = 13.2 days
c) t90 = 6.1 days
First-Order Reactions
3. A drug product (10.0 mg/mL) becomes ineffective after 25% degradation. The drug content
was found to be 8.2 mg/mL. If the drug is degraded by first-order.
Calculate
a) The expiration date on the label and
b) The half-life of the product.
Answer
a) t75 = 17.4 months
b) t1/2 = 0.0165 month
4. A drug product undergoes degradation by first-order. Using the following assay data,
calculate the rate constant and the half-life.
53
Time (month) 0 2 4 6 12 18 24
% 100 89.5 77.4 68.0 45.5 30.9 21.0

concentration
Answer
a) k = 0.0651 month
b) t1/2 = 16.5 months
Second-Order Reactions
5. The saponification of ethyl acetate by NaOH was carried out at 25°C. The initial
concentration of ethyl acetate and NaOH were 0.0100 M. The concentrations of NaOH after
50 min was determined as 0.00600 M. Calculate the second-order rate constant and half-
life of the reaction.
Answer
a) k = 1.03 M–1 min–1
b) t1/2 = 97.1 min
a. The reaction of a drug A with a reagent B was carried out at equal
concentrations of the reactants. The decrease in the concentrations of A
was determined spectrometrically as follows:
t (s) 0 100 200 300 400 500
[A] × 103 5.00 3.27 2.40 1.92 1.59 1.40

M
Prepare a graph of A versus t and determine the order of reaction using the half-life method.
Answer
Second-order reaction
6. The second-order rate constants, k2, for the alkaline hydrolysis of aspirin at 30, 40 and 50°C
are 0.0572, 0.106 and 0.192 M–1 s–1, respectively. What is the activation energy (Ea) in kcal
mole–1 and kJ mole–1 and the frequency factor A, in s–1 for the reaction?
Answer
Ea = 12.0 kcal mole–1 or 50.2 kJ mole–1.
A = 2.67×107 s–1.
7. The first-order rate constant for the degradation of a drug at 80°C was determined as
9.6×10–7 s–1. If the activation energy, Ea for the degradation is 24.5 kcal mole–1, what is the
rate constant at 60°C?
Answer
k2 = 1.18×10–7 s–1
8. The hydrolysis of a drug is independent of pH in the range of 2–7 in ortho-phosphate buffer.
The first-order rate constant in the pH range was determined as 6.26×10–6 s–1 at 80°C. The
activation energy, Ea, of the reaction at pH 6.0 is 24 kcal mole–1. Calculate the shelf-life at
25°C in ortho-phosphate buffer.
54
Answer
t90 = 3.5 months
Q10 Calculations
9. Calculate the Q10 factors by which the rate constants may change for a change of a 10°
around room temperature (20–30°C), for two reactions with activation energies of 12.0 and
24.0 kcal mole–1.
b) Calculate the factors by which the above rate constants may change for a 25 to 50°C
change.
Answer
a) Q ΔT = 5.4
b) Q ΔT = 30.0
10. The expiration period for a reconstituted product (Q 10 = 2.0) is 72 h when stored in a
refrigerator at 5°C. Calculate the expiration period when the product is stored at room
temperature.
Answer
t90 (25°) = 18 h
11. An aqueous drug solution stored at room temperature (25°C) showed a shelf-life of 10 days.
Find the shelf-life when the solution is stored at 15°C (cold room) and at 8°C (refrigerator),
if the Q10 value is 2.0.
Answer
t90 (15°) = 20 days
t90 (5°) = 40 days
The shelf-life will be increased from 10 days to 40 days on storing the solutions in
refrigerator.
The problems included in this section have been selected from text books (Connors et al.,
1986; Hadjiioannou et al., 1993; Sinko, 2011; Florence and Attwood, 2006; Loftsson, 2014).
55
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59
60
CHAPTER – 3
CHEMICAL STABILITY
3.1 INTRODUCTION
The stability is an essential quality characteristic of drug products. It is considered as the
most important factor in relation to a drug substance for development into a therapeutically active
dosage form. The assessment of the chemical and physical stability of a product is carried out
during the preclinical formulation studies, process development and packaging evaluation. The
efficacy and safety of a product is based on the stability characteristics of the active ingredients
and excipients.
Knowledge of the specific chemical functional groups of a drug molecule may enable the
prediction of its degradation pathways and a possible approach to its stabilization. The selection
of an appropriate packaging system is necessary to ensure the chemical and physical stability of
the product during the storage period and use. The assessment of the stability of drug substances
and drug products is a mandatory requirement by regulatory agencies.
The chemical stability of drug products involves the assessment of the chemical integrity
and labeled potency of all the ingredients and that any change should be within the specified limits.
Several accounts of the chemical stability of drug substances and drug products are available
including monographs (Connors et al., 1986; Carstensen and Rhodes, 2000; Yoshioka and Stella,
2000; Baertschi, 2005; Loftsson, 2014), books (Lintner, 1973; Racz, 1989; Guillory and Poust,
2002; Florence and Attwood, 2006; O’Donnell and Bokser, 2006; Sinko, 2011) and reviews
(Carstensen, 1974; Mollica et al., 1978; De Ritter, 1982; Grit and Crommelin, 1993; Bastin et al.,
2000; Waterman and Adami, 2005; Blessy et al., 2014).
3.2 STUDY OF THE CHEMICAL STABILITY OF A DRUG
The study of the chemical stability of a drug substance requires a consideration of the
following factors:
 The solubility of the drug in aqueous and organic solvents.
 The spectral characteristics of the drug molecule.
 The ionization behavior (pKa) of the drug molecule.
 The sensitivity of the drug to environmental factors, excipients and medium characteristics.
 Chemical degradation pathways.
 Structural characteristics of chemical degradants.
 Toxicity of chemical degradants.
 A validated stability-indicating method for the assay of intact drug and degradants.
If a drug undergoes hydrolysis, oxidation or photolysis reaction, the following sequence of
steps is involved in this study:
 Verification of degradation by a certain mode of reaction using chromatographic and
spectrometric techniques. Thin layer chromatography (TLC) pattern and UV and visible
spectral changes provide an indication of the degradation of the compound.
61
 Separation, isolation, purification and characterization of the degradation products using
chromatographic (e.g. HPTLC, HPLC, electrophoresis), spectrometric (UV-vis, FTIR,
NMR, MS, GC/MS, LC/MS) and other techniques.
 Separation of degradation products from the parent compound by appropriate extraction
methods and confirmation by HPLC and UV-vis spectrometry. The separation may also be
achieved directly by HPLC.
 Development of a specific (stability-indicating) analytical method for the assay of the intact
drug in the presence of degradation products and any interfering substances present in
degraded solutions.
 Evaluation of the kinetics of degradation of the drug and determination of its shelf-life (t90).
 Establishment of stability protocol for the drug product under specified storage conditions
(i.e. temperature / relative humidity / light exposure) according to ICH Guidelines.
 Screening of the degradation products for their potential toxicity.
3.3 CHEMICAL DEGRADATION REACTIONS
The drug substances are chemical entities that possess diverse molecular structures and
different functional groups. They may undergo degradation reactions in aqueous and organic
solvents through various pathways depending upon the factors causing degradation. The major
modes of drug degradation are:
 Hydrolysis
 Oxidation
 Decarboxylation
 Elimination
 Isomerization
 Dimerization
 Epimerization
 Dehydration
 Dehydrogenation
 Dehalogenation
These reactions are described as follows:
3.3.1 Hydrolysis
Hydrolytic degradation in aqueous solution and in liquid dosage forms is among the most
common reactions destabilizing the drugs that contain ester, amide, imide, carbamate, lactone,
nitrile and carbohydrate groups. A large number of drugs are susceptible to acid and/or alkaline
hydrolysis such as aspirin, paracetamol, sulfacetamide, indomethacin, procaine, digoxin,
riboflavin, lincomycin, chloramphenicol, penicillins, cephalosporins and benzodiazepenes. The pH
of the medium plays an important role in the hydrolysis of drugs (see Section 2.5.4).
3.3.1.1 Hydrolysis of esters
The ester compounds undergo hydrolysis through nucleophilic attack of water or OH– ions
on the ester group.
62
Acetylsalicylic acid (Aspirin)
Aspirin (3.1) is the most common example of the hydrolytic degradation of an ester
compound. It undergoes hydrolysis in aqueous solution to form salicylic acid (3.2) and acetic acid
(3.3). The reaction is accelerated with an increase in temperature (Fersht and Kirby, 1967).
O O
OH O
H2O OH
+ H3C
O OH
OH
H3C O
(3.1) (3.2) (3.3)
Procaine
The most important reaction involved in the degradation of procaine (3.4) is hydrolysis. It
leads to the formation of 4-aminobenzoic acid (3.5) and diethylaminoethanol (3.6). The rate of the
reaction is influenced by the ionization of the molecule (pKa 8.05) (Higuchi et al., 1950).
C2H5
O O CH2 CH2 N O OH
C2H5
H2O C2H5
+ HO CH2 CH2 N
C2H5
NH2 NH2
(3.4) (3.5) (3.6)
3.3.1.2 Hydrolysis of amides
Compounds containing an amide bond are less susceptible to hydrolysis compared with
those containing an ester bond. This is because of the fact that the carbonyl carbon of the amide
bond has a lower electrophilic character.
Paracetamol
Paracetamol (3.7) is hydrolyzed in aqueous solution to form 4-aminophenol (3.8) and
acetic acid (3.3) (Koshy and Lach, 1961).
NHCOCH 3 H2N
O
H2O
+ H3C
OH
OH HO
(3.7) (3.8) (3.3)
Sulfacetamide
Sulfacetamide (3.9) in aqueous solution is hydrolyzed to form sulfanilamide (3.10) and
acetic acid (3.3) (Meakin et al., 1971). Sulfanilamide (3.10) undergoes oxidation to yield 4,4’-
azobenzenedisulfonamide (3.11) which is further oxidized to 4,4’-azoxybenzenedisulfonamide
(3.12) on exposure to light. These reactions are accompanied by the formation of a yellow to
reddish brown color (Ahmad and Ahmad, 1981, 1989; Ahmed et al., 2016).
63
SO2NHCOCH 3 H2NO 2S
O
H2O
+ H3C
OH
NH2 H2N
(3.9) (3.10) (3.3)
SO2NH 2 SO2NH 2
H2NO 2S
oxidation oxidation
N N O
N N
H2N
SO2NH 2 SO2NH 2
(3.9) (3.11) (3.12)

3.3.1.3 Hydrolysis by ring opening
The hydrolysis of a drug molecule by ring opening could occur by the cleavage of the C–N
bond.
Riboflavin
Riboflavin (vitamin B2) (3.13) undergoes base-catalyzed hydrolysis by cleavage of the
isoalloxazine ring to give 1,2-dihydro-6,7-dimethyl-2-keto-I-D-ribityl-quinoxaline-3-carboxylic acid
(β-keto acid) (3.14) and 6,7-dimethyl-4-D-ribityl-2,3-dioxo-1,2,3,4-tetrahydroquinoxaline (flavor-
violet) (3.15) (Surrey and Nachod, 1951; Ahmad et al., 1973). The degradation reaction is
accompanied by the loss of absorption of riboflavin at 445 nm and is accelerated by an increase
in temperature.
64
CH 2OH
HO H
HO H
HO H
H2C
H3C N O
NH
H3C N
O
(3.13)
OH-
O2 OH-
CH 2OH
CH 2OH
HO H
HO H
HO H
HO H
HO H
HO H
H2C
H2C
H3C N O
H3C N O
H3C N O
H3C N COOH H
(3.14) (3.15)
65
Norfloxacin
Norfloxacin (3.16), a fluoroquinolone antibacterial agent, is susceptible to hydrolytic
degradation by piperazine ring cleavage in the alkaline solution to form the products 3.17 and 3.18.
The reaction occurs in the presence of light (Ahmad et al., 2015).
O O O
O
F
F
OH
OH
hv HN N
N N
+ OH- H3N
+
H2N
(3.16) (3.17) CH3
CH3
-CH2CH2NH2
O O
F
OH
+
H3N N
CH3
(3.18)
3.3.2 Oxidation
The oxidative degradation of drugs is another widely occurring reaction in the presence of
oxygen or an oxidizing agent. Exposure of the drug to atmospheric oxygen during manufacturing,
storage or use can adversely affect the drug content by oxidation reactions (see Section 2.5.8.2).
Many drugs undergo oxidation including ascorbic acid, vitamin A, glucose, morphine,
hydrocortisone, methyldopa, aldehydes, phenols, unsaturated compounds, thiols, phenothiazenes
and polyenes. The pH of the medium may affect the rate of oxidation reactions as a result of
ionization and change in the redox potential of the species involved.
Ascorbic acid
The degradation of ascorbic acid (vitamin C) (3.19) in aqueous solution under aerobic
conditions results in the oxidation of the molecule to dehydroascorbic acid (3.20). The
dehydroascorbic acid undergoes hydrolysis to form diketogulonic acid (3.21) in alkaline solution
(Blaug and Hajratwala, 1972).
66
O O O
O HOHC
HOHC oxidation
CH 2OH CH 2OH
OH O O
HO
(3.19) (3.20)
Hydrolysis
HOOC
C O
C O
CHOH
CHOH
CH 2OH
(3.21)
Morphine
The major degradation reaction of morphine (3.22) in aqueous solution is oxidation by air
and light. The oxidation products of morphine include pseudomorphine (noxydimorphine) (3.23)
and morphine N-oxide (3.24) (Yeh and Lach, 1961).
HO
O
N CH3
HO
(3.22)
oxidation
N CH3
HO
O
N CH3 O
OH OH
HO
(3.23) (3.24)
67
Phenols
Phenols (3.25) undergo facile oxidation reactions. The hydroxyl group is strongly electron
donating to the phenyl ring which is oxidizable. Abstraction of the proton gives a stable radical
which then reacts with molecular oxygen. The deprotonation of phenol at higher pH to the
phenolate anion strongly catalyzes auto-oxidation reaction (base-catalyzed auto-oxidation). The
phenolate anion is an effective nucleophile that can react with electrophilic species at either the
oxygen or the ortho or para positions. Phenolic compounds are oxidized in the presence of Fe 3+
or Cu2+ ions (Baertschi, 2005).
OH O O O
O
CH
-H. HC
(3.25) CH
OH- -e- oxidative reactions, especially at ortho and

para positions of phenolic compounds
-
O
3.3.3 Decarboxylation
A drug possessing a carboxyl group may degrade by decarboxylation reaction under
certain conditions.
4-Aminosalicylic acid
The major degradation reaction of 4-aminosalicylic acid (3.26) in aqueous solution is
decarboxylation which leads to the formation of 3-aminophenol (3.27). The reaction is faster in the
acid medium compared to that of the alkaline medium in which the molecule is in the ionized form
(Connors et al., 1986).
O OH
OH
OH
-CO2
NH2
H2N
(3.26) (3.27)
3.3.4 Elimination
Elimination reactions involve the removal of two or more substituent from a molecule either
in one step or two steps. The one step mechanism is known as E2 reaction (bimolecular) and the
two step mechanism is known as E1 reaction (unimolecular).
Trimelamol
Trimelamol (N2,N4,N6-trimethylol-N2,N4,N6-trimethylmelamine) (3.28), a synthetic
carbinolamine-containing antitumor drug, undergoes degradation by two major pathways. One
degradation pathway involves the loss of hydroxylmethylene groups by elimination of
68
formylaldehyde to form the parent compound, trimethylmelamine (3.29). The products have been
determined by HPLC to evaluate the kinetics of the reaction (Jackson et al., 1991).
HOH 2C CH3 H CH3
N N
N N N N
CH3 -HCHO H3C CH3
H3C
N N N N N N
CH 2OH CH 2OH H H
(3.28) (3.29)
3.3.5 Isomerization
Isomerization reactions involve the transformation of one molecule into another having
exactly the same atoms but with a different arrangement.
Cephalosporins
Cephalosporins are known to undergo isomerization of the double bond involving the ∆3
position (3.30) to the ∆2 position (3.31).
H2N S H2N S
O O
N S N
1 2 S
NH NH 1 2
isomerization
N N 3
N 3
N
O 4 X O X
OCH 3 OCH 3 4
COOX COOX
∆3-isomer ∆2-isomer
(3.30) (3.31)
3.3.6 Dimerization
It is a chemical reaction in which two molecular subunits are joined, resulting in the
formation of a dimer.
Nalidixic Acid
Nalidixic acid (3.32) undergoes dimerization on thermolysis by decarboxylation to form a
dimer (3.33).
H3C CH2
N N CH3
O
O
COOH
thermolysis
H3C N N -CO2 O
H3C N N
H2C CH3
H2C CH3
(3.32) (3.33)
69
3.3.7 Epimerization
The epimerization process involves the changing of one of the chiral center in a molecule
to form another molecule called epimer. An epimer molecule differs from the other molecule (its
diastereomer) by only one chiral center. Epimers are not mirror images of each other and have
multiple sterogenic centers.
Ergotamine
Ergotamine (3.34) undergoes acid-catalyzed reversible epimerization at C–8 and C–2′
positions of the molecule (3.35) in the absence of air and light. The epimerization at C–8 occurs in
the lysergic acid part of the molecule at pH 3.8 in the temperature range of 30–60°C. The reaction
at C–2′ takes place in the cyclic tripeptide part of the molecule at pH 3.6 in the temperature range
of 50–80°C (Ott et al., 1966). Both isomers are detectable in ergotamine tartrate parenteral
solutions.
CH3
CH3
O OH O OH
H NH O
N H NH O
8 2
H N
2
N 8 H
O N
N O O
CH2 N O
H
CH3 H CH2
CH3
H
H
HN
HN
(3.34) (3.35)
3.3.8 Dehydration
Dehydration is a chemical reaction that involves the loss of a water molecule from the
reacting molecule.
Glucose
Glucose (3.36) undergoes dehydration reaction to form 5-(hydroxymethyl)-2-furaldehyde
(3.37) on heating with hydrochloric acid (Wolfrom et al., 1948).
CH 2OH
O
HO OH HOH 2C CHO
O
OH OH
(3.36) (3.37)
Batanopride Hydrochloride
In acidic media (pH 2–6) batanopride hydrochloride (3.38), an antiemetic drug, is
degraded by intramolecular cyclization followed by dehydration to form 2,3-dimethylbenzofuran
(3.39) (Nassar et al., 1992).
70
H5C2 H5C2
N CH2 CH2 NH O N CH2 CH2 NH O
H5C2 O H5C2
O O
CH3
CH3 CH3
Cl Cl
CH3
NH2 NH2
(3.38) (3.39)
3.3.9 Dehydrogenation
Dehydrogenation is a chemical reaction that involves the removal of hydrogen from a
molecule.
2- Aminofluorene
2-Aminofluorene (3.40) undergoes oxidative dehydrogenation to 2-nitro-9-fluorenone
(3.41) in acetonitrile using potassium iodide-tert-butyl hydroperoxide (KI–TBHP) as catalytic
system at 80 0C (Kumar et al., 2011).
KI-TBHP
NH2 NO 2
O
(3.40) (3.41)
3.3.10 Dehalogenation
A reaction involving the removal of a halogen atom from a molecule.
Norfloxacin
Norfloxacin (3.16) undergoes defluorination in neutral aqueous solution to form the
product (3.42) (Fasani et al., 1999).
O O
F COOH COOH
hv
N -F- N
N N
HN HN
CH3 CH3
(3.16) (3.42)
3.4 CHEMICAL STABILITY/DEGRADATION STUDIES
Several studies have been conducted to evaluate the chemical stability and degradation
of drug substances and drug products. Some of these studies are presented as follows.
3.4.1 Aqueous Solution
A kinetic study of the alkaline hydrolysis of 7,8-dimethyl-10-(formylmethyl)isoalloxazine
(FMF) (3.43), an intermediate product in the photodegradation of riboflavin, has been conducted
in the pH range 9 to 12. FMF leads to the formation of lumichrome (LC) (3.44) and lumiflavin (LF)
(3.45) in alkaline solution with second-order rate constants of 0.348 and 0.063 M–1 s–1 at pH 9 and
0.068 and 0.132 M–1 s–1 at pH 12, respectively. FMF and the hydrolytic products, LC and LF, have
71
been identified chromatographically and determined by a multicomponent spectrometric method.
LC and LF were extracted from the degraded solutions with chloroform at pH 2.0 and determined
by a two-component method at 356 and 445 nm. FMF was determined directly at 385 nm in the
aqueous phase. The molar concentrations of these compounds were used to evaluate the kinetics
of the reaction (Ahmad et al., 1980).
High-performance liquid chromatography (HPLC) has been used to study the chemical
stability of 5-aza-2′-deoxycytidine in the whole pH range. It undergoes fast reversible degradation
to form N-(formylamidino)-N′β-D-2-deoxyribofuranosylurea which further degrades to 1-β-D-2′-
deoxyribofuranosyl-3-guanylurea in alkaline solution. The kinetics of the degradation reactions has
been studied. The degradation of 5-aza-2′-deoxycytidine in alkaline solution is similar to that 5-
aza-cytidine. The intermediate product in the reaction is most stable in the neutral solution stored
at low temperature (Lin et al., 1981).
Cefoxitin sodium undergoes specific acid-base catalyzed hydrolysis of the ester group and
the β-lactam ring in aqueous solution. The apparent first-order rate constants for the hydrolytic
reaction at pH 3 to 9 have been determined. Under these pH conditions cefoxitin sodium shows
about 10% loss in two days at 25°C. The amorphous form of the drug is less stable than the
crystalline form (Oberholizer and Brenner, 1979).
The chemical stability of ranitidine hydrochloride in aqueous solution at different pH values
and temperatures has been studied using a HPLC method. The percent degradation of the drug
increases with a decrease in pH of the medium and an increase in temperature. The results
indicate that the degradation of ranitidine is a specific acid-catalyzed reaction (Teraoka et al.,
1993).
CHO
CH2
H3C N N O
NH
H3C N
(3.43) O
OH- OH-
CH3
H
H3C N N O H3C N N O
NH NH
H3C N H3C N
O O
(3.44) (3.45)
3.4.2 Pharmaceutical Preparations
Insulin preparations stored at different temperatures have been found to undergo
hydrolytic degradation. The degradation is rapid in acid media as a result of deamidation at residue
AsnA21 and is slow in the neutral media due to deamidation at residue Asn B3. The degradation rate
of insulin at residue B3 varies with temperature and preparation. A reduction in B3 transformation
has been observed for crystalline insulin compared to that of the amorphous form. In certain
72
crystalline suspensions cleavage of the peptide bond A8–A9 takes place. The hydrolytic
degradation of insulin involves the participation of an imide intermediate in the reaction.
Preparations containing rhombohedral crystals along with free zinc ions undergo hydrolysis of the
peptide chain only (Brange et al., 1992a). The storage of insulin preparations at 4–45°C leads to
the formation of covalent, high molecular weight products mainly, the covalent insulin dimers. In
the preparations containing protamine, covalent insulin-protamine products are formed. The
formation of oligo compounds and polymers also takes place at >25°C by parallel or consecutive
reactions. Temperature exerts a pronounced effect on the formation of different products in insulin
preparations. The dimer formation occurs between molecules within hexameric units present in all
types of insulin preparations and the formation of dimers is greater in preparations containing
glycerol (Brange et al., 1992b).
The lyophilized proteins and peptides contain sugars and polyols as bulking agents and
lyoprotectants but the reducing sugars have been found to react with proteins. The recombinant
human relaxin in lyophilized preparations reacts with glucose, used as excipient, to undergo fast
covalent modification. The LC/MS and tryptic mapping of the protein showed that one degradation
pathway involves covalent adduct formation of glucose with the side chain amino groups of the
protein (i.e. Lys and Arg) by Maillard reaction. The other pathway leads to Ser degradation from
C-terminal of the β-chain of proteins. The latter reaction occurs predominantly in the solid state
and involves the reaction of glucose with Ser hydroxyl group and hydrolysis of Trp–Ser amide
bond through a cyclic intermediate product. Mannitol (polyhydric alcohol) and trehalose
(nonreducing sugar) do not undergo such reactions with relaxin (Li et al., 1996).
A study has been carried out to determine: 1) the relation between chemical stability, aging
state and global molecular motion and 2), the molecular mobility in multicomponent systems. It
also envisaged to find out whether annealing a glass below its transition temperature (Tg) has any
effect on its chemical stability and to determine if the degradation rate couples with global
relaxation times determined by calorimetric method and/or with T1 and T1rho relaxation times
determined by solid state NMR spectrometry. In this study the chemical degradation of lyophilized
aspartame/sucrose and aspartame/trehalose (1:10, w/w) preparations has been investigated to
evaluate the impact of annealing on their chemical stability by the application of stretched time
kinetics. The results supported the hypothesis that molecular mobility for structural relaxation is
affected by thermal transitions. Such an effect is critical for chemical stability and annealing results
in the stabilization of the preparations (Luthra et al., 2008).
Pseudolatexes of biodegradable polyesters, poly (D, L-lactide) and poly (ε-caprolactone),
are used as aqueous coating material for sustained release dosage forms. A study has been
conducted out to determine the effect of surfactant, temperature, pH and particle size on the
hydrolytic degradation of these polymers in the form of colloidal dispersions. The nonionic
surfactant has no effect on the stability of the dispersion. Storage of dispersions in unbuffered
solution for one year at 5°C showed small changes in molecular weight of the polymers. Rapid
hydrolytic degradation of the dispersions was observed at 37°C. The polymers stored at pH 1.65
at 37°C underwent enhanced degradation while these were stable at pH 1.65 at 5°C for 4 months
(Coffin and McGinety, 1992).
The effect of spray drying and processing conditions on the residual moisture content and
biochemical stability of inhalation protein powders has been investigated. The mannitol-formulated
powders of a humanized monoclonal antibody (anti-IgE) and recombinant human
deoxyribonuclease (rhDNase) have been prepared by spray drying and the residual moisture and
moisture uptake determined by thermal gravimetric analysis and gravimetric moisture sorption
isotherm, respectively. The main degradation product of the protein, the protein aggregate,
observed on long-term storage, was determined by size exclusion HPLC. The results showed that
spray-dried powders with about 3% moisture, equivalent to freeze-dried powder, could be obtained
by high temperature spray-drying. At a RH of air lower than 50% during processing and storage,
the powders maintain aerosol performance (fine particle fraction). The powders on storage under
dried conditions show better long-term biochemical stability of the proteins (Maa et al., 1998).
73
The effect of surface charge on the degradation kinetics of methyl paraben, used as a
model solute in oil-in-water emulsions, has been studied. The surface charge is varied by adding
phosphatidylglycerol (anionic surfactant) or stearylamine (cationic surfactant) to a intravenous lipid
emulsion that was stabilized using egg phospholipid. The rates of hydrolytic degradation (pH 8.0)
in aqueous phase, oil phase, interface and aqueous micellar phase have been determined using
a four-phase kinetic model. The degradation rate in aqueous phases depends on zeta potential as
a result of surface charge on the pH of microenvironment of oil drops (surface activity). The rate
of hydrolysis of methyl paraben depends on the pH of microenvironment and on the pH of the bulk.
The hydrolysis rate is inversely proportional to the partition coefficient of methyl paraben. The
surface charge effect is greater with a small partition coefficient and smaller with a large partition
coefficient (Pongcharoenkiat et al., 2002).
A study has been conducted to determine the impact of drying methods on the stability of
dried vaccine preparations. A sucrose-based preparation of a live attenuated virus vaccine of
parainfluenza strain as such and that containing a surfactant was dried by freeze drying, spray
drying and foam drying methods. Differential scanning calorimetry, specific surface area analysis
and electron microscopy were used to characterize the dry powders. The preparations were stored
at 4, 25 and 37°C and the rate constants for degradation were determined. The spray dried
preparation showed the highest specific surface area (~2.82 m2g–1) in the absence of surfactant
and the foam dried preparation showed the lowest specific area (~ 0.1 m 2g–1) in the presence and
absence of surfactant. Electron microscopic measurements indicated the highest surface
coverage in spray dried preparation and lowest in foam dried preparation without surfactant. The
vaccine showed highest stability at 25 and 37°C in foam dried preparation with surfactant and
lowest stability in spray dried preparation without surfactant (Abdul-Fallah et al., 2007).
The chemical stability of rabeprazole sodium (proton-pump inhibitor) in simulated intestinal
fluid (pH 6.8) in the presence of certain excipients such as Brij 58 (nonionic surfactant), Poloxamer
188 (nonionic copolymer), Cremophor RH40 (solubilizer), Gelucire 44/14 (nonionic surfactant) and
PEG 6000 at 37 and 60°C has been studied. The main degradation product, thioether-rabeprazole,
has been identified by LC/MS and rabeprazole and its degradation product determined by HPLC.
Rabeprazole degrades by first-order kinetics and the rate constants at 37 and 60°C are 0.75 and
2.78 h–1, respectively, without the presence of excipients. The addition of excipients has been
found to improve the stability of rabeprazole. The greatest stabilizing effect has been observed in
the presence of Brij 58 which reduced the rate constants for degradation at 37 and 60°C to 0.22
and 0.53 h–1, respectively. It has been concluded that the presence of suitable excipients in
rabeprazole preparations enhances its stability in intestinal tract, resulting in maximum
bioavailability (Ren et al., 2008).
The effect of pH, suspending agents and temperature on the suspensions of ibuprofen
powder and microspheres has been studied by an accelerated stability protocol using a HPLC
method. The suspensions were found to be stable in different suspending agents on storage for a
period of 3 months at 23, 37, and 45°C. The dissolution stability of microspheres prepared from
an optimized formulation (17% drug loading) showed that suspensions of ceresine wax
microspheres stored at 37°C give faster release of the drug than that at 23°C. The microsphere
suspensions in syrup stored at 37°C showed faster dissolution rates than those suspended in
methyl cellulose. This could be due to an interaction between microsphere constituents and syrup.
Microcrystalline wax microsphere suspensions give better dissolution stability than those of
ceresine wax microspheres. At higher pH the drug release is faster from suspended microspheres.
The dissolution stability of microsphere is not significantly affected by the particle size (Adeyeye
and Price, 1993).
74
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78
CHAPTER – 4
PHOTOSTABILITY
4.1 INTRODUCTION
The sensitivity of many drug substances and drug products to light (British
Pharmacopoeia, 2016; United States Pharmacopeia, 2016; European Pharmacopoeia, 2015) can
lead to photochemical degradation resulting in potency loss, altered efficacy and undesirable
biological effects. This is an indication of the low quality of manufactured products. There are
several modes of photodegradation of drugs depending on the chemical structure, spectral
characteristics and photoreactivity of the compound as well as the irradiation wavelengths and the
stress conditions employed. The degradation products may be inactive and/or toxic to the
physiological system. The study of photodegradation reactions can provide useful information for
the stabilization of drug products. The evaluation of the photostability of drugs and drug products
is an essential component of formulation development. Photostability testing has to be conducted
on the drug substances and drug products according to the guideline of International Conference
on Harmonization (ICH, 1996). This ensures the quality, strength and freedom from any phototoxic
effects on the administration of photolabile drugs.
In the study of the photostability of drugs and their products it is necessary to develop a
knowledge of the principles of photochemistry, to understand the mode of degradation, to interpret
the stability data, to achieve reliable results and to draw meaningful conclusions. The study of the
photodegradation reactions of drug substances is a prerequisite to evaluation of the photostability.
Several monographs (Albini and Fasani, 1998; Tonnesen, 2004; Piechocki and Thoma,
2007), detailed accounts (Connors et al., 1986; Greenhill and McLelland, 1990; Moan, 1996;
Beijersbergen van Henegouwen, 1981,1997; Beaumont, 1999; Carstensen, 2000; Yoshioka and
Stella, 2000; Tonnesen, 2002; Fasani and Albini, 2005; Ahmad and Vaid, 2006) and reviews
(Sugden, 1985; Tonnesen, 2001; Boreen et al., 2003; Kullavanijaya and Lim, 2005; Vinod et al.,
2015; Ahmad et al., 2016a) have been published on the photochemistry, photostability,
phototoxicity, photoprotection and photostability testing of drugs and drug products for the benefit
of research workers involved in the field.
4.2 PHOTOSTABILITY AND RELATED ASPECTS
4.2.1 Photostability
The photostability of a drug may be defined as the response of a pharmaceutical
compound or a formulated product on exposure to radiation from sunlight, ultraviolet (UV) light or
visible light (or UV-visible light) in the solid and liquid state that leads to a chemical or physical
change.
The response of the drug to light absorption and excitation can be considered in terms of
photodegradation (photolysis) reactions through the formation of free radicals or photosensitization
reactions by intermolecular energy transfer. These reactions involve primary (photochemical) and
secondary (chemical) processes to give the final products (Ahmad et al., 2016a).
4.2.2 Effects of Photoinstability
The photoinstability of a drug may lead to the following changes :
4.2.2.1 Chemical and physical changes
 Loss of potency and efficacy.
79
 Alteration in physical characteristics.
 Appearance of color, turbidity or sediment.
 Evolution of gas and obnoxious smell.
 Formation of toxic photoproducts.
 Photocatalytic effects of metal contaminants.
 Variation in dissolution profile.
 Loss of package integrity.
4.2.2.2 Biological effects on administration
 Change in bioavailability.
 Toxicity of photoproducts.
 Adverse biological reactions.
4.2.2.3 Light induced side effects through interaction with endogenous substances
 Phototoxic reactions (chlorpromazine, psoralen, tetracycline).
 Photoallergic reactions (4-aminobenzoic acid, sulfonamides, thiazides).
 Photosensitization reactions (oestrogens, chloramphenicol, ethanol).
These aspects have been discussed by Tonnesen (2004), Beijersbergen van
Henegouwen (1997), Moan (1996), Epstein and Wintroub (1985), Harber et al. (1982), Moyal and
Fourtanier (2004) and Ahmad et al. (2016a).
The pharmacist should advise patients to avoid sunlight and use protective creams to
minimize the effects of light exposure.
4.2.3 Objectives of Photostability Studies
The photosensitivity and photoinstability of a large number of drugs and adjuvants require
a knowledge of their photochemical behavior to evaluate:
 Intrinsic photostability characteristics.
 Physical and chemical changes on exposure to light.
 Photodegradation pathways and mechanisms.
 Prediction of shelf-life of drug products.
 Need for measures during manufacturing, labeling, packaging, transportation and storage
to overcome the effects of light exposure.
 Need for modification of formulation parameters.
 Efficacy of stabilizing agents in photostabilization.
 Designing of appropriate packaging.
 Light induced biological effects.
4.2.4 Industrial Awareness on Photostability
There is a need to create awareness of the importance of photostability studies and
photostability testing of drugs and drug products among the technical workers of pharmaceutical
industry. This would motivate them to conduct such studies and testing on their photosensitive
products and to adopt appropriate measures in industrial processes to overcome photostability
problems. This is necessary to safeguard the interest of the consumer.
80
In view of the ICH QIB Guideline (1996), it is essential to conduct photostability studies
and photostability testing on new drugs and drug products to ensure product quality. This requires
a knowledge of the following aspects.
 Solubility of the drug in aqueous and organic solvents.
 Spectral characteristics of the drug molecule.
 Sensitivity of the drug molecule to UV and visible light.
 Mode of photodegradation and nature of photoproducts.
 A validated stability-indicating assay method to determine the contents of intact drug and
photoproducts in degraded material.
 Drug photoreactivity and stabilization.
4.3 PHOTOCHEMISTRY
Photochemistry is the study of the chemical and physical effects of light absorption and
electronic excitation resulting from the interaction of electromagnetic radiations with matter. The
electronic excitation of molecules takes place by the absorption of light in the UV and visible region.
4.3.1 Basic Laws of Photochemistry
Grottus and Draper Law: It states that only the absorbed light is photochemically active.
Stark and Einstein Law: It states that a molecule absorbs one quantum of light and from the
resulting excited state all the primary processes arise.
Noyes et al. (1956) have defined the primary photochemical process as: “The primary
photochemical process comprises the series of events beginning with the absorption of a photon
by a molecule and ending either with the disappearance of that molecule or with its conversion to
a state such that its reactivity is statistically no greater than that of similar molecules in thermal
equilibrium with their surroundings.”
4.3.2 Stages of Photochemical Reactions
The majority of photochemical reactions proceed in stages that involve:
 Absorption of electromagnetic radiation by a molecule and promotion to electronically
excited states.
 Occurrence of primary photochemical reactions through the electronic excited states.
 Occurrence of secondary (dark) reactions by the transformation of excited state species
(e.g. free radicals) to stable products.
4.3.3 Role of Photochemistry in Photostability Studies
The photodegradation reactions of drug substances may follow several pathways to form
different products. The elucidation of the mechanisms leading to these pathways requires a
thorough understanding of the nature and type of the photochemical reactions involved. This would
largely depend on the presence of certain functional groups, physical characteristics (light
absorption, pKas, solubility, etc.) and photoreactivity of the compound. The assessment of the
photostability of pharmaceutical compounds is based on the study of all those factors that
determine the rates and mechanisms of the underlying photochemical reactions.
4.4 PHOTOCHEMICAL REACTIONS
The photochemical reactions of organic molecules including a vast majority of drugs occur
by the absorption of specific wavelengths in the UV and visible region (200–700 nm) with energies
up to about 300 kcal/mole. This leads to the excitation of π and nonbonding (n) electrons in
molecules containing chromophoric groups (e.g. C=C, C=O, C=N) by π–π* and n–π* electronic
transitions. The asterisk (*) denotes the anti-bonding molecular orbitals with the electron in the
81
excited state. This may be followed by the loss of energy by heat, light emission (fluorescence and
phosphorescence) or free radical formation. The stabilization of the unpaired electron in the excited
state by delocalization would facilitate the formation of free radicals. The free radicals may react
to form stable products. The photochemical degradation of organic compounds involves various
mechanisms depending upon the chemical nature and photoreactivity of the compounds. In the
study of photochemical reactions a strict control of experimental conditions including the
wavelengths and intensity of light is required to obtain reliable results. This is particularly important
in kinetic studies. The number of photons absorbed by the reactants can be determined by
chemical actinometry. The details of the photochemistry of organic (Horspool and Armesto, 1992;
Horspool and Lenci, 2004; Turro et al., 2010) and drug molecules (Beijersbergen van
Henegouwen, 1981; Albini and Fasani, 1998; Tonnesen, 2004; Ahmad and Vaid, 2006) are well
documented.
4.4.1 Regions of UV, Visible and Sunlight Radiation
The regions of UV, visible and sunlight radiation involved in the photochemical reactions
are:
UVA : 320–400 nm
UVB : 290–320 nm
UVC : 200–290 nm
Visible : 400–700 nm
Sunlight : It includes UVA, UVB and visible radiations.
The majority of photochemical reactions of drug molecules take place by UVA, UVB and
visible radiations.
4.4.2 Important Chemical Functions for Photoreactivity in Organic Molecules
The presence of following chemical functional groups in organic molecules (Albini and
Fasani, 1998) is necessary for the occurrence of photochemical reactions.
 C = C double bond (oxidation / isomerization).
 C = O carbonyl group (reduction / fragmentation).
 C6H5NO2 nitroaromatic group / radical (intermolecular hydrogen abstraction/
rearrangement to nitrile esters).
 C6H4Cl2 aryl chloride (homolytic / heterolytic dechlorination).
 Compounds containing a weak C–H bond (photo-induced fragmentation via a hydrogen
atom transfer or electron-proton transfer).
 Sulfides, alkenes, polyenes and phenols (highly reactive with singlet oxygen
photochemically formed from ground state triplet oxygen).
hv
Triplet oxygen (3O2) Singlet oxygen (1O2)
4.4.3 Photophysical Processes

