CONTENT. i iv ix. SVKM s NMIMS, School of Pharmacy and Technology Management

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1 CONTENT Chapter No. Title Page No. Abbreviations List of Figures List of Tables 1 Introduction Practical aspects Stability-indicating assay method (SIAM) Regulatory aspects Techniques employed Identification and characterization of degradation products Decision trees for respective stress conditions Pharmaceutical applications Drug candidate synthesis and screening Metabolites identification and bioanalysis Impurity identification and profiling Stability studies of drug substances and drug formulations 20 2 Literature and Patent Review 22 3 Research Envisaged and Plan of Work Research envisaged Plan of work 37 4 Drug Profile Nelfinavir mesylate Linezolid Lacosamide Ritonavir 45 5 Experimental Methods Materials Instrumentation General procedures 48 i iv ix

2 5.4 Stress studies, HPLC method development and validation of drug substances MS/TOF and MS n studies on drugs LC-MS/TOF studies and characterization of degradation products Stress studies, HPLC method development and validation of drug products 73 6 Results and Discussion Nelfinavir mesylate Development of stability indicating HPLC method Degradation behavior Method validation MS/TOF, MSn and LC-MS/TOF studies Postulated structures of degradation products Postulated degradation pathway of the drug Stability study of nelfinavir mesylate tablets Linezolid Development of stability indicating HPLC method Degradation behavior Method validation MS/TOF, MSn and LC-MS/TOF studies Postulated structures of degradation products Postulated degradation pathway of the drug Stability study of linezolid tablets Lacosamide Development of stability indicating HPLC method Degradation behavior Method validation MS/TOF, MSn and LC-MS/TOF studies Postulated structures of degradation products Postulated degradation pathway of the drug Isolation and characterization of degradation products (DP-I and 156

3 DP-II) Toxicity study of DP-I and DP-II Stability study of lacosamide tablets Ritonavir Development of stability indicating HPLC method Degradation behavior Method validation MS/TOF, MSn and LC-MS/TOF studies Postulated structures of degradation products Postulated degradation pathway of the drug Stability study of ritonavir tablets Summary and Conclusions References Appendices 10 Publications

4 Abbreviations Abbreviations µg Microgram µl Microliter ACN ADME API API AR ASEAN AUC CE CE-MS CPMP Acetonitrile Absorption distribution metabolism elimination Active pharmaceutical ingredient Atmospheric pressure ionization Analytical reagent Association of Southeast Asian Nations Area under curve Capillary electrophoresis Capillary electrophoresis-mass spectrometry Committee for Proprietary Medicinal Products CYP Cytochrome P-450 DAD DART DESI DMPK DP ESI FT-ICR GC GC-MS h Diode array detection Direct analysis in real time Desorption electrospray ionization Drug metabolism and pharmacokinetics Degradation product Electrospray ionization Fourier transform-ion cyclone resonance Gas chromatography Gas chromatography-mass spectrometry hour i

5 Abbreviations H/D H2O2 HCl HIV HPLC HPTLC ICH IND IP LC-MS LC-NMR m/z MeOH mg ml mm MRM MS n N NaOH NDA nm NMR PDA Hydrogen/deuterium Hydrogen peroxide Hydrochloric acid Human immunodeficiency virus High performance liquid chromatography High performance thin layer chromatography International Conference on Harmonization Investigational New Drug Indian Pharmacopoeia Liquid chromatography-mass spectrometry Liquid chromatography-nuclear magnetic resonance Mass to charge Methanol Milligram Milliliter Millimole Multiple reaction monitoring Multi-stage mass spectrometry Normality Sodium hydroxide New Drug Application Nanometer Nuclear magnetic resonance Photodiode array ph Power of hydrogen ii

6 Abbreviations pka RDB Rf RH RPLC RSD RT SD SIAM SRM TLC TOF TPD UPLC US-FDA USP UV VIS Acid dissociation constant Ring double bond Retention factor Relative humidity Reverse phase liquid chromatograph Relative standard deviation Room temperature Standard deviation Stability indicating assay method Selective reaction monitoring Thin layer chromatography Time of flight Therapeutic Product Directorate Ultra performance liquid chromatography United States Food and Drug Administration United States Pharmacopoeia Ultraviolet Visible iii