The various photophysical processes involved in the absorption and dissipation of light
energy (Eq. 4.1) – (Eq. 4.7) have been described by Moore (2004). This may be followed by
photochemical processes to form free radicals and subsequently the final products (Eq. 4.8) – (Eq.
4.11).
hv
82
Absorption Ao 1A (excited singlet state) (4.1)
Internal conversion 1A ic Ao (ground singlet state) (4.2)
Fluorescence 1A hv Ao + hv´ (4.3)
Photoionization 1A A•+ + e– (4.4)
1A isc 3A
Intersystem crossing (excited triplet state) (4.5)
3A ic
Internal conversion Ao (ground singlet state) (4.6)
Phosphorescence 3A Ao + hv″ (4.7)
Radical formation 3A + Ao A•+ + A•– (ionic radicals) (4.8)
-H+
A•+ A• (oxidized radical) (4.9)
+H
A•– AH• (reduced radical) (4.10)
Final products 2AH• AH2 + Ao (4.11)
A molecule in the ground state (Ao) on the absorption of photons of specific wavelengths
in the UV or visible region is promoted to the excited singlet state ( 1A) in which the electron spins
remain anti-parallel (Eq. 4.1). The molecule in the excited singlet state with life time of nanosecond
can dissipate its energy by different physical process and thus deactivated. This could happen by
internal conversion (ic) (Eq. 4.2), a non-radiated transition to the ground state, or by photon
emission (fluorescence) to return to the ground state (Eq. 4.3). The excess energy in an excited
state can also be dissipated as heat on collision with neighboring molecules by vibrational
relaxation (VR). Since in the excited singlet state a reduction in the ionization potential of the
molecule takes place, it is easy to remove the electron in the excited state than it is in the ground
state of the molecule. This occurs in the presence of an electron acceptor as a result of
photoionization (Eq. 4.4), particularly in the molecules having an anionic state. Another process
that can occur from the excited singlet state is by intersystem crossing (isc) to the metastable
excited triplet state (3A) in which the electron spins are parallel (Eq. 4.5). The isc has a high
efficiency for molecules that are photochemically active. The excited triplet state with life-times of
the order of microsecond to seconds has a greater probability of interaction with other molecules
and undergoes photochemical reaction. Alternatively, it can return to the ground state by another
ic (Eq. 4.6) or by the emission of phosphorescence (Eq. 4.7). Further photochemical processes
involving the excited triplet state may lead to the formation of cationic and anionic radicals (Eq.
4.8) which can be converted to neutral oxidized radicals (Eq. 4.9) and neutral reduced radicals
(Eq. 4.10). The neutral free radicals may react to form the final products (Eq. 4.11). The excited
triplet state is a more powerful electron donor or acceptor than the ground state of a molecule. All
these processes mostly occur within a span of nanoseconds to seconds.
4.5 PRIMARY PHOTOCHEMICAL REACTIONS
The study of primary photochemical reactions of molecules involving electronically excited
states, their interactions (e.g. electron/proton transfer) and decay processes have been conducted
using flash photolysis and related techniques. These are described in the following sections.
4.5.1 Flash Photolysis
The technique of flash photolysis was developed to study fast reactions by Norrish and
Porter (Porter, 1950) who were awarded Nobel Prize in chemistry for this discovery in 1967 *. This
* The Principal author (I.A.) has the privilege of working with Professor Lord George Porter on laser
flash photolysis studies of redox reactions of photosystem II D1/D2 cytochrome b559 reaction
centers of higher plants at Imperial College, London on a Royal Society Fellowship during the
period 1989–1990.
83
technique took considerable time for its further development, instrumentation and applications in
the study of excited state reactions.
Flash photolysis involves the exposure of a compound in solution to an intense flash of
light of very short duration (of the order of microseconds, 10 –6s) to initiate a chemical reaction by
producing excited state species (i.e. singlets and triplets) and thereby free radicals that lead to
stable products.
The technique has the limitations of nonuniform light intensity and the duration of flash.
These factors have been overcome by the development of laser flash photolysis.
4.5.2 Laser Flash Photolysis
This technique uses laser (Light Amplification by Stimulated Emission of Radiation)
flashes to generate excited states of a molecule and to study the formation and decay of excited
singlet and triplet states and free radicals. A laser beam is produced by supplying excitation energy
to a substance (e.g. rod of ruby) to produce a large number of excited state atoms that can release
spontaneous energy with the emission of photons.
Pulsed lasers provide emission of radiation for periods of very short duration [microsecond
(µs) to picoseconds (ps)] to detect excited state species and to follow photochemical processes
having half-lives up to picoseconds (ps, 10–12 s) to femtoseconds (fs,10–15 s).
Laser flash photolysis is one of the most effective methods of studying the rates of
reactions of transient species such as the excited singlet and triplet states, free radicals and ions
formed in chemical and biological systems. The technique is used for the study of very fast
photochemical reactions occurring up to the fs time scale. The 1999 Nobel Prize in chemistry was
awarded to Professor Ahmad Zewail of California Institute of Technology for the study of very fast
chemical reactions using ultra-short laser flashes on the time scale of fs where these reactions
actually occur.
4.5.3 Two-Laser Flash Photolysis
The technique of two-laser flash photolysis is used to study the fate of bimolecular
reactions involving an upper excited state of a molecule (A**) in solution competing with a fast
unimolecular deactivation process of a lower excited state (A*). A high-intensity radiation source
is required to produce high concentrations of A* and A** species by using two lasers of different
wavelengths sequentially (Turro et al., 2010).
The first laser gives rise to the lower excited state
hv
A A* (4.12)
This is achieved by the excitation of the ground singlet state (So) of a molecule to the
excited singlet state (S1). S1 is converted to the lower excited triplet state (T 1) by intersystem
crossing (isc).
isc
S1 T1 (4.13)
The second laser selectively excites T 1 into an upper level by the process
hv
T1 T2 (4.14)
This is achieved by the excitation of T1 to the upper triplet state (T2) termed above as A**.
The use of a tunable laser allows the selection of the photons of the second wavelength
corresponding to the absorption maximum of A*. Examples of the application of this technique
include the study of the α–cleavage of carbonyl (e.g. benzil) and halo-aromatic compounds (e.g.
2-bromonaphthalene) through a biphotonic process. The photostability of 2-bromonaphthalene
shows that T1 is not reactive towards C–Br bond cleavage. When the molecule undergoes two-
laser (i.e. two photon) flash photolysis, it results in the loss of bromine atom. The details of this
technique and its applications to the study of photochemical degradation of organic compounds
are given by Turro et al. (2010).
84
4.5.4 Time-Resolved Spectroscopy
The absorption spectra of transient species (i.e. excited singlet and triplet states) formed
in the photochemical reactions with life-times in the time scale of µs to fs are determined using
time-resolved spectroscopic techniques.
The details of all these techniques and their applications to the study of the kinetics and
mechanisms of photochemical reactions of drug substances are presented by Navaratnum (2004).
Some examples of the applications of laser flash photolysis in drug degradation studies include
flavin electron transfer reactions (Ahmad and Tollin, 1981a), flavin triplet quenching and
semiquinone formation (Ahmad and Tollin, 1981b), primary processes in the laser flash photolysis
of L-ephedrine (Navaratnum et al., 1983), primary processes in the photochemistry of fenbufen
(Navaratnum and Jones, 2000), primary photophysical properties of ofloxacin (Navaratnum and
Claridge, 2000), laser and flash photolytic studies on the effect of solvents and solutes on excited
singlet and triplet states of N,N,N′-tetramethyl paraphenylene diamine (TMPD) (Richards and
Thomas, 1970), laser flash photolysis of tolmetin (Sortino and Scaiano, 1999a), photophysical
properties of rufloxacin (Sortino et al., 1999b), photodegradation of formylmethylflavin (Heelis et
al., 1980), laser flash photolysis studies of electron transfer between semiquinone and fully
reduced free flavin and horse heart cytochrome c (Ahmad et al., 1981) and the cytochrome c-
cytochrome oxidase complex (Ahmad et al., 1982), multiple radical pair states in photosystem 2
reaction centers (Booth et al., 1991), electron transfer oxidation of tryptophan and tyrosine (Lu and
Lin, 2002), time–resolved thermal lensing and phosphorescence studies on photosensitized
molecular oxygen formation (Redmand and Braslavsky, 1988), and nanosecond time-resolved
resonance raman and absorption studies of the photochemistry of chlorpromazine (Sarata et al.,
2000).
4.5.5 Excited State Reactions
Moore (2004) has discussed the reactions occurring directly from the excited states
(singlet and triplet) and are briefly described in this section.
An excited state molecule (A*) can react with another molecule (Q) to form a complex,
called exciplex (A*Q) in the excited state. The molecule Q is a quencher (deactivator of an excited
state) of fluorescence (by deactivation of excited single state) or phosphorescence (by deactivation
of excited triplet state). Quenching normally takes place by intermolecular energy transfer or
electron transfer between A* and Q.
If the molecule A has a high concentration, then in the excited state (A*) it can interact with
another molecule in the ground state to form A*A species, called an excimer. In both cases the
formation of an exciplex and an excimer gives rise to a bathochromic shift in the fluorescence
emission of the molecule (A). The polycyclic aromatic hydrocarbons exhibit excited state
complexes. Exciplex formation may occur in concentrated solutions of drug molecules or in solid-
state mixtures leading to electron transfer to the drug molecule, the quencher or both.
Photoaddition reactions have been reported to proceed via exciplex formation with the quencher
molecule chemically bound to the drug molecule. An example of these reactions includes the
photoaddition of riboflavin (RF) in the presence of divalent ions such as HPO42– or SO42– ions.
These anions form a complex with RF in the excited state [RF * HPO42–] and catalyze the
photoaddition reaction. They also quench the fluorescence of RF. The photoaddition of RF leads
to the formation of cyclodehydroriboflavin (CDRF) (Schuman Jorns et al., 1975; Ahmad et al.,
2004a, 2005, 2006) according to the following reaction.
RF+HPO42– [RF*HPO42–] CDRF (4.15)
complex
A molecule in the excited state is considered as a more powerful electron donor or
acceptor than it is in the ground state. It can react with a quencher molecule in the following
manner:
A*+ Q A*Q A+• + Q–• (4.16)
85
A*+ Q A*Q A–• + Q+• (4.17)
The Eqs. (4.16) and (4.17) represent the oxidative and reductive quenching of A,
respectively. The quencher molecule is reduced or oxidized in the reaction. The electron transfer
processes may involve the formation of an exciplex in the presence of a quencher in polar solvents.
An example of these reactions is the electron transfer to the flavin excited triplet state ( 3F) (Eq.
4.18), conversion of [F–•] and [F+•] radicals to neutral reduced [FH•] (Eq. 4.19) and oxidized radicals
[F•] (Eq. 4.20), and the oxidation of the flavin semiquinone (FH.) by oxidized flavin radical (F+.) (Eq.
4.21), oxidized 2,6-dimethylphenol radical (PHO.) (Eq. 4.22) and by 2,5-dichlorobenzoquinone
(QN) (Eq. 4.23), studied by laser flash photolysis. The bimolecular rate constants for triplet
quenching by 2,6-dimethylphenol and flavin semiquinone yields are dependent on dielectric
constant and viscosity of the medium (Ahmad and Tollin, 1981a). The triplet quenching and
electron transfer reactions of flavins (F) are expressed as follows.
3F +F F–• + F+• (4.18)
F–• + H+ FH• (4.19)
F+• + –H+ F• (4.20)
FH• + F+• 2F + H+ (4.21)
FH´ + PHO• F+ PHO– + H+ (4.22)
FH´ + QN F+ QN–• + H+ (4.23)
4.5.6 Photosensitized Reactions
Photosensitization is the involvement of molecular species called photosensitizer to bring
a photochemical change by light absorption and electron or energy transfer to another molecular
species which does not absorb light. The photosensitizer does not directly participate in the
photochemical reaction. The majority of these reactions occur by photosensitized oxidation. These
reactions involve two mechanisms termed as Type I and Type II depending upon the nature of
oxidation.
4.5.6.1 Type I: Free radical mechanism
In this mechanism the excited state (singlet or triplet) of the sensitizer (S *) reacts with the
substrate to give free radicals through hydrogen atom or electron transfer.
4.5.6.2 Type II: Mechanism involving singlet oxygen
This mechanism involves the reaction of the excited state of the sensitizer (S*) with
molecular oxygen (3O2) to give rise to singlet oxygen (1O2). The singlet oxygen reacts with the
substrate to give oxidation products.
The Type I and Type II reactions may occur independently or simultaneously depending
on the chemical nature and the reactivity of the substrate and the sensitizer, reaction medium,
oxygen content and the affinity of the sensitizer and the substrate. These factors have been
discussed by Henderson and Dougherty (1992). Under specific experimental conditions one type
of photosensitized reaction may dominate the other type. An example of photosensitized reaction
would be described with reference to riboflavin (vitamin B2) (RF). It strongly absorb in the visible
region at 444 nm (British Pharmacopoeia, 2016), and participates in a number of photosensitized
reactions involving different substrates (e.g. SH) by Type I and Type II mechanisms (Ahmad and
Vaid, 2006; Silva and Quina, 2006; Garcia et al., 2006). Silva and Quina (2006) have described
these reactions and are presented with further explanations.
Type 1 mechanism
hν 1RF
RF formation of excited singlet state (4.24)
isc
1RF 3RF transformation to excited triplet state (4.25)
3RF+SH RF–•+ SH+• formation of radical ions (4.26)
86
RF–•+ SH+• RFH• + S• formation of free radicals (4.27)
RF–•+ O2 RF+ O2–•formation of superoxide radical anion (4.28)
2RFH• RF+RFH2 formation of oxidized and reduced molecules
(4.29)
RFH2 +O2 RF+ H2O2 formation of hydrogen peroxide (4.30)
H2O2 + O2–• OH–+OH•+O2 formation of hydroxyl ion and radical
(4.31)
S•+H2O2/O2–•/OH– Sox formation of oxidation products of substrate
(4.32)
SH+•+H2O2/O2–•/OH– Sox formation of oxidation products of substrate
(4.33)
Type 2 mechanism
hν 1RF
RF formation of excited singlet state (4.34)
1RF isc 3RF transformation to excited triplet state (4.35)
3RF+3O RF+1O formation of singlet oxygen by energy transfer
2 2
(4.36)
oxidation
SH+1O2 SOOH formation of oxidation products of substrate
(4.37)
In the above equations, RF, 1RF and 3RF represent RF molecule in the ground state,
excited singlet state and excited triplet state, respectively; RF –•, RFH• and RFH2 are the radical
anion, the free radical and the reduced form of RF; SH is the reduced substrate and SH +•, S• and
Sox represent the radical cation, free radical and oxidized form of the substrate, respectively.
4.6 PHOTODEGRADATION REACTIONS
A large number of drug substances are sensitive to light and undergo photodegradation
by various pathways on exposure to light. These reactions may proceed through free radical
intermediates and could involve more than one step to form the final products. The major modes
of photodegradation reactions are as follows:
 Photoaddition (e.g. riboflavin).
 Photoaquation (e.g. cyanocobalamin).
 Photodealkylation (e.g. chloroquine).
 Photodecarboxylation (e.g. amino acid).
 Photodehalogenation (e.g. meclofenamic acid).
 Photodimerization (e.g. primaquine).
 Photoelimination (e.g. mefloquin).
 Photodehydrogenation (e.g. nifedipine).
 Photo-induced hydrolysis (e.g. sulfacetamide).
 Photoisomerization (e.g. chlordiazepoxide).
 Photooxidation (e.g. ascorbic acid).
 Photopolymerization (e.g. 2-hydroxyethyl methacrylate).
 Photo-induced rearrangement (e.g. benzydamine)
87
 Photoreduction followed by oxidation (e.g. riboflavin).
 Photoinduced ring cleavage and other reactions (e.g. fluoroquinolones).
In some photodegradation reactions, more than one pathway may be involved such as in
the case of sulfacetamide, hydrolysis is followed by oxidation, in the case of riboflavin, reduction
is followed by oxidation and in the case of furosemide, oxidation is followed by reduction. The
photodegradation of drug substances may also occur by simultaneous (parallel) reactions to give
two or three products or by consecutive reactions involving an intermediate species to give the
final product. These reactions may involve zero, first or second-order kinetics (see Chapter 2).
Several examples of the photodegradation reactions of durg substances involving different
mechanisms have been reported (Albini and Fasani, 1998; Carstensen, 2000; Yoshioka and
Stella, 2000; Fasani and Albini, 2005; Tonnesen, 2002, 2004; Ahmad and Vaid, 2006; Sinko,
2006). The photostability and related aspects of drug substances and drug products have been
dealt by many workers (Lintner, 1973; Tonnesen, 1991, 2001, 2002, 2004; Tonnesen and Karlson,
1995, 1997; Tonnesen and Moore, 1993; Albini and Fasani, 1998, Fasani and Albini, 2005;
Piechocki and Thoma, 2007; Bhalekar et al., 2008). The phototoxic, phototherapeutic and
photosensitization effects of drugs have been reviewed by Magnus (1976), Beijersbergen van
Henegouwen (1981), and Moan and Juzenas (2004). Examples of some photodegradation
reactions are presented in this section.
4.6.1 Photooxidation Reactions
4.6.1.1 Photooxidation of benzaldehyde
The photooxidation of drugs by UV radiation involves a free radical mechanism. This has
been studied with reference to the photooxidation of benzaldehyde (Moore, 1976). In the free
radical chain process, a sensitizer (e.g. benzophenone) abstracts a hydrogen atom from the drug
molecule (Eq. 4.38). The free radical of the drug reacts with a molecule of oxygen (Eq. 4.39). The
chain reaction in propagated by removing a hydrogen atom from another molecule of oxidant a
hydroperoxide (Eq. 4.40). The hydroperoxide then reacts further by a nonradical mechanism to
form inert products (Eq. 4.41). The scheme showing initiation, propagation and termination steps
in the chain reaction involved in the photooxidation of benzaldehyde is presented in Fig 4.1.
Initiation .
CHO + hv CO + H
(4.38)
.
. Propagation CO 3
CO + O2
(4.39)
. .
CO 3 + CHO CO 3H + CO
Propagation
(4.40)
. Termination
2CO 3 inert products
(4.41)
Fig.4.1 Photooxidation of benzaldehyde.
88
4.6.1.2 Photooxidation of ascorbic acid
Ascorbic acid (vitamin C) (AH2) (4.1) on UV irradiation undergoes photooxidation to
dehydroascorbic acid (A) (4.3) through the ascorbyl radical anion (4.2) according to the reactions
shown below (Eq. 4.42).
H H
OH OH H
OH
HO HO
HO
O O O O O O
hv,-e-/-2H+ -e-
+e-/+2H+ +e- (4.42)
-
HO OH O O
O O
(4.1) (4.2) (4.3)

The photochemical reactions involved in the photooxidation of AH 2 may be described by
a general scheme (Ahmad et al., 2016b, Sheraz, 2009) as follows:
hv
AH2 [1AH2] (4.43)
[1AH2] [3AH2] (4.44)
[3AH 2] + AH2 AH•+ + AH•– (4.45)
+e
AH•+ AH• (4.46)
-e
AH•– AH• (4.47)
AH2 AH– + H+ (4.48)
AH• + AH• AH2 + A (4.49)
AH• + O2 A+ HO2• (4.50)
HO2• + AH– AH• + H2O2 (4.51)
According to this scheme, the ground state AH2 molecule is promoted to the excited singlet
state [1AH2] by the absorption of a photon of UV light (Eq. 4.43). The [1AH2] state may undergo
intersystem crossing (isc) to form the excited triplet state [ 3AH2] (Eq. 4.44). This state may react
with a ground state AH2 molecule to produce cationic [AH•+] and anionic [AH•–] ascorbyl radicals
(Eq. 4.45). These radicals may be converted to neutral radicals by gaining (Eq. 4.46) or losing an
electron (Eq. 4.47). AH2 is ionized in water to form an ascorbyl ion [AH –] (Eq. 4.48). The ascorbyl
radicals [AH•] may react to give AH2 and dehydroascorbic acid [A] molecules (Eq. 4.49). The [AH•]
radicals can be oxidized to form peroxyl [HO 2•] radicals (Eq. 4.50) which on interaction with AH–
ions may form [AH•] radicals and H2O2 (Eq. 4.51). [AH•] may further take part in the reaction.
4.6.2 Photoreduction Reactions
4.6.2.1 Photoreduction of riboflavin
A detailed study of the photoreduction reactions of riboflavin (RF) (4.4) in aqueous solution
has been made by Ahmad et al. (1981a, 1990, 2004b, 2006, 2008, 2011, 2013, 2014a) and other
workers (Cairns and Metzler, 1971; Heelis, 1982, 1991; Holzer et al., 2005; Insinka-Rak et al.,
2012, 2014; Sheraz et al., 2014). RF on light absorption is promoted to the excited singlet state
[1RF] (Eq. 4.52) followed by its conversion to the excited triplet state [ 3RF] (Eq. 4.53) which leads
to the formation of leucodeuteroflavin [RFH2] by intramolecular photoreduction (Eq. 4.54). [RFH 2]
is oxidized to formylmethylflavin (FMF) (4.5) as an intermediate product in the reaction (Eq. 4.55).
FMF is hydrolyzed to lumichrome (LC) (4.6) in acid solution (Eq. 4.56) and to LC and lumiflavin
(LF) (4.7) (Eq. 4.57) in alkaline solution (Ahmed et al, 1980, 2004b). It is also oxidized to
carboxymethylflavin (CMF) (4.8). The rate of photodegradation of RF is faster at higher pH due to
the sensitivity of RF excited triplet state [3RF] to alkaline hydrolysis. The chemical structures of RF
89
and photoproducts are shown in Fig. 4.3. The mechanism of photodegradation of RF by
photoreduction (Ahmad and Vaid, 2006) is represented by the following reactions:
RF [1RF] (4.52)
[1RF] [3RF] (4.53)
[3RF] RFH2 (4.54)
O2
RFH2 FMF + side-chain products (4.55)
FMF hv LC + side-chain products (4.56)
FMF H+ , OH– LC + LF + side-chain products (4.57)
4.6.3 Photodealkylation Reactions
4.6.3.1 Photodealkylation of riboflavin
It has been suggested that RF may be degraded by photodealkylation reaction which may
lead to the formation of LC directly through the excited singlet state [1RF] (Song, 1971).
RF [1RF] LC (4.58)
4.6.4 Photoaddition Reactions
4.6.4.1 Photoaddition of riboflavin
RF also undergoes photodegradation in the presence of divalent ions such as HPO 42– and
SO42– ions by the photoaddition reaction to form cyclodehydroriboflavin (CDRF) (4.9). The
appearance of the peak around 410 nm in the absorption spectra of photodegraded solutions of
RF is due to the formation of CDRF in the reaction (Ahmad et al., 2004a). The photoaddition of RF
occurs via the RF–HPO4–2 complex, which creates sterically favorable condition for C (9)/(2′α)
interaction (Eq. 4.59) (Schuman Jorns et al, 1975). The involvement of excited singlet state [ 1RF]
in this reaction has been suggested on the basis of quenching experiment. The presence of
HPO42– ions may facilitate the reorientation of C–2′ hydroxyl group to affect photoaddition. The
autoxidation of dihydroflavin intermediate leads to the formation of CDRF (Eq. 4.60). The
photoaddition of RF is expressed by the following reactions.
HPO42– hv
RF RF–HPO42– [1RF] (4.59)
complex
autoxidation
[1RF] [Dihydroflavin] CDRF (4.60)
The kinetics of simultaneous photoreduction and photoaddition reactions of RF has been
studied by Ahmad et al. (2004a).
4.6.5 Photoaquation Reaction
4.6.5.1 Photoaquation of cyanocobalamin
Cyanocobalamin (vitamin B12) is sensitive to light and its photochemical conversion to
hydroxocobalamin (vitamin B12b) takes place in aqueous solution (Connors et al., 1986; Ahmad et
al., 1992). The photolysis of B12 takes place according to the following reaction.
hv, H2O
[Co3+ CN] [Co3+ OH] + CN– (4.61)
B12 B12b
H+
[Co3+ OH] [Co3+ OH2]+ irreversible oxidation products
OH–; pKa= 7.8
B12b B12a (4.62)
In the photolysis process the CN– group with its full complement of electrons is replaced
by a water molecule without causing any change in the valency of cobalt (Eq. 4.61). B 12b exists in
equilibrium with aquocobalamin (B12a) in aqueous solution (Eq. 4.62). This reaction takes place
by the absorption of light leading to π–π* transition in the corrin ring. The photolysis reaction is pH
dependent with the lowest rate in the pH range of 6–7.
90
4.6.6 Photodegradation of Moxifloxacin
Moxifloxacin (MF) (4.10) is an important fluoroquinolone antibacterial agent. It undergoes
several photodegradation reactions under acid and alkaline conditions (Ahmad et al., 2014b).
These reactions are described as follows.
4.6.6.1 Acid Solution
MF (4.10) on UV excitation undergoes hydroxylation of the piperidine ring to form the
products (4.11, 4.12). The product (4.12) is then degraded by photooxidation of the pyrrole ring in
the diazabicyclononane side chain give the products (4.13 and 4.14). The product (4.14)
undergoes further reaction by the cleavage of the diazabicyclononane side chain to produce the
quinolone derivative (4.15) as the final product. The rate and extent of formation of these products
depends on the pH and acid-base equilibria in the region (Fig. 4.4).
4.6.6.2 Alkaline Solution
MF (4.10) on light absorption undergoes hydroxylation and photooxidation of the pyrrole
ring to form product (4.11) and on oxidation of piperidine ring in the side chain to give the product
(4.14). This is followed by cleavage of the diazabicyclononane side chain of the product to form
quinolone derivative (4.15), as in the case of acid solution. However, the detection of only three
products in alkaline solution indicates that the reaction is faster in the alkaline solution compared
to that of the acid solution. This could be due to the greater reactivity of any intermediates involved
in the process to form the detected products. The mode of photodegradation of MF is similar in
acid and alkaline media as a result of the specific acid-base catalysis in the whole pH range (Fig.
4.5).
CH 2OH
H OH
H OH
CHO
H OH
CH2
CH2
H3C N N O
H3C N N O
NH
NH H3C N
H3C N
O
O
(4.4) (4.5)
CH3
H
H3C N N O H3C N N O
NH NH
H3C N H3C N
O O
(4.6) (4.7)
CH 2OH
COOH (HOHC) 2
CH2 CH
O CH2
H3C N N O
H3C N N O
NH
H3C N NH
H3C N
O
(4.8) (4.9)
O
Fig. 4.3 Chemical structures of riboflavin and photoproducts.
91
O O O O O O
F F F
OH OH OH
hv
N N hydroxylation N N + N N
O -
+
+
O O
N H3C N +
H2 N H3C
H2 HO H2
(4.10) (4.11) (4.12)

hydroxylation/oxidation
O O
F
OH
N N
+
O
N H3C
O H2
(4.13)
oxidation
O O
F
OH
O O
F
O N N
OH
+
O
+ N
H3N N clevage of diazabicyclononane H3C
O H2
O side chain
H3C
(4.14)
(4.15)
Fig. 4.4 Proposed pathway for the photodegradation of MF in acid solution.

O O
F
O O
F OH
OH
hv N N
N -
N hydroxylation +
O
N OH
O
+ H2
N H3C (4.11)
H2
(4.10)
hydroxylation/oxidation
O O
F
OH
O O
F O N N
OH O
+
+
clevage of diazabicyclononane N H3C
H3N N O H2
side chain
O
H3C
(4.14)
(4.15)
Fig. 4.5 Proposed pathway for the photodegradation of MF in alkaline solution.
92
4.6.7 Other Photodegradation Reactions
The details of other photodegradation reactions of drugs (photodealkylation,
photodecaroxylation, photodehalogenation, photodimerization, photoelimination,
photodehydrogenation, photo-induced hydrolysis and photoisomerization) are described by
Ahmad et al. (2016a).
4.6.8 Photochemical Interactions
Many drugs present in combination in a product may undergo chemical interactions to
affect the stability of the individual components. The photochemical interactions of ascorbic acid
(AH2) with riboflavin (RF), nicotinamide (NA), and α–tocopherol (TP) in cream formulations have
been studied by Ahmad et al. (2012) and are described in this section.
4.6.8.1 Interaction of riboflavin with ascorbic acid
The interaction of RF with the ascorbyl ion (AH–) may be represented by the following
reactions proposed by Silva and Quina (2006):
[RF] [1RF] (4.52)
isc
[1RF] [3RF] (4.53)
[3RF] + AH– RF– • + AH• (4.63)
AH• + O2 A + HO2– (4.64)
HO2–+ AH– H2O2 + AH• (4.65)
RF, on the absorption of a photon of light, is promoted to the excited singlet state [ 1RF]
(Eq. 4.52) and may undergo intersystem crossing (isc) to form the excited triplet state [ 3RF] (Eq.
4.63). The [3RF] may react with the ascorbyl ion [AH–] to generate the ascorbyl radical (AH•) (Eq.
4.63) (Kim et al., 1993). The ascorbyl radical reacts with oxygen to give dehydroascorbic acid [A]
and peroxyl radical (HO2–) (Eq. 4.64). This radical may interact with ascorbyl ion to generate further
ascorbyl radicals (Eq. 4.65). These radicals may again take part in the sequence of reactions to
form A. The role of RF in this reaction is to act as a photosensitizer in the oxidation of AH 2 to A.
4.6.8.2 Interaction of nicotinamide with ascorbic acid
NA is known to form a 1:1 complex with ascorbic acid (Guttman and Brooke, 1963). The
complexation of NA and AH2 may result from the donor-acceptor interaction between AH2 (donor)
and NA (acceptor) as observed in the case of tryptophan and NA (Florence and Attwood, 2006).
The interaction of NA and AH2 can be expressed by the following reactions.
hv
NA [1NA] (4.66)
isc
[1NA] [3NA] (4.67)
[3NA] + AH2 NAH + AH• (4.68)
2 AH• A + AH2 (4.48)
2NAH + O2 2NA + H2O2 (4.69)
In the presence of light NA is promoted to the excited singlet state [1NA]
(Eq. 4.66) and is
then converted to the excited triplet state [3NA] by intersystem crossing (isc) (Eq. 4.67). The
interaction of [3NA] with AH2 may cause reduction of NA [NAH] to form the ascorbyl radicals [AH •]
(Eqs. 4.68) which are oxidized to dehydroascorbic acid [A] (Eq. 4.48). The NAH may be oxidized
to NA and H2O2 (Eq. 4.69).The proposed reactions suggest that on photochemical interaction AH2
undergoes photosensitized oxidation in the presence of NA indicating that the photostability of
ascorbic acid is affected by NA.
93
4.6.8.3 Interaction of α–tocopherol with ascorbic acid
TP is an unstable compound and its oxidation by air results in the formation of an epoxide
which than produces a quinone that is inactive (Connors et al., 1986). TP is destroyed by sunlight
and artificial light emitting the wavelengths in the UV region (Ball, 2006). It interacts with other
antioxidants such as ascorbic acid (AH2) according to the following reactions
TP–O• + AH TP + AH• (4.70)
2AH• A + AH2 (4.48)
TP + AH• TP–O• + AH2 (4.71)
The tocopheroxyl radical (TP–O·) reacts with AH2 to regenerate TP and form the ascorbyl
radical (AH·) (Eq. 4.70). This radical undergoes further reactions as described by equations (Eq.
4.48) and (Eq. 4.71) (Traber, 2007). It may disproportionate back to A and AH 2 (Eq. 4.48) or react
with TP to produce again the TP–O· radical and AH2 (Eq. 4.71). Thus in the presence of light TP
leads to the oxidation of AH2 which is ultimately regenerated in the reaction (Davies et al., 1991).
Ascorbic acid has the ability to act as a co-antioxidant with the TP–O· to regenerate TP (Packer et
al., 1979, 2002). In this manner AH2 and TP act synergistically to function in a redox cycle to
stabilize AH2.
4.6.8.4 Interaction of nicotinamide with riboflavin
The photochemical interaction of NA with RF has been studied by Ahmad et al. (2016c)
and is represented by the following reactions.
hv
RF [1RF] (4.52)
[1RF] isc [3RF] (4.53)
[3RF] + RFox RFH• +RFox• (4.72)
oxidation
2RFH• RFox + RFH2 (4.73)
RFH2 FMF + side chain products (4.55)
FMF hv LC + side chain products (4.56)
hydrolysis
FMF LC + LF + side chain products (4.57)
RFH2 + NA FMF + NAH (4.74)
2NAH + O2 NA + H2O2 (4.75)
The RF, in the ground state, absorbs light and is excited to the singlet state [ 1RF] (Eq.
4.52) which may be converted to the excited triplet state [3RF] by intersystem crossing (isc) (Eq.
4.53). The interaction of [3RF] with a ground state [RF] molecule leads to the formation of a
semiquinone radical [RFH•] and an oxidized [RFox•] radical (Eq. 4.72). The disporportination of two
semiquinone radicals results in the formation of an oxidized [RF] and a reduced [RFH 2] molecule
(Eq. 4.73). [RFH2] is oxidized to give formylmethylflavin [FMF] (Eq. 4.55) which undergoes
hydrolysis to yield lumichrome [LC], lumiflavin [LF] and side chain products (Eq. 4.56 and 4.57).
NA (electron acceptor) may undergo photochemical interaction with a [RFH 2] molecule to form
[FMF] and a reduced [NAH] (Eq. 4.74). The [NAH] molecule is oxidized to NA (Eq. 4.75). In this
manner NA accelerates the rate of photodegradation of RF in aqueous solution.
4.6.8.5 Interaction of ascorbic acid with cyanocobalamin
The study of the photochemical interaction of ascorbic acid [AH 2] with cyanocobalamin
[Co3+ CN] has been conducted by Ahmad et al. (2016d). The reactions involved in the interaction
can be expressed as follows.
hv
[Co3+ CN] 1[Co3+ CN] (4.76)
1[Co3+ isc 3[Co3+
CN] CN] (4.77)
94
AH2 AH– + H+ (4.78)
3[Co3+ CN] + AH– [Co2+] + AH● + CN– (4.79)
AH● A• – + H+ (4.80)
3[Co3+ CN] ⃰ + A●– [Co2+] + A● + CN– (4.81)
O2, OH–
[Co2+] [Co3+ OH] (4.82)
[Co2+] O2 Corrin ring cleavage oxidation products (4.83)
AH● + AH● AH2 + A (4.84)
The ground state B12 molecule [Co3+ CN] absorbs light and is promoted to the excited
singlet state 1[Co3+ CN] (Eq. 4.76). This may be converted to the excited triplet state 3[Co3+ CN] by
intersystem crossing (isc) (Eq. 4.77). The formation of a corrin triplet has been observed on the
basis of phosphorescence quenching. AH2 on ionization gives ascorbyl ions (AH–) (Eq. 4.78). The
3[Co3+ CN] may react with AH– ions and reduced to B 2+ ●
12r form [Co ] along with a AH radical (Eq.
● ●– 3 3+
4.79). AH may deprotonate to form A anion radical (Eq. 4.80). The [Co CN] could also react
with the A●– anion radical to form [Co2+] and a A● radical (Eq. 4.81). The [Co2+] form of B12 can
either be oxidized to B12b [Co3+OH] (Eq. 4.82) and/or undergo oxidative degradation to corrin ring
cleavage products (Eq. 4.83) depending on AH 2 concentration. Two AH● may combine to give a
reduced [AH2] and an oxidized [A] molecule (4.84).
95
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CHAPTER – 5
PHYSICAL STABILITY
5.1 INTRODUCTION
Drug substances and drug products may undergo physical and chemical changes during
storage as a result of environmental factors and chemical interactions. Physical stability
significantly contributes to the chemical stability of the products. It is necessary to consider the
physical stability of pharmaceuticals, in addition to chemical stability and the stability in biological
fluids (in vivo), to not only ensure their quality during the shelf-life period but also to maintain their
organoleptic properties for consumer acceptance. The physical properties of a drug such as
melting point, particle size and solubility depend on its physical state (e.g. crystalline or
amorphous), and any change in this property could affect the physical stability of the material.
Physical instability may be considered as any change in the physical state of a formulation during
preparation or storage. The study of a change in the physical characteristics of drugs and
excipients gives an indication of variations in the quality attributes of the product. Physical stability
is a key factor in product integrity in the dosage forms. It may alter the dissolution profile and
bioavailability of the drug. The dissolution rate may be considered as a measure of physical
stability. Changes in physical stability may influence the chemical stability of drugs and lead to an
acceleration of the degradation processes in the products. Therefore, appropriate measures
should be taken to maintain the physical stability of the products.
5.2 Analytical Techniques in the Study of Physical State
Various analytical techniques have been used for the characterization of the physical state
of drug substances and excipients and to study the effect of any variations on their stability. These
techniques have also been applied to the quantitative analysis of active ingredients and are briefly
described as follows.
5.2.1 Thermal Methods
5.2.1.1 Thermogravimetric analysis (TGA)
It involves the measurement of change in sample weight as a function of temperature
and/or time. A thermobalance continuously records the weight loss or gain of a sample as a
function of time. It is used to determine the thermal stability of a material and the fraction of volatile
components present.
5.2.1.2 Differential scanning calorimetry (DSC)
It is a modern and accurate technique used in the analysis of solid formulations. DSC
involves the measurement of difference in heat capacity between the sample and a reference as
a function of temperature or temperature. It can be used to monitor the energy released or
absorbed through chemical reactions occurring during the heating process.
5.2.1.3 Differential Thermal Analysis (DTA)
It involves the measurement of difference in temperature between the sample and a
reference as a function of temperature. The changes on heating the sample include melting, phase
transition, sublimation and decomposition.
5.2.1.4 Microcalorimetry
It is used to study the kinetics of chemical degradation of drug substances. The heat flow
produced in a degradation reaction follows a certain order of reaction. The thermal conductivity
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detector can detect small amount of degradation at room temperature such as that involved in the
slow solid-state degradation of drugs.
5.2.1.5 Isothermal calorimetry
All physical and chemical processes are accompanied by heat exchange with their
surroundings. In this technique the sample is maintained under isothermal conditions within a
microcalorimeter. When a chemical reaction occurs a temperature gradient is formed between the
sample and its surroundings. The resulting heat flow between the sample and its surroundings is
measured as a function of time. The technique is used for the characterization and stability
assessment of different physical forms of a drug or a product.
5.2.1.6 Dilatometry
Dilatometry is a thermoanalytical method used to measure the shrinkage or expansion
over a controlled temperature range (up to 1000°C). It is used to measure the rate of chemical
reactions such as changes in molar volume in polymerization reactions and rates of phase
transformations.
5.2.1.7 Hot-stage microscopy
It involves the measurement of changes in a crystal on temperature variation and provides
useful information on solid-state transitions.
5.2.2 Spectroscopic Methods
5.2.2.1 Vibrational spectroscopy
Vibrational spectroscopy is a collective term used to describe infrared (IR) and Raman
spectroscopy. It involves the measurement of vibrational energy levels associated with the
chemical bonds in a compound. It is used for the characterization and structure determination of
drug substances and to study the interactions occurring within a sample.
5.2.2.2 Fourier transform infrared (FTIR) spectroscopy
FTIR spectroscopy is used as a finger print technique for the characterization of the
polymorphs of a compound. It can also be used to determine the quality of a sample, composition
of a mixture and the nature of molecular interactions.
Attenuated total reflectance (ATR) is used in conjunction with FTIR (ATR–FTIR)
spectroscopy to enable the samples of a drug to be examined directly in the solid or liquid state.
ATR uses the property of total internal reflection resulting in an evanescent wave (that tends to
vanish). A beam of infrared light is passed through the ATR crystal in such a way that it reflects it
at least once off the internal surface in contact with the sample.
5.2.2.3 Diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS)
It is a technique that collects and analyzes IR radiations scattered by fine particles and
powders. Sampling is fast and easy because little or no sample preparation is required. It is used
for the identification of raw material, elucidation of crystal properties of polymorphs and quantitative
analysis of drug mixtures.
5.2.2.4 Solid state nuclear magnetic resonance (SSNMR) spectroscopy
The solid state NMR spectroscopy is a well established technique for the characterization
of the exact structure and differentiation of materials such as the polymorphs and solvates. It is
also used for the study of their interactions with adsorbed species (e.g. carbon dioxide, water).
5.2.2.5 Dynamic light scattering (DLS)
DLS is used to measure the size of particles at the submicron level. It monitors the
Brownian motion of particles suspended in a liquid with light scattering. The larger the particle, the
slower the Brownian movement is observed. It is also used to measure the zeta potential (surface
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charge) of a particle and to determine the molecular weight of organic compounds. DLS is also
referred to as Photon Correlation Spectroscopy (PCS).
5.2.2.6 X-ray powder diffraction (XRPD)
It measures the average spacing between the layers or rows of atoms in a molecule. It is
used for the characterization and identification of polycrystalline phases of a compound. The main
use of this technique is to identify components in a sample by a search/match method on
comparison with known diffraction patterns. The technique can also be applied to the quantitation
of different phases.
5.2.2.7 Single crystal X-ray diffraction (XRD)
XRD is used to determine the orientation and the structural features of a single crystal for
characterization.
5.2.3 Other Techniques
5.2.3.1 Polarized light microscopy
It is a very useful technique in the study of polymorphism for particle characterization such
as the size, shape and aggregation.
5.2.3.2 Particle electrophoresis
It is a widely used technique for the measurement of zeta potential. An electric field is
applied across a sample (e.g. suspension) which induces charged particles to move. The velocity
of the particle is dependent on its zeta potential. The measurement of zeta potential is necessary
for the prediction of formulation stability and interactions. It can also be used as a simple method
of quality control.
This chapter deals with a brief description of the various aspects of physical stability of
drugs and drug products. Detailed accounts of the physical stability of drug substances (Yoshioka
and Stella, 2000), extemporaneous preparations (Jackson and Lowey, 2010; Haywood and Glass,
2013) and physical testing of drug products (Carstensen, 2000) are available for further study.
Several reviews have been published on the physical stability of amorphous and crystalline states
( Berglund et al., 1990;Saleki-Gerhardt et al., 1994; Hancock and Zografi, 1997; Craig, 1999; Yu
et al., 2001; Vippagunta et al., 2001; Babu et al., 2012), solid dispersions (Qian et al., 2010; Kalia
and Poddar, 2011; Kapoor et al., 2012; Kumavat et al., 2013), emulsions (Zografi, 1982),
nanoparticles (Wu et al., 2011) and physical transformations (Morris et al., 2001; Zhou et al., 2009;
Bhattacharaya and Syrayanarayanan, 2009).
5.3 CHANGES IN PHYSICAL STABILITY
Drug substances and adjuvants are usually manufactured in the solid form and exist in the
amorphous state or in different crystalline states (polymorphs). The amorphous drug in most cases
is not stable and may gradually change to a thermodynamically more stable crystalline form. It
may also undergo hydration or dehydration process during storage. The changes in the physical
stability of liquid dosage forms may lead to a change in appearance, formation of precipitates,
formation of polymorphs of low solubility, flocculation and sedimentation, drug adsorption on to the
container surface and microbial growth. The change in physical stability of solid dosage forms may
affect characteristics such as appearance (e.g. color, shape), mechanical strength (e.g. tablet
hardening, softening), content uniformity (e.g. suspensions), and dissolution rate and
bioavailability. The major cause of all these factors is phase transition occurring in the material. It
may involve polymorphic transition, solvation and desolvation, salt and salt exchange, and
amorphization and devitrification (reversion to crystalline form). Phase transition can occur through
solid state, melt, solution or solution mediated mechanisms. Pharmaceutical processes including
comminution, compaction, granulation, drying and coating may lead to partial or complete phase
transition resulting in the physical destabilization of the material.
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5.4 FACTORS AFFECTING PHYSICAL STABILITY
Most of the multicomponent systems used in the formulation of drug products do not
assume a state of thermodynamic equilibrium and, therefore, undergo transitions to achieve a
state of equilibrium. These transitions are accompanied by a decrease in the free energy of the
system and are influenced by the following factors.
5.4.1 Internal Factors
Formulations of drug substances often contain additives and excipients and may involve
potential drug–drug and drug–excipients interactions and compatibility problems. These could lead
to changes in the physical and chemical stability of the system. Two or more drugs present in a
product may also react with each other and thus cause a change in the physicochemical
characteristics of the product.
5.4.2 External Factors
The storage of pharmaceutical products at high temperature may cause transmission of
the thermal activation energy to the system to make it thermodynamically unstable. This may lead
to physical changes such as those observed in appearance, crystalline structure, consistency,
viscosity, homogeneity, dispersion, firmness and disintegration of solids. The changes in the
physical state may also include drying of semisolid dosage forms, liquefaction of eutectic mixtures
of powder due to low melting point, and cementing of tablets, etc.
Solid dosage forms on storage under humid conditions may lead to the absorption of
moisture resulting in changes in the mechanical strength of the tablets. The change in mechanical
strength is a function of moisture uptake of the tablet, the moisture permeability of the package
and the humidity conditions employed. Physical stability of solid pharmaceuticals is also affected
by the plasticizing effect of water probably due to an increase in molecular mobility. Amorphous
drugs (e.g. indomethacin, nifedipine, lamotrigine mesylate) show decreased values of glass
transition temperature (Tg) and increased crystallization on the absorption of moisture. Tg and NMR
relaxation-based critical mobility temperature (Tmc) both are useful parameters for the
measurement of molecular mobility. Tmc of an amorphous drug is generally lower than Tg indicating
that the glassy pharmaceutical solids show significant molecular mobility even at temperature
below Tg (Yoshioka and Stella, 2000).
5.4.3 Amorphous State
The non-crystalline state of solids is known as the amorphous state. These solids do not
possess long-range order characteristics of a crystal and have no unit cells. They appear to behave
like super-cooled liquids that show the arrangement of a molecule in a random order similar to that
of the liquid state. Solids in the amorphous state exhibit properties that are different from those of
the crystalline state of the same substance. They do not have a melting point as observed in the
case of crystalline materials possessing a crystal lattice that collapses on melting.
The Tg is characteristic of the amorphous solids. It represents the temperature at which
an amorphous material changes its physical character from a glass-like solid into a more mobile
rubber like state. Tg is a measure of the stability of the amorphous state of a drug. The physical
stability of amorphous solids increases with an increase in Tg. The use of an amorphous drug in a
dosage form leads to an increase in the rate of dissolution and consequently the bioavailability of
the drug (Bauer, 2009).
The drugs and adjuvants in the amorphous state have generally greater solubility in water
than that of the crystalline state, which has a lower ground state free energy (∆G) compared to the
amorphous state. Therefore, the drugs in the later state would convert to the thermodynamically
more stable crystalline state on storage. According to Yoshioka and Stella (2000) this change may
lead to drastic variations in release characteristics of the drug which would alter its clinical efficacy
and toxicological effect. Examples of conversion of amorphous state of drugs to crystalline state
during storage include nifedipine (Uekama et al., 1992), oxyphenbutazine (Matsuda and
Kawaguchi, 1986) and furosemide (Matsuda et al., 1992). The characteristics and significance of
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the amorphous state in pharmaceutical systems have been reviewed in detail by Hancock and
Zografi (1997) whereas their preparation, characterization and stabilization has been discussed
by Yu (2001).
Mahlin and Bergstrom (2013) studied the physical stability of the amorphous state of fifty
structurally diverse drugs using DSC and XRPD methods. The thermal properties and molecular
weight of drugs were used to develop predictive methods of physical stability. Drugs with molecular
weight greater than 300 g/mole were expected to transform into their amorphous state by spray
drying and melt-cooling technology. The Tg and molecular weight were used to predict the physical
stability of the material on storage for one month for the glass-forming compounds. A strong
sigmoid relationship has been found between the crystallization temperature and physical stability
of the drugs. These observations are useful in the development of amorphous formulations of
drugs.
The amorphous state stability of ketoprofen and flurbiprofen has been studied by thermal
analysis. The amorphous forms of these compounds were obtained by super-cooling of the melt
in a DSC apparatus and subjected to storage for sixty days. The measurement of the enthalpy
(∆H), the distribution of molecular relaxation times (Tm) and Tg of the amorphous samples indicated
that flurbiprofen has greater physical stability at any aging temperature compared to that of
ketoprofen. The values of ∆H and Tm of flurbiprofen were found to be greater than those of
ketoprofen. Both amorphous drugs were suggested to be classified as “fragile” (Hoti et al., 2012).
A study has been conducted to evaluate commonly calculated parameters of the
amorphous state of different drugs in term of their predictive capabilities of physical stability. It
included the determination of configurational heat capacity (Cp) and rate dependence of Tg of the
material. The amorphous samples were heated at 1 K/min from 50°C below to 30°C above the Tg.
The ∆Cp was calculated as the difference between Cp of the amorphous and the crystalline states
and the configuration thermodynamic properties, entropy (∆S), enthalpy (∆H) and Gibbs free
energy (∆G) were also determined. The results indicated that all the drugs are fragile glass
formers, however, variations in the degree of fragility with a group of drugs (acetaminophen,
cefuroxime axetil, donepezil HCl, indomethacin, lacidipine, nifedipine, salsalate, simvastatin,
tolbutamide and troglitazone) were observed. Below the Tg, fragility showed no linear correlation
with amorphous stability, and the strong glass formers could form more stable glasses. It has been
observed that below Tg no clear relationship between the various factors and physical stability
exists. Above Tg, ∆S showed the largest correlation with stability, however, the stability above Tg
can not necessarily be related to the physical stability below Tg and, therefore, ∆S may only serve
as a limited predictive parameter of physical stability (Graeser et al., 2008).
Lobmann et al. (2011) developed a co-amorphous system to enhance the physical stability
and dissolution rate of drug substances. It was applied to a combination of non-steroidal anti-
inflammatory drugs (NSAIDs), naproxen and indomethacin. The co-amorphous binary phase of
these drugs was prepared at molar ratios of 2:1, 1:1 and 1:2 by quench cooling, and the physical
stability was studied at 277.15 and 298.15 K under dry conditions using XRPD analysis. FTIR was
used to detect molecular interaction between the two drugs and DSC to assess Tg. The results
indicated that naproxen in combination with indomethacin is converted to the co-amorphous form.
FTIR spectra suggested the formation of a heterodimer between the two drugs. A sample at 1:1
ratio of the drugs remained in the amorphous form while those at 1:2 and 2:1 ratios resulted in the
recrystallization of these drugs upon storage. The dissolution testing of the co-amorphous form
showed an increase in the dissolution rate of both drugs with a synchronized release for the 1:1
blend. This approach can be adopted to overcome the problem of formulation of poorly-soluble
crystalline drugs so as to increase their solubility and dissolution rate.
5.4.4 Dosage Forms
The physical stability of the amorphous drugs in various dosage forms has been studied
by several workers and is presented in the following sections.
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5.4.4.1 Solid dispersions
The physical stability of four alcohol-free spironolactone suspensions stored at 4, 25 and
40°C over a period of sixty days has been studied. The rheological behavior, particle size variation
and optical characteristics were used to predict long-term physical stability of the suspensions. All
the suspensions were coarse dispersion with particle size greater than 1 µm. Sedimentation was
found to occur in one suspension and flocculation of the drug in the other three suspensions (syrup
base). The high viscosity of these suspensions made it difficult to achieve homogeneous
redispersion. It affected their dissolution profile that was the slowest in comparison to the other
suspension. There was no change in color or odor of the suspensions during storage at all the
three temperatures. A very slight increase in particle size distribution (PSD) was observed after
the storage period (Bernal et al., 2014). PSD is a critical parameter that affects the bioavailability
and pharmacokinetics of the product (Kulshreshtha et al., 2010). Optical analysis of the
suspensions was carried out to detect destabilization of the suspensions. This technique is used
to assess the physical stability of the system without diluting or modifying the formulation
(Gonzalez-Mira et al., 2010).
The evaluation of the physical stability of clopidogrel oral suspension indicated isomeric
conversion of the active S–form of the drug to the inactive R–enantiomer. A 1.5% and 3.0%
conversion of the S–form to R–enantiomer took place after storage for four days at 4 and 25°C,
respectively. On the basis of these results an expiry date of one month under refrigeration
conditions and two week at 25°C has been recommended (Mihaila et al., 2012).
The amorphous solid dispersions of quinapril HCl and citric acid (1:1 to 1:6) prepared by
colyophilization have been subjected to degradation in citrate buffer at 80°C and Tg values
measured by DSC. The rate of reaction showed low dependence at pH 2.49 on the Tg of the
sample. However, the rate decreased on an increase in Tg value at pH ≥ 2.75. The rate of
degradation was found to increase with pH at a constant Tg value of about 70°C. The degradation
of quinapril HCl is a function of the relative concentrations of quinapril and its zwitterionic form. At
higher pH values with a higher concentration of zwitterionic form the rate of the reaction is strongly
affected by the Tg of the mixture and hence, the molecular mobility. At the lower pH, with a higher
concentration of the non-ionized quinapril molecule, the degradation is less sensitive to Tg
probably due to a different mechanism involved (Li et al., 2002).
Solid dispersions have been shown to possess a potential to increase the release rates of
poorly water soluble drugs. Most of the drug candidates in pharmaceutical pipeline today are either
poorly soluble or water-insoluble. To meet this challenge, various processes have been developed
to increase the solubility, dissolution rate and bioavailability of active ingredients of pharmaceutical
formulations belonging to Biopharmaceutics Classification System (BCS) II and IV. Out of the
various formulations (solid dispersion, nanoformulations, lipid formulations, cyclodextrin
complexes, etc.), solid dispersion is a vital area of research in the development of pharmaceutical
formulations. Specifically, it involves the development of formulations with a high drug loading
and/or containing drugs with a high tendency to crystallize (indicated by high melting point of >
290°C) (Brough and Williams, 2013). Such dispersion is basically a simple two-component system
of drug polymer interaction in the solid state, where the drug and the polymer act as a solute and
solvent, respectively. The ultimate success of a solid dispersion is determined by its performance
on dissolution after oral administration. The general strategy behind almost all the solubilization
technologies is the so called “spring- and – parachutes” concept (Guzman et al., 2007). According
to this concept for solid dispersions, drug should first dissolve along with the soluble polymer matrix
to create a supersaturated solution (“the spring”), after which super-saturation should be
maintained long enough for drug absorption (“the parachutes”) to take place.
The major problem with most of the solid dispersions for oral use is that they form a
supersaturated drug solution when they come in contact with the aqueous environment of GIT.
Drugs in this state have a tendency to precipitate rapidly before being absorbed (causing poor
bioavailability). Recently such work has been carried out to evaluate suitable polymers that are
able to maintain a supersaturated drug concentration in vivo for an extended period of time to allow
optimal absorption. Fortunately, few polymers specifically some cellulose derivatives are known
106
to possess this ability and they include hydroxypropyl methyl cellulose (HPMC) and hydroxypropyl
methyl cellulose acetate succinate (HPMC AS), vinyl polymers such as polyvinylpyrrolidone (PVP)
and polyvinyl pyrrolidone-co-vinyl acetate (PVPVA) (Xu and Dai, 2013). The mechanism how the
polymer prolong drug super-saturation is still not fully understood.
A study has been carried out to evaluate the effect of certain formulation parameters, i.e.
solvent evaporation, temperature, drug: PVP ratio and PVP molecular weight on the physical
stability of the amorphous solid dispersion of piroxicam. The results showed that the evaporation
temperature has the highest effect in inhibiting the nucleation of piroxicam, while piroxicam:PVP
ratio has the highest effect in decreasing the crystal growth. The influence of increasing
evaporation temperature and piroxicam:PVP ratio are in the same order of magnitude to increase
the physical stability of dispersions. The PVP molecular weight showed a minor effect in
decreasing the crystal growth of piroxicam in PVP matrix. The studies were carried out using
polarized light microscopy (Wu et al., 2011).
Yang et al. (2010) developed a kinetic model to predict the physical stability of amorphous
drug–polymer solid dispersions on recrystallization. The kinetics of recrystallization was
determined by DSC for amorphous efavirenz–PVP solid dispersion stored at controlled
temperature and relative humidity. The kinetic model was used to determine the recrystallization
rate constant and the microscopic geometry of crystal growth. Temperature was found to affect
the drug recrystallization rate constant according to the Arrhenius relationship, while the rate
constant increased linearly with relative humidity. PVP content inhibited the recrystallization
process by increasing the crystallization activation energy and decreasing the equilibrium
crystallinity.
FTIR spectroscopic imaging has been applied to study the physical stability of solid
dispersions of poorly water-soluble drugs in polyethylene glycol (PEG) and their dissolution in
water. The amorphous nifedipine was found to crystallize within PEG–8000 for formulations
containing 10% drug. The crystallization of the drug within the polymer matrix reduced its rate of
dissolution. FTIR imaging in the ATR mode provided information on the mechanism of the
dissolution of nifedipine from solid dispersions in water-soluble polymers which is helpful for the
optimization of manufacturing of these formulations (Chan and Kazarian, 2004).
The effect of thermal methods (e.g. melt method) on the polymorphic changes in the
formulation of solid dispersion of candesartan cilexetil with polyethylene glycol 8000 has been
studied. DSC, XPRD, FTIR and HPLC have been used to evaluate the polymorphic changes in
the final formulation of the drug. DSC indicated the shift of endothermic peak of the formulation
toward the lower temperature. XPRD showed the relative degree of crystallinity as 0.645. FTIR
indicated a shift in the peaks of the drug due to polymorphic changes. HPLC showed the in vitro
release of candesartan cilexetil from the solid dispersion within 10 min. It has been concluded that
the preparation of this formulation at high temperature may result in polymorphic changes in the
drug (Thirupathi et al., 2014).
5.4.4.2 Semisolid dispersions
The physical stability of a semisolid dispersion of piroxicam into hard gelatin capsules
prepared with Gelucire 44/14 (a methyl acetate derivative), labrasol and excipients such as
microcrystalline cellulose (MCC), mannitol and lactose (α–monohydrate) has been studied. The
master dispersion containing only Gelucire 44/14, 20% w/w, and labrasol, 80% w/w, was stored at
5±3°C in a refrigerator while the modified dispersion with the excipients (2% w/w), were kept at
25±2°C / 60±5% RH in a climatic chamber for one year. Dissolution tests were carried out in media
at different pH, on the freshly prepared dispersions and on those stored for three, six and twelve
months. FTIR and DSC studies confirmed the existence of piroxicam in the amorphous state in all
the dispersions under the specified storage conditions for one year (Karatas and Bekmezci, 2013).
5.4.4.3 Creams
The colloidal stability of alcoholic emulsion creams stored for six months at ambient
temperature has been studied. It was found that the size of fat droplets significantly affects the
stability of creams during storage. Dispersion of about 80% of the lipid fraction by pressure
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homogenization to the size below 20 µm prolonged the stability of the system by two months. The
enrichment of the creams with 0.1 % each of lecithin and sodium caseinate imparted full physical
stability to the creams during the storage period as a result of an increase in lipid phase dispersion
and delay in coalescence. The addition of oxidized sterol (0.5 %) decreased the mobility of
dispersed phase droplets and protected against coalescence and cream formation. A decrease in
conductance of the creams was also observed during storage, the largest decrease (from 99 to 72
µS/cm) being in the presence of oxidized starch which increased the viscosity of the emulsion.
There was no correlation between emulsion conductance and droplet size in the dispersed phase
(Tarko and Tuszynski, 2007; Floury et al., 2000).
The physical stability of theophylline cream (o/w) is affected by the presence of
preservatives. The addition of preservatives in the cream stored for forty nine days at room
temperature did not show a change in physical characteristics. While the cream without
preservatives exhibited a few signs of dryness and color change. The growth of microorganisms
in the cream can cause separation of fatty material and thus affect its physical stability. Good
homogenization technique may decrease the effect of heat on the stability of the cream (Abdul
Hussain et al., 2009).
Physical changes in the water-in-oil creams of ascorbic acid have been observed when
stored at 301°C for 3 months. The creams showed changes in color, creaming and phase
separation (breaking), with time. All the physical changes were found to be affected by the
formulation factors such as type of emollient and humectant, pH and viscosity of the medium, and
specific gravities of the liquids used in the formulation of the creams (Sheraz et al., 2014).
5.4.4.4 Liposomes
The physical stability of uncoated and chitosan-coated liposomes (1% w/w, soy lecithin)
incorporating polyphenol-rich grape seed extract (0.1% w/w) has been studied. Both types of
liposomes showed good physical stability during storage for eight days. It was confirmed by the
measurement of particle diameter using DLS and the determination of polydispersity index (PDI)
values that did not change during storage. PDI is considered as an indicator of the broadness of
particle size distribution. The uncoated liposomes showed the smallest PDI (0.2), indicating that
the solution was monodispersed whereas the coated liposomes were found to be polydispersed.
After the storage period uncoated liposomes (empty) showed a broad particle size distribution as
a result of the oxidative degradation of unsaturated fatty acids in phospholipids. The measurement
of zeta-potential of all the liposomes using particle electrophoresis did not show any change during
storage. Zeta-potential is a measure of the surface charge of the particles and affects the repulsive
colloidal interactions. It gives an indication of the physical stability of coated liposomes (Gibis et
al., 2013; Laye et al., 2008; Panya et al., 2010). In another study carried out on the physical stability
and drug release of cholesterol derivatives in liposomes revealed a positive charge at a pH
between 3 and 10 as indicated by zeta-potential. It was further revealed that cholesterol liposomes
have better physical stability compared to that of cholesterol without liposomes (Yang et al., 2013).
5.4.4.5 Proteins
The development of protein pharmaceuticals involves the study of their physical stability
under normal and stress conditions. According to Chang and Yeung (2010), the physical stability
of the majority of proteins can be expressed in terms of resistance to unfolding forces because
aggregation and/or precipitation can occur when the structural change results in a less soluble
conformational state. Conformational changes in proteins occur as a result of the conversion of
their biologically active forms to non-active and/or inactive conformations. The resistance to
unfolding (thermodynamic stability) depends on various forces that contribute to the folding of
proteins. These forces result from covalent bonds, electrostatic interaction, hydrophobic
interactions, hydrogen bonds and van der Waal interactions (Dill, 1990; Guo et al., 2006).
5.4.5 Crystalline State
Crystalline state of the matter is the state in which the molecules are packed in a defined
order that is repeated throughout its particles in the system. The physical stability of solid drugs is
influenced by their crystalline state. The crystalline drugs have lower ground state free energy and
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higher free energy of activation (∆G) with lower reactivity. ∆G gives the difference in free energy
between the reactant state and the activated state. The different crystalline forms of the same drug
(polymorphs) have different free energies and may undergo transition from one form to the other.
Polymorphic transition in drugs may occur during storage under different conditions of temperature
and humidity and thus alter their critical properties such as the solubility and the dissolution rate.
This may affect the efficacy of the drug in a formulation.
Crystalline drugs although are known to be more stable but possess a problem of low
solubility and bioavailability. Therefore, often various methods are employed to modify the physical
state properties of the active crystalline drug and enhance its solubility and bioavailability. These
methods may include alteration in crystal structure, drug–polymer complexation, formation of solid
dispersions, formulation of drug with lipophilic bases, particle size reduction, salt formation, etc.
The techniques used for such alteration may include solvent evaporation, solidification of melt,
mechanical activation of crystalline mass, rapid precipitation from solution i.e., during spray drying
or freeze-drying, etc. One problem often encountered during the preparation of amorphous solids
from their crystalline form is their reconversion to the parent state. In order to avoid such problem
often hydrophilic polymers are added in the dispersions. Hydrophilic polymers are known to retard
recrystallization of amorphous forms by their antiplasticizing effect. Such polymers may also forms
a complex with the drug and increase its Tg. Storage of amorphous blends below their Tg and
protection from plasticizers such as moisture can retard the chances of recrystallization. Huang
and Dai (2014) have reviewed the various aspects of solid dispersion and drug–polymer interaction
for poorly soluble drugs.
Tolfenamic acid is a crystalline drug that belongs to the fenamate family of NSAIDs. Its
amorphous form has been prepared either by freeze-drying with chitosan (Ahmed et al., 2013) and
polyacrylic acid (Sheraz et al., 2015) or by solvent evaporation technique with polyurethane
(Istanbullu et al., 2013). It has been found that the transformation of crystalline state to amorphous
form is not only limited to the technique employed but also depends on the properties such as the
ratio and molecular weight of the polymer, pH of the medium and storage conditions employed. In
case of tolfenamic acid the molecular weights of chitosan and polyacrylic acid showed to affect the
conversion from the crystalline state to the amorphous form whereas low molecular weight
polymers showed better conversion than the high molecular weight polymers. Similarly, pH has
also been shown to play an important role in the transformation of physical state properties of
tolfenamic acid. The pH values near to the pKa values of the polymers have shown better
conversion into the amorphous state with lesser amount of the polymer required. This could be
due to the better miscibility of the drug with the polymer thus resulting in better interaction between
the two compounds (Ahmed et al., 2013; Sheraz et al., 2015). In the case of solvent evaporation
technique used for the preparation of films of tolfenamic acid with polyurethane, it was observed
that the solvent employed for the evaporation also affects the degree of conversion from the
crystalline to the amorphous state. More amorphous tolfenamic acid was formed in films where
only tetrahydrofuran was used as compared to films prepared with a mixture of tetrahydrofuran
and ethanol (Istanbullu et al., 2013). No recrystallization was observed in any of the samples
prepared with chitosan, polyacrylic acid or polyurethane when stored in a desiccator for a period
of 3 months.
Many water-soluble crystalline compounds after micronization have poor physical stability
on exposure to moisture. It results in caking and severe aggregation which can be detrimental to
the performance of their pharmaceutical products. It has been observed that micronization gives
rise to amorphous regions into the crystalline material that cannot be determined by the XRPD
method. These amorphous regions transform, due to surface sintering and recrystallization, at
relative humidity well below the deliquescent point. The characterization of micronized solids can
be carried out using microcalorimetry (Bystrom, 1990).
5.4.6 Polymorphism
Polymorphism can be defined as the existence of a solid material (e.g. drug substance) in
more than one form or crystalline structure known as polymorph. The polymorphs can be classified
into two types as monotropes (a polymorph unstable at all temperature and pressures, e.g. glyceryl
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stearates) and enantiotropes (a polymorph reversibly converted into another form by changing
temperature or pressure, e.g. sulfur). This classification is based on the stability of the polymorphs
over a range of temperatures or pressures below their melting points at a constant atmospheric
pressure. The transition temperature is expressed as the temperature at which two polymorphs
possess identical free energies (∆G), can coexist together and have same solubilities in a solvent.
In a certain temperature range below the solid melting temperatures, a polymorph having the lower
free energy that corresponds to the lower solubility is considered as the thermodynamically stable
form.
The crystalline structure of a compound exerts a profound effect on its solid-state
properties. For a given material, the heat capacity, conductivity, volume, density, viscosity, surface
tension, diffusivity, crystal hardness, shape and color, refractive index, electrolyte conductivity,
melting and sublimation properties, latent heat of fusions, heat of solvation, solubility, dissolution
rate, enthalpy of transition, phase diagrams, stability, hygroscopicity and rate of reactions are all
affected by the nature of the crystal structures. The differences in solid state properties of alternate
crystal forms could give rise to measurable differences in the properties of pharmaceutical systems
(Brittain, 1999, 2002a). Various aspects of polymorphism of pharmaceutical compounds have
been discussed by Borka and Haleblian (1990), Brittain (1999), and Singhal and Curatalo (2004).
The different types of polymorphism are described as follows:
5.4.6.1 Conformational polymorphism
The polymorphism resulting from different conformers of the same molecule is called
conformational polymorphism. For example, the existence of probucol, a cholesterol lowering drug,
into two polymorphic forms. The molecular symmetry of the molecule is lost in the structure of form
1. The less symmetrical conformer (form 2) is more stable with lower activation energy than form
1 (Gerber et al., 1993).
5.4.6.2 Solvatomorphism
A solvatomorph can be defined as a crystalline solid in which solvent molecules are
included in the structure through the existence of positional substitution at positions that are site
specific and related to other solvent molecules through translational symmetry. It may also involve
the incorporation of water into a crystal lattice. For example, ampicillin crystallizes in one trihydrate
and at least two anhydrate forms. The transition temperature for the two forms in the presence of
water is 42°C, where as the trihydrate is formed when crystallization is conducted below this value
and the anhydrates are formed on crystallization at temperatures exceeding 42°C (Boles and
Girven, 1976).
5.4.6.3 Packing polymorphism
Polymorphism that exists as a result of difference in crystal packing of molecules is termed
as packing polymorphism. Single-crystal X-ray crystallography has been used to determine the
structures of organic molecules. The structure of resorcinol (1,3-dihyrdoxybenzene) showed that
the crystalline material corresponded to that ordinarily formed at room temperature was termed as
the α–form (Robertson, 1936). Later, it was found that the α–form undergoes transformation into
a denser crystalline form when heated about 74°C. The structure of this form (denoted as β–form)
was completely different from that of the α–form. The crystal structures of the two forms showed
that resorcinol is locked into a single confirmation and that each form is characterized by a different
form of hydrogen bonding. The α–form exhibits a relatively open architecture maintained by a
spiraling array of hydrogen bonding that ascends through the various plains of the crystal. The
effect of the thermally induced phase transformation is to remove the open arrangement of the α–
form by a more compact and parallel arrangement of the molecule to give the β–form. The crystal
change leads to an increase in the crystal density on passing from the α–form to the β–form. The
molecular packing existing in the β–form is typical of hydrocarbon than that of a hydroxylic
compound such as resorcinol (Robertson et al., 1938).
110
5.4.6.4 Pseudopolymorphism
The pharmaceutical compounds are often crystallized using different solvents. During this
process the solvent molecules may be incorporated into the crystal lattice in a fixed ratio. This
leads to the formation of co-crystals, termed as solvates. If the crystallization is carried out using
water the crystals are termed as hydrates. These crystalline forms of the compound are called
pseudopolymorphs and the phenomenon is known as pseudopolymorphism.
5.4.6.5 Forced polymorphism
In the study of the polymorphs an attempt is made to induce or force polymorphism in drug
substances. It does not imply that any polymorphic forms observed will necessarily be present
during the drug development processes or on stability evaluation. Under forced polymorphism the
forms obtained may not appear during the manufacturing process or stability evaluation. However,
if these forms are found during drug development it would be easier to detect them and to
determine their influence on the physical stability of the drug and the product.
5.4.7 Characterization of Polymorphs
The polymorphs of crystalline pharmaceutical solids can be identified by using various
analytical techniques (Haleblian, 1975; Stagner and Guillory, 1979; Ford and Timminis, 1989; Wu
et al., 1994; Brittain, 1995, 1997, 1999; Threlfali, 1995; Bougay, 2001). The most widely used
techniques for the physical characterization of solid materials (polymorphs and solvates) include
crystallography, microscopy, thermal analysis, solubility determination, vibrational spectroscopy
and NMR spectrometry (Brittain, 2002b). The major criterion for the existence of different types of
polymorphs is the observation of semiequivalence of their crystal structures by XRPD analysis. A
very important method for the characterization of polymorphs is microscopy. It can show variations
in the habits of different crystal structures and this is useful in the characterization of polymorphs
(Haleblian, 1975). The hot-stage microscopy and thermal microscopy are extensively used
techniques for the characterization of polymorphs and solvates. These techniques involve the
observation of changes during the heating and cooling of a few mg of the substance or any
crystalline material on a microscopic slide (McCorne, 1957). The thermal microscopic studies of a
large number of pharmaceuticals have been conducted (Kuhnert-Brandstalter, 1971).
Thermal analysis methods have been used for the characterization of polymorphs (Ford
and Timminis, 1989). These methods involve the determination of a physical property of the drug
substance as a function of an externally applied temperature. In these methods the physical
property and the sample temperature are automatically measured and the sample temperature is
varied at a predetermined rate. These techniques are widely used in pharmaceutical industry for
the characterization of compound purity, polymorphism and excipients compatibility (Giron, 1986).
The most commonly used methods of thermal analysis include TGA, DSC and DTA. These
techniques provide information on phase transformation as a function of temperature (Brittain,
2000).
The relative stability of the polymorphic forms of drugs can also be studied by thermal
analysis. Melting temperatures of the compounds can be used to establish the relative order of the
stability of their polymorphic forms and any interphase conversions involved. In the case of
auranofin the anhydrous polymorphic form of the molecule is most stable as indicated by the
melting point and heat of fusion data (Lindenbaum et al., 1985). DTA thermograms of the two
forms of chloroquin diphosphate have shown that one form is pure but the other form is a mixture
of two polymorphs (van Aerde et al., 1984). A DTA study of the dissolution of three crystalline
forms of spironolactone, in conjugation with XRPD, showed differences in the behavior of the drug
(Salole and Al-Sarraj, 1985). DSC analysis of the polymorphic compounds has the advantage that
the area under DSC peak is directly proportional to the heat absorbed or evolved on heating. The
peak area integration gives the enthalpy of the reaction (∆H) and throws light on the
thermodynamic behavior of the system (Brittain, 2002b).
FTIR spectrometry has been used to differentiate and characterize the polymorphic forms
of drug substances. The spectra of the two forms of ranitidine HCl show difference in the region
above 3000 cm–1 and in the regions 2300–2700 cm–1 and 1570–1620 cm–1 (Cholertou et al., 1984).
111
The different crystalline forms of zenoterone have been found to give characteristic absorption
bands in the IR region which can be used for the identification of these forms (Rocco et al., 1995).
The polymorphic changes in tolfenamic acid has been studied using FTIR spectrometry (Jabeen
et al., 2012; Mattei and Li, 2012; Sheraz et al., 2015)
The ATR–FTIR spectrometry has been used for the identification and quantitation of two
polymorphs of aprepitant (an antagonist) for chemotherapy–induced emesis. The spectra of the
powdered samples of the polymorph pair were obtained over the wavelength range 700–1500 cm–
1. Significant spectral differences between the two polymorphs at 1140 cm –1 have been observed
that indicate that this technique can be used for definitive identification of the polymorphs. The
quantification of the polymorphic form of the drug was carried out using a calibration plot of peak
ratio of the second derivative of absorbance spectra against the weight percent of form II in the
mixture. The polymorphic purity results obtained by ATR–FTIR spectrometry were in good
agreement with the prediction made by XRPD analysis (Helmy et al., 2003).
DRIFTS coupled with partial-least-squares (PLS) data analysis has been applied for the
determination of the components of solid state mixtures of ephedrine and pseudoephedrine. The
cross-validated standard errors of prediction of 0.74 wt% in the concentration range of 0–50 wt%
and 0.11 wt% in the concentration range 0–50 wt% have been obtained (Dijiba et al., 2005). The
technique coupled with artificial neural networks (ANNs) in two versions (ANN-raw and ANN-pca),
support vector machines (SVMs), lazy learning (LL) and PLS regression has been used to quantify
carbamazepine crystal forms in ternary powder mixtures (I, III and IV). The analysis has been
carried out in the IR spectral regions of 675–1180 and 3400–3600 cm–1. The results indicate that
all the selected algorithms perform better than the PLS regression with a root mean squared error
of prediction (RMSEP) of 3.0–8.2%. (Kipouros et al., 2006).
The two polymorphs of famotidine have been determined by DSC and FTIR
microspectroscopy. The results show that the raw material of the drug consists of form B. The
intensity of the IR absorption band of the B form at 3505 cm –1 gradually decreases with the grinding
time, while two new IR bands at 3451 and 1671 cm –1 for famotidine form A slowly appear. The
peak intensity ratio of 3451/3505 cm –1 linearly increases with the grinding time suggesting that the
grinding process could induce polymorphic transformation of famotidine from form B to form A by
a zero–order process (Lin et al., 2006).
The two polymorphic forms (I and II) of fluconazole have been prepared by crystallization
in dichloromethane and characterized using DSC, TGA, XRPD, solubility and DRIFTS. DRIFTS
has also been used to study the kinetics of the transformation of polymorph II (metastable form)
to polymorph I (stable form) under different isothermal temperatures. The application of 18 solid-
state reaction models showed that the Prout-Tompkins model provides the best fits for
transformation. The activation energy (Ea) value derived from the rate constants of the model was
found to be 329 kJ mol–1 (Obaidat et al., 2010).
Solid state NMR (SSNMR) spectrometry has been employed for the qualitative
differentiation of polymorphs or solvates. The technique shows differences in their molecular
conformation as a result of crystallographic vibrations. The crystal structure of one form of
fosinopril sodium shows a most stable phase which is different from that of its metastable phase
(Brittain et al., 1993). The SSNMR spectrometry has also been applied to determine the phase
composition of anhydrate and dihydrate forms of carbamazepine (Suryanarayanan and
Widemann, 1990). The SS13C–NMR spectra of the polymorphs of furosemide show a greater
molecular mobility and disorder in its form II, compared with the rigid and uniformly ordered
structure of form I (Doherty and York, 1988).
The polymorphic form of clopidogrel hydrogen sulfate (HSCL) (an antiplatelet agent) in
solid dosage forms can be verified by SSNMR spectrometry. Such structural characterization of
the polymorph could assist in the development of new pharmaceutical formulations containing
HSCL and also in the identification of its counterfeit drugs (Pindelska et al., 2015). The micro- or
nano crystalline proteins can be studied by magic-angle spinning (MAS)–SSNMR spectroscopy.
The technique is used to provide atomic-resolution insight into the structure of the molecule when
single crystals cannot be studied by XRD method. Slight differences in the local chemical
112
environment around the proteins including the cosolvent and the buffer indicate whether single
crystal is formed by a protein. It has been observed that several formulations of the microcrystals
of the protein GBI give very high quality of SSNMR spectra. The polymorphs of the protein have
been characterized by XRPD and NMR assignments have been made. The technique has
potential utility in the study of the formulation of industrial and therapeutic proteins (Schmidt et al.,
2007).
The applications of SSNMR spectrometry in the characterization of pharmaceutical solids
including drug substances and solid dosage forms have been reviewed (Tishmack et al., 2003).
This technique is generally used for:1) studying structure and conformation, 2) analyzing molecular
motions (relaxation and exchange spectrometry), 3) assigning resonances (spectral editing and
two-dimensional correlation spectrometry) and 4) measuring internuclear distances.
5.4.8 Pharmaceutical Implications
The physical stability of drug substances (amorphous or crystalline) and drug products
involves the study of variations in their physical state over a period of time. Most of the drug
substances are crystalline in nature and may occur in the form of different polymorphs. The study
of polymorphism, crystallization and characterization of the polymorphs is an important aspect of
preformulation work in drug development. The investigation of the solid state properties and their
changes in drug substances could enable the selection of a polymorph that is thermodynamically
most stable. The polymorphs of drug substances can show variations in solubility and dissolution
rates that could result in nonequivalent bioavailability of their polymorphic forms. It is, therefore,
necessary to evaluate polymorphism in drug substances to ascertain the role of their polymorphic
forms in the development of formulations. A drug may exist in more than one polymorphic form,
one of which may be more stable than the others and could be preferred for the formulation of a
product. However, if a metastable form has higher solubility, better release characteristics and
reasonable stability over a period of time, it may be used for development work.
The poorly water-soluble drugs are generally formulated in their amorphous state. This
state possesses a higher internal energy, enhanced molecular motion and better thermodynamic
properties than those of the crystalline state. These characteristics lead to enhanced solubility as
well as dissolution rate. However, the amorphous drugs tend to crystallize during manufacturing,
storage or administration. It is, therefore, necessary to apply methods for the stabilization of
amorphous drugs to take advantage of their enhanced solubility and dissolution rate in the
formulation of solid dosage forms.
113
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CHAPTER – 6
SOLID STATE STABILITY

6.1 INTRODUCTION
The chemical degradation of drug substances in the solid state is a subject of great interest
to pharmaceutical scientists because a large number of drugs have been developed as solid
dosage forms. Several workers have dealt with the stability of drugs in the solid state (Connors et
al., 1986; Byrn et al., 1999; Santos, 1999; Carstensen, 1974, 2000; Yoshioka and Stella, 2000;
Bastin et al., 2000; Guillory and Poust, 2002; Florence and Attwood, 2006; Zhou et al., 2009) and
many reviews have been published (Koenigbauer et al., 1992; Lai and Topp, 1999; Phipps and
Mackin, 2000; Huang and Tong, 2004; Yoshioka and Aso, 2007; Zhou, 2009) to understand the
characteristics and any transitions in the solid state. The chemical degradation of drugs in the solid
state and in solid dosage forms is more complex than that occurring in the liquid media. The
formulation of a drug in a complex matrix in solid dosage forms creates the possibility of interaction
between the drug and the excipients that may give rise to incompatibility and stability problems. In
addition to this external factors such as moisture and temperature also affect the stability of solid
drugs and dosage forms. Guidelines on stability studies have been provided by regulatory
authorities (ICH, 2003; WHO, 2009; FDA, 2014; EMEA, 2003).
An understanding of the solid state properties of a drug and their impact on its stability is
an essential component of the drug development process. The characterization of the solid states
of a drug and the selection of the best form in term of stability and performance is the first step
before subjecting the active pharmaceutical ingredient (API) to further studies. The physical state,
thermal behavior and polymorphism are important characteristics that affect the stability of a drug
in a formulation.
The stability of a solid drug may often depend on the state in which it is present in a dosage
form. Drugs in the amorphous state have the advantage of higher solubility, faster dissolution and
greater bioavailability. However, the stability of the amorphous drugs is lower than those that exist
in the crystalline state. Drugs in the amorphous state may be affected by moisture which leads to
plasticization of the amorphous form resulting in a decrease in the stability of the drug. These
aspects have been discussed in chapter 5. Moisture may also participate in the degradation
reactions (such as hydrolysis, hydration, isomerization, etc) to destabilize the drug.
The chemical stability of amorphous drugs can be improved if binary molecular mixtures
(solid molecular dispersions) of the drugs are prepared using excipients such as polyvinyl
pyrrolidone (PVP) (6.1), a proton acceptor, which forms hydrogen bonding with the drug to stabilize
it. On the other hand, dextrans (6.2) that act as proton acceptor as well as proton donor, can be
used to stabilize a drug that possess both characteristics.
(6.1) (6.2)
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Solid state degradation reactions may involve phase transformation, dehydration,
desolvation and chemical degradation by oxidation, cyclization, solvolysis, hydrolysis,
deamidation, etc. Knowledge of these reactions in a pharmaceutical system would enable the
pharmaceutical scientist to take necessary steps to prevent these reactions and thus enhance the
stability of the drugs in solid dosage forms.
The degradation of a drug in the solid state has been found to mainly occur in the solution
phase involving the solvent layers in contact with the solid phase. The solvent may come from
various sources as described by Connors et al. (1986).
 A melt from the drug or an ingredient of the formulation with a low melting point.
 Residual moisture or solvent from wet granulation.
 Moisture absorbed onto the excipients such as starch, lactose, microcrystalline cellulose.
 Adsorbed atmospheric moisture.
 A solvate or hydrate losing its bound solvent with time or temperature variations.
The solid state degradation of drug substances may also occur at high temperatures in
the absence of water vapors.
The design of the solid state degradation study of a drug requires knowledge of the
important variables (such as particle size of the crystal, stresses created in the crystal, trace
impurities in the crystal) involved in the degradation. The experimental setup should have provision
for the control of temperature and water vapor pressure during the degradation reaction along with
a method of proper homogenous sampling of the degraded material in the mixture to ensure the
accuracy of results. The degradation of the polymorphic forms of a drug may occur differently due
to a difference in their physical characteristics. All these factor may influence the results of a solid
state degradation study.
The experimental methods used in a kinetic study of solid state drug degradation involve
the application of techniques such as reflectance spectroscopy, X-ray diffraction, thermal methods,
microscopy, dilatometry, gas pressure-volume analysis and other techniques (see Chapter 5). The
treatment of solid state reaction data, temperature effects on solid state reactions and application
of Arrhenius equation, equilibria involved in solid state degradation and use of van’t Hoff equation
for a drug in the hydrate form in equilibrium with its dehydrated form, have been described
(Monkhouse and Van Campen, 1984).
6.2 TOPOCHEMICAL REACTIONS
The chemical reactions occurring by deformations in the solid crystalline state are termed
as topochemical reactions. These reactions have specific requirements to occur and depend on
the order of molecular packing in a crystal lattice. A thermal or photo-induced molecular
rearrangement (i.e., bond angle and distance) in the solid state would lead to a chemical reaction
in the crystal lattice. The nature and magnitude of this reaction would depend on the intensity of
the external stimuli. In topochemical reactions, the products are different from those formed in the
liquid state. The chemical reactivity in the solid state is determined by the crystal structure of a
compound. Any defects or strains in the crystal surface produce sites of high energy that are
involved in the initiation of a chemical reaction. Crystalline disorders are the main cause of the
susceptibility of a solid compound to chemical degradation.
The degree of crystallinity of a drug may be affected by manufacturing processes (milling,
granulation, compaction, etc). This would influence the reactivity of the material. The rate of a
chemical reaction in the solid state may be enhanced by an increase in the surface area as a result
of smaller particle size of the crystals. This would increase the magnitude of crystal defects and
hence an increase in the rate of reaction.
4-Aminosalicylic acid undergoes dimerization in the crystalline state and occurs in the form
of a dimer as shown in Fig. 6.1. It involves the formation of a hydrogen bond between carboxyl
120
groups and an intramolecular hydrogen bond between hydroxyl group at 2 position and oxygen
atom of the carboxyl group (Pothisiri and Carstensen, 1975).
Fig. 6.1 Solid crystalline state dimer of 4-aminosalicylic acid, a and c indicate directions in the
arrangement of crystals.
6.3 CHEMICAL DEGRADATION REACTIONS
The chemical degradation of drug substances in the solid state may occur by the following
reactions.
6.3.1 Solvolysis
It is a major reaction occurring in the solid state degradation of drugs by the participation
of a solvent. It also includes the hydrolysis of a compound such as acetylsalicylic acid (aspirin)
(6.3) to give salicylic acid (6.4) and acetic acid (6.5). The acceleration of the reaction with time has
been attributed to the formation of the degradation products. These products lower the pH of the
sorbet moisture layer, that further catalyses the degradation of aspirin. It undergoes acid catalysis
at low pH (Yang and Brooke, 1982).
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O O
OH O
H2O OH
+ H3C
O OH
OH
H3C O
(6.3) (6.4) (6.5)
It also involves the decarboxylation of a compound such as 4-aminosalicylic acid (6.6) at
high temperature to form 3-aminophenol (6.7). The reaction occurs both in the absence and
presence of moisture (Kornblum and Sciarrone, 1964).
O OH
OH OH
heat
+ CO2
NH2 NH2
(6.6) (6.7)
Generally the greater the solvation in the crystal, the lower is the solubility and dissolution
rate. Thus solvated and non-solvated forms of poorly soluble drugs may exhibit differences in
bioavailability. Anhydrous form of ampicillin is absorbed to a greater extent from hard gelatin
capsule or aqueous suspension than the trihydrate form of ampicillin (Hill et al., 1972).
6.3.2 Oxidation
It involves the reaction of a drug in the solid state with molecular oxygen. The reaction can
proceed slowly by auto-oxidation in the presence of oxygen. Unsaturated fats undergo auto-
oxidation to initially form hydroperoxides which on further oxidation give low molecular weight fatty
acids. These acids impart the typical odor to fats.
The auto-oxidation of a compound occurs through the initiation, propagation and
termination steps to form the oxidation products. It involves the participation of free radicals and
oxygen to complete the reaction. The various steps in auto-oxidation may be described by the
following equations.
Initiation
A A● (6.1)
A● + SH A–H + S● (6.2)
Propagation
S●+ O2 SOO● (6.3)
SOO●+ SH SOOH + S● (6.4)
Termination
S ●+ S● S–S (6.5)
S●+ SOO● SOOS (6.6)
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In this process the free radicals may be formed by hemolytic cleavage of the chemical
bond of an initiator (A) (6.1). The free radical of a compound (SH) may be formed on the abstraction
of a hydrogen atom by the free radical formed in the initiation step (A●) (6.2). The newly formed
free radical (S●) then reacts with oxygen to produce a peroxy free radical (SOO ●) (6.3). This free
radical abstracts a hydrogen atom from another molecule of SH which is in turn oxidized to produce
a hydroperoxide (SOOH) along with the formation of another free radical of the compound (S ●)
(6.4). The chain reaction is an auto-oxidation (autocatalytic) process that continues until the
termination of the free radicals ((6.5) and (6.6)). The oxidation of several molecules of a compound
can be caused by the participation of a single free radical in the reaction.
Examples of solid state oxidation of drugs include ascorbic acid (6.8) (Willson et al., 1996),
and excipient-induced oxidation of a cyclic heptapeptide (6.9) in lyophilized formulation. The
reducing sugar impurities in mannitol act as oxidizing agent in the reaction (Dubost et al., 1996).
NH
HO
NH NH2 O
HO O OH
O O H
N O
HN N
HO O
H
HN
O
N
HO OH S O N O
H
S
NH
H2N O
(6.8) (6.9)
6.3.3 Deamidation
Peptide and protein drugs are often formulated in the solid state to achieve stabilization.
However, these agents can undergo degradation and inactivation during storage. These reactions
are affected by temperature, moisture content, excipients and the physical state of the formulation
(amorphous versus crystalline). A major reaction undergone by peptides and proteins is
deamidation of amino acid moieties (Lai and Topp, 1999). The deamidation of L-asparagine (6.10)
in polypeptides by a nonenzymatic reaction has been studied (Li et al., 2005a; Yang and Zubarev,
2010). It gives rise to L-succinimide (6.11) followed by the formation of L-aspartate (6.12) and
other compounds.
O O O
CH3
NH2 NH
deamidation NH hydrolysis
NH OH
HN CH3 HN HN
CH3 O CH3 O CH3 O
(6.10) (6.11) (6.12)
The effect of sucrose and mannitol on the deamidation kinetics of some model peptides
has been studied (Li et al., 2005b). An automatic computerized technique for the quantitative
determination of the deamidation rates of proteins has been developed. It has been found that a
large number of proteins undergo deamidation reactions (Robinson, 2002).
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6.3.4 Pyrolysis
Pyrolytic degradation of a compound involves thermally induced bond rupture in the solid
state in the absence of a solvent or moisture. Fluconazole (6.13) crystals undergo pyrolytic
degradation at 290°C. Pyrolysis-GC/MS study of the reaction has shown the formation of
hexafluorobenzene (6.14) as a degradation product. On pyrolysis at 500 and 750°C the
degradation products of fluconazole have been detected in gaseous state. The nature of pyrolysis
products depends on the temperature used for the reaction (Moura et al., 2010).
N
N N
F
HO N N o
290 C
N
F F F
(6.13) (6.14)
Another example of the pyrolytic degradation of a drug is polymethyl silsesquioxane. It is
degraded in the presence of nitrogen at 900°C to give silica, silicon oxycarbide and traces of
amorphous carbon (Ma et al., 2002).
6.3.5 Photolysis
A drug in the solid state may undergo photolytic degradation on exposure to light in the
presence or absence of a solvent. A solid dosage form like a tablet or capsule may also be affected
by light. In this case photolytic degradation may occur on the surface of the formulation. It may be
accompanied by the appearance or fading of color. Examples of solid state photolytic degradation
of drugs include the polymorphic forms of furosemide (6.15) (De Villiers et al, 1992) and
indomethacin (6.16) (Matsuda et al., 1980). Photodegradation assessment of ciprofloxacin,
moxifloxacin, norfloxacin and ofloxacin in the presence of excipients from tablets has been made
by HPLC–MS/MS and DSC methods (Hubicka et al., 2013).
O
Cl
O
H3CO OH O
CH3
O
NH S
N
NH2
O
HO
O
Cl
(6.15) (6.16)
The photodegradation of colors added to the tablets has been studied. It has been found
that this occurs due to a surface phenomenon that results in the fading of colors of tablets to a
depth of about 0.3 mM. Prolonged light exposure does not affect deeper into the tablet coating
and thus the drug content remains stable (Lachman et al., 1961). The photostability of
indomethacin in gelatin capsules depends on the capsule shell thickness and on the concentration
of opacifier (titanium dioxide). The gelatin films and indomethacin tablets on exposure to a 400 W
mercury vapor lamp for 2 h developed color. A good linear relationship has been found between
color difference values and square root of exposure time at different concentrations and
thicknesses. The rate of coloration is directly proportional to the transmission of films over the
wavelength range involved in the photodegradation of indomethacin (Mastsuda et al., 1980).
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6.4 FACTORS AFFECTING STABILITY IN THE SOLID STATE
6.4.1 Moisture
The presence of moisture and water content in a dosage form can affect its stability. An
increased exposure of the dosage form to atmospheric moisture or from that of the excipients has
been found to produce deleterious effect on the stability of active ingredients. Attempts should be
made to select excipients in accordance with the chemical nature of the drug to be formulated and
to minimize the exposure of the dosage form to excessive moisture during manufacturing and
storage. The moisture content of some excipients commonly used in tablet formulations is reported
in Table 6.1.
The majority of the excipients reported in Table 6.1 possess a considerable amount of
moisture content at higher RH. This moisture may be present in the loosely bound or strongly
bound form. If this moisture comes in contact with the drug, it would be destabilized. Tingstad and
Dudzinski (1973) have studied the effect of moisture on the solid state stability of drugs. To
minimize the effect of moisture they suggested the use of highly sealed containers, determination
of the amount of water present in the dosage form and use of a separate sealed ampoule for each
assay and water determination. This would avoid the disturbance of water equilibration when the
container is opened. Genton and Kesselring (1977) have found a linear relationship between log
k for the solid state degradation of nitrazepam and the %RH. The stability of drugs in solid dosage
forms can be studied by subjecting them to the temperature and RH conditions recommended in
ICH guideline (ICH, 2003).
Table 6.1 Moisture content of commonly used tablet excipients at 25°C on storage at
different relative humidities (RH) (Callahan et al., 1982).
% Equilibrium moisture content (EMC) at 25°C
Excipient (USP /NF grade) RH
33% 75% 100%
Anhydrous calcium phosphate <0.1 <0.1 7.0
Spray dried lactose 0.5 1.0 21.5
Magnesium stearate 3.1 3.5 –
Microcrystalline cellulose 3.7 8.1 –
Polyethylene glycol 3350 <0.3 2.0 62.2
Pregelatinized starch 7.8 14.7 36.4
Corn starch 8.0 14.4 16.5
Povidone 12.2 27.8 –
6.4.2 Temperature
Temperature is known to affect the stability of drugs in solid dosage forms. However, other
factors may complicate the evaluation of results under the following conditions (Connors et al.,
1986).
 Humidity is not simultaneously controlled.
 One of the ingredients, the drug or the excipients has a low melting point.
 One of the ingredients has loosely bound water and alterations in temperature change
the degree of binding of the water to the excipients.
 One of the ingredients of the dosage form is present in the form of a hydrate or solvate
that loses its bound solvent on changes in temperature.
 The solid dosage form is stored in different types of containers, open or closed and
permeable or hermetic, that may affect the stability in different ways.
The thermal degradation of vitamin A esters and other derivatives in the solid state has
been studied by observing changes in crystallinity by melting point determination. It has been
125
concluded that the degradation of these compounds depends on their melting point and that the
stability increases with an increase in the melting point (Table 6.2). The degradation at 50°C
follows an apparent first-order kinetics (Guillory and Higuchi, 1962). It has been suggested that
the degradation occurs only in the liquid phase on the surface of the crystal. The fraction of the
drug, that undergoes degradation, is a function of the melting point of the crystalline solid and can
be expressed by Eq. (6.7).
2.303 R
log X1 = (1/T – 1/Tm) (6.7)
–∆Hf
where X1 is the mole fraction of the compound in the melt form or the amount of the liquid phase,
∆Hf is the molar heat of fusion, R is the gas constant, T is temperature and T m is the melting point
of the pure solid compound in degree Kelvin. Under these conditions the rate of the degradation
reaction is proportional to X 1 and a linear relationship is observed between log k and Tm–1, where
k is the apparent zero-order rate constant.
Table 6.2 A Comparison of the apparent zero-order rate constants (ko) for the degradation
of various vitamin A derivatives at 50°C and their melting points.
Vitamin Aa derivatives ko × 102 mol h–1 Melting point (°C)
Acetate 27.0 57–58
Phthalimide-N-acetate 4.80 111–112
Nictomate 2.50 93–94
3,4,5-Trimethoxybenzoate 1.40 85–86
Succinate triphenyl guanidine salt 0.76 140–140.5
Benzhydrazone 0.38 181–182
a All the compounds do not follow the same pattern which may be due to differences in their
crystal structure and other factors.
6.5 DRUG INTERACTIONS
The drug may undergo drug-drug and drug excipient (additive) interactions in a solid
dosage form. The stability of a drug can be affected by the excipients which may act as surface
catalysts, alter the pH of the moisture layer or directly react with the drug. The potential influence
of excipients on the drug bioavailability is well known which results by virtue of the formation of
poorly soluble, non-absorbable drug-excipient complexes, for example, between tetracyclines and
dicalcium phosphate used as a diluent. Thus the excipients in solid dosage forms must comply
with the requirements of FDA monograph (21 CFR 330.1 (e)), regarding the stability of solid
dosage forms.
Racz (1989) has dealt with the drug-additive and additive-additive interactions in detail.
These interactions may vary with the nature of the additives. The different types of additives used
in the formulation of solid dosage forms include anion-active (negatively charged) additives (acrylic
acid polymers such as carbapol 934, ionic hydrocolloids, sodium alginate), cation-active (positively
charged) additives (quaternary ammonium salts, benzalkonium chloride, cetylpyridinium chloride),
amphoteric additives (proteins, gelatin) and non-active additives (polyvinyl pyrolidone (PVP)),
methyl cellulose (MC) and derivatives, polyethylene glycols (PEG), polyvinyl alcohol (PVA) and
starches). Drug interactions with different additives may decrease the stability by chemical
degradation or improve the stability (for example, by complexation). The drug-drug interaction may
occur in combination products. Aspirin (6.17) has been found to undergo reaction with
acetaminophen (paracetamol) (6.18) to form its acetyl ester (6.19) and salicylic acid (6.20) (Koshy
et al., 1967).
126
O O
O NH O
CH3 NH CH3
OH OH
+ +
O OH
OH H3C O
H3C
O
O
(6.17) (6.18) (6.19) (6.20)
6.6 KINETICS OF SOLID STATE DEGRADATION