7 List of figures List of Figures Fig Fig Fig Fig Fig Fig Fig Fig Fig Fig Fig Fig Fig Fig Fig Fig Fig Decision tree for performing stress studies for hydrolytic degradation under acid and alkali conditions. Decision tree for performing stress studies for hydrolytic degradation under neutral conditions. Decision tree for performing stress studies under oxidative conditions. Decision tree for performing stress studies for photolytic degradation. Block diagram showing plan of proposed research work. Structure of Nelfinavir mesylate. Structure of Linezolid. Structure of Lacosamide. Structure of Ritonavir. Chromatogram showing nelfinaivr mesylate (drug) and its degradation products (I-III) contained in a mixture of stress samples. Chromatograms showing nelfinavir mesylate and its hydrolytic degradation products Key: (a) unstressed sample, (b) acid hydrolysis, (c) alkaline hydrolysis, (d) neutral hydrolysis. Chromatogram showing nelfinavir mesylate and its degradation behavior in oxidative stress condition. Chromatogram showing nelfinaivr mesylate and its photolytic degradation products (II, III). Key: (a) photoacid, (b) photobase, (c) photoneutral (d) photosolid samples exposed to 8500 Lx. h fluorescent and 0.05 W/m 2 UV light. Chromatogram showing nelfinavir mesylate under thermal stress condition. Calibration plot for nelfinavir mesylate. Line spectrum of nelfinavir mesylate obtained in MS/TOF study. Fragmentation pathway of nelfinavir mesylate along with the exact masses of the fragments. iv

8 List of figures Fig Fig Fig Fig Fig Fig Fig Fig Fig Fig Fig Fig Fig Fig Fig Fig Line spectra of degradation products (DP-I-III) obtained in LC-MS/TOF studies. Fragmentation pathway of DP-I. Fragmentation pathway of DP-II and DP-III. Degradation pathway of nelfinavir mesylate. Chromatogram showing nelfinaivr mesylate tablets (drug) and its degradation products (I-III) contained in a mixture of stress samples. Chromatograms showing hydrolytic degradation products of nelfinavir mesylate tablets. Key: (a) unstressed sample, (b) acid hydrolysis, (c) alkaline hydrolysis, (d) neutral hydrolysis. Chromatogram showing oxidative degradation products of nelfinavir mesylate tablets. Chromatogram showing nelfinavir mesylate tablets under photolytic stress conditions. Key: (a) photoacid, (b) photobase, (c) photoneutral (d) photosolid samples exposed to 8500 Lx. h fluorescent and 0.05 W/m 2 UV light. Chromatogram showing nelfinavir mesylate tablets under thermal stress condition. Calibration plot for nelfinavir mesylate tablets. Chromatogram showing linezolide (drug) and its degradation products (I-III) contained in a mixture of stress samples. Chromatograms showing linezolid (drug) and its hydrolytic degradation products (II, III). Key: (a) unstressed sample, (b) acid hydrolysis, (c) alkaline hydrolysis, (d) neutral hydrolysis. Chromatogram showing linezolid and its degradation behavior in oxidative stress condition. Chromatogram showing linezolid and its degradation behavior in photolytic conditions. Key: (a) photoacid, (b) photobase, (c) photoneutral (d) photosolid samples exposed to 8500 Lx. h fluorescent and 0.05 W/m 2 UV light. Chromatogram showing of linezolid under thermal stress condition. Calibration plot for linezolid. v