The kinetics of thermal degradation of a compound in the absence and presence of
moisture or solvents in the solid state has been described by Ng (1975) and Carstensen (1974),
and is summarized by Connors et al. (1986).
Most of the thermal degradation reactions can be expressed by Eq. (6.8)
dx/dt = k α 1–p (1–α) 1–q (6.8)
where α is the concentration of fractional degradation, and k is a composite rate constant
which includes a term for N0, the number of potential degradation nuclei sites on the solid. The
quantities p and q are the parameters related to the mechanism of the degradation reaction with
limits in the range of 0–1.
Eq. (6.8) has been developed on the basis of the fact that the degradation of a compound
initially occurs at nuclei (stress points, imperfections, dislocation points) on the crystal surface. The
rate of degradation (dx/dt) is directly proportional to α, the fractional degradation. This is because
of the fact that the actual act of degradation induces an increase in the rate of degradation through
an increase in stress or dislocation in the crystal that results in an increase in the number of nuclei
undergoing degradation. The dependence of the rate of degradation on the increase in the crystal
stress is given by p.
If p = 0, then α 1–p = α, which shows that the rate is directly proportional to α. The term (1–
α)1–q is used to describe the degradation behavior of the drug. If both p and q are unity, Eq. (6.8)
is reduced to Eq. (6.9) indicating an overall zero-order kinetics.
dx/dt = k (6.9)
If p = 0 and q = 1, Eq. (6.9) is expressed in the form of Eq. (6.10)
dα/dt = kα (6.10)
In this case the degradation has been found to follow as apparent exponential kinetics. If
p and q both are equal to zero, the reaction can be expressed by Eq. (6.11).
dα/dt = k α (1–α) (6.11)
Eqs. (6.8)–(6.11) originally developed for the study of kinetics of thermal degradation of
drugs can also be applied to moisture dependent reactions.
The single component drugs in the solid state in a pharmaceutical system undergo
degradation by zero-order or first-order reaction. It is difficult to determine the exact order of a
reaction unless the reaction is carried out up to an adequate number of half-lives to find whether
it follows zero-order or first-order kinetics (see Chapter 2).
The kinetics aspects of chemical degradation of solids and solid dosage form have been
discussed by Florence and Attwood (2006), Yoshioka and Stella (2000) and Carstensen (2000).
127
6.7 SOLID STATE STABILITY STUDIES
Several studies on various aspects of solid state stability have been conducted to
investigate the structural features of different solid state forms, kinetics of degradation and effect
of excipients on the stability of drug substances. Some of these studies are presented in the
following sections.
6.7.1 Structural Studies
Multistep methods have been developed for the screening of physical and chemical
stability and/or reactivity of new drug candidates. The physical reactivity test is used to provide
information on the existing solid-state form in relation to the thermodynamically stable form. A
method to find the most stable form of the drug has been described. In the case of polymorphism
a search for additional polymorphs is made and different solid phases are characterized. Special
tests for the characterization of hydrates and anhydrous forms are reported (Berglund et al., 1990).
Thermoanalytical methods and non-thermal methods such as XRPD and IR spectrometry
have been used to study the structural changes of crystalline forms of moxifloxacin HCl (6.21)
stored under different conditions of relative humidity (0%, 40%, 75% and 90% RH) for a period of
one month. After the storage period at 0% and 40% RH there was no change in the crystalline
structure of the drug showing good physical stability of the material. However, in samples stored
at 75% and 90% RH, a hydrated crystalline form has been identified (Julio et al., 2015). The study
indicated that crystalline forms of moxifloxacin are not stable at higher RH.
O O
F
OH
H H
N N N .HCl
O
H3C
H
(6.21)
A solid-state stability study of the β-lactam antibiotic, meropenem (6.22), has been
conducted using UV, FTIR and Raman spectrometry. The optimum molecular geometry, harmonic
vibrational frequencies, IR intensities and Raman scattering characteristics have been determined
according to the density-functional theory (DFT). The differences between the observed and
scaled wave number values of peaks in the FTIR and Raman spectra made it possible to detect
non-degraded and degraded samples of the drug. Molecular electrostatic potential (MEP), front
molecular orbitals (FMOs) and the gap potential between highest occupied molecular orbital
(HOMO) and lowest unoccupied molecular orbital (LUMO) have been determined to enable the
interpretation of the results (Criclecka-Piontek et al., 2013).
OH CH3
H H O
O
S
N CH3
N
O O N
H H3C
HO
(6.22)
128
6.7.2 Kinetic Studies
Isothermal calorimetry has been applied to determine the rate of solid state room
temperature degradation of drug substances. This technique involves measurement of the rate of
heat output of a compound at several elevated temperatures and determination of its rate of
degradation at a single temperature as well as the activation energy. The solid state stability of
phenytoin, triamterene, digoxin, tetracycline, theophylline and diltiazem has been studied by this
method (Koenigbauer et al., 1992).
The chemical stability of ranitidine HCl (6.23) in the solid state at various temperatures
has been studied by a HPLC method. The critical relative humidity (CRH) of the bulk powder was
found to be ~ 67% RH. The amount of water adsorbed by the powder above the CRH is
proportional to the RH value. The percent degradation of the drug powder at 60–70% RH is higher
than that observed above 70% RH. Ranitidine HCl powder is unstable around the CRH (Teraoka
et al., 1993).
CH3 NO 2
N O NH
H3C S CH3
NH
(6.23)
The evaluation of the effect of ‘microenvironmental pH’ on the stability and dissolution of
solid dosage forms has gained considerable importance. The microenvironmental pH has shown
significant impact on the stability of drugs which are affected by the pH of the solution. The kinetics
of degradation of such drugs is dependent on the microenvironmental pH of the solid material. The
use of pH modifiers is an effective method to modulate the microenvironmental pH so as to improve
the stability of solid dosage forms. The selection of the appropriate pH modifier, its concentration
and method of incorporating the modifier is important to enhance the stability of the drug (Badawy
et al., 2007).
The degradation kinetics of asparagine (6.10) in two model peptides has been studied at
50°C at pH 7 in the presence and absence of 5% sucrose (6.24) or mannitol (6.25) and at 50°C
and 30% RH in solid samples lyophilized from the solutions. Solid formulations have been
characterized using Karl Fischer coulometric titration, TGA, DSC, FTIR and solid-state NMR
spectrometry. Asparagine showed similar pseudo first-order rates for deamidation in solution and
in the absence of sucrose and mannitol. The addition of 5% sucrose or mannitol was found to
decrease the rates up to 17%. The model peptides degraded 2 to 80 fold more slowly in the solid
formulations of sucrose and mannitol than those in 5% solutions of these carbohydrates. Mannitol
formulations were found to be largely amorphous immediately after lyophilization with some
crystalline like structures while sucrose formulations remained amorphous after lyophilization and
storage. Sucrose stabilized the peptides against deamidation in the solid state (Li et al., 2005b).
O
NH2
NH
HN CH3
CH3 O
(6.10)
129
(6.24)
OH OH
OH
HO
OH OH
(6.25)
Differential scanning calorimetry (DSC) has been used to study the kinetics of thermal
degradation of several derivatives of glycine (i.e., 3,5-disubtiutied tetrahydro-2H-1,3,5-thiadiazine-
2-thione, THTT, and derivatives) (6.26) in the solid state to serve as an amino acid and peptide
drug model. The two DSC peaks indicated the melting and degradation of the compounds,
respectively. The Augin, Benret and Kissinger equations were used to determine the activation
energy of the degradation reaction carried out up to 300°C, the activation energy of melting and
the enthalpy (∆H) of the compounds. The study has been used to evaluate the relative stability of
the compounds and the most stable prodrug that possesses the highest activation energy and the
longest shelf-life (Abdol-Elrahman et al., 2002).
O
H3C
N N
OH
S S
(6.26)
The stability of 1,3,5-triazine (6.27), a corticotrophin releasing factor inhibitor, has been
studied in solid formulations and the structure of degradants elucidated by LC/MS and NMR
spectrometry. The degradation of 1,3,5-triazine involves hydrolysis of the triazine ring and hydroxy
substitution of amino group on the triazine ring followed by its hydrolysis. The stability of the
compound is dependent on the manufacturing process and degradation is more rapid in
amorphous regions formed during the process. The degradation rate in tablet formulations is
enhanced under high humidity (Badawy et al., 2009).
NH2
N N
H2N N NH2
(6.27)
130
The stability of freeze-dried liposomes of different lipid composition containing trehalose
as a lypoprotectant has been investigated. The dry cakes of liposomes were exposed to different
temperatures for 30 min and the retention of carboxyfluorescein and average vesicle size after
rehydration were examined by DSC. FTIR was employed to study the acyl chain order and
interaction between trehalose molecules and phospholipid head groups. All lipid compositions of
liposomes showed induction of leakage, suppression of onset bilayer melting transition
temperature (Tm) and enhancement of the interaction between sugar and phospholipids below the
glass tranisition temperature (Tg). These changes were accompanied by melting transition of the
bilayers. It has been concluded that for liposomes freeze-dried in trehalose the temperature range
of bilayer melting is a better indicator than the Tg for maximum temperature exposure of liposomes
for short period of time (30 min) (Van Winden and Crommelin,1999).
Cyclodextrin (CD) (6.28) has been used to prepare inclusion complexes with drugs in the
solid state. The drug–CD complexes have greater stability and potential advantage in dosage form
design such as layered formulations. The 2D heteronuclear and homonuclear correlation solid-
state NMR (SSNMR) involving 1H, 13C, 19F and 31P nuclei has been used to investigate drug–CD
interactions in these complexes that involve dipolar interactions between nuclei within the drug
and CD molecules. The technique provides information on the inclusion of drug within the CD
cavity in powder samples of drug complexes of dipivoxil, voriconazole, dexamethasone and
prednisolone. SSNMR can be used for the characterization and quantitative analysis of solid drugs
and their complexes (Vogt and Strohmier, 2012).
(6.28)
The chemical degradation pathways of amorphous solids can be determined by the
relative mobilities of potential reactants. The molecular dynamic simulations of amorphous glasses
of PVP containing small amounts of water, ammonia and a small peptide (6.29) over a period of
100 ns have been used to monitor aging process of PVP segments and embedded solutes. Tg
values have been obtained by observing changes in slopes of the volume/temperature profiles and
the internal energy/temperatures profiles for the inherent structures on cooling at different rates.
Determination of molecular trajectories below Tg show temporal and spatial heterogenicity in the
polymer and solute mobility, with each molecule showing different relaxation behavior for
translational, rotational and/or conformational motions. The data have been used to study the
degradation of the peptide by deamidation (Xiang and Anderson, 2004).
O R O R O R
H2N NH NH OH
NH NH NH
R O R O R O
(6.29)
131
6.7.3 Effect of Excipients
It is important to understand the role of excipients with a high affinity for water in a
formulation exposed to moisture. In this context the effect of polyvinyl pyrrolidone (PVP) and RH
on the solid state stability of anhydrous theophylline has been studied by moisture uptake, XRPD,
HPLC and FTIR spectrometry. The physical mixtures of anhydrous theophylline and PVP were
stored at room temperature at various humidities and the physical and chemical changes
monitored. A hypothesis has been presented to explain the role of amorphous polymeric excipients
and the associated mobility of water. The mechanism of protection of hydration of theophylline
(6.30) by PVP involves a desiccant action. The efficiency of this action is dependent upon the
amount of water in the system and the kinetics of reaching the equilibrium moisture content (EMC)
(Kesavan and Garnet, 1996).
O
H
H3C N
N
N N
O
CH3
(6.30)
The effect of amorphous bulking agents on the chemical stability of freeze-dried drugs has
been studied. PVP, dextrans of different molecular weight and lactose have been used as bulking
agents and sucrose as an acid sensitive compound. Lyophiles of the bulking agent and sucrose
at 10:1 (w/w) ratio were examined by XRPD, DSC and Karl Fisher titration. The amount of sucrose
inversion in lyophiles stored at 60°C was determined by HPLC. It has been observed that the
bulking agent has a major impact on both the solid-state acidity (measured by Hamrnett acidity
function) and the degradation rate. The values of degradation rate constants are higher for dextran
lyophiles (more acidic) that those of PVP and sucrose (less acidic). The Hamrnett acidity function
can be used to predict the order of stability of acid-sensitive drugs in lyophiles prepared with
different bulking agents (Lu et al., 2009).
6.7.4 Effect of Aging
The term “aging” is used to express the physical instability of pharmaceutical dosage
forms. It is a process through which changes in the disintegration and/or dissolution properties of
dosage forms are caused by delicate alterations in the physicochemical characteristics of the inert
or active ingredients in the dosage forms. As the disintegration and dissolution of the drugs may
be rate-determining steps in their absorption, any changes in these processes due to aging of
dosage forms could affect the bioavailability of the product (Guillory and Poust, 2002). Several
studies have been carried out on the aging of the excipients and solid dosage forms. Some of
these studies are presented in this section.
The physical aging of PVP K25 on storage has been studied by positron lifetime
spectroscopy and scanning electron microscopy. The transition of PVP K25 from glassy state (at
25°C/ 55% RH) to completely plasticized wet rubbery state (at 25°C/ 75% RH) is not uniform. A
slow anomalous structure is formed on storage at 65% RH. It has been found that the actual water
content and storage conditions determine the size distribution of free volume holes in the material.
Under high humidity conditions a hydrogen bound “network” is formed between the polymer chains
and the water molecules (Suvegh and Zelko, 2002).
The influence of aging on the release of salbutamol sulfate from oral formulations (lipid
matrices) prepared with Gelucire® as a lipid excipient has been studied. The release profiles of the
drug from the capsule showed dependence on the type of Gelucires, indicating a fast release from
Gelucire 35/10, a slow release from Gelucire 46/07 and a slower release from Gelucire 48/09.
Differential scanning colorimetric studies of the physical state of the drug in different matrices have
132
shown aging effects on storage. It has been concluded that a higher effect of aging on the capsules
is indicated by a faster rate of dissolution (San Vicente et al., 2000).
The effect of aging on acetaminophen tablets prepared by wet granulation using povidone
or pregelatinized starch as binder on storage at 40°C/ 52% RH and 40°C/ 94% RH for eight weeks
has been studied. At 40°C/ 52% RH the tablets showed an increase in hardness and at 40°C/ 94%
RH a decrease in hardness. The pregelatinized starch granulated tablets showed a lower effect of
changes in hardness by humidity than the povidone granulated tablets. The disintegration of
tablets with both of these binders slowed down with an increase in humidity. A considerable slow
down in the dissolution of the tablets was observed at 40°C/ 94% RH compared to that at 40°C/
52% RH. The tablets containing pregelatinized starch were less affected by humidity than those
containing povidone (Sarisuta and Parrott, 1988).
The tablets prepared by wet granulation have been found to be affected by the moisture
content of granules on aging. The evaluation of changes in hardness, disintegration and drug
release of tablets prepared by direct compression of different bases with variable moisture content
has been made. Tablet with high initial moisture content showed an increase in hardness on
storage depending upon the physical properties of the base and the absolute moisture content.
Hardness increase resulted in an increase in disintegration time and a decrease in drug release.
The moisture uptake of tablets enhanced the disintegration time as well as the drug release. The
tablets prepared with lactose as a base with variable initial moisture content were highly resistant
to any changes on storage (Molokhia et al., 1987).
The effect of aging on the stability of glibenclamide (GB) / β-cyclodextrin (CD) systems
and CD–complexed GB tablets has been investigated using IR spectrometry and X-ray diffraction
analysis. The results indicated that the physicochemical properties of the tablets are not affected
even after storage for four years. However, the crystallinity of the physical mixture of GB/CD
decreases with aging. The effect of aging on the dissolution of GB in tablets can be overcome by
preparing a GB/CD complex in the tablet dosage form (Babu and Pandit, 1999).
The effect of humidity aging on hardness, disintegration and dissolution of Ca3 (PO4)2–
based tablet with variable moisture content has been evaluated. It has been found that a decrease
in the disintegration time, an increase in the dissolution rate and no change in the hardness of the
tablets with higher initial moisture content occurs on aging under low humidity. On the contrary, a
decrease in hardness, an increase in disintegration and a decrease in dissolution rate of tablets
with lower initial moisture content occurs on aging under high humidity conditions. The physical
characteristics of the tablets are affected by the moisture content of tablet granulation at
compression time and moisture uptake on aging during storage (Chowhan and Amaro, 1979).
The influence of aging on the dissolution of phenylbutazone tablets has been studied. The
dissolution rate of old tablet batches has been found to decrease gradually with aging. A similar
effect is produced by subjecting the tablet to higher temperatures. This effect may be related to
the subcoat layer of the sugar coating of the tablet that strongly adheres to the tablet core and thus
causes a slowdown in its disintegration (Barrett and Fell, 1975).
133
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138
CHAPTER – 7
FORCED DRUG DEGRADATION

7.1 INTRODUCTION
A consideration of the chemical stability of drug substances is of fundamental importance
to the formulator since it affects the quality, efficacy and safety of drug products. It is necessary to
know any change in the chemical stability of a drug substance, with time, under the influence of
environmental factors such as air, moisture, heat, light and chemical factors such as pH, solvent,
buffer. This can be achieved by performing stress testing or forced degradation studies on drugs
and drug products. The ICH (International Conference on Harmonization) Q1A (R2) Guideline
(ICH, 2003) states the object of stress testing of new drug substances as follows:
“Stress testing of the drug substance can help identify the likely degradation products,
which can in turn help establish the degradation pathways and the intrinsic stability of the molecule
and validate the stability indicating power of the analytical procedures used. The nature of the
stress testing will depend on the individual drug substance and the type of drug product involved”
The ICH guideline makes further recommendation as follows:
“Stress testing is likely to be carried out on a single batch of the drug substance. It should
include the effect of temperatures (in 10°C increments (e.g., 50°C, 60°C, etc.) above that for
accelerated testing), humidity (e.g., 75% RH or greater) where appropriate, oxidation and
photolysis on the drug substance. The testing should also evaluate the susceptibility of the drug
substance to hydrolysis across a wide range of pH values when in solution or suspension.
Photostability testing should be an integral part of stress testing. However, it may not be necessary
to examine specifically for certain degradation products if it has been demonstrated that they are
not formed under accelerated or long term storage conditions. Results from these studies will form
an integral part of the information provided to regulatory authorities”
The requirements for ICH Q1B Guideline (ICH, 1996a) on photostability testing are as
follows:
“Forced degradation testing studies are those undertaken to degrade the sample
deliberately. These studies, which may be undertaken in the development phase normally on the
drug substances, are used to evaluate the overall photosensitivity of the material for method
development purposes and/or degradation pathway elucidation”
Forced degradation of new drug substances and drug excipients is considered as a
degradation process that has been carried out at conditions that are more severe than those of
the accelerated conditions. It enables the establishment of the degradation pathways,
determination of the chemical structure of the degradation products, assessment of intrinsic
stability of drug substances and the validation of stability-indicating assay methods. An
understanding of the chemical reactivity of molecules during forced degradation studies is helpful
in the development of different dosage forms. Forced degradation studies are also considered as
integral part of the drug development process. The pharmaceutical industry performs forced
degradation studies on drugs during the preformulation stage to enable the selection of appropriate
active ingredients and excipients, product characterization, compatibility assessment, assay
development and formulation development and optimization. Since these studies provide
information on the mode of degradation of a drug and the products formed, it can be used for the
improvement of the manufacturing process and in the design of suitable packaging. Forced
degradation studies require the application of stability-indicating assay methods for the accurate
determination of the intact drug in the presence of degradation products (see Chapter 11). Data
139
on forced degradation studies are required for New Drug Application (NDA) for registration
purpose. The development, methodology, application and regulatory aspects of forced
degradation studies of drug substances and drug products have been reviewed (Reynolds, 2004;
Reynolds et al., 2002; Ngwa, 2010; Maheswaran, 2012; Singh and Rehman, 2012; Roge et al.,
2013, Singh et al., 2013; Jain and Basniwal, 2013; Charde et al., 2013; Hotha et al., 2013; Blessy
et al., 2014; Deokate and Gorde, 2014; Shete et al., 2014; Rawat and Pandey, 2015; Tamizi and
Jouyban, 2016).
7.2 OBJECTIVES
The objectives of forced degradation studies of new drug substances and drug products
have been described by Blessy et al. (2014) and are as follows:
 Establishment of degradation pathways of drug substances and drug products.
 Differentiation of degradation products of the drug in the drug products from those formed
from the non-drug product in a formulation.
 Determination of the chemical structures of degradation products.
 Determination of the intrinsic stability of a drug substance in a formulation.
 Elucidation of the degradation mechanism of the drug substances and drug products such
as oxidation, hydrolysis, thermolysis, photolysis, isomerization.
 Development of the stability-indicating assay method of the drug substances.
 Understanding of the chemical properties of drug molecules.
 Development of more stable formulations.
 Determination of degradation profiles of the drug substances similar to that observed in a
formal stability study under ICH conditions.
 Solution of stability–related problems of drug substances.
They may also include:
 Identification of impurities related to drug substances or excipients.
7.3 FACTORS INVOLVED IN DEGRADATION

7.3.1 Degradation Conditions
The stress conditions selected for the forced degradation study of a drug or product should
be considered according to its degradation behavior during manufacturing, storage and use
(Jenke, 1996). A general protocol of forced degradation conditions used for drugs or products
(Blessy et al., 2014) is presented in Fig. 7.1. The normally used stress conditions in a forced
degradation study include acid/base hydrolysis, oxidation, thermolysis and photolysis to achieve
10% degradation. These conditions have been described by Ngwa (2010) and are summarized in
Table 7.1.
7.3.2 Degradation Limits
It is important to set the limits of degradation of a drug or product that are acceptable in
forced degradation studies. The degradation limits of 5, 10 and 20% have been considered as
acceptable for the validation of chromatographic methods (Carr and Wahlich, 1990; Jenke, 1996;
Reynolds et al., 2002). Protocols for forced degradation studies of drugs and products could be
different as a result of variations in drug concentrations, matrices and other factors. A maximum
period of 14 days of stress testing in acid/base solution, and 24 days in peroxide solution has been
recommended to produce stressed samples in a forced degradation study (Klick et al., 2005). It is
necessary to avoid over-stressing of a sample that may lead to the formation of a secondary
140
degradation product not observed in formal studies on the shelf-life stability of a drug. On the other
hand, under stressing a sample may not produce sufficient amount of the degradation products
(Maherwaran, 2012). These factors should be taken into consideration in the design of a forced
degradation study to achieve the desired objectives.
Forced degradation study
Drug Substance Drug product
Solid Solution/Suspension Solid Solution/Suspension
Semisolid
Photolytic
Photolytic Acid/Base hydrolysis Photolytic
Thermal
Thermal Oxidation Thermal
Thermal/Humidity Photolytic
Thermal/Humidity Oxidative
Oxidative Thermal
Thermal/Humidity
Fig. 7.1 Stress conditions used for the degradation of drug substances and drug products.
Table 7.1 Widely used conditions for conducting forced degradation a.
Reaction Condition Storage
Hydrolysis Control drug (water) 40°C, 60°C
0.1 M HCl ‫״‬
0.1 M NaOH ‫״‬
Acid / Base control (without drug) ‫״‬
pH 2, 4, 6, 8 ‫״‬
Oxidation 3% H2O2 25°C, 60°C
H2O2 control ‫״‬
Thermolysis Heat (stability chamber) 60°C
‫״‬ 60°C / 75% RH
‫״‬ 80°C
‫״‬ 80°C / 75% RH
Heat control Room temperature
Photolysis Light 1 (ICH option 1) –
Light 2 (ICH option 2) –
Light control –
a The studies can be carried out at a drug concentration of 1mg/ml (Bakshi and Singh, 2002). This
concentration is considered sufficient to detect even the minor degradation products by analytical
methods such as HPLC. The sampling during a degradation reaction may be done at suitable
intervals depending upon the nature and the rate of reaction for 1 to 7 days or more.
7.3.3 Method of Analysis
In forced degradation studies it is necessary to use an assay method that is stability-
indicating. A stability-indicating method is a validated quantitative analytical method used to
determine the concentration changes in a drug or product, with time, without interference from
degradation products, impurities and excipients (FDA, 2000; ICH, 2005). The specificity of the
method can be confirmed by its application to samples that have undergone stress testing. The
development of stability-indicating assay methods for application to pharmaceutical systems has
been discussed by many workers (Ahmad, 1985; Bakshi and Singh, 2002, Ruan et al., 2006;
141
Smela, 2005; Aubry et al., 2009; Annapurna et al., 2012). A detailed treatment of the stability-
indicating assay methods has been presented in Chapter 11.
7.4 FORCED DEGRADATION STUDIES IN THE DEVELOPMENT OF

DRUGS
Hawe et al. (2012) have discussed the relevance of forced degradation studies in different
phases of drug development with recommendations for selecting suitable conditions. The ICH Q5C
guideline (ICH, 1996b) states that forced degradation studies can help in: 1) the assessment
whether accidental (or intended) exposure to conditions other than those proposed, for example,
during transportation or storage, is deleterious to the product and, 2) the evaluation of analytical
method as indicator of product stability.
Forced degradation studies are important in formulation development to identify the stable
formulation. The degradation behavior of a drug or a product under particular stress conditions,
such as temperature or light, can be correlated with the proposed storage conditions. The stability
of test formulations under specified forced degradation conditions could be compared and the most
stable and robust formulations may be selected for further development.
7.5 ANALYTICAL TECHNIQUES USED IN FORCED DEGRADATION
STUDIES
Forced degradation studies can only be carried out with the help of suitable analytical
techniques for the characterization of the degradation products and for the assay of the drug and
degradation products under the intended stress conditions. The application of these techniques is
essential for the detection and determination of the degradation products to assess the stability of
the drug substance or drug products. The various analytical techniques used during the forced
degradation studies are summarized in Table 7.2. The application of various chromatographic
methods in forced degradation profiling of a large number of drugs has been reported by Jain and
Basniwal (2003).
Table 7.2 Application of analytical techniques in forced degradation studies.
Technique Type
Spectroscopy Ultraviolet, infrared, Raman, nuclear magnetic resonance,
mass, fluorescence, circular dichroism,
Chromatography Size exclusion, HPLC (reversed phase, ion exchange),
HPLC–mass spectrometry (HPLC–MS), UPLC, UPLC–
mass spectrometry (UPLC–MS)
Electrophoresis Sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE), capillary electrophoresis,
capillary electrophoresis–mass spectrometry (CE–MS)
7.6 FORCED DEGRADATION STUDIES
Several forced degradation studies have been carried out on drug substances and drug
products. Some of these studies are presented in this section.
A forced degradation study of rebamipide in bulk and in tablet dosage form has been
conducted. The drug and tablet (extract) solutions were subjected to acid and alkaline hydrolysis,
hydrogen peroxide oxidation, thermolysis and photolysis and the drug contents were determined
using a newly developed validated stability-indicating RP–HPLC assay method. A comparison of
the conventional degradation using reflux and microwave assisted degradation showed that the
microwave radiation can be used to enhance force degradation under hydrolytic conditions. The
drug was stable to acid hydrolysis and oxidative, thermolytic and photolytic degradation. However,
on alkaline hydrolysis rebamipide (7.1) underwent amide bond (C–N) cleavage to form 4-
chlorobenzoic acid (7.2) and (2-oxo-1,2-dihydroquinolone-4-yl) alanine (7.3) (Sonawane and Gide,
2011).
142
O OH
O OH O
OH NH2
NH
alkaline O
hydrolysis +
Cl
Cl N O
H
N O
H
(7.1) (7.2) (7.3)

The forced degradation behavior of lumivudine (7.4) under stress conditions of hydrolysis
(acid, base, neutral) oxidation, thermolysis and photolysis according to ICH guideline Q1 A (R2)
(ICH, 2003) has been studied. Lumivudine is stable in neutral solution and unstable in acid and
alkaline solutions. It undergoes extensive oxidative degradation and is stable to heat and light.
Five degraded products of the drug have been separated by LC and identified by LC–MS/TOF
methods (Bedse et al., 2009).
NH2
O N
O
HO
S
(7.4)
The chemical structures of the forced degradation products of tamsulosin (7.5), an α 1–
adrenorecpetor antagonist, have been determined by a gradient HPLC combined with quadrupole
time-of-flight electrospray ionization tandem mass spectrometry (LC/Q–TOF–ESI–MS/MS)
method. Tamsulosin was found to degrade under hydrolytic (base and neutral), oxidative,
thermolytic and photolytic conditions. Twelve degradation products of the drug have been
identified in the study (Namdev et al., 2014).
O
H2N NH
S
O
O O
H3C CH3
O
OH
(7.5)
The forced degradation of clobetasol 17-propionate (7.6) under different stress conditions,
i.e. acid, base, neutral hydrolysis, oxidation, thermolysis and photolysis has been studied using a
validated stability-indicating RP–HPLC method. The drug undergoes extensive degradation in
strong base and under oxidative conditions (Fauzee and Walker, 2013).
143
Cl
O
CH3
HO O
CH3
CH3 H
O
CH3
F H
O
(7.6)
The forced degradants of carisbamate (7.7) have been separated by a RP–HPLC method
and characterized by ESI–MS, 1H and 13C NMR, MS/MS and 2D NMR (Cosy and HSQC)
spectrometry. These products result from acid/base hydrolysis, hydrogen peroxide oxidation,
thermolysis and photolysis under stress conditions (Rao et al., 2013).
OH
H2N O
O
Cl
(7.7)
The stability of crystolepine HCl (7.8) under various stress conditions (acid, alkali, neutral,
light, dry heat and oxidation at different temperature) has been studied. The drug is highly sensitive
to oxidative conditions and is stable in acid and neutral solutions. Exposure to light and dry heat
at 60°C for 12 h did not affect the drug concentration in the samples (Kuntworbe et al., 2013).
H3C
+
N
-
N
(7.8)
The dry heat forced degradation of buserelin (7.9), a GnRH agonist peptide drug used in
cancer therapy, has been carried out in the solid state by exposing the powder to high
temperatures for prolonged periods. The assay of the drug and its degradants was performed by
a stability-indicating UPLC–photodiode array (PDA) method. The statistical evaluation of different
solid state kinetics models indicated the application of the Ginstling-Brounshtein model to the data.
No significant degradation was observed under hot melt extrusion conditions, i.e. 5 min at 100°C
and 125°C (D’Hondt et al., 2014).
144
O
NH N
NH N
H
O
O
NH O
OH
NH
NH
O
NH O
OH HN CH3
O CH3
O CH3
NH
H3C NH O
O CH3
CH3 O
NH
N
H2N
NH2
(7.9)
145
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148
CHAPTER – 8
PACKAGING EFFECTS ON STABILITY

8.1 INTRODUCTION
The regulatory authorities and pharmaceutical manufacturers have to pay great attention
to the stability of drug products to ensure their shelf-lives and delivery to the consumer with the
highest quality attributes. One of the essential components in this effort is the packaging of the
products. The design of a package for a particular product depends on the stability profile of the
product. The packaging development and integrity plays a major role in maintaining the stability of
the individual solid and liquid dosage forms. The stability of a product depends on the efficacy of
the packaging material to preserve its chemical and physical characteristics during the storage
period.
The container-closure system is an important component of packaging that can affect the
stability of a product. The packaging material and system must be suitable for its intended use and
should adequately protect the product from deterioration and tampering. It should be compatible
with the dosage form and should be composed of materials that are considered safe for use
specially the child resistant containers (WHO, 2009). The photostability characteristics of new drug
substances and products should be evaluated to show that, on exposure to light the product and
packaging materials do not result in any undesirable change (ICH, 1996).
An understanding of the factors influencing drug stability and the application of modern
packaging techniques could enable the development of suitable packaging materials. An
appropriate design of a stability program including different batches of a finished product in various
strengths and involving different types of packaging material can be implemented by the
application of factorial designs such as matrixing and bracketing. Several authors have dealt with
the subject of pharmaceutical packaging and its various aspects (Croce et al., 1986; Harburn,
1991; Dean et al., 2000; Byett, 2002; Soroka, 1996, 2002; Sinha et al., 2006; Yam, 2009) and a
number of reviews have been published on the selection, role and design of packaging and its
effect on the chemical and photostability of drug products (Akala, 2010; Reed et al., 2003;
Templeton et al., 2005; Waterman and MacDonald, 2010; Sacha et al., 2010; Sabah et al., 2014).
Guidelines on the packaging of pharmaceutical products are also available for the industry (FDA,
1995; WHO, 2002; United States Pharmacopeia, 2016; British Pharmacopoeia, 2016).
8.2 DEFINITION
Several definitions of packaging are described in the literature.
World Health Organization (2002):
“Packaging may be defined as the collection of different components (e.g. bottle, vial,
closure, cap, ampoule, blister) which surround the pharmaceutical product from the time of
production until its use”.
Soroka (1996):
“Packaging is a coordinated system of prepared goods for transport, distribution, storage,
sales and use. It is a complex, dynamic, scientific, aesthetic and business function, which in its
most fundamental form, contains, protects, preserves, provides convenience and informs the
concerned people within the acceptable environmental constraints”.
Sinha et al. (2006):
149
“Pharmaceutical packaging is a means of providing protection, presentation, identification,
information, convenience, compliance and compatible unit that maintains the integrity and stability
of the product”.
8.3 TYPES
The packaging material used for pharmaceutical products can be divided into two types.
8.3.1 Primary Packaging Material
It consist of bottles, containers, ampoules, vials, tubes etc and contains the product. It
provides protection to the product from any change against environmental factors. The primary
packaging material should be compatible and should not undergo any chemical interaction with
the product resulting in leaching of the components, absorption of the material and adsorption of
the drug on the surface of container. The primary packaging for a product (e.g., container and
closure) must be suitable for the specific purpose because it is in direct contact with the product.
8.3.2 Secondary Packaging Material
It consists of cartons, boxes, drums etc. to hold the primary packagings of the product.
The secondary packaging material provides protection against external factors as well as the
physical and mechanical stress during transportation and shipment. It also provides strength for
stacking in the ware house.
8.4 FUNCTIONS
Packaging is an integral part of the drug development process, in particular, the stability
assessment program. It plays a significant role in protecting the drug product from chemical and
physical changes caused by the environmental factors. These changes may occur under ambient
or accelerated storage conditions. The various functions of the primary packaging material to
preserve product stability against environmental factors are given in Table 8.1.
A major route of the chemical degradation of drugs in liquid preparations involves
hydrolytic processes as a result of change in pH, for example, in esters and amides. This can be
controlled by the use of Type 1 glass containers (borosilicate glass, highly resistant to chemical
action) for parenterals or Type II glass containers (chemically treated soda glass, high hydrolytic
resistance) for acid or neutral aqueous preparations (British Pharmacopoeia, 2016). The choice of
these containers depends on the nature of the preparation and its sensitivity to hydrolytic
degradation. The light sensitivity of drug substances (e.g. vitamins, steroids, alkaloids,
fluoroquinolones, NSAIDs) and drug products requires an effective packaging system for
protection against photochemical damage. The various Pharmacopoeias (British Pharmacopoeia,
2016; United States Pharmacopeia, 2016; European Pharmacopoeia, 2015) have prescribed
conditions for the use of containers (e.g. light-resistant) and storage (e.g. protected from light) for
light-sensitive drugs and products. These can be protected from light by the use of opaque or
amber colored containers. Amber glass is effective against UV and visible light (<470 nm). The
opaque secondary packaging also provides protection from light.
Table 8.1 Packaging preservation of product stability against environmental factors.
Factor Packaginga
Ultraviolet, visible and sunlight Light-resistant containers
(200 – 800 nm) (opaque or amber)
Temperature Plastic containers
(heat resistant)
Atmospheric gases Hermetic/air-tight containers
(oxygen, carbon dioxide)
Moisture/vapors tight/air-tight containers
Particulate matter well-closed/tight containers
Microbial containments tight containers
150
aThe secondary packaging also provides a certain degree of protection from light, heat,
moisture, gases, microbes, etc.
8.5 SELECTION
The selection of packaging material for a drug product is based on its efficacy and
performance characteristics to preserve the quality, strength, and safety of the product. It takes
into consideration the nature of the product, the chemical and physical characteristics of the
product, the protective requirements of the product and its marketing needs. It should be
compatible with the product, possess high protective efficacy against environmental factors, be
chemically non-reactive and non-toxic and should have aesthetic appeal. The use of substandard
packaging material could lead to the destabilization of the product as a result of different stress
conditions, drug-container interactions and chemical degradation (Sabah et al., 2014). According
to Sinha et al. (2006) the choice of the packaging material for a pharmaceutical product depends
on the following factors.
 Dosage form desired, e.g. syrup, tablet, creams.
 Degree of required protection.
 Compatibility of the material with the dosage forms.
 Presentation and aesthetics.
 Consumer convenience, e.g. size, weight of dosage form.
 Method of filling.
 Method of sterilization.
 Method of distribution-hospital, pharmacy, retailer.
 Capacity of packaging-small, bulk for pharmacies, OPD.
 Required shelf-life.
8.6 PACKAGING STUDIES
Several studies have been conducted to evaluate the effect of packaging on the stability
of drug products during storage under different conditions (e.g. humidity, temperature, light). These
studies have been helpful in the design of suitable packaging material to meet a particular
requirement in order to achieve optimum stability of the product. Some of these studies are
presented in the following sections.
8.6.1 Solid Dosage Forms
The effect of packaging on the storage of strip sealed carbamazepine tablets (Tegretol
and Tegral) under different temperature/humidity conditions has been studied. The tablets in
original strips were stored at 40°, 50° and 60°C for 6, 3 and 1 month, respectively at 75% RH. The
tablets removed from their strips and placed in closed bottles were also exposed to 40°C / 97%
RH for 5 min daily for 1 month. The tablet performance was examined by dissolution using a HPLC
method. The results indicated that the stress conditions used do not affect the stripped Tegretol
tablets while dissolution of tegral tablets stored at 50–60°C / 75% RH for 6 months is increased.
The tablets stored at 40°C / 97% RH for 1 month showed similar effects. They were hardened and
contents dissolved to the extent of about 7% in 60 min. Finlepsin tablets, dispersed in bottles, were
also exposed to 25° and 40°C / 97% RH for 5 min daily by removing the caps for 1 month. Under
these conditions, the effect of humidity has been found to be more drastic at 40°C than that of
25°C with a decrease in dissolution rate. All the brands of carbamazepine tablets stored under
different stress conditions remained chemically stable (Al-Zein et al., 1999).
The efficacy of different packages used to evaluate the chemical stability of the tablets of
a new moisture sensitive drug stored under accelerated conditions for 6 months has been
151
determined. The values of equilibrium moisture content (EMC) have been found to be 2.3, 2.4 and
2.9% at 25°C / 60% RH, 30°C / 60% RH and 40°C/75% RH, respectively. The permeation of the
containers (mg per blister per day) increased in the order: cold-form aluminium blister (0.001),
aclar blister (0.008), cyclic olefin blister (0.040) and polyvinyl chloride blister (0.259). The drug
contents after storage of the product in different packages for 6 months at 40°C / 75% RH were:
cold-form aluminium blister (100%), high density polyethylene container (99%), aclar blister (97%),
cyclic olefin blister (91%), and polyvinyl chloride blister (84%). The stability of the packaged
product has been predicted on the basis of EMC, degradation rate of unpackaged product, and
moisture barrier characteristics of the packages (Allinson et al., 2001).
The physical and chemical stability of fixed dose combinations (FDC) of the anti-
tuberculosis drugs, rifampicin, isonizaid, pyrazinamide and ethambutol, stored for 3 months under
ICH accelerated conditions (40°C / 75% RH) in packaged and unpackaged tablets forms has been
studied. After three months the unpackaged tablets showed severe physical and chemical
changes compared to those observed in the packaged products. An interaction between isoniazid
and rifampicin in the stored tablets was also found that could affect the potency loss of these drugs.
It has been suggested that these tablets should be packed in moisture barrier packaging to
eliminate the effect of moisture on the stability of the products (Bhutani et al., 2004).
A study has been carried out to determine the effects of temperature and humidity on the
stability of aspirin and ascorbic acid in different tablet matrices stored in various packagings. The
stability of the drugs was found to vary according to a relation between the hardness and moisture
sorption of the tablets. The packaging used were evaluated on the basis of the parameters: 1) the
ratio of residual concentration of the tabletted drug in closed containers to the ratio of residual
concentration of the drug in exposed tablets (stability ratio), and 2) the moisture uptake of tablets
in closed containers. The results indicated the superiority of cellophane and aluminium foil strip
single dose packagings as moisture barriers to well-closed glass or plastic container under the
intermediate-to-high water vapor pressure conditions employed during the storage (Lee et al.,
1965).
The stability of paracetamol tablets packed in polycoated paper, cellophane,
PVC/AC/aluminium foil and PVC / PVDC / aluminium foil has been studied under different storage
conditions for six months. The shelf-lives of the tablets at 25°C / 75% RH in these packages were
18.2, 17.1, 19.1 and 23.0 months, respectively. Thus PVC / PVDC / aluminium foil offered best
protection to the tablets compared to the other packagings (Ahmad and Shaikh, 1993).
Paracetamol tablets in these packagings showed an increase in disintegration time on increasing
the temperature from 25–45°C at 75% RH. The tablets packaged in PVC / PVDC / aluminium foil
were least affected by changes in disintegration time during storage (Ahmad and Shaikh, 1994a).
The tablets stored in these packagings at 25°C / 75% RH showed a loss in hardness from 5 to
10% and at 45°C / 75% RH, from 10 to 39% (Ahmad and Shaikh, 1994b). The PVC / PVDC /
aluminium foil packaging gave best protection to tablets against moisture on storage under
different RH conditions (Ahmad and Shaikh, 2003).
8.6.2 Liquid Dosage Forms
The stability of various injection dilutions of taxol (0.3, 0.6, 0.9 and 1.2 mg/ml in 50%
polyoxyethylated castor oil and 50% dehydrated ethanol ) in infusion solutions (5% dextrose or
0.9% sodium chloride solution) stored in 100 ml glass bottles, polyvinyl chloride (PVC) infusion
bags and polyolefin containers at 20–23°C for 24 hours has been studied. The drug content was
determined by a stability-indicating HPLC method and the clarity was observed by visual
inspection. The drug did not show any loss in 24 hours. All the solutions became hazy initially.
Solutions in PVC bags developed greater haze with time compared to those in glass and polyolefin
containers. The haze in solutions stored in PVC bags was identified as being due to the leaching
of di(2-ethylhexyl) phthalate (DEHP) used as a plasticizer. The formation of DEHP was not
observed in solutions stored in glass and polyolefin containers. The results indicated that the taxol
solutions stored in different containers are chemically stable over a period of 24 hours (Waugh et
al., 1991).
152
The chemical degradation of ceftazidine in intravenous solutions (40 mg/ml) stored in 100
ml polypropylene (PP) bags and polyvinyl chloride (PVC) bags and in glass containers filled with
5% dextrose or 0.9% sodium chloride solution, at 20 and 35°C for 20 hours has been studied.
Ceftazidine and its main degradation product, pyridine, were assayed by a HPLC method. The
degradation of the drug was greater in PP and PVC bags than that in the glass bottles. Solutions
stored in PP bags were more stable compared to those in PVC bags. The results showed that
glass containers are better than the PP and PVC bags for the storage of ceftazimide solutions at
different temperatures (Arsene et al., 2002).
The stability of beclofen (10 mg/ml), diltiazem HCl (12 mg/ml), dipyridamole (10 mg/ml)
and flecainide acetate (20 mg/ml) in extemporaneously compounded oral liquids has been
determined. These liquids were prepared in a 1:1 mixtures of Ora-Sweet and Ora-Plus and Ora-
Sweet SF and Ora-Plus (Paddock Laboratories, USA) and cherry syrup and stored in 100 ml
amber and clear polyethylene terephthalate containers, three each at 5 and 25°C in the dark for
60 days. The drug content of each preparation was determined by a stability-indicating HPLC
method. At the end of the storage period it was found that beclofen, diltiazem HCl, dipyridamole
and flecainide acetate solutions retained an average of 92% of the initial concentration at both 5
and 25°C. There was no change in appearance, odor or pH of the solutions. All the containers
were found to provide good stability to these drugs in oral liquid preparations (Allen and Erickson,
1996).
The stability of a number of drugs in under filled plastic and glass containers has been
evaluated. The drugs were reconstituted according to the manufacturers’ instructions and then
added to 50 ml dextrose injection (5%) in PVC bags and glass partial-filled bottles. All admixtures
were stored at 25°C, unprotected from light and the drug content determined over 24 hours by a
HPLC method. Methotrexate, leucovorin calcium, cytarabine, dactinomycin, mithramycin,
vinblastin sulfate, cyclophosphamide and dacarbazine were stable (10% or no change in 24 hours)
in plastic and glass containers. Doxorubicin and fluorouracil were found to be more stable in plastic
containers than the glass containers. The t90 values of the drugs are reported in Table 8.2.
It has been suggested that carmustine and bleomycin sulfate should be administered only
in glass containers in which these drugs are more stable. Mitomycin dissolved in 0.9% NaCl
injection is more stable in plastic container while it is not stable in 5% dextrose injection (Benvenuto
et al., 1981). The stability of carboplatin (3.2 mg/ml in 5% glucose infusion solution) stored in
polyethylene, polypropylene and glass containers at 25, 40 and 60°C has been studied using a
HPLC method. The degradation of carboplatin follows an apparent first-order kinetics that does
not depend on the nature of the container. The application of Arrhenius equation indicated a <2%
loss in the concentration of the drug at room temperature in one month (Prat et al., 1994).
Table 8.2 t90 Values of drugs in plastic and glass containers.
Drug Container t90 value (h)
Doxorubicin glass 40
Fluorouracil glass 7
plastic 43
Vincristine sulfate glass 10
Bleomycin sulfate plastic 0.7
Carmustine plastic 0.6
A comparison of the adsorption effects of antineoplastic drugs on low density polyethylene
(LDPE) containers, glass containers and PVC bags has been made. The therapeutic doses of
common cytotoxic drugs, carboplatin, carmustine, cytarabine, dacarbazine, fluorouracil,
gemcitabine, melphalan, methotrexate and vinorelbine, were placed in the containers filled with
0.9% isotonic sodium chloride solution and 5% dextrose infusion solution. The containers were
stored in the dark at 4 and 25°C for 168 hours and the drug contents were determined by a HPLC
method. Carmustine did not adsorb in LDPE and glass containers at 4°C. However, a little loss in
the concentration was observed at 25°C. A greater loss of the drug was noted in PVC bag.
Dacarbazine and melphalan also showed a loss in the concentration that was independent of the
153
type of container. The other drugs did not show any loss in concentration. The stability of the drugs
in these containers appeared in the order: glass < LDPE < PVC (Beitz et al., 1999).
The stability of the antineoplastic drug, docetaxel, in infusion solutions has been studied
after: 1) reconstitution of the injection concentrate and 2) further dilution in 0.9% sodium chloride
and 5% dextrose solution, on storage in polyolefin containers and PVC bags. The HPLC analysis
indicated that reconstituted docetaxel solutions were stable to the extent of 95% or more for four
weeks at 4 and 25°C. The diluted solutions (0.3 mg/ml and 0.9 mg/ml) were also stable at a level
of 95% or more for four weeks in polyolefin containers at 25°C. However, docetaxel in dilute
solutions stored in PVC bags showed precipitation after the 5th day. The leaching of DEHP from
PVC bags by docetaxel infusion solutions, with time, was also observed (Thiesen and Karmer,
1999).
The photostability of a compound (lyophilized product reconstituted solution (0.365 mg/ml
in 20 ml of 3.3% dextrose/0.3% NaCl)) has been studied at 25°C under combined UV-visible light
(8.1 klx visible and 4.3 W/m 2 UV light) using a photostability chamber. The concentration of the
active material and degradant (formed by photoisomerization) was determined by a stability-
indicating HPLC method. The results showed that the lyophilized product and the reconstituted
solution degrade to the extent of 0.09 and 0.29% per klx–h, respectively. The drug solution before
lyophilization (manufacturing) and post lyophilization (secondary packaging) degraded to the
extent of 0.017 and 0.014% per klx–h, respectively. The amount of combined UV-visible light
exposure to achieve 0.1% photodegradation of reconstituted solution in amber-vial was 3.5 h and
of lyophilized product was 10.8 h (Templeton et al., 2005).
The antihypertensive 1,4–dihydropyridine drugs are sensitive to light and are dispensed
in solid dosage forms. However, the solutions of these drugs have been stabilized by using
photoprotective polyethylene terephthalate (PET) containers. The solutions of felodipine in blue
PET containers are completely stabilized for 6 h when exposed to stress irradiation conditions
using a Xenon lamp. On the contrary, the t90 of the drug in glass containers has been found to be
24 min. The study shows that the polymeric containers are effective as packaging material for the
photoprotection of liquid preparation of these drugs (DeLuca et al., 2016).
8.7 STABILITY PREDICTION IN PACKAGED PRODUCTS
A consideration of the stability of drug products must take into account the packaging since
it affects the shelf-life of the product. Packaging plays several roles in improving or worsening the
shelf-life. The packaging effects on the stability of the product include: 1) altering the movement
of volatile/gaseous materials between inside and outside of the package and 2) providing
leachable and extractable impurities into a dosage form. Packaging slows down the equilibration
of the external humidity with the active ingredient inside the packaging. The water-impermeable
packaging (e.g. glass bottles, foil-foil blisters) prevents and transfer of moisture to the product. In
this case the equilibrium relative humidity (ERH) inside the packaging will be a function of the
moisture content of the drug product as packaged and the adsorption tendency of the product at
a give temperature. In the case of water-permeable packaging (e.g. plastic bottles and blisters)
moisture will enter or leave the package at a rate that depends on the moisture vapor transmission
rate (MVTR) that is a function of the packaging material, the thickness of the package, the surface
area of the package and the difference between RH inside and outside the packaging. As the
moisture difference between external and internal environments becomes closer the moisture
transfer rates slow down (Waterman, 2009).
8.8 STABILITY TESTING
The stability testing should be conducted on the dosage form packaged in container-
closure system proposed for marketing (including, as appropriate, any secondary packaging and
container label). Any studies carried out on the drug product outside its immediate container or in
other packaging material can form a useful part of the stress testing of the dosage form or can be
considered as supporting information, respectively (ICH, 2003).
154
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CHAPTER – 9
STABILIZATION
9.1 INTRODUCTION
Drug substances are sensitive to environmental factors and drug products may undergo
chemical and physical degradation reactions during formulation, production, storage and
shipment. The degradation may be prevented and the shelf-lives of the products may be enhanced
by adopting appropriate stabilization methods. The application of these methods would depend on
the nature of the dosage form, type of the degradation reaction and the sensitivity of the active
ingredient to factors such as oxygen, moisture, temperature, light, pH, buffer, ionic strength,
solvent, etc. The common approaches to minimize degradation and to achieve stabilization of drug
products include:
 Optimization of formulation parameters (e.g. excipients, particle size, pH, solvent, buffer)
with respect to the stability of the drug.
 Control of drug-drug and drug-excipient interactions.
 Control of environmental factors.
 Use of appropriate stabilizers and coating agents.
 Nitrogen purging during production and packaging for oxidizable products.
 Use of appropriate packaging material.
 Use of recommended storage conditions.
The various methods for the stabilization of drug products have been described by
Connors et al. (1986), Racz (1989), Thoma (1996), Yoshioka and Stella (2000), Eccleston (2002),
Imp-Ensep (2002), Sinko (2011) and reviewed by Connors et al. (1997), Yu (2001), Challa et al.
(2005), Piechocki and Thoma (2007), Rasheed et al. (2008), Bhattacharya and Syrayanarayanan
(2009), Laitinen et al. (2013), Sheraz et al. (2015) and Ahmad et al. (2016).
9.2 PREVENTION OF DEGRADATION REACTIONS
9.2.1 Common Degradation Reactions
9.2.1.1 Hydrolysis
The hydrolytic reactions can be minimized by:
 Adjustment of pH to an optimum value.
 Use of buffers at a low concentration.
 Use of appropriate cosolvents.
Example: Hydroylsis of chlordiazepoxide
Chlordiazepoxide (9.1) is hydrolyzed by ring cleavage to form a benzophenone derivative
(9.3) through the participation of a lactam intermediate (9.2) by specific acid-base catalysis.
157
NHCH 3 H O
N N NH2
H2O
O
N N
Cl Cl
O O
(9.1) (9.2) (9.3)

The stabilization of chlordiazepoxide can be achieved by the adjustment of pH to an
optimum value of 2.0. It can also be stabilized by using a separately packaged solvent to prepare
a solution by reconstitution of the lyophilized compound at the time of the use of the product
(Maulding et al., 1975).
9.2.1.2 Oxidation
The oxidation reactions can be minimized by:
 Exclusion of oxygen.
 Adjustment of pH (increase in oxidation potential by decrease in pH).
 Use of antioxidants.
Example: Oxidation of ascorbic acid
Ascorbic acid (9.4) is easily oxidized to dehydroascorbic acid (9.5) in aqueous solutions
in the presence of air. The rate of oxidation is increased with pH due to the formation of ascorbyl
ion (AH–) as an intermediate in the reaction.
H
OH H
HO OH
O HO
O
oxidation O O
HO OH
O O
(9.4) (9.5)
The stabilization of ascorbic acid can be achieved by the adjustment of pH to 2.0–3.0 to
form the nonionized species of the molecule (Blaug and Hajratwala, 1972).
9.2.1.3 Photolysis
The photolysis (photodegradation) reactions can be minimized by
 Use of amber/opaque containers.
 Use of stabilizers.
 Use of UV and visible absorbers.
Example: Photooxidation of menadione
Menadione (vitamin K3) (9.6) undergoes photooxidation in aqueous solution on irradiation
with UV light to form 2-methyl-2,3-epoxy-1,4-naphthaquinone (9.7).
158
O O
CH3 CH3
hv
O
pH 6-12
O O
(9.6) (9.7)
Menadione can be stabilized in aqueous solution by the adjustment of the pH in acidic
region. It can also be stabilized in the presence of various electron donors. The stability of the drug
increases with an increase in the concentration of the electron donor (Vire et al., 1980).
9.2.2 Prevention of Degradation Reactions Involving Steric Structural Variations
In addition to common degradation reactions described above, other chemical
transformations involving steric structural variations may occur during the manufacture and
formulation of drug substances.
9.2.2.1 Cyclization
Example: Cyclization of diclofenac sodium
Diclofenac sodium (9.8) undergoes intramolecular cyclization in acid solution in which the
molecule exists in the nonionized state. The H3O+ ion-catalyzed degradation of the intermediate
product (9.9) leads to the formation of a lactam as the final product (9.10) (Palomo et al., 1999).
O
C H3O+ -
OH O
+
NH NH OH
Cl Cl
Cl Cl
(9.8)
N O +
N OH
+
H + Cl Cl
Cl Cl
(9.10) (9.9)
159
The rate determining step in the reaction is the H3O+ ion-catalyzed degradation of the
intermediate product (9.9). A change in pH towards the neutral region would lead to the
stabilization of the molecule.
9.2.2.2 Dimerization
Example: Dimerization of amoxicillin
Amoxicillin (9.11), a penicillin derivative, undergoes dimerization reaction at higher
concentrations in the pH range of 8.0–10.0 while hydrolysis of the molecule occurs at lower
concentrations. In the dimerization reaction two molecules of amoxicillin undergo interaction. One
molecule undergoes nucleophilic attack of the α-amino group of the side chain on β-lactam
carbonyl group of the other molecule to form a dimer (9.12) (Bundgaard, 1977).
OH
OH
S CH3
CH CO NH CH S CH3
NH2
CH3 + CH CO NH CH
N - CH3
O COO NH2 N -
O COO
(9.11)
Dimerization
OH
S CH3
CH CO NH CH
CH3
NH2 C HN -
O COO
NH S CH3
CH CO NH
CH3
C N
-
O COO
OH (9.12)
The dimerization of amoxicillin can be prevented by a decrease in concentration and
increase in pH of the solution. The injectable preparations of amoxicillin may be used in a buffered,
lyophilized form to prevent the dimerization.
9.2.2.3 Epimerization
Example: Epimerization of tetracycline
Tetracycline (9.13) solutions undergo epimerization to form 4-epitetracycline (9.14) during
storage. The epimer is more toxic than the tetracycline (Yuen and Sokolski, 1977).
160
(H 3C) 2N
H3C OHH N(CH 3)2
H3C OH H
OH
OH
HO CONH 2
HO CONH 2
OH O OH O
OH O OH O
(9.13) (9.14)
Epimerization of tetracycline can be minimized by adjusting the pH values of the solutions
below 2.0 or above 9.0.
9.2.2.4 Racemization
Example: Racemization of epinephrine
The optically active compounds such as epinephrine (9.15) can be converted from one
form to the other (9.16) in aqueous solution under certain conditions (Hellberg, 1955).
OH
HO HO CH3
HN
H H
HN
HO CH3 HO
OH
(9.15) (9.16)
The racemization of epinephrine can be prevented by adjusting the pH of the solution in
the pH range of 3.5–5.5. Complexation of adrenaline with boric acid also leads to an increase in
the stability of the drug.
9.2.2.5 Polymerization
Example: Polymerization of ampicillin
The concentrated aqueous solutions of sodium ampicillin (9.17) (10–25 %, w/v) for IM
administration undergo change to form high molecular weight ampicillin polymers when stored at
room temperature and pH values in the range of 8.0–10.0 (Racz et al., 1989).
NH2
H H
NH S CH3
H CH3
O N
O O
H
HO
(9.17)
The polymerization of ampicillin can be prevented by maintaining the pH of the solution in
the pH range of 3.0–6.0.
9.3 METHODS OF STABILIZATION
The important methods for the stabilization of drug substances are as follows.
9.3.1 Temperature Control
The rates of chemical degradation reactions involving drug substances are normally
proportional to the number of collisions per unit time. An increase in the number of collisions with
161
an increase in temperature results in an enhanced degradation of drugs. The relation between the
rate constant of degradation and the temperature is given by Arrhenius equation (see Section
2.5.1). The higher the activation energy, Ea (energy required to transfer a molecule from the ground
state to the transition state), the more difficult is for a molecule to undergo degradation. An increase
in the stability of drug products can be achieved by a decrease in temperature above the frozen
conditions. The storage conditions of drug substances and drug products are given in the
pharmacopoeias.
9.3.2 Cyclodextrin Complexation
Cyclodextrin (CDs) are cyclic oligosaccharides that consist of six (α–CD), seven (β–CD),
or eight (γ–CD) units of dextrose. These have lipophilic inner cavities and hydrophilic out surfaces.
The chemical structure of CDs (6.28) is presented in section 6.7.2. CDs may interact with a large
number of drugs and form noncovalent inclusion complex. This may lead to the stabilization of
drug molecules. CDs may interact with a large number of drugs and form noncovalent inclusion
complex. This may lead to the stabilization of drug molecules. CDs complexation could also
improve the water solubility and bioavailability of drugs. The application of CDs in drug stabilization
(Loftsson and Brewester, 1996; Yoshioka and Stella, 2000), in drug delivery (Challa et al., 2005;
Rasheed et al., 2008) and as excipients (European Medicine Agency, 2014) have been reported.
CDs have been found to improve the stability of a number of labile drugs against
dehydration, hydrolysis, oxidation and photolysis, resulting in an increase in the shelf-life of these
drugs (Loftsson and Brewster, 1996). The rate of degradation of labile drug can be slowed down
by inclusion into the CD cavity. The rate of a drug (1:1 complex) in CD solution is the average of
the degradation rates of the free drug and the drug–CD complex (Rasheed et al., 2008).
Equilibrium binding of the drug and CD to form a 1:1 complex can be expressed as
Drug + CD Drug:CD complex
For a 1:1 complex, the following equation can be used to determine the equilibrium binding
or association constant, K, from the slope of the linear portion of the curve.
S0 (1–
Ka:b = slope)
Slope
Where S0 is the intrinsic solubility of the drug under the condition of the study. The binding
constants for many drug–CD complexes range from 100 to 20000 M–1. A 1:100 dilution of the
solution as an injection or dilution in the stomach and intestine can reduce up to 70% of the
complex drug to free drug (Challa et al., 2005). Since the hydrolysis of CDs encapsulated drugs is
slower than the free drugs. The stability of the drug–CD complex (i.e. magnitude of stability of
constant of complex) is an important factor in the stabilization of the drug (Kang et al., 2003; Ma
et al., 2000; Dwivedi et al., 1999).
Some examples of the CD complexation effect on the improvement of the stability of drugs
include thermal stability of diclofenac sodium in the solid state by β–CD (Cwiertnia et al., 1999),
photostability of promethazine by HP–β–CD or DM–β–CD (Lutika, 2002). Photostability of
doxorubicin by HP–β–CD or HP–γ–CD (Brewester et al., 1992) shelf-life (4 years) of glibenclamide
by β–CD (Babu and Pandit, 1999). Stability against hydrolysis of benzylpenicillin by HP–β–CD
(Pope et al., 1991), and stability against intramolecular cyclization of quinaril in solid state by β–
CD of HP–β–CD (Li et al., 2002).
9.3.3 Polymer complexation
The polymer-based amorphous solid dispersions offer a major development in the
formulation of poorly water soluble drugs. Polymers are inert, hydrophilic drug carrier matrices that
have the advantage of greater stability and solubility. The drug-polymer interactions involve
hydrogen bonding, higher structural relaxation time and delayed crystallization kinetics that results
162
in the stabilization of the drug. The polymer may also cause steric and structural effects to cause
a greater stability to the drug (Thompson et al., 2006; Kothari et al., 2015; Baghel et al., 2016).
The physical stability and molecular mobility of drug-solid dispersions are affected by drug-
polymer hydrogen bonding interactions. A study of solid dispersion of nifedipine with
polyvinylpyrrolidone (PVP), hydroxypropylmethyl cellulose (HPMC), and poly(acrylic acid) (PAA)
has indicated that the hydrogen bonding, structural relaxation time and crystallization kinetics are
in the order: PVP> HPMC> PAA. PVP showed the highest amount of drug hydrogen bonding to
the polymer and the highest resistance to crystallization (Kothari et al., 2015).
Multiple nanosuspensions of drugs such as azodicarbonamide, fenofibrate, griseofulvin,
ibuprofen, and phenyl butazone have been stabilized by using the combination of a nonionic
cellulosic polymer (hydroxymethyl cellulose) and an anionic surfactant (sodium dodecyl sulfate).
The physical stability of wet-milled drug nanosuspensions is enhanced when the surfactant
concentration is optimum to overcome the Ostwald ripening (Li et al., 2011). The reduction of size
of pharmaceutical suspensions to µm and nm scale, to achieve increased dissolution rate, creates
the problem of particles agglomeration. This has been overcome by the use of biocompatible
polymers. These polymers stabilize the suspensions by imparting surface-active steric and kinetic
stability through an increase in the viscosity and change to non-newtonian rheological properties
(Romanski et al., 2011)
The peptide drugs are known to undergo chemical degradation by hydrolytic reactions.
The degradation can be prevented by complexation of the peptide with a polymer such as PVP. It
has been suggested that the stabilization of the peptide is due to the formation of the secondary
structure in which the polymer exerts steric effect depending on its size and structural effect leading
to an increase in the distance between reacting atoms within the peptide (Thompson et al., 2006).
9.3.4 Use of Stabilizers
Stabilizers are generally used to protect a drug from chemical degradation in a dosage
form. These include antioxidants, complexing agents and chelating agents. The most commonly
used antioxidants are sodium sulfite, sodium bisulfite, sodium metabisulfite, α-tocopherol, ascorbic
acid, acetylcysteine, butylated hydroxytoulene (BHT), butylated hydroxyanisole (BHA), propyl
gallate in a combination of 0.1–0.2%. The complexing agents include caffeine and cyclodextrins.
The common chelating agent for metal ion contaminants is sodium editate.
9.3.5 Liposomal Formulation
Liposomes are microscopic and submicroscopic phospholipid vesicles having a bilayer
membrane structure. These drug delivery systems provide protection against chemical,
photochemical and biological degradation. The stability of drugs in liposomes is affected by
liposomal composition, entrapment efficacy and drug-lipid interactions (Michaelis et al., 2005).
Several drugs have been stabilized against chemical and photodegradation by entrapment in
liposomes such as riboflavin (Loukas et al., 1995a,b; Ahmad et al., 2015a), doxorubicin (Bandak
et al., 1999), fluoroquinolones (Vazquez et al., 2001; Budai et al., 2008, Ahmad et al., 2016),
amlodipine (Ragno et al., 2003), barnidipine (Ioele et al., 2014), tretinoin (Ioele et al., 2005) and
local anesthetics (Habib and Rogers, 1987, 1989).
9.4 CHEMICAL AND PHOTOSTABILIZATION STUDIES
The chemical and photostabilization of different drugs and dosage forms have been
studied by several workers. Some examples of these studies are presented in the following
sections.
9.4.1 Chemical Stabilization
9.4.1.1 Amorphous Drugs
There is an increasing number of new therapeutically active pharmaceutical compounds
with low water solubility. This has created problems in the formulations of their oral dosage forms.
The formation of stabilized amorphous forms of poorly water soluble compounds can help to
163
increase the solubility, dissolution rate and bioavailability of these compounds. The stabilization of
amorphous drugs and related aspects have been reviewed by many workers (Laitinen et al., 2013;
Kawabata et al., 2011; Qian et al., 2010; Bhattacharaya and Syrayanarayanan, 2009; Janssen
and Van der Mooler, 2009; Yu, 2001; Leirner and Dressman, 2000; Serajuddin, 1999; Craig et al.,
1999; Hancock and Zografi, 1997).
The formulation of solid polymer dispersions is considered as the best method for the
stabilization of amorphous drugs and the enhancement of their dissolution rate. However,
alternative methods of stabilization of amorphous drugs have been suggested (Laitinen et al.,
2013). These methods are based on the formulation of co-amorphous mixtures of small molecules
and the use of mesoporous silicon and silicon-based carriers. These approaches have been found
to be useful in the stabilization of amorphous drugs.
The amorphous state of a drug is unstable (e.g. nifedipine, furosemide, novobiocin) on
thermodynamic considerations since it tends to revert back to the crystalline state with time. It has
been observed that the storage of amorphous material at Kauzmann temperature (TK) (the
temperature at which entropy of the supercooled liquid is equal to that of the crystalline material)
gives good physical stability to the material. TK is taken as the maximum temperature for the
storage of amorphous formulations (Yu, 2001; Kaushal et al., 2004; Kaushal and Bansel, 2008).
It has been observed that the storage of unstable amorphous drugs (e.g. nifedipine,
furosemide, novobiocin) at TK provides good physical stability to such drugs (Graeser et al., 2009)
Several factors affect the crystallization of amorphous state (Marsac et al., 2006; Kushal
and Bansel, 2008; Bhugra and Pickel, 2008; Grzykowska et al, 2010) and include:
 Thermodynamic (configurational entropy, enthalpy or Gibbs free energy, ∆G).
 Kinetic (molecular mobility, glass transition temperature (Tg), or structural relaxation time
is an indication for this).
 Molecular (e.g. hydrogen binding) interactions.
 Moisture content.
 Method and condition of preparation.
The main factor governing the physical stability of the amorphous state of a drug is
molecular mobility. The highest physical stability is shown by the compounds that have high Tgs,
high configurational entropy barriers, high TKs and low molecular motilities (Zhou et al., 2002;
Laitinen et al., 2013).
9.4.1.2 Binary Co-Amorphous Mixtures
It is well known that the addition of certain excipients, such as surfactants, anti-plasticizers
and other inhibitors of crystallization can lead to the stabilization of amorphous drugs. The binary
amorphous systems have been found to possess a potential for the improved stability of drugs.
Small molecules such as citric acid, sugars, urea and nicotinamide have been used as carriers for
the stabilization of amorphous drugs in solid dispersions (Lu and Zografi, 1998; Ahuja et al., 2007;
Masuda et al., 2012). The forces involved in complex formation include van der Waals forces,
dipole-dipole interactions, hydrogen bonding, Coulomb forces and hydrophobic interactions
(Yoshioka and Stella, 2000).
Examples of the binary co-amorphous mixtures include indomethacin/ranitidine/ citric acid,
acyclovir/citric acid paracetamol/citric acid anhydrate and naproxen/cimetidine. These mixtures
are stabilized by hydrogen bonding interactions and possess a greater shelf-life than that of the
drug alone. Indomethacin has been stabilized with cimetidine by salt formation that also leads to
an increase in the stability of the drug (Laitinen et al., 2013).
164
9.4.1.3 Solid Dosage Forms
∆9-tetrahydrocannabinol hemisuccinate (THC–HS) has been stabilized in polymeric
matrix systems using a hot-melt process at low temperature. The addition of vitamin E succinate
to the prodrug greatly reduced the degradation of THC–HS during the polyethylene matrix
production at 80°C. A combination of vitamin E succinate and Noveon AA–1 (a high molecular
weight acrylic acid polymer cross linked with divinyl glycol) gives best stabilization to the prodrug
system during production and storage at 4°C. The degradation of THC–HS is minimized in the
acidic medium (Munjal et al., 2006).
The stabilization of certain enzymes has been achieved by chemical modification. α-
Amylase has been stabilized by covalent linkage to the anionic polysaccharide, carboxymethyl
cellulose. The modified enzyme has improved thermal and pH stability compared to the native
enzyme. The conjugate shows more resistance to the action of denaturing agents such as urea
and sodium dodecyl sulfate (Villalonga et al., 1999). Acetylcholinesterase is inactivated on
chemical modification by thiosulfinate allicin on reaction with the buried cysteine (Cys 231).
Circular dichroism (CD) spectral measurements have shown that the inactivation of the enzyme
can be reversed by reaction with glutathione. The half-life of allicin modified enzyme at room
temperature is ~100 min. The transition of the modified enzyme can be prevented by divalent
cations, Ca2+, Mg2+ and Mn2+ for >24 h at room temperature. Differential scanning calorimetry
(DSC) has been used to confirm the stabilization of the modified enzyme by divalent cations
(Millard et al., 2003).
The interactions between drugs (e.g. indomethacin) and the surface of excipients such as
Neusilin (a synthetic magnesium alumina metasilicate) on amorphization by co-grinding the
mixture imparts physical stability to the drug during storage (Bahl and Bogner, 2006). The silanol
rings present on the surface of Neusilin make it a potential proton donor as well as proton acceptor.
The hydrogen bonding between silanol rings and the drugs are also involved in the stabilization of
drugs including quinapril HCl, acelofenac and other acidic drugs (Gupta et al., 2003; Hailu and
Bogner, 2009).
The control of the environmental factors in the stabilization of some drug substances in
the solid state or solid dosage forms is given in Table 9.1.
9.4.1.4 Liquid Dosage Forms
Cyanocobalamin (vitamin B12) is degraded in liquid multivitamin preparations by
interaction with other vitamins. The degradation of cyanocobalamin can be greatly reduced by the
addition of complex cyanides (e.g. potassium ferrocyanide, potassium cobalcyanide, potassium
cuprocyanide) or iron salts (e.g. ferrous sulfate, ferrous gluconate, ferric ammonium citrate). The
stabilization of cyanocobalamin is more effective with complex cyanides than with the iron salts
under aerobic conditions. The complex cyanides are also effective in protecting cyanocobalamin
against UV light (Zuck and Conine, 1963).
The stabilization of cyanocobalamin in liquid multivitamin preparations can also be
achieved by the use of α-hydroxynitriles of their esters (9.18). The stabilizing effect is due to the
degradation of α-hydroxynitrile into hydrogen cyanide and the corresponding aldehydye. α-
hydroxynitrile also protects cyanocobalamin from degradation on exposure to UV light for short
periods of time (Conine and Zuck, 1963).
1
R
HO CN
R2
(9.18)
A study of the degradation of cyanocobalamin and hydroxocobalamin (vitamin B12b) in the
presence of ascorbic acid at pH 1–8 has been conducted. Cyanocobalamin is degraded to
hydroxocobalamin which is further degraded to corrin ring oxidation products. Both of these
165
compounds degrade by an apparent first-order kinetics and t1/2 values range from 13.7 to 137.5 h
and 2.5 to 87.5 h, respectively. The second-order rate constants for the interaction of
cyanocobalamin and hydroxocobalamin with ascorbic acid are 0.05 to 0.28×10–2 and 1.10 to
30.08×10–2 M–1s–1, indicating a greater effect of ascorbic acid on the degradation of the later
compound. Both compounds can be stabilized in the presence of ascorbic acid in the acid region
around pH 2.0. Cyanocobalamin is stable in the absence of ascorbic acid in the pH range 6 to 7
that is suitable for pharmaceutical formulations (Ahmad et al., 2014).
The use of antioxidants and stabilizers in the stabilization of drug substances in the dosage
forms is given in Table 9.2.
Table 9.1 Stabilization of drug substances in solid state/solid dosage form a.
Drug Drug dosage form Prevention/control
Paracetamol in combination with Tablets Moisture and alkali
aspirin and codeine phosphate
4-aminosalicylic acid solid Moisture and elevated
temperature
Amoxicillin Crystalline powder High humidity and temperature
Amphotericin Powder Light and air
Ampicillin Powder Interconversion of
hydroxyethyl and unhydrated
forms, humidity and
temperature
Ascorbic acid Solid High moisture content
Aspirin Solid Minimizing contact with water,
basic substances (e.g.
carboxylic salts) and
nucleophiles (e.g. amines and
hydroxyl groups)
Aztreonam Lyophilized powder Moisture, light and high
temperature
Diethylpropion HCl solid Moisture and light
a Selected from monographs in Connors et al. (1986).
166
Table 9.2 Stabilization of drug substances in liquid dosage form a
Drug substance Degradation pH of Addition of Antioxidant /
reaction maximum stabilizer
stability
Paracetamol Hydrolysis 5.0–7.0
4-Aminosalicylic Decarboxylation 9.2–9.7 Na2S2O5, to prevent color
acid formation
Aminobarbital Hydrolysis Low pH
Ampicillin Hydrolysis 5.8 Addition of alcohol to lower
dielectric constant of solution
to enhance stability
Aspirin Hydrolysis 2.5
Atropine Hydrolysis 3.5
5-azacytidine Hydrolysis 2.5 EDTA, NaHSO3
Aztreonam Hydrolysis 6.0
Benzylpenicillin Hydrolysis 6.75 Improved stability in
suspension form
Carbenicillin Hydrolysis 6.5
Cephradine Hydrolysis 2.0–5.0
Chlordiazepoxide Hydrolysis 2.0–3.5 Protection from light
Cholecalciferol Oxidation Ethylgalate, BHT improved
stability in syrup form
Clindamycin Hydrolysis 4.0
Cyanocobalamin Cyclization 4.5–5.0 EDTA, citric acid, cysteine
Cytrabine Deamination 6.9
Diazepam Hydrolysis 5.0 Enhanced stability in mixed
aqueous solution
Erythromycin Hydrolysis 7.0–7.5
5-Flurouracil Hydrolysis 9.0
Meperidine Hydrolysis 4.0
6-Mercaptopurine Oxidation 2.0–8.0 Protection from light and
moisture
Methotrexate Hydrolysis 7.0 EDTA
α-Methyldopa Oxidation 5.0–6.0 Protection from light
Morphine Oxidation 3.0–5.0 Protection from light,
NaHSO3, Na2S2O5, EDTA
Nystatin Oxidation 7.0 BHA, BHT, propyl gallate
Oxazepam Hydrolysis 5.0–6.0
Phenylbutazone Hydrolysis/oxidation 6.0–7.0
Procaine Hydrolysis 3.0–4.0 Polysorbate 80
Promethazine Oxidation 2.0–3.0 EDTA
Sulfacetamide Hydrolysis 5.0–9.0
Thiamine HCl Oxidation 2.0 EDTA
a Selected from monographs in Connors et al. (1986).
9.4.2 Photostabilization
9.4.2.1 Solid and Semisolid Dosage Forms
The photosensitive drugs such as danurubicin, dihydroergotamine, haloperidol,
furosemide, nifedipine and nitrofurazone may undergo photodegradation when their dosage forms
are exposed to light during the manufacturing process and handling by the end user. The
photostabilization of these drugs may be achieved by the application of the principle of
photoprotection by spectral overlay. It involves the use of suitable colorants or excipients absorbing
daylight in the region that corresponds to the absorption characteristics of the individual drug. This
is applicable to tablets or topical dosage forms (Thoma and Klimek, 1991).
167
The photostabilization of light sensitive drug products such as nifedipine tablets can be
achieved by film coating with TiO2 (9–29%) to impart opacity. Scanning electron microscopy, used
to evaluate film thickness of the tablets (2–15% weight, increase), showed the thickness in the
range of 24–145 nm. The uncoated and film coated tablets were exposed to 4.4 klux light for 21
days. The results showed that the coated tablets (29% TiO2) at a thickness of 145 µm provided
good protection to the drug from photodegradation compared to that of the uncoated tablets
(Bechard et al., 1992).
The uncoated sorivudine and nifedipine tablets have been stabilized against
photodegradation using iron oxides that absorb UV-light. The 10 mg wet granulated tablets
containing 0.2% yellow iron oxide were exposed to room light or 400 foot-candle light for a fixed
period of time. The assay of the drugs in the tablets showed that the uncoated tablets containing
iron oxide were more stable in light compared to those with no stabilizer. The addition of 0.2% iron
oxides (black, yellow and red) to the uncoated tablets showed greater protection from light (>11%)
compared to a film coated tablet. A combination of yellow and red iron oxides was more effective
as light protectant that the use of single stabilizer (Desai et al., 1994).
Boric acid (BA) has been used to stabilize ascorbic acid (AH 2) against UV light in o/w
cream formulations prepared using different humectants and emulsifiers. The apparent first-order
rate constants (kobs) for the photodegradation of AH2 in creams range from 0.42–1.20×10–3 min–1
compared with those of 0.59–1.30×10–3 min–1, in the absence of BA. The second-order rate
constants for the interaction of AH2 and BA are in the range of 2.61–6.02×10–3 M–1 min–1 indicating
the inhibitory effect of BA on the degradation of the vitamin. The nature and amount of the
humectant and emulsifier and the physical properties of creams influence the extent of
stabilization. The Photostabilization of AH2 in creams involves the formation of a complex between
AH2 and BA (Ahmad et al., 2015b).
9.4.2.2 Liquid Dosage Forms
The photosensitive drugs can be stabilized by complex formation between the drug and
certain agents. Riboflavin has been stabilized by complexation with caffeine. The complex form of
riboflavin is stable in aqueous solution around pH 6.0 and is suitable for pharmaceutical
formulations (Ahmad et al., 2009). Caffeine complexation involves the formation of stacking
complexes (Evstigneev et al., 2005). The complex formation between the ribityl side chain of
riboflavin and boric acid leads to the photostabilization of the vitamin in aqueous solution (Ahmad
et al., 2008)
168
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174
CHAPTER – 10
STABILITY OF HERBAL DRUGS AND PRODUCTS