9 List of figures Fig Fig Fig Fig Fig Fig Fig Fig Fig Fig Fig Fig Fig Fig Fig Fig Line spectrum of linezolid obtained in MS/TOF study. Fragmentation pathway of linezolid along with the exact masses of the fragments. Line spectra of degradation products (DP-I-III) obtained in LC-MS/TOF studies. Fragmentation pathway of degradation products (DP-I, DP-II and DP-III). Degradation pathway of linezolid. Chromatogram showing linezolid tablets (drug) and its degradation products (I-III) contained in a mixture of stress samples. Chromatograms showing hydrolytic degradation products (II and III) of linezolid tablets. Key: (a) unstressed sample, (b) acid hydrolysis, (c) alkaline hydrolysis, (d) neutral hydrolysis. Chromatogram showing oxidative degradation product (I) of linezolid tablets. Chromatogram showing linezolid tablets under photolytic stress conditions. Key: (a) photoacid, (b) photobase, (c) photoneutral (d) photosolid samples exposed to 8500 Lx. h fluorescent and 0.05 W/m 2 UV light. Chromatogram showing linezolid tablets under thermal stress condition. Calibration plot for linezolid tablets. Chromatogram showing lacosamide (drug) and its degradation products (I-II) contained in a mixture of stress samples. Chromatograms showing lacosamide and its hydrolytic degradation products. Key: (a) unstressed sample, (b) acid hydrolysis, (c) alkaline hydrolysis, (d) neutral hydrolysis. Chromatogram showing lacosamide and its degradation behavior in oxidative stress condition. Chromatogram showing lacosamide and its photolytic degradation products (I, II). Key: (a) photoacid, (b) photobase, (c) photoneutral (d) photosolid samples exposed to 8500 Lx. h fluorescent and 0.05 W/m 2 UV light. Chromatogram showing lacosamide under thermal stress condition. Fig Calibration plot for lacosamide. vi

10 List of figures Fig Fig Fig Fig Fig Fig Fig Fig Fig Fig Fig Fig Fig Fig Fig Fig Line spectrum of lacosamide obtained in MS/TOF study. Fragmentation pathway of lacosamide along with the exact masses of the fragments. Line spectra of degradation products (DP-I-II) obtained in LC-MS/TOF studies. Fragmentation pathway of DP-I and DP-II. Degradation pathway of lacosamide. Line spectrum of isolated degradation products. Key: (a) DP-I; (b) DP-II. Chromatogram showing lacosamide tablets (drug) and its degradation products (I-II) contained in a mixture of stress samples. Chromatograms showing hydrolytic degradation products of lacosamide tablets. Key: (a) unstressed sample, (b) acid hydrolysis, (c) alkaline hydrolysis, (d) neutral hydrolysis. Chromatogram showing lacosamide tablets under oxidative stress conditions. Chromatogram showing lacosamide tablets under photolytic stress conditions. Key: (a) photoacid, (b) photobase, (c) photoneutral (d) photosolid samples exposed to 8500 Lx. h fluorescent and 0.05 W/m 2 UV light. Chromatogram showing lacosamide tablets under thermal stress condition. Calibration plot for lacosamide tablets. Chromatogram showing ritonavir (drug) and its degradation products (I-X) contained in a mixture of stress samples. Chromatograms showing ritonavir and its hydrolytic degradation products. Key: (a) unstressed sample, (b) acid hydrolysis, (c) alkaline hydrolysis, (d) neutral hydrolysis. Chromatogram showing degradation behavior of ritonavir in oxidative stress condition. Chromatogram showing ritonavir under photolytic stress conditions. Key: (a) photoacid, (b) photobase, (c) photoneutral (d) photosolid samples exposed to 8500 Lx. h fluorescent and 0.05 W/m 2 UV light. Fig Chromatogram showing ritonavir under thermal stress condition. vii