10.1 INTRODUCTION
Interest in herbal drugs from natural sources has grown in recent years and herbal
products are being used as alternative and complementary medicines worldwide. Herbal drugs
have been utilized from time immemorial and are still part of modern medicine. Some of the active
ingredients derived from natural products and used as drugs are anthaquinones (cascara),
artemesinin (artemisia), atropine (nightshade), colchicine (autumn crocus), digitoxin (foxglove),
diosgenin (Mexican yam), morphine (opium poppy), podophyllin (mayapple), quinine (cinchona
bark), reserpine (Indian snakeroot), taxol (Pacific yew), vincristine (periwinkle) and many
antibiotics (Der Marderosian and Riedlinger, 2006).
The herbal products may contain either a single or a mixture of several herbal ingredients
often five to ten or more in a single formulation in the solid, semisolid or liquid dosage form. This
creates the possibility of medium effects and interactions between different active ingredients and
with excipients to cause the degradation of the individual components. This may lead to stability
problems affecting the potency and efficacy of active ingredients individually as well as the overall
biological activity of the products. Therefore, proper care is required in the handling, drying and
storage of herbal drugs to maintain their potency, safety and efficacy.
The commonly used dosage forms of herbal drugs include powdered material, spray dried
extract powdered material and freeze-dried powdered material either used as such or mixed with
excipients to formulate as tablets and capsules. Other dosage forms include creams, ointments,
semisolid preparations, liquid preparations, syrups, liquid extracts, tinctures, etc. The whole or
powdered bulk material, on drying in the oven or under sunlight as such or encapsulated, may be
affected by environmental factors such as air, moisture, heat, light and microbes and thus lose
potency. The most common reaction undergone by chemical constituent of herbal drugs is
oxidative degradation. However, the hydrolytic, photolytic or other modes of degradation may also
occur depending on the nature of the drug. The herbal products are standardized to ensure the
presence of the desired amount of active ingredients in the single or polyherbal formulation
manufactured in different dosage forms before marketing.
Herbal drugs are playing an important role in the treatment of a wide range of ailments.
They are generally considered safe, however, some of the herbal drugs may contain toxic
constituents with undesirable side effects. The importance and use of herbal drugs have been
described by Majno (1975), Fransworth et al. (1985), Bukhari et al. (1987), Gilani et al. (1992),
Bisset (1994), Barl (1997), Duke and Martinez (1994), Roberts and Tyler (1997), Bouldin et al.
(1999), Fabricant and Fransworth (2001), Phillips (2002), Sagar et al. (2003), Bodeker et al.
(2005), Barnes et al. (2007), Tapas et al. (2008), Andreescu et al. (2008).
10.2 DEFINITIONS
A plant drug or herbal medicine has been defined by WHO (1993) as “a plant-derived
material or preparation with therapeutic or other human health benefits which contains either raw
or processed ingredients from one or more plants. In some traditions, materials of inorganic or
animal origin may also be present”.
Herbal drugs, processed herbal drugs, herbal drug preparations and herbal drug extracts
have been defined by British Pharmacopoeia (2016) as follows:
175
10.2.1 Herbal Drugs
“Herbal drugs are mainly whole, fragmented, or broken plants, parts of plants, algae, fungi
or lichen, in an unprocessed state, usually in dried form but sometimes fresh. Certain exudates
that have not been subjected to a specific treatment are also considered to be herbal drugs. Herbal
drugs are precisely defined by the botanical scientific name according to the binominal system
(genus, species, variety and author)”.
Whole describes a herbal drug that has not been reduced in size and is presented, dried
or undried, as harvested, for example: dog rose, bitter funnel or sweet funnel, Roman chamomile
flower.
Fragmented describes a herbal drug that has been reduced in size after harvesting to
permit ease of handling, drying and/or packaging; for example: cinchona bark, rhubarb, passion
flower.
Broken describes a herbal drug in which the more-fragile parts of the plant have broken
during drying, packaging or transportation, for example: belladonna leaf, matricaria flower, hop
strobile.
Cut describes a herbal drug that has been reduced in size, other than by powdering, to
the extent that the macroscopic description in the monograph of the herbal drug can no longer be
applied. When a herbal drug is cut for a specific purpose that results in the cut herbal drug being
homogenous, for example when cut for herbal teas, it is a herbal drug preparation.
Herbal drugs are obtained from cultivated or wild plants. Suitable collection, cultivation,
harvesting, drying, fragmentation and storage conditions are essential to guarantee the quality of
herbal drugs.
10.2.2 Processed Herbal Drugs
“Processed herbal drugs are obtained by subjecting herbal drugs to traditional processing
methods. Processed herbal drugs are defined precisely by the botanical scientific name according
to the binomial system (genus, species, subspecies, variety, and author) and plant part”.
Processed Herbal Drugs are obtained by subjecting herbal drugs to specific types of
processing according to traditional processing methods. These traditional processing methods
have the potential to alter the physical characteristics and/or chemical constituents of herbal drugs.
Traditional processing methods may require the addition of processing aids to the herbal drug, for
example, honey, vinegar, wine, milk and salt. The additional processing aids used should be of a
suitable quality or of pharmacopoeial quality where a monograph exists. The method of traditional
processing is provided under the production section in individual monographs.
10.2.3 Herbal Drug Preparations
Herbal drug preparations are defined as the homogenous products obtained by subjecting
herbal drugs to treatments such as extraction, distillation, expression, fractionation, purification,
concentration or fermentation.
10.3.3 Herbal Drug Extracts
Herbal drug extracts are liquid (liquid extraction preparations), semisolid (soft extracts and
oleoresins) or solid (dry extracts) preparations obtained from Herbal drugs using suitable solvents.
An extract is essentially defined by the quality of the herbal drug, by its production process
(extraction solvent(s), method of processing, etc.) and by its specifications.
Standardized extracts are adjusted to a defined content of one or more constituents with
known therapeutic activity. This is achieved by adjustment of the extract with inert excipients or by
blending batches of the extract.
Quantified extracts are adjusted to one or more active markers, the content of which is
controlled within a limited, specified range. Adjustments are made by blending batches of the
extract.
176
Other extracts are not adjusted to a particular content of constituents. For control
purposes, one or more constituents are used as analytical markers. The minimum content for
these analytical markers is given in an individual monograph in British Pharmacopoeia (2016).
10.3 QUALITY CONTROL METHODS
The application of quality control methods in the assessment of quality, determination of
the potency of active ingredients and detection of impurities is necessary to assure good
manufacturing practices, quality control, safety, efficacy and stability of the herbal products. The
following quality control methods are normally applied to the herbal material.
10.3.1 Herbal Products
Tests of identity, purity, foreign matter, loss on drying, water content, pH, heavy metals,
total ash, acid insoluble ash, extractable matter, swelling index, bitterness value, microbial
contamination and assay of active ingredients.
10.3.2 Essential Oils
Test of identity (chromatographic profile), relative density, refractive index, optical rotation,
fatty acids and resinified oils, freezing point, acid value, peroxide value, foreign esters and residue
on evaporation.
10.3.3 Herbal Extracts
Test of identity, relative density, water content, solvent content, loss on drying, dry residue,
residual solvents, heavy metals, microbiological quality, aflatoxins B 1, ochratoxin A, pesticide
residues and assay of active ingredients.
Quality control methods for herbal drugs have been described by WHO (1998), British
Pharmacopoeia (2016), EMEA (1998, 2005), Eskinazi et al. (1999), Capasso et al. (2000) and
Ahmad and Usmanghani (2003) and discussed by Barl (1997), Bauer (1998), De Smet (1999),
Tsai (2001), Gaedcke and Steinhoff (2002), Mukherjee (2002), Phillips (2002), Liang et al. (2004),
Wani (2007), Kunle (2012), Zhang et al. (2012), Bele and Khale (2013) and Azmir et al. (2013).
The analytical methods used for the isolation, separation, characterization, determination
and stability studies of herbal drugs, degradation products and contaminants are given in Table
10.1.
10.4 FINGERPRINT ANALYSIS OF HERBAL DRUGS
Herbal drugs are cultivated in a certain region or different areas of the world. Even though
herbal drugs may belong to the same species, the quality and efficacy may be different according
to the growing conditions such as climate and soil, based on the geographical origin. Therefore,
rapid, sensitive and accurate analytical methods are required to determine the correct contents of
the major constituents of herbal drugs and to discriminate them according to the geographical
origin (Woo et al., 1999).
Fingerprint analysis is an accepted method for the assessment of the quality of Traditional
Chinese Medicines (TCM) or herbal drugs by WHO (2001). A fingerprint can be considered as a
chemical profile that represents the chemical composition of the samples of TCM or the herbal
drugs. Chromatographic fingerprint analysis using CE, GC, GC–MS, HPTLC, HPLC and HPLC–
MS (Peishan, 2001; Drasar and Moravcova, 2004; Gong et al., 2004; Xie, 2005; Lu et al., 2005;
Xie et al., 2006, 2007; Yin and Qian, 2007; Chen et al., 2007; Jiang et al., 2007) has been applied
to the authentication of plant species, origin of Chinese herbs, quality standards to ensure
consistency and stability of herbal drugs, assessment of raw material and in-process assay and
the detection of adulterants in herbal drugs.
The absorption spectra such as ultraviolet (UV) (Ni et al., 2009), infrared (IR) (Cao et al.,
2002; Xu et al., 2002; Zhou et al., 2003; Xu et al., 2005; Yang et al., 2009), near infrared (NIR)
(Woo et al., 1999; Scafi and Pasquini, 2001; Laasonen et al., 2002; Sun et al., 2010), Fourier
transform infrared (FTIR) (Ayiguli et al., 2006; Chen et al., 2007;Jin et al., 2008; Li et al., 2006; Pei
et al., 2008; Wu et al., 2008; Cai et al., 2009; Cheung et al., 2009), nuclear magnetic resonance
177
(NMR) (Kang et al., 2008) and mass spectra (MS) (Cai et al., 2002), fingerprint analyses have
been used as effective techniques for the identification of cultivation areas of herbs, counterfeit
drugs and drugs in multicomponent matrices, pattern recognition for discrimination of herbal drugs
and processing and quality control of herbal drugs. Small changes in test samples may be detected
by variations in fingerprints for the differentiation of herbal drugs.
10.5 STORAGE
The plant material used as drugs is normally stored in glass containers or as alcoholic or
aqueous extracts to preserve the active ingredients and enhance shelf-life. The herbal drugs
should be stored under appropriate storage conditions to ensure potency, efficacy and safety. The
British Pharmacopoeia (2016) prescribes storage conditions for herbal drugs and products in terms
of protection from light, moisture and heat and use of well-filled, air tight and light-resistant
containers. The recommended storage for herbal drugs is at a temperature ≤25°C and when frozen
at or below –18°C. In the case of certain drugs (e.g. Sterculia granules) storage in a dried place is
recommended and for certain drugs (e.g. Tolu balsam) storage in powder form is not
recommended. The storage conditions of some herbal drugs and products are given in Table 10.2.
Table 10.1 Analytical methods for the study of herbal drugs.
Methods Applications
Extraction Methods
Liquid–liquid phase Extraction of hydrophobic
components in the organic phase
Liquid–solid phase Separation of mixtures of different

molecules
Chromatographic Methods
Thin-layer chromatography (TLC) Separation, isolation and
High-performance thin-layer chromatography (HPTLC) determination of constituents of
High-performance liquid chromatography (HPLC) plant materials and herbal drugs
(normal and reversed phase)
Gas-liquid chromatography (GLC)
Electrophoresis
Capillary electrophoresis (CE) Separation, isolation and
Gel electrophoresis (GE) determination of constituents of
plant materials and herbal drugs
Spectroscopic Methods
Ultraviolet spectrometry (UV) Structural and quantitative
Visible spectrometry (Vis) analysis
Infrared spectrometry (IR)
Fourier transform spectrometry (FTIR)
Nuclear magnetic resonance spectroscopy (NMR)
Mass spectrometry (MS)
GC–mass spectrometry (GC–MS)
HPLC–mass spectrometry (HPLC–MS)
Spectrofluorimetry
Circular dichorism (CD) Structural and quantitative
Optical rotatory dispersion (ORD) analysis
X-ray Diffractometry (XRD) Structural analysis
Atomic absorption spectrometry (AAS) Structural analysis
Structural analysis
Elemental analysis
178
Table10.2 Sensitivity and storage of some herbal drugs and productsa (British
Pharmacopoeia, 2016).
Method of Sensitivit
Herbal Drug Major Constituents Storage
Analysis y
Essential oils Terpenes (mono- and GLC Light Well-filled, air tight
sesqui-terpenes) containers,
protected from
light
Herbal Teas Light Protected from
light
Barbados Hydroxyanthracene Spectrometric Air tight containers
aloes derivatives 512 nm
Cape aloes Hydroxyanthracene Spectrometric Air tight containers
derivatives 512 nm
Angelica Z-ligustilide LC Moisture Protected from
sinensis root moisture
Star Anise oil Pseudoisoeugenol, GLC Heat Temperature
2-methylbutyrate ≤25°C
Azadirachta Tetranortriterpenoids LC Moisture Protected from
indica leaf (salannin, moisture
azadirachtin-A)
Belladona leaf Alkaloids Acid-base Air tight containers
powder (hyoscyamine) titration
Berberis Berberine LC Moisture Protected from
aristata moisture
Fresh Bilberry Anthocyanins Spectrometric Heat
(chrysanthemin) 528 nm When frozen, at or
below
– 18°C
Black current Redox Light Well-filled
syrup titration container
protected from
light
Caraway oil Essential oil GLC Heat Temperature
(β-myrcene, carvone) ≤25°C
Cardamom oil Essential oil GLC Light Well closed
(β-myrcene, carvone) container
protected from
light
Cassia oil Essential oil GLC Heat Protected from
(trans- heat
cinnamaldehyde,
trans-2-methoxy-
cinnamaldehyde,
coumarin, eugenol
Cinnamon Essential oil GLC Heat Protected from
bark oil (cineole, linalol, β- heat
caryophyllene, trans-
cinnamic aldehyde,
eugenol
Ceylon Cineloe, linalol, β- GLC Heat Protected from
cinnamon leaf caryophyllene, trans- heat
oil cinnamic aldehyde,
eugenol
179
Clove oil Essential oil GLC Heat Protected from
(β-caryophyllene heat
eugenol, acetyl
eugenol)
Colophony Thymol, linalol Do not reduce to a
powder
Table 10.2 continued
Coriander oil Essential oil GLC Heat Temperature
(terpenes and ≤25°C
camphor)
Digitalis leaf Cardenolic glycosides Spectrometric Moisture Protected from
540 nm moisture
Dill oil Carvone Titration with Light Well-filled
KOH in 90% containers,
ethanol protected from
light
Eucalyptus oil Essential oil GLC Heat Temperature
(α-α-pinene, ≤25°C
limonene, 1,8-cineole)
Bitter fennel Essential oil GLC Moisture Protected from
(anethole, fenchone) moisture
Bitter-fennel Essential oil GLC Heat Temperature
fruit oil (α-pinene, trans- ≤25°C
anethole, fenchone,
estragol)
Bitter-fennel Essential oil GLC Heat Temperature
Herb oil (α-pinene, limonene, ≤25°C
fenchone, trans-
anethole)
Sweet fennel Essential oil GLC Moisture Protected from
(Anethole) moisture
Fig Moisture Protected from
moisture
Ipecacuanha Moisture Protected from
root powder moisture
Ispaghula Moisture Protected from
husk granules moisture
Juniper oil Essential oil GLC Heat Temperature
(α- and β-pinene, ≤25°C
sabinene, β-myrcene,
limonene,
terpinen-4-ol
Lavender oil Essential oil GLC Heat Temperature
(1,8-cineole, ≤25°C
3-octanone, linalol,
linalyl acetate,
α-terpineol)
Lemon oil Essential oil GLC Heat Temperature
(β-pinene, sabinene, ≤25°C
limonene, γ-
terpinene, geranial)
Terpeneless Aldehydes Titration with Light Well-filled
lemon oil KOH in 60% container
ethanol protected from
light
180
Liquorice root Glycyrrhizic acid LC Moisture Protected from
for use in moisture
TCMb
Mandarin oil Essential oil GLC Heat Temperature
(α- and β-pinene, β- ≤25°C
myrcene, limonene,
γ-terpinene)

Matricaria oil Essential oil GLC Heat Temperature
(chamazulene, α- ≤25°C
bisabolol, bisabolol
oxides)
Dementholize Essential oil GLC Heat Temperature
d mint oil (limonene, cineole, ≤25°C
menthone,
isomenthone,
menthol, pulegone,
carvone)
Myrrh Anethole, thymol Do not store in
Tincture plastic container
Nutmeg oil Essential oil GLC Heat Protected from
(α-and β-pinene, heat
sabinene, car-3-ene,
limonene, γ-
terpinene, myristicine,
terpinen-4-ol)
Orange oil Essential oil Keep in well-filled
(aldehydes) container,
protected from
light
White Peony Paeoniflorin LC Moisture Protected from
root moisture
Peppermint Essential oil GLC Heat Temperature
oil (limonene, 1,8- ≤25°C
cineole, menthone,
menthol,
menthofuranisomenth
one, pulegone)
Gastro- Peppermint oil GLC Light Protected from
resistant (limonene, 1,8- light
peppermint oil cineole, menthone,
capsules menthol,
menthofuran,
isomenthone,
pulegone)
Peru balsam Esters Gravimetric Light Protected from
(benzyl benzoate, light
benzyl cinnamate)
Phellodendro Berberine, palmatine LC Moisture Protected from
n amurense moisture
bark
Phellodendro Berberine LC Moisture Protected from
n Chinese moisture
bark
181
Dwarf Pine oil Essential oil GLC Heat Inert containers,
(α-and β-pinene, Temperature
camphene, car-3-ene, ≤25°C
β-myrcene, limonene,
β-phellandrene,
terpinolene,
p-cymene)
Rosemary oil Essential oil GLC Heat Temperature
(α- and β-pinene, ≤25°C
camphene, β-
myrcene, limonene,
cineole, camphor,
borneol)

Sage oil Essential oil GLC Heat Temperature
(linalol, linalyl acetate, ≤25°C
α-terpineol,
germacrene)
Scutellariae Baicalin LC Moisture Protected from
baicalensis moisture
root
Senna fruit Hydroxyanthracene Spectrometric Moisture Protected from
glycosides 515 nm moisture
Senna Sennsides LC Moisture Air tight containers
granules
Senna leaf Hydroxyanthracene Spectrometric Moisture Protected from
glycosides 515 nm moisture
Spearmint oil Essential oil GLC Light Keep in a well-
(limonene, cineole, closed containers,
menthone, protect from light
isomenthone,
menthol, carvone)
Sterculia Gum Acid-base Moisture Stored in a dry
granules (volatile acid) titration place
Stramonium Alkaloids Acid-base Moisture Protected from
leaf (hyocyamine and titration moisture
hyocine)
Prepared Alkaloids Acid-base Moisture Stored in air tight
stramonium (hyocyamine, titration containers
hyocine)
Thyme oil Essential oil GLC Heat Temperature
(β-myrcene, ≤25°C
γ-terpinene,
p-cymene, linalol,
thymol, carvacrol)
Tolu balsam Oleo-resin Acid-base Do not store in
(cinnamic acid) titration powder form
Turpentine oil Essential oil GLC Heat Temperature
(α-and β-pinene, ≤25°C
camphene, car-3-ene,
limonene, longifolene,
β-caryophyllene)
182
Withania Withaferin A and LC Moisture Protected from
somnifera withanolide A moisture
root
a For many herbal drugs and products storage conditions are not mentioned.
bTCM, Traditional Chinese Medicine.
10.6 PHOTOSENSITIVITY REACTIONS OF HERBS

The term photosensitivity is used to describe an adverse biological reaction occurring as
a result of the action of sunlight on a herbal product. The reaction may be phototoxic, photoallergic
or photosensitization. Herbs can cause photosensitivity reactions to skin on exposure to sunlight
(Ernst, 2000; Ernst et al., 1998; Palanisamy et al., 2003). These reactions result in inflammation
of the skin with redness similar to sunburn and other disorders (Table 10.3).
Table 10.3 Herbs causing skin sensitivity on exposure to sunlight.
Herb Reaction caused to skin
Essential oils redness,
(lime, lemon, orange, bergamot, grapefruit, ginger, hyperpigmentation,
cumin, angelica root) used topically in aromatherapy blistering
St. John’s wort itching erythema
Kava drink photosensitive dermatitis
(Pacific island)
Yohimbe (containing yohimbine alkaloid) photosensitive dermatitis
ingestion
Some light sensitive herbal drugs are reported in Table 10.4.
Table 10.4 Some light sensitive drugs and products.
Natural Compounds Amphotericin,, ammoidin, atropine, emetine, cyanocobalamin,
ephedrine, ergocalciferol, erythromycin, folic acid, reserpine,
retinol, riboflavin, steroids
Plant Products Essential oils, fixed oils, ginseng dry extract, peru balsam,
cardamom fruit, podophyllum resin
Animal Products Hard fat
10.7 STABILITY OF HERBAL DRUGS AND PRODUCTS
Stability is an important consideration in the assessment of the quality, efficacy and safety
of herbal products. The chemical constituents of the herbal ingredients of these products may
undergo various degradation reactions during production, storage and use. The stability of herbal
products has been reviewed by Thakur et al. (2011), Deepa and Kannappan (2008), Bankoti et
al. (2012), Dawar et al. (2013), Hou et al. (2013) and Noor-ul-Basar et al. (2013). Some stability
studies of herbal drugs and products are reported in the following sections.
10.7.1 Photodegradation of Herbal Drugs
Many herbal drugs are sensitive to sunlight and artificial light and undergo
photodegradation to form inactive or toxic products (Table 10.5).
183
Table 10.5 Photodegradation of herbal drugs by sunlight.
Drug/Material Use Photoproducts
Triclosan antimicrobial dibenzodichlorodioxin
agent (more toxic)
Fenpropathrin pyrethroid decarboylated and ester bond cleavage
insecticide products
Azadirachtin insecticide photodegradation products involving
(Azadirachdica indica fruit) tigolyl moiety
(Neem plant)
Chlorophyll oxidative products (hematinic acid,
(leaves of higher plants e.g. methyl ethyl maleimide, methyl vinyl
barley) maleimide dialdehyde)
Membrane proteins food photooxidation products (formation
(containing tryptophan) material indicated by loss of tryptophan
fluorescence at 290 nm)
Reserpine oxidative products (3-dehydroreserpine
and lumireserpine)
Ephedrine 2,5-diphenyl-3,4-dimethyl oxazolidine
Riboflavin formylmethylflavin, , lumichrome,
lumiflavin
Some examples of the photodegradation of herbal drugs are as follows:
Ephedrine
Ephedrine (10.1) in aqueous solution on exposure to sunlight and UV light in the presence
of air is oxidized to benzaldehyde (10.2) which condenses with the unreacted ephedrine to form
3,4-dimethyl-2,5-diphenyl-1,3-oxazolidine (10.3). This product is biologically inactive (Khan et al.,
1975).
HO H3C O
NH
CH3
hv
(10.1) (10.2)
HO H3C H3C CH3

O
N
NH
CH3 O
+
(10.1) (10.2) (10.3)
Reserpine
Reserpine (10.4) in aqueous solution and chloroform on exposure to light undergoes
photooxidation to form 3,4-dehydroreserpine (10.5) which is further oxidized to 3,4,5,6-
tetradehydroreserpine (lumireserpine) (10.6) (Wright and Tang, 1972).
184
O O CH3
N H
O O CH3
O CH3 + H
NH
H3C O
N H O CH3
O
hv H3C
H H O O
N H
O H3C O H H
O O
H3C O H3C
O O
H3C H3C
O
H3C
(10.4) (10.5)
O O CH3
+ H
NH
O CH3
H3C O
N H
O H H O
O H3C
O
H3C
O
H3C
(10.6)
Riboflavin
Riboflavin (vitamin B2) (10.7) on photodegradation in aqueous solution gives rise to
formylmethylflavin (10.8) as an intermediate product which is hydrolyzed to lumichrome (10.9) and
lumiflavin (10.10) (Ahmad and Rapson, 1990; Ahmad et al., 2004).
CH 2OH
H C OH
H C OH
H C OH CHO
CH2 CH2
H3C H3C N N O
N N O
hv
NH NH
N H3C N
H3C
O (10.8) O
(10.7)
H+/OH-
OH-
CH3
H
H3C N N O H3C N N O
NH NH
H3C N N
H3C
O O
(10.9) (10.10)
185
Quinine
Quinine (10.11) on UV irradiation in aqueous solution leads to the formation of 6-methoxy-
quinoline-4-ylmethyl-oxonium (10.12) as the final photoproduct (Yadav et al., 2013).
H
H2C
+
N O H2 H
HO CH3
CH3
H
O O
hv
N
N
(10.11) (10.12)
10.7.2 Chemical Degradation of Herbal Drugs
Many drugs are sensitive to air and pH and undergo chemical degradation by oxidation,
hydrolysis and other reactions in aqueous solution. Some examples of the chemical degradation
of drugs are as follows:
Erythromycin
Erythromycin (10.13) in acidic solutions undergoes acid-catalyzed dehydration reaction by
the loss of one molecule of water to form anhydroerythromycin (10.14) (Atkins et al., 1986).
CH3
N(CH 3)2 CH3
O N(CH 3)2
HO
H3C OH HO
CH3
HO H3C O CH3
O HO
O CH3 O
HO O O CH3
H3C CH3 H+
H5C2 -H2O H3C CH3
O O O CH3 H5C2
CH3 O O O CH3
CH3
O
O
OH
H3C OCH 3 OH
H3C OCH 3
(10.13) (10.14)
Digitoxin
Digitoxin (10.15) is degraded by acid-catalyzed hydrolysis at pH 1 to 2 to give digitoxigenin
(10.16) and other products (Peters et al., 1978).
OH
OH
O O O O
CH3 O O
HO CH3
CH3
CH3
O
CH3 CH3 H
CH3 H
H+
O
O H OH
H OH
HO
HO O H
H
(10.15) (10.16)
186
Morphine
Morphine (10.17) in aqueous solution undergoes oxidation in the presence of air to give
pseudomorphine (oxydimorphine) (10.18) and other products (Yeh and Lach, 1961).
OH
HO
CH3
N
O
O2 O
N CH3
N CH3
HO O
O
OH OH
(10.17) (10.18)
Atropine
Atropine (10.19) is degraded by H+ ion catalyzed hydrolysis in aqueous solution to form
tropine (10.20) and tropic acid (10.21) (Kirchhoff et al., 2004).
H3C N
H
CH 2OH H3C N CH 2OH
H HO
O
H+ +
O OH O
(10.19) (10.20) (10.21)
Pilocarpine
Pilocarpine (10.22) undergoes hydrolysis in alkaline solution to form isopilocarpic acid
(10.23) (Bundgaard and Hansen, 1982; Zoppi et al., 2012).
H3CH2C CH3 H3CH 2C CH3
N N
OH- CH 2OH
O O N O OH N
(10.22) (10.23)
Ginseng Saponins
The ginseng saponins, ginsenosides Rg1, Re and Rb1 have been found to degrade under
mild acidic conditions to form prosapogenins which have been identified by 13C–NMR
spectroscopy. Rg1-prosapogenins II is a mixture of ginsenoside Rh1 and its C–20 isomer, formed
by the hydrolysis and epimerization at C–21. Rg1-prosapogenin III is a C–25, 26 hydrated
derivative of Rg1-prosapogenin II. Re-prosapogenin II has been shown to be a mixture of
ginsenoside Rg2 and its C–20 epimer and Re-prosapogenin III as a C–25, 26 hydrated derivative
of Re-prosapogenin II (Han et al., 1982).
10.8 STABILITY/DEGRADATION STUDIES OF HERBAL DRUGS IN FORMULATIONS
Acid-base titrimetry and NMR spectroscopy have been used to study the kinetics of OH
ion-catalyzed hydrolysis and epimerization reactions of pilocarpine in ophthalmic solutions. The
pseudo-first-order rate constants and activation energies of the reactions have been determined.
187
Epimerization of pilocarpine is the major pathway of degradation of the drug that involves the
formation of a carbanion stabilized by resonance with the enolate hybrid. The rate of epimerization
to isopilocarpine is temperature dependent that may affect the stability of pilocarpine in ophthalmic
solutions on sterilization by heat (Nunes and Brochmann-Hanssen, 1974).
There is a high demand for optically pure drugs for the preparation of stable herbal
formulations with a chiral quality of the desired isomer. In a study the effects of cyclodextrins (CDs)
and derivatives on the kinetics of racemization and hydrolysis of
(–)-(S)-hyoscyamine and (–)-(S)-scopolamine has been investigated. The stability tests involved
the chromatographic determination of the enantiomer composition and degradation products. All
CDs, except α–CD, have been found to slow down the racemization and hydrolytic reactions of
these alkaloids depending on the pH and temperature. The drug–CD complexation results in the
inhibition of the OH– ion and/or H2O attack on the drug molecule to cause the degradation
reactions. The formation of a soluble 1:1 drug–CD complex has been confirmed by NMR
spectroscopy (Blaschke et al., 1993).
The effects of microwave (12 and 15 min at 1100 W) and conventional heating (36 and 45
min at 230 OC) on refined and virgin olive oil have been studied. The amount of oxidative and
hydrolytic degradation of different oils has been determined by high performance size-exclusion
chromatography. The results show that the formation of polar compounds of triglyceride
oligopolymers and oxidized triglycerides are more than 26% after the most intense treatment. The
microwave heating results in a higher amount of oxidative degradation. The polar compounds have
an adverse effect on human health (Caponio et al., 2002).
The epimerization of ergot alkaloids in rye flour after baking cookies and then subjecting
them to an in vitro digestion model using salivary, gastric and duodenal juices has been studied.
The toxic (R)-epimers and inactive (S)-epimers of several ergot alkaloids were determined by a
HPLC method with fluorescence detection. A 2–30% degradation of different alkaloids has been
observed with an increase in epimeric ratio towards the (S) epimer. The % degradation to the (R)-
epimer was found to increase after the digestion of cookies. The results show selective toxification
of ergotamine and ergosine in duodenal juice (intestinal tract) which should be taken into
consideration in use of the product (Markel et al., 2012).
The microemulsions are used to simultaneously deliver flavor oils and lipophilic bioactive
compounds in beverages. In this context, the delivery of β-carotene in microemulsions formulated
with peppermint oil and a blend of Tween 20 and sunflower lecithin has been studied. The poorly
water-soluble and oil-soluble β-carotene dissolved in microemulsions with particle size less than
10 nm was found to be stable during storage at room temperature for 65 days. The addition of β-
carotene does not change the flow properties and Newtonian viscosity of microemulsions. The
degradation of β-carotene in these emulsions during storage and thermal treatment at 60–80°C
follows first-order kinetics. The antioxidant property of peppermint oil and excess of lecithin
protects β-carotene from degradation. These microemulsions may have applications in the
manufacture of transparent beverages (Chen and Zhong, 2015).
Other studies on the stability of herbal formulations include the stability of terpenes in
lemon oil (Nguyen et al., 2009), herbal capsules with different ingredients (Bankoti et al., 2012),
ointments containing eucalyptus oil (Dawar et al., 2013), herbal antihypertensive formulations
containing reserpine (Deore et al., 2013; Sandhya et al., 2014) and herbal cream containing
embelin (Bele and Khale, 2011).
10.9 STABILITY TESTING OF HERBAL PRODUCTS
Stability testing is carried out to provide evidence of variations in the quality of drug
products with time under the influence of environmental factors such as temperature, humidity and
light for a period of 6 to 12 months. These studies are necessary to recommend storage conditions
and to assign a shelf-life to the product. The storage conditions used for the stability testing of drug
products (ICH, 2003) are given in Table 10.6. The standard conditions for the photostability testing
of drug substances and products are described in ICH (1996) (see Chapter 12, Section 12.7).
188
Table 10.6 Storage conditions for stability testing of drug substances.
Minimum time period
Study Storage condition covered by data at
submission
Long-terma 25±2°C / 60±5% RH 12 months
or
30±2°C / 65±5% RH
Intermediateb 30±2°C / 65±5% RH 6 months
Accelerated 40±2°C / 75±5% RH 6 months
aIt is up to the applicant to decide whether long term stability studies are performed at 25±2°C /
60±5% RH or 30±2°C / 65±5% RH
b If 30±2°C / 65±5% RH is the long-term condition, there is no intermediate condition.
The stability testing of pharmaceutical active ingredients (Vipul and Devesh, 2012),
stability studies of ayurvedic health supplements (Deepa and Kannappan, 2012), and Unani
(herbal) formulations (Noor-ul-Basar et al., 2013) have been conducted.
10.10 HERB–DRUG INTERACTIONS
The popularity of herbal products in the treatment of diseases is increasing worldwide.
However, it requires an understanding of the potential interactions between herbs and prescribed
drugs if administered concurrently. The likelihood of herb-drug interactions could be higher than
drug-drug interactions since the drugs usually contain a single chemical ingredient, while herbal
products contain mixtures of pharmacologically active constituents (Fugh-Berman and Ernst,
2001). Many herbs and drugs are therapeutic at one dose and toxic at another. Herb-drug
interactions could lead to an increase or decrease in the pharmacological and toxicological effects
of the either component. In some cases synergistic therapeutic effects may affect the dosing of
long-term medications e.g. herbs that decrease glucose concentration in diabetes could cause
hypoglycemia on combination with conventional drugs (Fugh-Berman, 2000).
Clinical studies have shown that the use of St John’s wort with certain drugs lowers their
serum concentrations, e.g. digoxin (Johne et al., 1999), phenprocoumon (Maurer et al., 1999),
indinivir (Piscitelli et al., 2000) and amitriptyline (Roots et al., 2000). The chewing of Latha edulis
(Khat) affects the pharmacokinetics of single-dose ampicillin and reduces its bioavailability in the
system (Attef et al., 1997). The plasma concentrations of prednisolone are increased by the use
of liquorice (Chen et al., 1990) which also potentiates the vasoconstrictor response of
hydrocortisone (Teeluksingh et al., 1990). Denshen interferes with platelet function and decreases
the elimination of warfarin. Ginkgo and garlic are also known to interfere with the platelet function
and cause bleeding even in the absence of treatment with anticoagulants (Chan et al., 1995). The
interactions of herbal supplements containing coumarin derivatives, and possessing antiplatelet
and anticoagulant properties, with aspirin and other non-salicylate non-steroidal anti-inflammatory
drugs (NSAIDs) (e.g. ibuprofen, flurbiprofen, diflunisal, naproxen, Ketorolac, ketoprofen and
meclofenamate) results in the reduction of platelet aggregation (Abebe, 2002). The interaction of
St. John’s wort with conventional drugs has been reviewed in detail (Mills et al., 2004).
The interaction of herbal drugs and conventional drugs need further studies to understand
the mechanisms of their interactions. It would be advisable not to use both types of drugs
concurrently to avoid any adverse effects. Some of the adverse effects of herbal products are
reported in Table 10.7. The adverse effects of herbal medicines have been dealt by De Smet
(1995, 2004), De Smet et al. (1997) and Tyagi and Delanty (2003). The interactions between these
drugs and conventional drugs may also cause potency loss of either or both of the drugs and thus
affect their efficacy and bioavailability.
189
Table 10.7 Adverse effects of herbs and herbal products.
Active
Herb Drug Adverse effects
constituents
Karela Chlorpropamide decreased glucose
concentration in blood
Liquorice Glycyrrhazin Prednisolone decrease plasma
(Glycyrrhiza clearance
glabra) Glycyrrhetinic Hydrocortisone
acid potentiation of cutaneous
Oral contraceptives vasodilator response
hypertension, edema
Salbokinto Prednisolone increased prednisolone
(Asian herbal concentration
mixture)
Shankahapushpl Phenytoin decreased phenytoin
(Ayurvedic mixed concentration
herb syrup)
Tamarind Aspirin Increased aspirin
bioavailability
Yohimbine Tricyclic hypertension
antidepressants
190
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CHAPTER – 11
STABILITY-INDICATING ASSAY METHODS

11.1 INTRODUCTION
Stability studies are an integral part of drug development process in pharmaceutical
industry. The assay method used in stability studies must be specific and stability-indicating for
the drug. It should be capable of separating and determining the drug and the degradation products
as well as major impurities. The reliability and specificity of the assay method must be
demonstrated on the pure drug and on its degradation products. A determination of the kinds and
amounts of various contaminants and degradation products in drug substances and formulated
products is a measure of both product stability and Good Manufacturing Practices (GMP).
The pharmacopoeial assays do not necessarily take into account the presence of various
contaminants and degradation products. This also applies to the assay of certain classes of
compounds, e.g. barbiturates, salicylates, steroids, sulfonamides, penicillins, which contain a
common nucleus as well as the main functional groups involved in the assay. Each class frequently
possesses similar physical characteristics which interfere with the specificity of the assay for a
given compound. The presence of degradation products further complicates the system. Thus
stability-indicating assay methods are required to deal with the analysis of individual drugs in the
presence of degradation products and related compounds. This is necessary to achieve accurate
assay results for the drug and to set the limits of degradation products in drug products.
11.2 DEFINITIONS
FDA Guideline (1987): The stability-indicating methods are “quantitative analytical
methods that are based on the characteristic structural, chemical or biological properties of each
active ingredient of a drug product, and that will distinguish each active ingredient from its
degradation products so that the active ingredient content can be accurately measured”.
FDA Guideline (1998): The stability-indicating methods are “validated quantitative
analytical methods that can detect the changes with time in the chemical, physical or
microbiological properties of the drug substance and drug product, and that are specific so that
the contents of active ingredient, degradation products, and other components of interest can be
accurately measured without interference”.
ICH Guideline (2003) states the following about the application of stability-
indicating method in stress testing “Stress testing of the drug substance can help identify the
likely degradation products, which can in turn help establish the degradation pathways and the
intrinsic stability of the molecule and validate the stability-indicating power of the analytical
procedures used. The nature of the stress testing will depend on the individual drug substance
and the type of drug product involved”.
11.3 DEVELOPMENT OF ANALYTICAL METHODOLOGY FOR ASSAY OF A
DRUG COMPOUND
The development of specific analytical methodology for the assay of a drug compound is
based upon the exploitation of its structural features and physicochemical characteristics to show
a particular response (e.g. light absorption or light emission, electrooxidation or electroreduction,
change in current or potential, etc.) on the application of an analytical technique. A drug may exhibit
a single physicochemical characteristic or multiple characteristics that can be made a basis for its
determination. This is followed by a careful assessment of all the parameters involved in its
quantitation to achieve optimum conditions for the assay of the drug. An example of the
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physicochemical characteristics of a drug such as riboflavin (vitamin B2) to be considered as a
basis of the development of its analytical methods is as follows.
CH 2OH
HO C H
HO C H
HO C H
CH2
H3C N N O
NH
H3C N
O
Fig. 11.1 Chemical structure of riboflavin.
Physicochemical characteristics Assay Method
Light absorption at 444 nm Spectrophotometric assay (British Pharmacopeia,
2016)
Fluorescence emission at 530 nm Spectrofluorimetric assay (United States
Pharmacopeia, 2016)
Redox system E0 –0.185 V Potentiometric titration (Lowe and Clark, 1956)
Redox system E1/2 –0.47 V Polarographic assay (Ke, 1957)
Metal complexation Spectrometric assay (Wade and Fritchie, 1963)
Photodegradation to lumichrome Photochemical assay (Ahmad et al., 2015)
Selective adsorption/partition Chromatographic assay (Gliszczynska-Swiiglo and
Koziolowa, 2000).
A similar approach may be adopted for the development of an assay method for a new drug.
11.4 DEVELOPMENT OF ANALYTICAL METHODOLOGY FOR STABILITY-
INDICATING ASSAY OF A DRUG COMPOUND
The development of a stability-indicating method would depend on the chemical
characteristics of the drug substance, its mode of degradation under specific stress conditions
(e.g. moisture, heat, light) and the nature of the degradation products. It may involve the following
steps.
 Verification of degradation under specific conditions, e.g. oxidation, hydrolysis,
thermolysis, photolysis, radiolysis, using chromatographic and spectroscopic methods
such as TLC and UV spectrometry.
 Separation and purification of degradation products by appropriate extraction and/or
chromatographic methods.
 Characterization of known/unknown degradation products by comparison of their spectral
characteristics (i.e. UV, IR, NMR, Mass) with those of authentic/ structurally related
compounds.
 Determination of specific analytical characteristics of the drug and its degradation
products (e.g. absorption wavelengths, ionization behavior, pH effects) suitable for their
assay in stored/stressed samples.
 Quantitation of the assay method based on the selection of a particular physicochemical
characteristic.
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 Assessment of the interference of degradation products in the assay of the parent drug.
 Validation of the assay method under the condition of use so as to meet the requirements
for its specific analytical application.
It may be necessary to screen the degradation products for their potential toxicity to ensure
the safety of the patient.
11.5 STABILITY-INDICATING SPECTROMETRIC ASSAY METHODS
The ultraviolet (UV) and visible spectrometric methods for the assay of a drug compound
are based on the measurement of absorbance of the compound in solution at the absorption
maximum (λmax) and determination of the concentration with reference to a calibration curve of
absorbance versus concentration. It can also be determined directly by Beer’s law relation using
the values of specific absorbance [A (1%, 1 cm), dlg–1cm–1] or molar absorptivity ( , M–1 cm–1) in
a certain concentration range where absorbance is proportional to concentration.
11.5.1 One-Component Assay
The Beer’s Law states that
A = abc (11.1)
Where ‘A’ is absorbance, ‘a’ is called absorptivity, ‘b’ is cell path length and ‘c’ is
concentration. When c is in percent, ‘a’ is expressed as specific absorbance ‘A’ and
A = A (1%, 1cm) bc (11.2)
When c is in M/L, a is expressed as molar absorptivity and
A = bc (11.3)
The concentration c of the solution can be calculated from the above relation as
c = A/A(1%, 1cm) b (11.4)
or
c = A/b (11.5)
11.5.2 Multicomponent Assay
The method of one component assay is not applicable to the assay of mixtures of
compounds or a drug and its degradation products due to overlapping of the absorption spectra
and mutual interference at the analytical wavelengths. In such cases the methods of
multicomponent spectrometric assay may be used which are capable of determining the
components of a mixture or a drug and its degradation products with high accuracy. The total
absorbance of the solution of a mixture of compounds at a particular wavelength is equal to the
sum of the absorbance of the individual components.
Atotal = A1 + A2 + ------------ + An = 1bc1+ 2bc2+----------+ nbcn (11.6)
Where the subscripts refer to absorbing components 1, 2………..n.
In the analysis of mixtures, the values of specific absorbance or molar absorptivities of the
compounds at the selected wavelengths (e.g. absorption maxima) are determined under the
experimental conditions used (i.e. pH, solvent, temperature, etc.) and the concentrations are
calculated using appropriate equations.
11.5.2.1 Two-component assay (additive absorbencies)
In the assay of a two-component system, the absorbance measurements are made at two
suitably selected wavelengths, λ1 and λ2, and if the light path remains constant, two simultaneous
equations may be written:
A1 = 1K1 1C + 2K1 2C (11.7a)
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A2 = 1K2 1C + 2K2 2C2 (11.7b)
where A1 is absorbance at wavelength λ 1
A 2 is absorbance at wavelength λ2
1K1 is absorptivity-cell path product for component 1 at λ 1
1K2 is absorptivity-cell path product for component 1 at λ2
2K1 is absorptivity-cell path product for component 2 at λ 1
2K2 is absorptivity-cell path product for component 2 at λ2
1C is concentration of component 1
2C is concentration of component 2
The solution of equations (Eq. 11.7a) and (Eq. 11.7b) for 1C and 2C is:
1C = (2K2.A1 – 2K1.A2) / (1K1. 2K2–2K1. 1K2) (11.8a)
2C = (1K1.A2 – 1K2.A1) / (1K1. 2K2–2K1. 1K2) (11.8b)
11.5.2.2 Three-component assay (additive absorbencies)
In this case the solution of three simultaneous equations is required which may be done
for the sake of convenience, using matrix method. Thus for measurements A1, A2, A3, at λ1, λ2, λ3
on a mixture of components 1, 2, 3 at concentration 1C, 2C, and 3C:
Wavelength Absorbance Absorbance sum
λ1 A1 = 1K11C + 2K1 2C + 3K1 3C
λ2 A2 = 1K2 1C + 2K2 2C + 3K2 3C
λ3 A3 = 1K3 1C + 2K3 2C + 3K3 3C
(11.9a)
The matrix equation is as follow:
A1 1K1 2K1 3K1 1C