11 List of figures Fig Fig Fig Fig Fig Fig Fig Fig Fig Fig Fig Fig Fig Calibration plot for ritonavir. Line spectrum of ritonavir obtained in MS/TOF studies. Fragmentation pathway of ritonavir along with the exact masses of the fragments. Line spectra of degradation products (DP-II-X) obtained in LC-MS/TOF studies. Fragmentation pathway of DP-II, DP-III, DP-IV, DP-V and DP-X. Fragmentation pathway of DP-VI, DP-VII, DP-VIII and DP-IX. Degradation pathway of ritonavir. Chromatogram showing ritonavir tablets (drug) and its degradation products (I-X) contained in a mixture of stress samples. Chromatograms showing hydrolytic degradation products of ritonavir tablets. Key: (a) unstressed sample, (b) acid hydrolysis, (c) alkaline hydrolysis, (d) neutral hydrolysis. Chromatogram showing oxidative stress degradation products. Chromatogram showing ritonavir tablets under photolytic stress conditions. Key: (a) photoacid, (b) photobase, (c) photoneutral (d) photosolid samples exposed to 8500 Lx. h fluorescent and 0.05 W/m 2 UV light. Chromatogram showing ritonavir tablets under thermal stress condition. Calibration plot for ritonavir tablets. viii

12 List of tables List of Tables Table 1.1. Table 2.1. Table 2.2. Table 2.3. Table 2.4. Table 5.1. Table 5.2. Table 6.1. Table 6.2. Table 6.3. Table 6.4. Table 6.5. Table 6.6. Table 6.7. Table 6.8 Table 6.9. Table Table Table Table Table Techniques employed in stability-indicating method Analytical methods reported in literature for determination of nelfinavir mesylate. Analytical methods reported in literature for determination of linezolid. Analytical methods reported in literature for determination of lacosamide. Analytical methods reported in literature for determination of ritonavir. MS/TOF parameters for nelfinavir mesylate and linezolid in positive ESI mode. MS/TOF parameters for lacosamide and ritonavir in positive ESI mode. Linearity data for nelfinavir mesylate. Intra-day and inter-day precision data for nelfinavir mesylate. Recovery data for nelfinavir mesylate. Peak purity data for nelfinavir mesylate and its degradation products. Interpretation of MS/TOF data of fragments of the drug. MS n fragmentation of nelfinavir mesylate. LC-MS/TOF data of DPs (I-III) along with their possible molecular formulae and major fragments. Stress conditions for optimum degradation. Linearity data for nelfinavir mesylate tablets. Intra-day and inter-day precision data for nelfinavir mesylate tablets. Recovery data for nelfinavir mesylate tablets. Peak purity data for nelfinavir mesylate tablets and its degradation products. Linearity data for linezolid. Intra-day and inter-day precision data for linezolid. ix

13 List of tables Table Table Table Table Table Table 6.20 Table Table Table Table Table Table Table Table Table Table Table Table 6.32 Table Table Table Table Table Recovery data for linezolid. Peak purity data for linezolid and its degradation products. Interpretation of MS/TOF data of fragments of the drug. MS n fragmentation of linezolid. LC-MS/TOF data of DPs (I-III) along with their possible molecular formulae and major fragments. Stress conditions for optimum degradation. Linearity data for linezolid tablets. Intra-day and inter-day precision data for linezolid tablets. Recovery data for linezolid tablets. Peak purity data for linezolid tablets and its degradation products. Linearity data for lacosamide. Intra-day and inter-day precision data for lacosamide. Recovery data for lacosamide. Peak purity data for lacosamide and its degradation products. Interpretation of MS/TOF data of fragments of lacosamide. MS n fragmentation of the drug. LC-MS/TOF data of DPs (I-II) along with their possible molecular formulae and major fragments. Stress conditions for optimum degradation. Linearity data for lacosamide tablets. Intra-day and inter-day precision data for lacosamide tablets. Recovery data for lacosamide tablets. Peak purity data for lacosamide tablets and its degradation products. Linearity data for ritonavir. x

14 List of tables Table Table Table Table Table Table 6.43 Table 6.44 Table Table Table Table Intra-day and inter-day precision data for ritonavir. Recovery data for ritonavir. Peak purity data for ritonavir and its degradation products. Interpretation of MS/TOF data of fragments of ritonavir. MS n fragmentation of the drug. LC-MS/TOF data of DPs (II-X) along with their possible molecular formulae and major fragments. Stress conditions for optimum degradation. Linearity data for ritonavir tablets. Intra-day and inter-day precision data for ritonavir tablets. Recovery data for ritonaivr tablets. Peak purity data for ritonavir tablets and its degradation products. xi