A2 = 1K2 2K2 3K2 2C (11.9b)
A3 1K3 2K3 3K3 3C
(AM) (ASM) (CM)
where
(AM) = Absorbance Matrix
(ASM) = Absorbance Sum Matrix
(CM) = Concentration Matrix
The solution of (Eq. 11.9b) for each concentration is carried out by replacing the
appropriate column in the absorbance sum matrix in its determinant form and dividing the resultant
by the absorbance sum matrix (ASM) again in its determinant form.
A1 2K1 3K1 1K1 2K1 3K1
1C = A2 2K2 3K2 1K2 2K2 3K2
A3 2K3 3K3 1K3 2K3 3K3
(11.10a)
1K1 A1 3K1
2C = 1K2 A2 3K2 (ASM)
1K3 A3 3K3
(11.10b)
1K1 2K1 A1
3C = 1K2 2K2 A2 (ASM)
1K3 2K3 A3
(11.10c)
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The matrices are then expanded by any convenient method e.g., for 1C using the top row
and Laplace’s method:
2K2 3K2 A2 3K2 A2 2K2
A1 – 2K1 + 3K1
2K3 3K3 A3 3K3 A3 2K3
1C =
ASM expanded
A1 (2K2 3K3 – 3K2 2K3) – 2K1 (A2 3K3 – 3K2 A3) + 3K1 (A2 2K3 – 2K2 A3)
1C =
ASM expanded
(11.11)
Similarly the matrices are expanded for 2C and 3C. For each determinant of a different set
of 1C, 2C and 3C, the top line of (Eq. 11.11) has to be computed a fresh, since A1, A2, A3 vary, whilst
ASM is always the same. This may be achieved by the application of programmed software.
11.5.3 Advantages
The multicomponent spectrophotometric methods, on application to the study of a stability
problem, have the following advantages over the chromatographic methods.
 Simultaneous determination of the drug and its degradation product(s) and confirmation
of its accuracy on the basis of the molar balance achieved (Ahmad et al., 1990; Ahmad
and Vaid, 2006; Sheraz et al, 2014).
 Elimination of interference due to minor contaminants by the application of correction
procedures for linear or nonlinear irrelevant absorption (Ahmad, 2013a, 2015; Arsalan,
2016).
 Immediate determination of the concentration of species involved in degradation at a
particular time as compared to that of GLC/HPLC method which takes considerable time
for detection after sample application and hence the possibility of a chemical change in
the mobile phase (e.g. on a tablet extract dilution ) or on the column during the separation
process. This may lead to erroneous analytical results in the stability evaluation of a
compound depending upon its sensitivity to assay conditions.
 Time required to complete an assay is much shorter than that of GLC/HPLC assay and
the technique is more suitable for kinetic work, if applicable.
 Cost of performing assays in terms of time, material and equipment is much less than that
involved in GLC/HPLC assays.
11.5.4 Applications
Several stability-indicating multicomponent spectrometric methods have been developed
for the simultaneous determination of a drug and its degradation products. An important application
of these methods is the evaluation of the kinetics of degradation reactions (Ahmad and Vaid, 2006,
Sheraz et al., 2014). This would be illustrated with reference to their application in chemical and
photodegradation studies. The details of the degradation reactions of some drug compounds are
as follows.
 Hydrolysis of aspirin (Khurshid, 2013) (Fig 11.2)
 Hydrolysis of procaine HCl (Al-Blewi et al., 2013) (Fig 11.3)
 Hydrolysis of riboflavin (Ahmad et al., 1973) (Fig 11.4)
 Hydrolysis of formylmethylflavin (Ahmad et al., 1980) (Fig 11.5)
 Thermolysis of reserpine (Ahmad et al., 1979) (Fig. 11.6)
 Hydrolysis and photolysis of sulfacetamide (Ahmad and Ahmad, 1981) (Fig. 11.7)
 Photolysis of riboflavin (Ahmad et al., 2004a) (Fig 11.8)
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 Photoaddition of riboflavin (Ahmad et al., 2004b) (Fig 11.9)
 Riboflavin sensitized photooxidation of ascorbic acid (Sheikh, 1996) (Fig 11.10)
Some other applications of stability-indicating multicomponent spectrometric methods in
the study of drug degradation reactions include the photolysis of riboflavin (Ahmad and Rapson,
1990), riboflavin-sensitized photolysis of cyanocobalamin (Ahmad and Hussain, 1992; Ahmad et
al., 2012), degradation of cyanocobalamin in the presence of ascorbic acid (Ahmad et al.,
2014a,b), and nicotinamide (Ahmad et al., 2003), buffer catalyzed photolysis of riboflavin (Ahmad
et al., 2008, 2014c), solvent effect on photolysis of formylmethylflavin (Ahmad et al., 2006, 2013b),
and divalent ions effect in the photolysis of riboflavin (Ahmad et al., 2010).
Stability-indicating spectrometric methods have also been employed for the assay of
norfloxacin (Taha et al., 1998), lisinopril (El-Yazbi et al., 1999), aceclofenac (El-Saharty et al.,
2002; Hasan et al., 2003), omeparazole, lensoparazole, pantoprazole (Wahbi et al., 2002),
oxicams (Taha et al., 2006) and vincamine (El-Bardicy et al., 2008) in the presence of degradation
products.
11.6 STABILITY-INDICATING THIN-LAYER CHROMATOGRAPHIC (TLC)
AND HIGH-PERFORMANCE THIN-LAYER CHROMATOGRAPHIC (HP–
TLC) ASSAY METHODS
Stability indicating TLC methods with densitometric detection have been developed for the
assay of aceclofenac in the presence of its main degradation product, diclofenac at 275 and 283
nm, respectively (El-Saharty et al., 2002; Hasan et al., 2003). Another application of the
development and validation of a HP–TLC method with densitometric detection is the determination
of bisacodyl in pharmaceutical tablets. The quantitative evaluation has been performed by
absorbance measurements of the zones of analyte at 254 nm using the reflectance mode
(Campbell and Sherma, 2003). The photostability testing of piroxicam using forced degradation by
exposing the sample solution to the artificial irradiation from a xenon source and sunlight has been
carried at 280 nm using a HPTLC/densitometry stability-indicating assay method (Bartsch et al.,
1999).
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Fig. 11.2 Hydrolysis of aspirin in alkaline solution.
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Fig. 11.3 Hydrolysis of procaine HCl in alkaline solution.
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Fig. 11.4 Hydrolysis of riboflavin at pH 11.0.
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Fig. 11.5 Hydrolysis of formylmethylflavin at pH 11.0.
[Reproduced from I. Ahmad et al. (1980), with permission].
206
Fig. 11.6 Thermolysis of aqueous soluiton of reserpine (pH 3.0) at 60°C.
207
Fig. 11.7 Hydrolysis and photooxidation of sulfacetamide in aqueous solution.
208
Fig. 11.8 Photolysis of riboflavin at pH 7.0.
[Reproduced from I. Ahmad et al. (2004b), with permission].
209
Fig. 11.9 Photoaddition reaction of riboflavin at pH 7.0.
[Reproduced from I. Ahmad et al. (2004b), with permission].
210
Fig. 11.10 Photolysis of ascorbic acid in the presence of riboflavin at pH 4.0.
211
11.7 STABILITY-INDICATING HIGH- PERFORMANCE LIQUID
CHROMATOGRAPHIC (HPLC) ASSAY METHODS
HPLC methods are the most widely used stability-indicating methods for the assay of drug
substances and formulated products in mixtures or in the presence of degradation products.
Several authors have dealt with the development and validation of stability-indicating HPLC assay
methods for applications in pharmaceutical industry, drug analysis, normal and forced degradation
studies and drug product testing (Ahmad, 1985; Weiser, 1998; Xu and Trissel, 1999; Hong and
Shah, 2000; Ruan et al., 2002; Bakshi and Singh, 2002; Shabir, 2003; Smela, 2005; Wen, 2006;
Aubry et al., 2009; Singh and Rehman, 2012). Hong and Shah (2000) have described in detail the
stages involved in the development and validation of HPLC stability-indicating assay methods.
11.7.1 Development of HPLC Stability-Indicating Assay Methods
Bakhshi and Singh (2002) have described the following steps in the development of HPLC
stability-indicating assay methods to meet regulatory requirements.
 Critical study of the drug structure to assess the likely decomposition route(s).
 Collection of information on physicochemical properties.
 Stress (forced decomposition) studies.
 Preliminary separation studies on stressed samples.
 Final method development and optimization.
 Identification and characterization of drug degradation products, and preparation of
standards.
 Validation of stability-indicating assay methods.
11.7.2 Applications
11.7.2.1 Drug mixture
Some applications of stability-indicating HPLC assay methods in the determination of drug
mixture include the assay of dipyridamoline injection (Zhand et al., 1997), aceclofenac and
diclofenac in pharmaceutical formulations (El-Yazbi et al., 1999), drug analysis (Xu and Trissel,
1999), ramipril and hydrochlorothiazide in dosage forms (Belal et al., 2001), prolocaine and
procaine drug combinations (Stroms et al., 2002), montilukast and loratidine in pharmaceutical
formulations (Radhakrishna et al., 2003), and non-steroidal anti inflammatory drugs (Dubroil-
Cheneau et al., 2011).
11.7.2.2 Stress testing / forced degradation studies
The applications of stability indicating HPLC assay methods in drug degradation studies
include photodegradation studies of pyroxicam (Bartsch, 1999), determination of aceclofenac in
presence of its degradation product, diclofenac (Hassan et al., 2003), assay of levofloxacin
(Ahmad et al., 2013c), moxifloxacin (Ahmad et al., 2014d) and norfloxacin (Ahmad et al., 2015) in
photolyzed solutions, assay of glimepride under hydrolytic stress condition (Kovarikova et al.,
2004), assay of doxophylline on hydrolytic degradation (Gupta et al., 2011), assay of rapamycin in
forced degradation studies (Oyler et al., 2012), assay of cefaclor in solid state degradation
(Dorman et al, 1997), and assay of atorvastatin and its stress degradation product (Shah et al.,
2008). The various applications of stability indicating assay methods in pharmaceutical stress
testing have been described by Baertschi (2005), Ruan et al. (2006), and Wen et al. (2006).
11.8 VALIDATION OF STABILITY-INDICATING ASSAY METHODS
Validation of a method is an integral process that is done simultaneously with method
development (Hibbert, 1999). It is carried out to make sure that an analytical procedure will be
suitable for its intended purpose. Validation in the simplest of meaning is defined as “the action of
proving” or “fitness / accuracy of work”. According to International Council on Harmonization (ICH,
212
2000) validation is defined as “a documented program that provides a high degree of assurance
that a specific process, method, or system will consistently produce a result meeting pre-
determined acceptance criteria”.
Basic requirements for the validation of any method include:
 Use of calibrated instrument.
 Well-characterized reference materials and chemicals with documented purity.
 Skilled worker.
A method cannot be validated if the instruments used are not properly calibrated. Timely
calibration of instruments is highly essential for accurate and reproducible results. Similarly, use
of high purity chemicals is also important for appropriate validation of any method. Sometimes
even a correct material may produce false results if it is not of the highest purity or at least of the
desired purity. This is because the impurities present in the material may interfere with the final
results. Moreover, all will be in vain if the worker is not well-trained or expert in the field. The worker
must have a knowledge of the basic use of the particular instrument or apparatus in order to record
the reading correctly. Due to this factor basic training on the instrument and availability of the
standard operating procedures (SOPs) are mandatory in any analytical laboratory.
Guidelines for method validation have been provided in detail by the ICH (2005). According
to their guidelines an analytical method must be evaluated for certain parameters which include:
 Linearity
 Range
 Accuracy
 Precision
 Repeatability
 Intermediate precision
 Reproducibility
 Specificity
 Sensitivity
 Detection limit
 Quantitation limit
 Robustness
The study of these parameters is of utmost importance for the validation of any analytical
method. It is possible that a certain method of analysis may not be applicable to other
systems/dosage forms of the same drug probably due to the interference by related substances
or excipients. This interference can only be determined or ascertained by studying all the validation
parameters stated above. A good example in such a case is that of aspirin for which different assay
methods are given in British Pharmacopoeia (2016) for pure form and for tablets. Depending on
the method of analysis some additional parameters may also be included such as system suitability
in case of HPLC which determines the retention time, theoretical plates, resolution and tailing
factor.
A brief detail of the validation parameters according to ICH (2005) is discussed as follows:
213
11.8.1 Linearity
Linearity of the method is determined by plotting a graph between the signal and
concentration or content of the analyte. A straight line indicates linear relationship between the
response of the analyte and its concentration or content. A minimum of five concentrations are
required to plot such graphs. This may be done by making appropriate dilutions from the stock
solution of the drug or separate weighing of synthetic mixtures of the drug product components. If
a linear relationship is observed visually then some statistical calculations should also be made to
evaluate the linearity. This may include determination of regression, correlation coefficient, slope,
intercept and their errors (Table 11.1). Such data also help in determining the absorption maxima,
molar absorptivity and A (1%, 1 cm) values from the curve. The regression line predicts or
estimates the values of y for x axis by comparing each value whether it is proportional or in range
with each other and with line whereas correlation coefficient measures the strength between
response (y-axis) and concentration (x-axis). Molar absorptivity is the slope of the curve whereas
the value of A (1%, 1 cm) is calculated from the formula [(ϵ×10)/molecular weight].
An example of such linearity can be seen in Fig. 11.11 where a plot has been constructed
between the absorbance and concentration of a drug which is further confirmed for linearity by
statistical calculations (Table 11.1). The overlay spectra of the drug (Fig. 11.12) show that the
signal or response of the analyte is directly proportional to its concentration over the studied
concentration range thus obeying Beer’s law. The overlay spectra of the drug also confirm the
uniformity in the absorption maxima which has been observed at 271 nm (Fig. 11.12). If in case
the linearity is not observed then the analytical response should be described by an appropriate
function of the concentration (amount) of an analyte in a sample (ICH, 2005).
Table 11.1 Analytical parameters for the validation of sulfacetamide sodium (Anwar,
2014).
max 271 nm
Concentration range 1.0–5.0×10–5 M (0.25–1.27 mg%)
Correlation coefficient (R) 0.99993
Molar absorptivity (ɛ) 1.69×104 M–1 cm–1
A (1%, 1 cm) 665
Slope 16900
Intercept 0.0300
Standard error of slope 0.0037
Standard error of intercept 0.0038
Standard deviation of intercept 0.0086
1.2
Absorbance
R² = 0.99986
0.8
0.4
0.0
0.0 1.0 2.0 3.0 4.0 5.0
Concentration (M×105)
Fig. 11.11 Calibration curve of sulfacetamide sodium in distilled water (Anwar, 2014).
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Fig. 11.12 Overlay UV spectra of sulfacetamide sodium in distilled water (Anwar, 2014).
11.8.2 Range
The working concentration range of the analyte is usually determined from the linearity
plots (Table 11.1). The range is selected on the basis of the linearity being observed between a
physical property and the concentration values. It also depends on the technique being employed
for example in case of absorbance measurements by UV-visible spectrometry, values in the region
of around 0.2–0.8 are recommended for highest precision (Hansen et al., 2012).
11.8.3 Accuracy
The difference between the true value and the analytical result is termed as accuracy. It
basically determines how close the analytical results are to the true value or labeled claim. The
lower the difference between the two values, lesser will be the error and higher will be the accuracy.
The drug or analyte may occur in pure form or in a product or mixture or with impurities. Accuracy
of the method is determined by adding known amounts of the drug in a solution within the linearity
range. A minimum of nine determinations are required for accuracy that contain three
concentrations in triplicate covering the specified range and is reported as %recovery or as a
difference between the mean and true value (Hansen et al., 2012). It is better to report the recovery
results with confidence intervals. Table 11.2 gives an example for the presentation of accuracy
data. In case of a new method the test results are statistically compared with the results of the
established or well-characterized method. An example for such a comparison is reported in Table
11.3.
Accuracy of any method should not be affected by impurities, excipients, and degradation
products. Accuracy is considered secondary in cases where linearity, precision and specificity of
the method have been well established.
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Table 11.2 Accuracy and precision of sulfacetamide sodium by the UV spectrometric
method at 95% confidence interval (Anwar, 2014).
Amount Amount Relative Precisio
Recover Mean recovery
added found accuracy error n
y (%)a (%)  SD
(M×105) (M×105) (%)b (%RSD)
1.00 1.01 100.59 +0.99
100.81 
1.00 0.99 99.88 –0.92 1.0533
1.0619
1.00 1.02 101.97 +1.15
3.00 2.98 99.41 –0.27
3.00 1.98 100.28 99.68  0.5211 +0.60 0.5228
3.00 1.97 99.34 –0.34
5.00 4.99 99.76 +0.15
5.00 4.97 99.41 99.61  0.1835 –0.20 0.1842
5.00 4.98 99.66 +0.05
100.03 
Mean = – 0.5868
0.5888
aRecovery (%) = (amount found / amount added) × 100, where amount found was calculated from
(mean absorbance of 3 determinations – intercept) / slope (Ahmed et al., 2013).
b Relative accuracy error (%) = (Recovery – Mean recovery) / (Mean recovery) × 100.
Table 11.3 Comparison of tolfenamic acid recovery by titrimetric, UV and FTIR
spectrometric methods (Ahmed et al., 2013).
% Accuracy ± SDa Relative
accurac P(F<=f P(T<=t
FTIRb UV Titration y error )d )d
(%)c
Pure TA
Height 99.91 ± 2.028 100.93 ± –1.01 0.117 0.360
–
Area 99.75 ± 2.601 1.056 –1.17 0.054 0.392
Clotam®
e
96.03 ± 2.709 –2.15 0.094 0.167
Height – 98.14 ± 1.310
96.41 ± 3.054 –1.76 0.065 0.296
Area
100.21 ± 100.93 ±
Pure TA – –0.71 0.425 0.337
1.167 1.056
Clotam®
e – 98.80 ± 1.878 98.14 ± 1.310 +0.67 0.251 0.543
a values represent a mean % recovery of 5 determinations ± standard deviation.

b Bands taken for peak height at 1438 cm –1 and peak area at 1530–1470. Clotam® values are of
the same bands of height and area.
c Relative accuracy error (%) calculated as [(FTIR or UV) – (titration) × 100] / (titration), where
(FTIR or UV) and (titration) values belong to their % accuracy.
dAt 95% confidence interval (P<0.05), the degrees of freedom (df) for one-tailed F test (n–1) are
df1=4 and df2=4 and for two-tailed t test (n1+n2–2) are df = 8.
eThe values of Clotam® represents the mean recovery for different concentration ranges by three
analytical methods.
11.8.4 Precision
Precision is the closeness of agreement between a series of measurements obtained from
multiple samples of the studied drug under prescribed conditions. Precision is expressed as
standard deviation (SD) or %relative standard deviation (%RSD) or coefficient of variation with a
216
confidence interval. Accuracy and precision can be explained through a figure (Fig. 11.13)
illustrating both parameters on a dart pattern. It is possible that a method may be precise but not
accurate or vice versa. The acceptance criterion for precision is very much dependent on the
method of analysis. A precision with RSD of <2% is generally considered good for analysis
whereas in case of pharmaceutical quality control a precision of <1% is considered better.
Precision acceptance level may increase up to 20% in case of biological samples where there is
a high degree of variations in results due to obvious biological factors.
Fig. 11.13 Illustration for accuracy and precision.

According to ICH (2005), precision can be further divided into:
11.8.4.1 Repeatability
Precision obtained under same operating conditions over a short interval of time by a
same worker is termed as repeatability. Generally, it is carried out on same equipment in the same
laboratory within a day. Repeatability should be assessed using a minimum of nine determinations
i.e. three concentrations in the specified range in triplicate. It is better to select three concentrations
over the selected specified range as low, middle and high concentration. Alternatively, it can also
be evaluated by determining a minimum of six readings at 100% of the test concentration.
11.8.4.2 Intermediate precision
Intermediate precision is also known as ruggedness (Hansen et al., 2012). It is the
expression of variations present within laboratory. It includes analysis on different days with
different analysts using different equipment. It is not considered important if reproducibility of a
method has been established.
11.8.4.3 Reproducibility
Reproducibility is extremely important for the standardization of an analytical procedure.
It is the variation in results between different laboratories in a collaborative study. It is due to the
reproducibility of analytical procedures that pharmacopeial methods are applicable all over the
world if applied correctly following the basic protocols.
217
11.8.5 Specificity
It is of tremendous significance to establish the specificity of an analytical method during
the validation studies. A method must be specific in presence of impurities, degradation products
and matrix components such as excipients in order to report data of the analyte of interest rather
than a combination of other constituents. A HPLC chromatogram of carvedilol solution after
exposure to sunlight along with its photodegradation products is shown in Fig. 11.14. In case if a
particular method is not specific for an analyte than a combination of two or more analytical
procedures is recommended for correct estimation. If a single method is required to be validated
for the specificity and there are certain interferences than use of multicomponent analysis is
recommended (Ahmad and Rapson, 1990; Sheraz et al., 2014). A good example in such case is
the analysis of riboflavin by UV-visible spectrometric method. Riboflavin gives four peaks at 445,
375, 265 and 220 nm (Fig. 11.15). Some of its degradation products are also known to absorb in
the same region thus interfering with the final analysis (Fig. 11.14). In such cases each substance
can be analyzed accurately by employing multicomponent spectrometric methods of analysis as
described in section 11.2.
Fig. 11.14 HPLC Chromatogram of carvedilol and its photodegradation products.
218
Fig. 11.15 Absorption spectra of riboflavin (——), cyclodehydroriboflavin (…....), formylmethylflavin
(------) at pH 2.0 in KCl–HCl buffer (Ahmed, 2009; Sheraz et al., 2014).
219
11.8.6 Sensitivity
11.8.6.1 Limit of detection (LOD)
LOD is the minimum amount of an analyte that can be detected but cannot be quantified
under the analytical conditions used with highest accuracy and precision. It is considered as a limit
test as only a certain limit of analyte i.e. below or above the level can be determined. There are
several approaches available for the determination of LOD based on both instrumental and non-
instrumental approaches:
 Visual evaluation
This approach is used for both instrumental and non-instrumental methods. LOD of the
sample is determined by adding known concentration of the analyte and thus visually establishing
the minimum concentration that can be detected.
 Signal-to-noise (S/N) ratio
This approach is applied to instrumental methods which exhibit baseline noise. It can be
measured by comparing signals from samples containing a minimum amount of the analyte with
blank samples. In this way a minimum concentration of an analyte can be detected reliably. A S/N
ratio of 2:1 or 3:1 is generally considered acceptable.
 Standard deviation of the response and the slope
The LOD or detection limit can be calculated using the following formula:
LOD = 3.3 × σ / S
where σ is the standard deviation of the response and S is the slope of the calibration
curve. The standard deviation can be calculated in different ways:
a) From the blank: The standard deviation of the responses can be determined by measuring
the magnitude of analytical background by analyzing an appropriate number of blank
samples.
b) From calibration curve: If a calibration curve is used for the determination of standard
deviation than the residual standard deviation of the regression line or of y-intercept of
regression line can be used.
LOD of visual evaluation and/or S/N ratio can be represented through chromatograms but in case
of standard deviations LOD is reported in the same unit as that the concentration of the analyte in
the sample.
11.8.6.2 Limit of quantitation (LOQ)
It is the minimum amount of an analyte that can be quantified under the analytical
conditions used with the highest accuracy and precision. The approaches available for the
determination of LOQ are similar to those as explained earlier in LOD. A S/N ratio of 10:1 is
generally considered acceptable. LOQ is generally thrice the detection limit and is calculated by
the following formula:
LOQ = 10 × σ / S
where σ is the standard deviation of the intercept and S is the slope of the calibration
curve.
11.8.7 Robustness
To remain unaffected by small but deliberate changes in the system of analysis is termed
as robustness. It is an important part of both method development and validation studies as it
determines the reliability of the method under small variations in method parameters. Some
common parameters that are usually studied for robustness include solution stability, extraction
time, temperature, pH of the system, wavelength, mobile phase composition, buffer composition,
flow rate, etc.
220
The analytical methods used for the assay of drug substances in formulated products,
drug mixtures and degraded samples should be stability-indicating and need to be validated
according to ICH (1995, 1996, 2000), FDA (2000), USP (2007) and cGMP (1998) guidelines.
Detailed accounts of the validation of stability-indicating assay methods are available in the
literature (Swartz and Krull, 1998; Brittain, 1998; Cuirizak, 1998; Hong and Shah, 2000; Bakshi
and Singh, 2002; Diana, 2009).
221
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226
CHAPTER – 12
REGULATORY ASPECTS OF STABILITY TESTING†

12.1 INTRODUCTION
Stability testing of pharmaceutical products is an essential component of drug
development process and is a regulatory requirement. It is carried out to establish storage
conditions and retest periods and to assign shelf-life and expiry dating to the product. Any change
in the stability characteristics of a product beyond an acceptable criterion would affect its quality
and further stability studies may be required to re-establish the product efficacy and safety. The
ICH Q1A (R2) guideline (ICH, 2003) states “the purpose of stability testing is to provide evidence
and how the quality of drugs substance or drug product varies with time under the influence of
variety of environmental factors such as temperature, humidity, and light, and to establish a retest
period for the drug substance or a shelf-life for the drug product and recommended storage
conditions”.
Stability testing involves a series of tests designed to provide evidence on how the quality
of a drug substance or drug product varies with time under the influence of a variety of
environmental factors such as temperature, humidity and light in order to establish re-test period
for drug substance (in exceptional cases e.g. for unstable drug substances, shelf-life is given) or
a shelf-life for drug product under specified packaging and storage conditions (WHO, 1996).
Stability of drug substances and drug products has been a concern of both pharmaceutical
industry and regulatory agencies throughout the world, as both groups aim to ensure that the
patient receives a safe and effective drug product throughout its claimed shelf-life.
Stability testing normally begins with short-term stress testing on the drug substance. The
information derived from stress testing can be used to establish a program for long-term testing
under accelerated and normal storage conditions. The design of the studies for the drug product
is based on a knowledge of the stability properties of the drug substance gained in stress testing
and long-term studies (Jeffs, 1999).
The design of stability testing program also takes into account the intended market and
the climatic conditions in the area in which the drug product will be used. For the purpose of
worldwide stability testing the world has been divided into four climatic zones (Schumacher, 1974;
WHO, 2006).
 Zone I–Temperate (Germany, Canada, Russia etc.).
 Zone II–Subtropical, with possible high humidity (Argentina, Nepal, South Africa etc.).
 Zone III–Hot / Dry (Botswana, Jordan, Chad etc.).
 Zone IVa–Hot / humid (Pakistan, South Africa, Nepal etc.).
 Zone IVb–Hot / Very humid (Indonesia, Cuba, Ghana etc.).
Since there are only few countries in zone I, therefore, to market products in temperate
climate zone it is always advised to conduct the studies on the conditions in zone II. Similarly
countries where certain regions lie in zone III and zone IV it is always advised to conduct stability
studies on conditions in zone IV. Furthermore these studies are conducted on the basis of mean
kinetic temperature which reflects the actual situation better than the measured mean temperature
†This chapter has been contributed by Dr. Saif-ur-Rehman Khattak, Ph. D., Director, Central Drugs
Laboratory, Karachi, Drug Regulatory Authority of Pakistan.
227
of the country. The mean climatic conditions, calculated data and derived storage conditions in
these zones are summarized in Table 12.1.
12.2 OBJECTIVES
Stability testing data are required in the drug development phase, approval phase and
post-approval period. The data serve different objectives in these phases.
12.2.1 The Development Phase
Both accelerated and real time studies are performed in the development phase.
Accelerated stability tests provide a means of comparing alternative formulations, packaging
materials, and/or manufacturing processes in short term experiments. Once the final formulation
and manufacturing process are established, the manufacturer carries out a series of accelerated
stability tests which enable the stability of the drug product to be predicted and its shelf-life and
storage conditions determined. Real-time studies are also started at the same time for confirmation
purposes.
12.2.2 The Approval Phase
The drug regulatory authority requires the manufacturer to submit information on the
stability of the product derived from tests on the final dosage form in its final container and
packaging. The data submitted are obtained from both accelerated and real-time studies.
Published and/or recently obtained experimental supporting stability data may also be submitted,
e.g. on the stability of active ingredients and related formulations. Where the product is to be
diluted or reconstituted before being administered to the patient (e.g. a powder for injection or a
concentrate for oral suspension), “in use” stability data must be submitted to support the
recommended storage time and conditions for these dosage forms. With the approval of the drug
regulatory authority a tentative provisional shelf-life (generally 2 years) is often established
provided that the manufacturer has undertaken by virtue of a signed statement to continue and
complete the required studies and to submit the results to the regulatory authority.
12.2.3 The Post-Approval Phase
Once the drug substance or drug product is approved, the manufacturer must carry out
ongoing real-time stability studies that permit the detection of any stability issue e.g. changes in
labels of degradation products. Additional stability studies are required whenever major
modifications are made to the formulation, manufacturing process, packaging or method of
preparation. The results of these studies must be communicated to the concerned drug regulatory
authorities.
Table 12.1 Mean climatic conditions: calculated data and derived storage conditions
(Grims, 1993).
Derived storage
Climatic Calculated data conditions
zone (For real-time studies)
°Ca °C MKTb %RHc °C %RH
I 20.0 20.0 42 21 45
II 21.6 22.0 52 25 60
III 26.4 27.9 35 30 35
IV 26.7 27.4 76 30 70
a Calculated temperatures are derived from measured temperatures, but all measured
temperatures of less than 19°C were set equal to 19°C.
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b Mean kinetic temperature – A single derived temperature that, if maintained over a defined period
of time, affords the same thermal challenge to a drug substance or drug product as would be
experienced over a range of both higher and lower temperature for an equivalent defined period.
c Relative humidity
12.3 DESIGN OF STABILITY STUDIES
Stability studies for a drug substance should be designed in such a way that they provide
all the information on the stability of the drug substance. For drug product the studies should be
designed in the light of the properties and stability characteristics of the drug substance and the
climatic conditions of the intended market zone.
12.3.1 Stress Testing
Stress testing or forced degradation studies are performed on drug substance with a view
to identify the potential degradation products which can in turn help establish the degradation
pathways and the intrinsic stability of the molecule and to validate the stability indicating power of
the analytical procedures used.
Stress testing is carried out generally on a single batch of the drug substance and the
nature of tests depends on the nature of the drug substance and the type of the drug product
involved. Generally it includes the effect of temperatures (In 10% increments above accelerated
storage conditions, e.g. 50°C, 60°C, etc.), humidity (75% RH or greater) and, where appropriate,
oxidation and photolysis on drug substance.
To evaluate susceptibility of the drug substance to hydrolysis in acidic or alkaline media
the stress testing program also conducts testing of the drug substance over a wide range of pH
values in solutions or suspensions (WHO, 2005).
Photostability (Forced photodegradation testing) is also an integral part of stress testing.
The intensity of light and the duration of exposure will vary depending on the photosensitivity of
the drug substance. Studies need to be stopped when extensive degradation is observed. The
influence of light is to be evaluated not only on solid drug substance but also on its solutions.
Stability of the drug substance in different solvents will also make part of the stress testing
program. The solvents that may be considered for such testing include generally those used in the
manufacture of the drug substance and particularly for crystallization in the last step of purification.
12.3.2 Selection of Batches
For drug substance both ICH and WHO stability guidelines (ICH, 2003; WHO, 2009)
require stability studies data to be provided on at least three primary batches. The batches should
be minimal in the size of pilot scale produced by the same synthetic route and method of
manufacture and procedure that simulate the final process to be used for commercial scale
batches.
For the drug product data from stability studies should be provided on at least three
primary batches (two of the three batches should be at least pilot scale batches and the third one
can be smaller, if justified). The primary batches should be of the same formulation, representative
of the manufacturing process and packaged in the same container closure system as proposed
for marketing. Where possible, the batches to be tested should be manufactured from different
batches of active ingredients.
Stability studies should be performed on each individual strength, dosage form and
container type and size of the drug product unless bracketing or matrixing is applied.
12.3.3 Container Closure System
Stability studies on drug substance or drug product should be conducted in the container
closure system that is same or simulates the packaging proposed for storage and distribution or
marketing.
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12.3.4 Test procedure and Test Criteria
Stability studies should include testing of those attributes of the drug substance or drug
product that are susceptible to change during storage and are likely to influence quality, safety,
and/or efficacy. The testing should cover, as appropriate, the physical, chemical, biological and
microbiological attributes. For drug products other tests like preservative content (e.g. antioxidant,
antimicrobial preservative) and functionality tests (e.g. for a dose delivery system) should also be
added to the testing program. Moreover, for drug products it may be appropriate to establish
release acceptance criteria and shelf-life acceptance criteria, however, the difference between the
shelf-life and release acceptance criteria should be justified based on the stability evaluation and
the changes observed on storage.
Validated stability-indicating analytical procedures should be applied. The need for the
extent of replication will depend on the results of validation studies (WHO, 2007).
12.3.5 Frequency of Testing
12.3.5.1 Long term or real-time studies
For drug substance or drug product with a proposed re-test period or shelf-life of at least
12 months, the frequency of testing at the long-term storage conditions should normally be every
3 month over the first year, every 6 month over the second year and annually thereafter through
the proposed re-test period or shelf-life.
12.3.5.2 Accelerated studies
At the accelerated storage conditions, a minimum of three time points, including the initial
and final time points (e.g. 0, 3 and 6 months), from a 6 months study is recommended.
12.3.5.3 Intermediate studies
When testing at the intermediate storage condition is called for as a result of significant
change at the accelerated storage condition, a minimum of four time points, including the initial
and final time points (e.g., 0, 6, 9, 12 months), from a 12-month study is recommended.
Note: Testing frequency can be reduced by using bracketing or matrixing, if justified.
Bracketing
The design of stability schedule such that only samples at the extremes of certain design
factors, e.g. strength and package size, are tested at all time points as in a full design. The design
assumes that the stability of any intermediate levels is represented by the stability of the extremes
tested. Where a range of strengths is to be tested, bracketing is applicable if the strengths are
identical or very closely related in composition (e.g. for a tablet range made with different
compression weights of a similar basic granulation or a capsule range made by filling different plug
fill weights of the same basic composition into different size capsule shells). Bracketing can be
applied to different container sizes or different fills in the same container closure system. A simple
bracketing design is shown in Table 12.2.
Matrixing
The design of a stability schedule such that a selected subset of the total number of
possible samples for all factor combinations is tested at a specified time point. At a subsequent
time point, another subset of samples for all factor combinations is tested. The design assumes
that the stability of each subset of samples tested represents the stability of all samples at a given
time point. The differences in the samples for the same drug products should be identified as, for
example, covering different batches, different strengths, different sizes of the same container
closure system, and, possibly in some cases, different container closure systems. A simple matrix
design is shown in Table 12.3.
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Table 12.2 Stability Protocol Design Using Bracketing (Helboe, 1992).
Dosage Strength/ Active Substance Lot (A, B, C)

Pack type
50 mg 75 mg 100 mg
A B C A B C A B C
Blister x x x (x) (x) (x) x x x
HDPE/ 15 x x x (x) (x) (x) x x x
HDPE/ 100 (x) (x) (x) (x) (x) (x) (x) (x) (x)
HDPE/ 500 x x x (x) (x) (x) x x x
(x) means that the sample is not tested at this time point.
Table 12.3 Stability Protocol Design Using Matrixing (Helboe, 1992).
Dosage Strength/ Active Substance Lot (A, B, C)

Pack type
50 mg 75 mg 100 mg
A B C A B C A B C
Blister x X (x) (x) x x x (x) x
HDPE/ 1 (x) X x x (x) x x x (x)
HDPE/ 2 x (x) x x x (x) (x) x x
(x) Means sample is not tested at this time point.
12.4 STORAGE CONDITIONS
Long-term, accelerated and where appropriate, intermediate storage conditions with a
minimum period data required at submission and total study period for drug substance and drug
product are detailed in sections 3.6.1–3.6.6. The general case applies if the drug substance or
drug product is not specifically covered by a subsequent section. Alternative storage conditions
can be used if justified.
12.4.1 General Case
If long-term studies are conducted at 25±2°C / 60±5% RH and “significant change” occurs
at any time during six months’ testing at the accelerated storage condition, additional testing at the
intermediate storage condition should be conducted and evaluated against significant change
criteria. In this case the initial application should include a minimum of six months data from a 12
month study at the intermediate storage condition (Table 12.4).
Significant change for a drug substance is defined as failure to meet its specification
whereas for a drug product it is define as:
 A 5% change in assay for the active substance(s) from its initial value or failure to meet
the acceptance criteria for potency when using biological or immunological procedures.
 Any degradation product exceeding its acceptance criterion.
 Failure to meet the acceptance criteria for appearance, physical attributes and
functionality test (e.g. color, phase separation, resuspendibility, caking, hardness, dose
delivery per actuation), however, some changes in physical attributes (e.g. softening of
suppositories, melting of creams) may be expected under accelerated conditions.
 Also as appropriate for the dosage form:
 Failure to meet the acceptance criteria for pH or
 Failure to meet the acceptance criteria for dissolution for 12 dosage units.
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Table 12.4 General Case (drug substance or drug product).
Storage Minimum time period covered

Study Total study period
condition by data at submission
Long-terma 25±2°C/ 60±5% a) Drug substance: a) Drug substance:
RH (Zone–II) 12 months (new drug Proposed re-test
or substance) or 6 months period or shelf-life
30±2°C / (existing stable drug
65±5% RH substance).
(Zone–IVa) b) Drug product:
or b) Drug product: Proposed shelf-life
30±2°C / 12 months (drug product
75±5% RH containing new drug substance)
(Zone–IVb) or 6 months (drug product
containing stable drug
substance and where no
significant change is observed
in the drug product stability
studies at accelerated and long-
term conditions for at least 6
months).
Intermediate 30±2°C / 6 months 12 months
b 65±5% RH
Accelerated 40±2°C / 6 months 6 months
75±5% RH
aWhether long-term stability studies are performed at 25±2°C / 60±5% RH or 30±2°C / 65±5% RH
or 30±2°C / 75±5% RH is determined by the climatic condition under which the drug substance or
drug product is intended to be stored. Testing at a more severe long-term condition can be an
alternative to testing condition, i.e. 25°C / 60% RH or 30 °C / 65% RH.
bIf 30±2°C / 65±5% RH or 30±2°C / 75±5% RH is the long-term condition there is no intermediate
condition.
12.4.2 Drug substance or drug product intended for storage in a refrigerator
Both accelerated and long term storage condition studies are conducted on drug
substance or drug product intended for storage in a refrigerator (Table 12.5) .
If significant change occurs between three and six months’ testing at the accelerated
storage condition, the proposed shelf-life should be based on the data available from the long-
term storage condition. If significant change occurs within the first three months’ testing at the
accelerated storage condition, a discussion should be provided to address the effect of short-term
excursions outside the label storage conditions, e.g. during shipment and handling.
Table 12.5 Drug substance or drug product intended for storage in a refrigerator.
Minimum time period
Total study
period
submission
Proposed re-test
Long-term 5±3°C 12 months
period or shelf-life
25±2°C / 60±5% RH
or
Accelerated
a 30±2°C / 65±5% RH 6 months 12 months
or
30±2°C / 75±5% RH
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a Whether accelerated stability studies are performed at 25±2°C / 60±5% RH or 30±2°C / 65±5%
RH or 30±2°C / 75±5% RH is based on a risk-based evaluation. Testing at a more severe long
term condition can be an alternative to storage testing at 25°C / 60% RH or 30°C / 65% RH.
12.4.3 Drug substance or drug product intended for storage in a freezer

For drug substance or drug product intended for storage in a freezer, the re-test period or
shelf-life should be based on the long-term data obtained at the long-term storage condition (Table
12.6). In the absence of an accelerated storage conditions for these substances or products testing
on a single batch at an elevated temperature (e.g. 5±3°C or 25±2°C or 30±2°C) for an appropriate
time period should be conducted to address the effect of short-term excursions outside the
proposed label storage condition e.g. during shipping or handling.
Table 12.6 Drug substance or drug product intended for storage in a freezer.
Minimum time period
Total study
period
submission
Proposed re-test
Long-term –20±5°C 12 months
period or shelf-life
12.4.4 Drug Products Packaged in Impermeable Containers
Sensitivity to moisture or potential for solvent loss is not a concern for drug products
packaged in impermeable containers that provide a permanent barrier to passage of moisture or
solvent. Thus stability studies for products stored in impermeable containers can be conducted
under any controlled or ambient relative humidity condition.
12.4.5 Drug Products Packaged in Semi-Permeable Containers
Aqueous-based products packaged in semi-permeable containers should be evaluated for
potential water loss in addition to physical, chemical, biological and microbiological stability. This
evaluation can be carried out under conditions of low relative humidity, as discussed below. Other
comparable approaches can be developed and reported for non-aqueous, solvent-based products
(Table 12.7).
Table 12.7 Drug products packaged in semi-permeable containers.
Minimum time
Maximum
period covered by
Study Storage condition study
data at
period
submission
Long-terma 25±2°C / 40±5% RH or 12 months Proposed re-
30±2°C / 35±5% RH test period or
shelf-life
Intermediate 30±2°C / 65±5% RH 6 months 12 months
Accelerated 40±2°C / not more than 25% RH 6 months 6 months
a Whether long-term stability studies are performed at 25±2°C / 40±5% RH or 30±2°C / 35±5%.
RH is determined by the climatic condition under which the drug substance or drug product is
intended to be marketed. Testing at 30°C / 35% RH can be an alternative to the storage condition
at 25°C / 40% RH.
A significant change in water loss alone at the accelerated storage condition does not
necessitate testing at the intermediate storage condition. However, data should be provided to
demonstrate that the pharmaceutical product would not have significant water loss throughout the
proposed shelf-life if stored at 25°C / 40% RH or at 30°C / 35% RH.
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For long-term studies conducted at 25±2°C / 40±5% RH, that fail the accelerated testing
with regard to water loss and any other parameter, additional testing at the “intermediate” storage
condition should be performed as described under the general case to evaluate the temperature
effect at 30°C.
A 5% loss in water from its initial value is considered a significant change for a product
packaged in a semi-permeable container after an equivalent of three months’ storage at 40°C and
not more than (NMT) 25% RH. However, for small containers (1 ml or less) or unit-dose products,
a water loss of 5% or more after an equivalent of three months’ storage at 40°C / NMT 25% RH
may be appropriate, if justified.
12.4.6 Drug Substance or Drug Product Intended for Storage Below –20°C
Drug substances or drug products intended for storage below –20°C should be treated on
a case-by-case basis.
12.5 PHOTOSTABILITY
The intrinsic photostability characteristics of new drug substances and products should be
evaluated to demonstrate that, as appropriate, light exposure does not result in unacceptable
change. Normally photostability testing is performed on a single batch of material, major variations
and change like formulation and packaging also warrant repetition of these studies.
A systematic approach to photostability testing is recommended covering, as appropriate,
studies such as:
 Tests on the drug substance.
 Tests on the exposed drug product outside of the immediate pack
and if necessary.
 Tests on the drug product in the immediate pack and if necessary.
 Tests on the drug product in the marketing pack.
The extent of drug product testing should be established by assessing whether or not
acceptable change has occurred at the end of the light exposure testing as described in the
Decision Flow Chart for Photostability Testing of Drug Products (Figure 12.1). Acceptable change
is a change within limits justified by the applicant.
12.5.1 Light Sources
The ICH guideline (ICH, 1996) mentions the light sources for photostability testing as
described under options 1 and 2.
Option 1
Any light source that is designed to produce an output similar to the D65/ID65 emission
standard such as an artificial daylight fluorescent lamp combining visible and ultraviolet (UV)
outputs, xenon, or metal halide lamp. D65 is the internationally recognized standard for outdoor
daylight as defined in ISO 10977 (1993). ID65 is the equivalent indoor indirect daylight standard.
For a light source emitting significant radiation below 320 nm, an appropriate filter(s) may be fitted
to eliminate such radiation.
Option 2
For option 2 the same sample should be exposed to both the cool white fluorescent and
near ultraviolet lamp.
 A cool white fluorescent lamp designed to produce an output similar to that specified in
ISO 10977(1993). The cool white fluorescent lamp covers the visible part of the spectrum.
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 A near UV fluorescent lamp having a spectral distribution from 320 nm to 400 nm with a
maximum energy emission between 350 nm and 370 nm; a significant proportion of UV
should be in both bands of 320 to 360 nm and 360 to 400 nm.
12.5.2 Testing Criteria
For drug substances, photostability testing should consist of two parts: forced degradation
testing and confirmatory testing.
The purpose of forced degradation testing studies is to evaluate the overall
photosensitivity of the material for method development purposes and/or degradation pathway
elucidation. This testing may involve the drug substance alone and/or in simple
solutions/suspensions to validate the analytical procedures. In these studies, the samples should
be in chemically inert and transparent containers. In these forced degradation studies, a variety of
exposure conditions may be used, depending on the photosensitivity of the drug substance
involved and the intensity of the light sources used. For development and validation purposes it is
appropriate to limit exposure and end the studies if extensive decomposition occurs. For
photostable materials, studies may be terminated after an appropriate exposure level has been
used. The design of these experiments is left to the applicant’s discretion although the exposure
levels used should be justified.
Confirmatory studies should then be undertaken to provide the information necessary for
handling, packaging and labeling. For the formal confirmatory studies, the overall light exposure
should not be less than 1.2 million lux hours, with an integrated near UV energy of not less than
200 watt hr/m2 (ICH, 1996, Beaumont, 1999).
For drug products photostability studies should normally be carried out in a sequential
manner starting with testing the fully exposed product then progressing as necessary to the
product in the immediate pack and then in the marketing pack. Testing should progress until the
results demonstrate that the drug product is adequately protected from exposure to light.
Normally, only one batch of drug substance or drug product is tested during the
development phase, and then confirmed on another single batch in case of clearly photostable or
photolabile drug substance. Testing of up to two additional batches may be made if the results of
the confirmatory study are equivocal.
For some products where it has been demonstrated that the immediate pack is completely
impenetrable to light, such as aluminium tubes or cans, testing should normally be conducted on
directly exposed drug product only.
It may be appropriate to test certain products such as infusion liquids, dermal creams, etc.,
to support their photostability in-use. The extent of this testing should depend on and relate to the
directions for use, and is left to the applicant’s discretion. The analytical procedures used should
be suitably validated.
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Fig.12.1 Decision flow chart for photostability testing of drug products.
12.5.3 Presentation of Samples
Care should be taken to ensure that the physical characteristics of the samples under test
are taken into account and efforts should be made, such as cooling and/or placing the samples in
sealed containers, to ensure that the effects of the changes in physical states such as sublimation,
evaporation or melting are minimized.
As a direct challenge for samples of solid drug substances, an appropriate amount of
sample should be taken and placed in a suitable glass or plastic dish and protected with a suitable
transparent cover if considered necessary. Solid drug substances should be spread across the
container to give a thickness of typically not more than 3 millimeters. Drug substances that are
liquids should be exposed in chemically inert and transparent containers.
Where practicable when testing samples of the drug product outside the primary pack,
these should be presented in a way similar to the conditions mentioned for the drug substance.
The samples should be positioned to provide maximum area of exposure to the light source. For
example, tablets, capsules, etc., should be spread in a single layer.
If direct exposure is not practical (e.g. due to oxidation of a product), the sample should
be placed in a suitable protective inert transparent container (e.g. quartz).
If testing of the drug product in the immediate container or as marketed is needed, the
samples should be placed horizontally or transversely with respect to the light source, whichever
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provides for the most uniform exposure of the samples. Some adjustment of testing conditions
may have to be made when testing large volume containers (e.g. dispensing packs).
12.5.4 Post Exposure Sample Analysis
At the end of the exposure period, the samples should be examined for any changes in
physical properties (e.g., appearance, clarity, or color of solution) and for assay and degradants
by a method suitably validated for products likely to arise from photochemical degradation
processes.
Where solid drug substance samples are involved, sampling should ensure that a
representative portion is used in individual tests. Similar sampling considerations, such as
homogenization of the entire sample, apply to other materials that may not be homogeneous after
exposure. The analysis of the exposed sample should be performed concomitantly with that of any
protected samples used as dark controls if these are used in the test.
Drug product samples of powder nature should be sampled in such a way that ensures
that a representative portion is used in individual tests. For solid oral dosage forms, testing should
be conducted on an appropriately sized composite of, for example, 20 tablets or capsules. Similar
sampling considerations, such as homogenization or solubilization of the entire sample, apply to
other materials that may not be homogeneous after exposure (e.g. creams, ointments,
suspensions etc.). The analysis of the exposed sample should be performed concomitantly with
that of any protected samples used as dark controls if these are used in the test.
12.5.5 Recommendation for Handling and Packaging
Depending on the extent of change special labeling or packaging may be needed to
mitigate exposure to light. When evaluating the results of photostability studies to determine
whether change due to light exposure is acceptable, it is important to consider the results obtained
from other formal stability studies in order to assure that the drug substance or drug product will
be within the proposed specifications during the re-test period or shelf-life.
12.6 EVALUATION OF TEST RESULTS
The purpose of stability study is to establish re-test period or shelf-life and storage
instructions for drug substances or drug products produced on commercial scale. During the study,
physical, chemical, biological and microbiological tests and other specific tests in case of dosage
forms (like dissolution rate for solid oral dosage forms), are conducted on a minimum number of
batches. The requested re-test period or shelf-life is granted without any statistical analysis, if
results of the study show very little degradation and very little variability from batch to batch and
within a batch.
In case of a drug product, a shelf-life of 24 months may be established provided the
following conditions are satisfied:
 The drug substance is known to be stable (not easily degradable).
 No significant changes have been observed during stability studies performed.
 Supporting data indicate that similar formulations have been assigned a shelf-life of 24
months or more.
 The manufacturer will continue to conduct long-term studies until the proposed shelf-life
has been covered and the results obtained will be submitted to the national medicines
regulatory authority.
Quantitative analysis of the data generally employs the concept of one-sided 95%
confidence limit of the quantitative attribute changing with time. For the purpose of quantitative
analysis the data on all batches can be combined into one if batch-to-batch variability is small,
however, if the data could not be combined then the overall shelf-life should be based on the
minimum time a batch can be expected to remain within the acceptance criteria.
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The nature of any degradation relationship will determine whether the data should be
transformed for linear regression analysis. Limited extrapolation of the long-term data from the
long-term storage condition beyond the observed range to extend the re-test period or shelf-life
can be undertaken if justified.
Any evaluation should cover not only the assay but also the levels of degradation products
and other appropriate attributes. Where appropriate, attention should be paid to reviewing the
adequacy of evaluation linked to drug product stability and degradation “behavior” during the
testing.
12.7 STABILITY REPORT
A stability report must be established for internal use, registration purposes, etc., giving
details of the design of the study, as well as the results and conclusions.
The results should be presented as both in the form of a table and a graph. For each
batch, the results of testing both at the time of manufacture and at different times during storage
should be given. A standard form should be prepared in which this can be summarized.
The stability of a given drug substance or drug product, and the proposed re-test period
or shelf-life and storage conditions, must be determined on the basis of these results.
12.8 STATEMENTS AND LABELING
A storage statement should be established for display on the label based on the stability
evaluation of the drug substance or drug product. Where applicable specific instructions should be
provided, particularly for drug substances or drug products that cannot tolerate freezing or
excursions in temperature. Terms such as “ambient conditions” or “room temperature” should be
avoided.
The recommended labeling statements for use, if supported by the stability studies, are to
be provided.
A re-test period for drug substance should be derived from the stability information, and a
re-test date should be displayed on the container label if appropriate.
12.9 STABILITY COMMITMENT
When the available long-term stability data on primary batches do not cover the proposed
re-test period or shelf-life granted at the time of approval, a commitment should be made to
continue the stability studies post-approval in order to firmly establish the re-test period or shelf-
life.
Where the submission includes long-term stability data on the number of production
batches covering the proposed re-test period or shelf-life, a post-approval commitment is
considered unnecessary. Otherwise one of the following commitments should be made:
 If the submission includes data from stability studies on the specified number of
production batches a commitment should be made to continue these studies through the
proposed re-test period or shelf-life.
 If the submission includes data from stability studies on fewer than the specified number
of production batches, a commitment should be made to continue these studies through
the proposed re-test period or shelf-life and to place additional production batches, to a
total of at least three, in long-term stability studies through the proposed re-test period or
shelf-life.
 If the submission does not include stability data on production batches, a commitment
should be made to place the first two or three production batches on long-term stability
studies through the proposed re-test period or shelf-life and on accelerated studies for six
months. The stability protocol used for long-term studies for the stability commitment
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should be the same as that for the primary batches, unless otherwise scientifically
justified.
12.10 ONGOING STABILITY STUDIES
After a marketing authorization has been granted, the stability of the drug substance or
drug product should be monitored according to a continuous appropriate program that will permit
the detection of any stability issue in the container closure system in which it is marketed. The
purpose of the ongoing stability program is to monitor the drug substance or drug product within
re-test period or over its shelf-life and to determine that the drug substance or drug product remains
and can be expected to remain within specifications under the storage conditions on the label. The
ongoing stability program should be described in a written protocol and results formalized as a
report.
The protocol for the ongoing stability program can be different from that of the initial long-
term stability study as submitted in the marketing authorization dossier provided that this is justified
and documented in the protocol (e.g. the frequency of testing or when updating to meet revised
recommendations).
For drug substance, at least one production batch per year should be added to the stability
monitoring program and tested at least annually to confirm the stability (WHO, 2007).
For drug product the number of batches and frequency of testing should provide sufficient
data to allow for trend analysis. Unless otherwise justified, at least one batch per year of product
manufactured in every strength and every primary packaging type, if relevant, should be included
in the stability program (unless none is produced during that year). The principle of bracketing and
matrixing designs may be applied if scientifically justified in the protocol (ASEAN, 2005).
In certain situations additional batches should be included in the ongoing stability program.
For example, an ongoing stability study should be conducted after any significant change or
significant deviation to the synthetic route, process or container closure system. Any reworking,
reprocessing or recovery operation should also be considered for inclusion in the case of drug
products (WHO, 2007).
Out-of-specification results or significant atypical trends should be investigated. Any
confirmed significant change, out-of-specification result or significant atypical trend should be
reported immediately to the relevant competent authorities in case of drug product whereas, in
case of drug substance, to the relevant drug product manufacturers also. The possible impact on
batches on the market should be considered in consultation with the relevant competent
authorities.
A summary of all the data generated, including any interim conclusions on the program,
should be written and maintained. This summary should be subjected to periodic review.
12.11 IN-USE STABILITY TESTING
The purpose of in-use stability testing is to provide information for the labeling on the
preparation, storage conditions and utilization period of multi-dose products after opening,
reconstitution or dilution of a solution, e.g. an antibiotic injection supplied as a powder for
reconstitution.
As far as possible the test should be designed to simulate the use of the drug product in
practice, taking into consideration the filling volume of the container and any dilution or
reconstitution before use. At intervals comparable to those which occur in practice appropriate
quantities should be removed by the withdrawal methods normally used and described in the
product literature.
The physical, chemical and microbial properties of the drug product susceptible to change
during storage should be determined over the period of the proposed in-use shelf-life. If possible,
testing should be performed at intermediate time points and at the end of the proposed in-use
shelf-life on the final amount of the drug product remaining in the container. Specific parameters,
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e.g. for liquids, semi-solids and preservatives, per ml content and effectiveness, need to be
studied.
A minimum of two batches, at least pilot-scale batches, should be subjected to the test. At
least one of these batches should be chosen towards the end of its shelf-life. If such results are
not available, one batch should be tested at the final point of the submitted stability studies.
This testing should be performed on the reconstituted or diluted drug product throughout
the proposed in-use period on primary batches as part of the stability studies at the initial and final
time points and, if full shelf-life, long-term data are not available before submission, at 12 months
or the last time point at which data will be available.
In general this testing need not be repeated on commitment batches.
12.12 Variations
Once the drug product has been registered, additional stability studies are required
whenever variations that may affect the stability of the drug substance or drug product are made
(WHO, 2007).
The following are examples of such changes:
 Change in the manufacturing process.
 Change in the composition of the drug product.
 Change of the immediate packaging.
 Change in the manufacturing process of a drug substance.
In all cases of variations, the applicant should investigate whether the intended change
will or will not have an impact on the quality characteristics of drug substances and/or drug
products and consequently on their stability. The scope and design of the stability studies for
variations and changes are based on the knowledge and experience acquired on drug substances
and drug products.
The results of these stability studies should be communicated to the concerned regulatory
authorities (WHO, 2015).
Foot Note
This chapter has been written in the light of ICH and WHO guidelines on stability testing
of drug substance and drug products. The readers are advised to consult the original guidelines
on stability testing in case they need more explanation on any specific area of the subject.
240
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Helboe P. new designs for stability testing programs Drug info J. 1992;26:629–634.
ICH. Harmonized Tripartite Guideline. (ICH Q1B): Photostability of Testing of New drug
substances and products, Geneva, Switzerland, 1996.
Jeffs P. The importance of stability testing in the registration of pharmaceutical products. In: Mazzo
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Illinois, USA, 1999.
Schumacher P. Aktuelle Fragenzur Haltbarkeit von Arzneimitteln. Current questions on drug
stability. PharmazeutischeZeitung. 1974;119:321–324.
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241
242
INDEX
1,3,5-triazine, 141 batch, 5, 6, 17, 149, 255, 261, 262, 264,

2-Aminofluorene, 62 267, 269, 270
2-hydroxyethyl methacrylate, 83 batches, 5, 143, 160, 194, 256, 257, 264,
4-aminosalicylic acid, 3, 59, 127, 128, 182 266, 267, 268, 269, 270
5-aza-cytidine, 63 benzaldehyde, 83, 84, 204
7,8-dimethyl-10- benzydamine, 83
(formylmethyl)isoalloxazine, 63 Binary Co-Amorphous Mixtures, 180
Accelerated studies, 257 bioavailability, 66, 73, 101, 104, 105, 107,
Accuracy, 237, 240, 241, 242 108, 111, 116, 124, 128, 134, 141, 177,
acetylsalicylic acid, 4, 128 179, 211, 212
additives, 105, 134 biochemical stability, 66
adjuvants, 73, 104, 106 biological efficacy, 1
admixtures, 1, 165 bracketing, 6, 160, 256, 257, 269
adverse biological effects, 3 Bracketing, 6, 257, 258
Adverse effects of herbs, 212 buffer, 3, 5, 28, 33, 36, 37, 44, 107, 116,
Aging, 141 149, 171, 226, 244, 246
air, 57, 61, 65, 92, 149, 162, 172, 182, 192, buserelin, 155
195, 198, 202, 204, 207, 208 candesartan cilexetil, 109
air-tight containers, 162 capillary electrophoresis, 153
amber/opaque, 173 capillary electrophoresis–mass
amide, 3, 41, 52, 53, 65, 153 spectrometry, 153
amides, 53, 161 carisbamate, 155
amino acid, 82, 130, 139 Catalysis, 34
amorphization, 4, 104, 181 Cefoxitin sodium, 63
amorphous drug, 104, 105, 109 ceftazidine, 164
Amorphous drugs, 105 Characterization of Polymorphs, 114
Amorphous Drugs, 179 chemical degradation, 4, 15, 65, 102, 124,
amorphous solids, 105, 111, 140 125, 126, 128, 134, 136, 140, 161, 162,
amorphous state, 104, 105, 106, 109, 112, 164, 177, 178, 179, 207
116, 124, 180 chemical factors, 149
Amorphous State, 105 Chemical Functions, 76
amoxicillin, 19, 174, 175 chemical kinetics, 8, 15
amphotericin, 3 Chemical kinetics, 15
ampicillin, 113, 128, 176, 177, 211 chemical reactions, 3, 15, 78, 101, 102, 126
ANALYTICAL METHODS, 5 chemical reactivity, 126, 149
analytical techniques, 101, 114, 153 chemical stability, 8, 50, 65, 101, 149
ANALYTICAL TECHNIQUES, 152 Chemical Stabilization, 179
antioxidants., 172 chlordiazepoxide, 83, 172
ascorbic, 3, 4, 20, 39, 41, 56, 57, 83, 85, chloroquine, 3, 82
91, 92, 110, 129, 164, 172, 179, 182, CHROMATOGRAPHIC (HPLC) ASSAY
184, 226, 235 METHODS, 236
ascorbic acid, 182 Chromatographic Methods, 197
Ascorbic acid, 57, 85, 92, 172, 182 Chromatography, 153
asparagine, 130, 138 ciprofloxacin,, 131
aspirin, 18, 20, 38, 41, 43, 52, 128, 164, circular dichroism, 153
182, 211, 212, 226, 227, 238 climatic conditions, 1, 7, 253, 254, 255
Aspirin, 52, 134, 182, 183, 212 climatic zones, 253
Atropine, 183, 208 clopidogrel, 107, 116
autoxidation, 58, 129 coalescence, 110
Batanopride Hydrochloride, 62 co-amorphous system, 107
coating, 39, 65, 104, 131, 143, 171, 184
243
comminution,, 104 degradation reactions, 6, 15, 23, 31, 33, 37,
compaction, 104, 126 40, 41, 51, 63, 124, 125, 135, 171, 173,
compatibility of drugs, 1 203, 209, 225, 226
DEGRADATION REACTIONS, 30, 51, 128,
Complex Chemical Reactions, 23 171
conditions., 3, 15, 25, 59, 142, 149, 150, DEGRADATION STUDIES OF HERBAL
152, 153, 154, 177, 181, 225, 242, 259 DRUGS, 209
conductance, 110 Dehalogenation, 51, 62
Conformational polymorphism, 113 dehydration, 3, 62, 104, 125, 177, 207
Consecutive Reactions, 27 Dehydration, 51, 61
consistency, 105, 195 Dehydrogenation, 51, 62
Container Closure System, 256 DESIGN OF STABILITY STUDIES, 255
container-closure system, 2, 160, 167 desolvation, 4, 104, 125
content uniformity, 104 Detection limit, 238
Creams, 2, 109 development process, 1, 5, 124, 149, 161,
critical mobility temperature, 105 220, 253
crystalline form, 4, 63, 104, 111, 113, 136 diclofenac sodium, 173, 178
crystalline state, 4, 105, 106, 111, 116, 124, Differential scanning calorimetry, 66, 101,
126, 127, 180 139, 181
Crystalline state, 111 Differential Thermal Analysis, 101
crystolepine, 155 Diffuse reflectance infrared Fourier
cyanocobalamin, 3, 35, 36, 82, 87, 181, transform spectroscopy (DRIFTS), 103
182, 203, 226 Digitoxin, 207
cyclization, 62, 125, 173, 178 Dilatometry, 102
Cyclization, 173, 183 Dimerization, 51, 60, 174
Cyclodextrin, 140, 177 disintegration of solids, 105
cyclosporine, 3 dispersion, 65, 105, 107, 108, 109, 110,
cytarabine, 3, 165 111, 178, 197
deamidation, 4, 64, 125, 130, 138, 140 dispersions, 65, 104, 107, 108, 109, 111,
Deamidation, 130 124, 178, 179, 180
decarboxlyation, 3 dissolution, 2, 5, 41, 66, 73, 101, 104, 105,
decarboxylation, 4, 59, 61, 128 107, 108, 109, 111, 112, 114, 116, 124,
Decarboxylation, 51, 59, 183 128, 137, 141, 142, 143, 163, 178, 179,
degradation, 1, 3, 5, 6, 8, 15, 19, 22, 23, 25, 259, 266
26, 27, 30, 31, 32, 33, 34, 35, 36, 37, 38, divalent anions, 26
39, 40, 41, 42, 43, 50, 51, 52, 54, 56, 57, DLS, 103, 110
59, 60, 63, 64, 65, 66, 72, 75, 79, 101, dosage forms, 1, 2, 3, 4, 5, 15, 33, 35, 36,
102, 107, 110, 124, 125, 128, 130, 131, 37, 39, 41, 52, 65, 101, 104, 105, 107,
133, 134, 135, 136, 137, 138, 139, 140, 116, 124, 125, 132, 133, 134, 137, 141,
141, 149, 150, 151, 152, 153, 154, 156, 149, 160, 162, 166, 179, 181, 182, 184,
161, 163, 164, 165, 171, 173, 174, 177, 192, 236, 238, 254, 266
178, 179, 181, 182, 184, 192, 195, 203, Dosage Forms, 107, 163, 164, 180, 181,
207, 209, 210, 220, 221, 222, 223, 225, 184
226, 236, 241, 243, 254, 255, 256, 259, dosage forms., 1, 3, 15, 33, 35, 36, 65, 101,
263, 266, 267 116, 124, 125, 133, 134, 137, 141, 149,
Degradation Conditions, 150 160, 162, 166, 179, 254
Degradation Limits, 151 dried preparation, 66
degradation pathway, 60, 149 drug adsorption, 4, 104
degradation pathways,, 6, 149 drug development, 1, 5, 113, 116, 124, 149,
degradation process, 5, 6, 149 152, 161, 220, 253, 254
degradation products, 3, 5, 6, 51, 72, 128, drug development process, 1
131, 149, 150, 151, 152, 153, 154, 195, drug interactions, 211
209, 220, 222, 223, 225, 226, 236, 241, DRUG INTERACTIONS, 134, 211
243, 254, 255, 267 drug manufacturers, 1
drug products, 1, 3, 4, 5, 6, 15, 17, 30, 33,
39, 50, 63, 72, 73, 74, 83, 101, 104, 105,
244
116, 149, 150, 151, 153, 160, 161, 163, expiration dating, 6, 7, 15, 17
166, 171, 177, 184, 210, 220, 253, 256, Expiration dating, 17
257, 261, 262, 264, 265, 266, 267, 269, External Factors, 3, 105
270 Extraction Methods, 197
drug substance, 5, 6, 30, 50, 112, 114, 149, famotidine, 115
150, 153, 220, 221, 253, 254, 255, 256, FDA, 5, 8, 124, 134, 152, 160, 220, 246
257, 258, 260, 261, 262, 263, 264, 266, FINGERPRINT ANALYSIS, 195
267, 268, 269, 270 finished products, 1
drug substances, 1, 2, 3, 6, 15, 50, 51, 63, flocculation, 104, 107
72, 75, 79, 82, 83, 101, 102, 104, 105, Fluconazole, 131
107, 113, 114, 116, 124, 125, 128, 136, fluorescence, 75, 77, 80, 153, 204, 210
137, 149, 150, 151, 153, 160, 161, 173, fluoroquinolones, 83, 161, 179
177, 181, 182, 183, 210, 211, 220, 236, flurbiprofen, 106, 211
246, 253, 262, 263, 265, 266, 267, 270 forced degradation, 6, 41, 149, 150, 151,
drug–CD complexes, 140, 178 152, 153, 154, 155, 226, 236, 237, 255,
drug–polymer solid dispersions, 109 263
drying, 65, 66, 104, 105, 106, 111, 192, Forced degradation, 149
193, 194 Forced polymorphism, 113
DSC, 101, 106, 107, 109, 114, 115, 131, formulation development, 6, 72, 150, 152
138, 139, 141, 181 formulation studies, 50
Dynamic light scattering (DLS), 103 formylmethylflavin, 21, 79, 86, 93, 204, 206,
electron microscopy, 66, 141, 184 226, 230, 244
Electrophoresis, 153, 197 Fourier transform infrared (FTIR)
Elimination, 51, 59, 225 spectroscopy, 102
emulsion creams, 109 Free radical mechanism, 81
emulsions, 66, 104, 210 Frequency of Testing, 257
enthalpy, 106, 112, 114, 139, 180 FTIR, 51, 102, 107, 109, 114, 115, 136,
entropy, 106, 180 138, 139, 141, 195, 197, 241
environmental, 1, 2, 6, 50, 101, 149, 161, FTIR,, 51
162, 171, 181, 192, 210, 253 functional groups, 50, 51, 75, 76, 220
environmental conditions, 2 furosemide, 4, 83, 106, 115, 131, 180, 184
environmental factors, 1, 6, 50, 101, 149, GC/MS, 51
161, 162, 171, 181, 192, 210, 253 General acid-base catalysis, 36
Enzyme Catalyzed Reactions, 28 Gibbs free energy, 106, 180
Ephedrine, 204 Ginseng Saponins, 209
epimerization, 3, 61, 175, 209, 210 glass containers, 161, 164, 165, 166, 195
Epimerization, 51, 61, 175, 176, 209 glucose, 3, 56, 65, 165, 211, 212
epinephrine, 3, 39, 176 Glucose, 61, 62
erythromycin, 3, 203 good manufacturing practices, 1, 194
Erythromycin, 183, 207 granulation, 104, 125, 126, 142, 257
Essential Oils, 194 Graphical method, 22
esters, 3, 21, 40, 52, 76, 133, 161, 181, 194 half-life, 17, 18, 22, 23, 40, 42, 43, 181
ethyl acetate, 20, 43 Half-life method, 22
etodolac, 3 hardness, 5, 41, 112, 142, 164, 259
etoposide, 3 heat, 75, 77, 101, 102, 106, 110, 112, 114,
European Pharmacopoeia, 7, 72, 161 133, 137, 149, 154, 155, 162, 192, 195,
excipients, 1, 3, 6, 50, 66, 101, 105, 109, 198, 199, 200, 209, 222
114, 124, 125, 130, 131, 132, 133, 134, heat capacity, 101, 106, 112
136, 141, 149, 150, 152, 171, 177, 180, Herbal Drug Extracts, 194
181, 184, 192, 194, 238, 241, 243 Herbal Drug Preparations, 194
Excipients, 140 herbal drugs, 8, 192, 193, 194, 195, 197,
exciplex, 80 198, 202, 203, 204, 211
excited, 39, 74, 75, 76, 77, 78, 79, 80, 81, Herbal Drugs, 193, 203, 207
82, 85, 86, 91, 92, 93 Herbal Extracts, 194
Excited State Reactions, 79 herbal products, 192, 194, 203, 211, 212
expiration dates, 15 Herbal Products, 194
245
high-performance liquid chromatography, 5 93, 103, 104, 108, 114, 131, 149, 152,
High-performance liquid chromatography, 154, 155, 160, 161, 162, 163, 165, 166,
63, 197 171, 173, 181, 182, 183, 184, 192, 195,
homogeneity, 105 198, 199, 200, 201, 203, 204, 205, 210,
homogenization, 110, 266 221, 222, 223, 253, 255, 256, 262, 263,
Hot-stage microscopy, 102 264, 266, 270
HPLC, 5, 41, 51, 60, 63, 64, 65, 66, 109, light sensitive drugs, 203
131, 137, 141, 152, 153, 154, 155, 163, Light Sources, 263
164, 165, 166, 195, 197, 210, 225, 236, Light-resistant containers, 162
238, 243, 244 Linearity, 237, 238
HPLC–mass spectrometry, 153, 197 Liposomal Formulation, 179
HPTLC, 51, 195, 197, 226 Liposomes, 110, 179
humidity, 1, 5, 6, 51, 105, 109, 111, 112, liquid dosage, 3, 4, 5, 33, 34, 35, 36, 37,
136, 137, 139, 142, 149, 163, 164, 166, 39, 52, 104, 160, 183, 192
182, 210, 253, 255, 261 Long term or real-time studies, 257
hydrolysis, 3, 4, 15, 20, 33, 38, 40, 41, 43, lumichrome, 63, 86, 93, 204, 206, 221
44, 51, 52, 53, 54, 57, 63, 64, 65, 66, 82, lumiflavin, 63, 86, 93, 204, 206
83, 86, 91, 93, 124, 125, 128, 139, 149, lumivudine, 154
150, 151, 153, 154, 155, 174, 177, 178, lyophilized compound, 172
207, 208, 209, 222, 229, 230, 256 lyophilized preparations, 65
Hydrolysis, 18, 19, 20, 21, 25, 51, 52, 53, lyophilized proteins, 65
54, 151, 171, 183, 226, 227, 228, 232 lyoprotectants, 65
hydrolytic degradation, 64, 65, 66, 236 manufacturer, 1, 5, 254, 267
ICH, 5, 6, 7, 8, 51, 72, 74, 124, 132, 149, matrixing, 6, 160, 256, 257, 269
150, 152, 154, 160, 163, 167, 210, 220, Matrixing, 6, 257, 258
237, 238, 242, 246, 253, 256, 263, 264, mechanical strength, 104, 105
270 Mechanism involving singlet oxygen, 81
ICH Q1B guideline, 6 meclofenamic acid, 3, 82
ICH Q5C guideline, 152 mefloquin, 82
ICH QIB Guideline, 74 Menadione, 173
imides, 3 METHODS OF STABILIZATION, 177
impurities, 125, 129, 150, 152, 166, 194, methyl paraben, 66
220, 237, 240, 241, 243 Michaelis constant, 30
indomethacin, 38, 52, 105, 106, 107, 131, microbial growth, 2, 4, 104
132, 180, 181 microbial growth., 4, 104
Industrial Awareness, 74 Microcalorimetry, 102
Insulin preparations, 64 microcrystalline cellulose, 109, 125
interactions, 1, 3, 77, 91, 101, 102, 103, moisture, 3, 4, 5, 41, 65, 105, 111, 112,
104, 105, 110, 111, 134, 140, 162, 171, 124, 125, 128, 130, 131, 132, 133, 134,
178, 179, 180, 181, 192, 211 135, 141, 142, 149, 162, 163, 164, 166,
Intermediate precision, 237, 243 171, 182, 183, 192, 195, 198, 199, 200,
Intermediate studies, 257 201, 202, 222, 261
Internal Factors, 3, 105 Moisture, 41, 124, 125, 132, 133, 162, 180,
intrinsic stability, 220 182, 198, 199, 200, 201, 202
IN-USE STABILITY, 269 moisture content, 132, 141, 142, 163, 166
Ionic Strength Effect, 37 Molecularity, 16
isomerization, 3, 41, 60, 76, 124, 150 Morphine, 57, 183, 208
Isomerization, 51, 60 moxalactam, 3
Isothermal calorimetry, 102, 137 moxifloxacin, 34, 35, 38, 87, 131, 136, 236
ketoprofen, 106, 211 MS, 51, 65, 66, 131, 139, 153, 154, 155,
Kinetic Studies, 137 195, 197
lamotrigine mesylate, 105 Multicomponent Assay, 223
LC/MS, 51 naproxen, 107, 180, 211
LC–MS/TOF methods, 154 new chemical entity, 1
light, 3, 5, 6, 39, 51, 53, 56, 57, 61, 72, 73, New Drug Application, 150
74, 75, 76, 78, 81, 82, 85, 86, 87, 91, 92, nicotinamide, 91, 92, 180, 226
246
nifedipine, 82, 105, 106, 109, 178, 180, 184 Phase transition, 104
NMR, 51, 65, 103, 105, 114, 115, 116, 138, Phenols, 58
139, 140, 155, 195, 197, 209, 222 phosphorescence, 75, 77, 79, 80
nonisothermal kinetics, 33 photoaddition, 26, 80, 86, 87
Norfloxacin, 56, 63 Photoaddition, 80, 82, 86, 226, 234
NSAIDs, 107, 111, 161, 211 Photoallergic reactions, 73
ofloxacin, 79, 131 photoaquation, 3
One-Component Assay, 222 Photoaquation, 82, 87
ONGOING STABILITY STUDIES, 268 Photochemical Interactions, 91
Order, 16, 17, 18, 19, 21, 23, 42, 43 photochemical process, 74
Order of Reaction, 16 photochemical reactions, 75, 76, 77, 78, 79,
orders of reaction, 15 85
oxidation, 3, 4, 15, 39, 40, 51, 53, 56, 57, Photochemistry, 74, 75
58, 76, 79, 80, 81, 82, 83, 87, 91, 92, photocyclization, 3
125, 129, 149, 150, 151, 153, 154, 155, photodealkylation, 3, 86, 91
172, 177, 182, 183, 207, 208, 222, 255, Photodealkylation, 82, 86
266 photodecarboxylation, 3
Oxidation, 20, 39, 51, 56, 129, 152, 172, Photodecarboxylation, 82
183 photodegradation, 3, 26, 34, 35, 37, 39, 63,
Oxidation reactions, 39 72, 74, 75, 79, 82, 83, 86, 87, 88, 89, 90,
Oxidizable drugs, 39 91, 93, 131, 166, 173, 179, 184, 203,
Oxygen, 39 204, 206, 225, 236, 243, 244, 256
oxyphenbutazine, 106 Photodegradation, 27, 73, 87, 91, 131, 203,
packaging, 1, 3, 5, 6, 8, 15, 39, 50, 74, 150, 204, 221
160, 161, 162, 163, 164, 166, 167, 171, photodegradation reactions, 3, 72, 82, 83
193, 253, 254, 256, 262, 264, 266, 269, Photodehalogenation, 82
270 Photodehydrogenation, 82
packaging development, 160 photodimerization, 3, 91
packaging material, 3, 160, 162, 163, 166, Photodimerization, 82
167 Photoelimination, 82
packaging materials, 1, 3, 160, 254 Photo-induced rearrangement, 83
PACKAGING STUDIES, 163 Photoinduced ring cleavage, 83
Packing polymorphism, 113 photoisomerization, 3, 91, 166
paracetamol, 52, 134, 164, 180 Photoisomerization, 83
Paracetamol, 53, 164, 182, 183 photolysis, 4, 15, 26, 28, 35, 36, 51, 72, 77,
Parallel Reactions, 25 78, 79, 80, 87, 149, 150, 151, 153, 154,
Particle electrophoresis, 104 155, 173, 177, 222, 226, 255
particle size, 3, 4, 65, 67, 101, 107, 110, Photolysis, 20, 21, 77, 78, 131, 152, 172,
111, 125, 126, 171, 210 226, 233, 235
Particle size, 4 photooxidation, 3, 39, 83, 85, 87, 173, 204,
Peptide, 130 205, 226, 232
pH, 3, 5, 33, 34, 35, 36, 39, 40, 42, 44, 52, Photooxidation, 83, 84, 85, 173
56, 58, 61, 62, 63, 64, 65, 66, 86, 87, 88, Photophysical Processes, 76
107, 109, 110, 111, 128, 134, 137, 138, photoreactivity, 72, 74, 75
149, 152, 161, 165, 171, 172, 173, 174, Photoreactivity, 76
175, 176, 177, 181, 182, 183, 184, 194, photoreduction, 3, 86, 87
207, 209, 222, 223, 229, 230, 231, 233, Photoreduction, 83, 86
234, 235, 244, 246, 256, 259 photosensitivity, 73, 149, 203, 256, 263
pharmaceutical manufacturers, 160 PHOTOSENSITIVITY REACTIONS, 203
Pharmaceutical Preparations, 64 photosensitization reactions, 72
pharmaceutical systems, 18, 40, 106, 112, Photosensitization reactions, 73
152 Photosensitized Reactions, 81
pharmaceuticals, 6, 15, 101, 105, 111, 114 photostability, 3, 6, 8, 72, 73, 74, 75, 79, 83,
pharmacists, 1 92, 132, 149, 160, 166, 178, 210, 226,
pharmacopoeias, 1, 7, 177 262, 263, 264, 265, 266
phase transition, 102, 104
247
Photostability, 3, 72, 73, 74, 75, 149, 178, Range, 237, 240
256, 263 ranitidine HCl, 41, 114, 137
PHOTOSTABILITY, 72, 262 ranitidine hydrochloride, 64
photostability testing, 6, 72, 74, 149, 210, rate constant, 16, 17, 18, 19, 20, 21, 22, 25,
226, 262, 263, 265 26, 30, 31, 33, 34, 36, 37, 38, 42, 43, 44,
Photostabilization, 184 109, 134, 135, 177
Phototoxic reactions, 73 rate–pH profiles, 34, 35
physical stability, 8, 50, 101, 104, 105, 106, rates, 15, 33, 37, 38, 40, 41, 66, 67, 75, 78,
107, 108, 110, 111, 114, 180 102, 108, 116, 130, 138, 140, 167, 177
physical state, 1, 101, 105, 111, 112, 116, Reaction Rate, 16
124, 130, 142 rebamipide, 153
Pilocarpine, 209 recombinant human deoxyribonuclease, 65
plastic containers, 165 recommended storage conditions, 3, 253
Plastic containers, 162 regulatory agencies, 1, 5, 50, 253
polyacrylic acid, 111 REGULATORY ASPECTS, 253
polydispersity index, 110 regulatory authorities, 124, 149, 160, 255,
polymer, 108, 109, 111, 140, 142, 178, 179, 270
181 regulatory requirement, 1, 253
Polymer complexation, 178 Repeatability, 237, 242, 243
Polymerization, 176 Reproducibility, 237, 243
polymorph formation, 4 Reserpine, 204, 205
polymorphic transition, 4, 104 reset, 6, 15
Polymorphism, 112, 113 re-test period, 5, 253, 257, 260, 261, 262,
polymorphs, 102, 103, 104, 111, 112, 113, 266, 267, 268
114, 115, 116, 136 riboflavin, 3, 20, 26, 27, 28, 31, 35, 37, 38,
polyols, 65 52, 54, 63, 80, 81, 82, 83, 86, 89, 91, 92,
polyurethane, 111 179, 184, 203, 221, 226, 229, 233, 234,
polyvinyl chloride (PVC) bags, 164 235, 243, 244
polyvinyl pyrrolidone-co-vinyl acetate, 108 Riboflavin, 35, 54, 184, 204, 206, 226, 243
polyvinylpyrrolidone, 108, 178 Robustness, 238, 246
Precipitation, 4 salt, 4, 41, 104, 111, 134, 180, 193
Precision, 237, 241, 242, 243 salt exchange, 4, 104
preservative, 4, 256 Secondary Packaging Material, 161
preservative activity, 5 Second-order Reaction, 20
preservatives/stabilizers, 1 Sedimentation, 107
primaquine, 4, 82 sedimentation,, 104
Primary Packaging Material, 161 Selection of Batches, 256
Procaine, 52, 183 Sensitivity, 74, 198, 238, 245, 261
prostaglandin E1 and E2, 3 shelf-life, 1, 2, 3, 4, 6, 7, 15, 17, 23, 30, 31,
protein drugs, 130 32, 33, 44, 51, 73, 101, 139, 151, 163,
proteins, 65, 66, 111, 116, 130, 134, 204 166, 177, 178, 180, 195, 210, 253, 254,
Proteins, 111 256, 257, 260, 261, 262, 266, 267, 268,
Pseudo First-order Reaction, 20 269, 270
Pseudolatexes, 65 Shelf-life calculation, 33
Pseudopolymorphism, 113 shelf-life., 1, 163, 166, 195, 253, 257, 266,
pyrolysis, 4, 27, 131 268, 269, 270
Pyrolysis, 130, 131 Single crystal X-ray diffraction (XRD, 103
QUALITY CONTROL METHODS, 194 Singlet oxygen, 39, 76
Quantitation limit, 238 solid dispersions, 109
quinapril, 107, 181 Solid dispersions, 107, 108
quinapril HCl, 107 solid dosage, 4, 5, 41, 104, 116, 124, 125,
Quinine, 206 131, 132, 133, 134, 136, 137, 141, 166,
QΔT calculation, 32 181, 182
Rabeprazole, 66 solid dosage forms, 5, 104, 124, 134, 137,
rabeprazole sodium, 66 141
Racemization, 176
248
solid state, 4, 8, 65, 103, 104, 108, 112, STABILITY-INDICATING HIGH-
115, 116, 124, 125, 126, 128, 129, 130, PERFORMANCE, 236
131, 132, 133, 135, 136, 137, 138, 139, STABILITY-INDICATING
140, 141, 155, 178, 181, 182, 237 SPECTROMETRIC ASSAY METHODS,
solid state degradation, 4, 125, 132 222
Solid state nuclear magnetic resonance stabilization, 8, 15, 30, 33, 50, 65, 72, 74,
(SSNMR) spectroscopy, 103 75, 106, 116, 130, 171, 172, 174, 177,
solid state stability, 8 178, 179, 180, 181, 182, 184
Solid state transitions., 4 Stabilizers, 179
solvation, 4, 104, 112, 128 stabilizers., 173
Solvatomorphism, 113 Stark and Einstein Law, 74
solvent, 3, 37, 38, 108, 111, 112, 113, 125, state degradation, 102
128, 131, 133, 149, 171, 172, 194, 223, STATE DEGRADATION, 135
226, 261 STATISTICAL APPLICATIONS, 6
solvent dielectric constant, 38 Statistical methods, 6
solvolysis, 4, 125 Steric Structural Variations, 173
Solvolysis, 128 storage conditions, 1, 2, 3, 5, 6, 7, 15, 17,
Specific acid–base catalysis, 34 51, 109, 111, 142, 149, 152, 161, 164,
specific acid-catalyzed reaction, 64 171, 177, 193, 195, 202, 210, 253, 254,
Specificity, 237, 243 255, 257, 258, 260, 261, 267, 268, 269
Spectroscopic Methods, 102, 197 STORAGE CONDITIONS, 258
Spectroscopy, 103, 153 storage period, 1, 5, 50, 107, 110, 136, 160,
stability, 1, 2, 3, 4, 5, 6, 7, 8, 15, 30, 33, 34, 165
35, 36, 37, 40, 41, 50, 51, 63, 64, 65, 66, stress conditions, 6, 41, 72, 111, 150, 152,
72, 74, 91, 101, 102, 104, 105, 106, 107, 153, 154, 155, 162, 163, 221
108, 109, 110, 111, 112, 113, 114, 116, stress testing, 6, 149, 151, 152, 167, 220,
124, 125, 132, 133, 134, 136, 137, 139, 237, 253, 256
140, 141, 142, 149, 150, 151, 152, 153, Stress testing, 149, 220, 236, 255
154, 155, 160, 161, 162, 163, 164, 165, Stress Testing, 255
166, 167, 171, 173, 176, 177, 178, 179, Structural Studies, 136
180, 181, 183, 192, 194, 195, 203, 209, substances, 1, 6, 15, 51, 72, 73, 83, 101,
210, 211, 220, 221, 225, 226, 236, 246, 104, 116, 149, 171, 177, 182, 238, 261,
253, 254, 255, 256, 257, 260, 261, 262, 262, 265, 270
266, 267, 268, 269, 270 Substitution method, 21
Stability, 1, 2, 3, 4, 5, 178, 203, 210, 220, sugars, 65, 180
226, 236, 253, 254, 255, 256, 258 sulfacetamide, 4, 41, 52, 82, 83, 226, 232,
STABILITY COMMITMENT, 268 238, 239, 240, 241
stability data, 6, 254 Surfactants, 40
STABILITY EVALUATION, 5 suspensions, 18, 19, 64, 66, 104, 107, 178,
STABILITY OF HERBAL DRUGS, 192, 203 256, 263, 266
STABILITY PREDICTION, 166 temperature, 1, 3, 4, 5, 6, 30, 31, 32, 33,
stability profile, 160 37, 38, 44, 51, 52, 54, 61, 63, 64, 65, 66,
stability program, 1, 160, 268 101, 102, 105, 106, 108, 109, 110, 111,
STABILITY REPORT, 267 112, 113, 114, 124, 125, 128, 130, 131,
stability schedule, 6 132, 133, 137, 139, 140, 141, 152, 155,
stability studies, 6, 124, 203, 211, 253, 254, 163, 164, 165, 166, 171, 176, 177, 180,
256, 268, 270 181, 182, 195, 209,뉐 210, 223, 246,
stability testing, 6, 8, 253, 270 253, 254, 255, 261, 262, 268
STABILITY TESTING, 5, 167, 210, 253 Temperature, 30, 64, 109, 133, 162, 177,
stability-indicating assay method, 5, 15, 74, 198, 199, 200, 201, 202
150, 226 tetracycline, 73, 137, 175, 176
stability-indicating assay methods, 5, 6, 8, tetracyclines, 3, 134
149, 152, 220, 236, 246 theophylline cream, 110
STABILITY-INDICATING ASSAY thermal gravimetric analysis, 65
METHODS, 220, 237 thermal methods, 109, 125, 136
Thermal Methods, 101
249
Thermogravimetric analysis, 101 UV-vis spectrometry, 51
Thermolysis, 21, 152, 226, 231 VALIDATION, 237
Thin layer chromatography, 51 Vibrational spectroscopy, 102
tight containers, 162, 198, 201, 202 viscosity, 4, 37, 80, 105, 107, 110, 112,
Time-Resolved Spectroscopy, 79 178, 210
Tolfenamic acid, 111 visible region, 3, 74, 75, 77, 81
toxicity, 2, 3, 15, 51, 222 vitamin, 3, 35, 39, 41, 54, 56, 57, 81, 85,
Toxicity, 50, 73 87, 133, 134, 173, 181, 182, 184, 206,
Traditional Chinese Medicines, 195 221
transformation, 60, 64, 75, 81, 82, 111, 113, water-in-oil creams, 110
114, 115, 125 WHO, 5, 8, 124, 160, 193, 195, 253, 256,
Trimelamol, 59, 60 257, 269, 270
Triplet oxygen, 76 X-ray powder diffraction (XRPD, 103
tryptic mapping, 65 XRPD, 106, 107, 112, 114, 115, 116, 136,
Two-component assay, 223 141
United States Pharmacopeia, 2, 7, 72, 160, zero-order reaction, 17, 18, 22
161, 221 α-pinene, 27, 199
UPLC–mass spectrometry, 153 α–tocopherol, 91, 92
UV and visible absorbers, 173 β-lactam, 40, 63, 136, 174
UV, Visible and Sunlight Radiation, 75
250

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STABILITY OF DRUGS

AND DRUG PRODUCTS

Iqbal Ahmad, T.I.

Muhammad Ali Sheraz

Faculty of Pharmaceutical Sciences

HIGHER EDUCATION COMMISSION

Lahore Karachi Peshawar Quetta

HEC – Cataloging in Publication (CIP Data):

Iqbal Ahmad, Dr.

Chemical and Photo stability of Drugs and Formulated Products

616.86 - dc23 2016

First Edition: 2016

Copies Printed: 500

Published By: Higher Education Commission – Pakistan

6 SOLID STATE STABILITY 101

8 PACKAGING EFFECTS ON DRUG STABILITY 131

10 STABILITY OF HERBAL DRUGS AND PRODUCTS 157

11 STABILITY-INDICATING ASSAY METHODS 179

12 REGULATORY ASPECTS OF STABILITY TESTING 209

2.1 Zero-order plot of A versus time. 15

1.1 Types and criteria for acceptable levels of stability. 2

Prof. Dr. Zahida Baqai

This monograph has been prepared to meet the requirements of

Table 1.1 Types and criteria for acceptable levels of stability

Table 1.2 Types of pharmaceutical dosage forms

Dosage form Phase Example

1.3 FACTORS INFLUENCING STABILITY

Fig. 2.1 Zero-order plot of A versus time.

Fig. 2.2 First-order plot of log A versus time.

Fig. 2.3. Second-order plot of 1/[A] versus time.

Rearranging and converting to the log form gives

2.4 Complex Chemical Reactions

k (a–xe) = kˊxe (2.22)

where xe is the value of x at equilibrium

Time (min) 0 20 35 50 65 80 100 ∞

Form the –2.303

kobs = k1 (1 + [C] / [B]) = k2 (1 + [C] / [B]) (2.30)

Q10 k(T1 + 10)

It is related to the activation energy, Ea

Table 2.3 Rate–pH profiles for the degradation of drugs.

kobs × 104 (min-1)

kobs × 107 (min-1)

triplet state singlet state

SO32– + M+ SO3–● + M● (2.76)

% 100 89.5 77.4 68.0 45.5 30.9 21.0

t (s) 0 100 200 300 400 500

[A] × 103 5.00 3.27 2.40 1.92 1.59 1.40

(3.9) (3.11) (3.12)

OH- -e- oxidative reactions, especially at ortho and

Triplet oxygen (3O2) Singlet oxygen (1O2)

4.4.3 Photophysical Processes

Fig.4.1 Photooxidation of benzaldehyde.

(4.1) (4.2) (4.3)

Fig. 4.3 Chemical structures of riboflavin and photoproducts.

(4.10) (4.11) (4.12)

Fig. 4.4 Proposed pathway for the photodegradation of MF in acid solution.

Fig. 4.5 Proposed pathway for the photodegradation of MF in alkaline solution.

SOLID STATE STABILITY

 Humidity is not simultaneously controlled.

(6.17) (6.18) (6.19) (6.20)

6.6 KINETICS OF SOLID STATE DEGRADATION

FORCED DRUG DEGRADATION

7.3 FACTORS INVOLVED IN DEGRADATION