Mutagenic Impurities

Andrew Teasdale, PhD, is a senior principal scientist with AstraZeneca and a member of ICH Q3C, Q3D, Q3E Expert working groups as well as an industry advisor to ICH M7. He received his doctorate in organic chemistry from Durham University. He is the inventor of the purge factor concept applied to risk assessment of mutagenic impurities and has authored over 30 papers on that subject.

• List of Contributors xixPreface xxiSection 1 The Development of Regulatory Guidelines for Mutagenic/Genotoxic Impurities – Overall Process 11 Historical Perspective on the Development of the EMEA Guideline and Subsequent ICH M7 Guideline 3Andrew Teasdale1.1 Introduction 31.1.1 CPMP – Position Paper on the Limits of Genotoxic Impurities –2002 41.1.1.1 Scope/Introduction 41.1.1.2 Toxicological Background 41.1.1.3 Pharmaceutical (Quality) Assessment 41.1.1.4 Toxicological Assessment 41.1.2 Guideline on the Limits of Genotoxic Impurities – Draft June 2004 51.1.3 PhRMA (Mueller) White Paper 61.1.4 Finalized EMA Guideline on the Limits of Genotoxic Impurities – June 2006 81.1.4.1 Issues Associated with Implementation 91.1.4.2 Control Expectations for Excipients 111.1.4.3 Control Expectations for Natural/Herbal Products 121.1.4.4 Identification of Potential Impurities 121.1.4.5 The Principle of Avoidance 121.1.4.6 The ALARP Principle 141.1.4.7 Overall 141.1.5 SWP Q&A Document 141.1.5.1 The Application of the Guideline in the Investigational Phase and Acceptable Limits for GIs Where Applied to Studies of Limited Duration 141.1.5.2 Application of the Guideline to Existing Products 151.1.5.3 Avoidance and ALARP 171.1.5.4 ICH Identification Threshold and its Relation to MI Assessment 171.1.6 FDA Draft Guideline 171.1.7 Other Relevant Guidance 171.1.7.1 Excipients 181.1.8 Herbals 181.1.9 ICH S9 181.1.10 Conclusions 19References 192 ICH M7 – Assessment and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk 21Andrew Teasdale and Raphael Nudelman2.1 Introduction 212.2 ICH M7 222.2.1 Introduction 222.2.2 Scope 222.2.2.1 Established Products 222.2.2.2 Anticancer Treatments 232.2.2.3 Nature of Therapeutic Agent/Excipients 232.2.3 General Principles 242.2.4 Considerations for Marketed Products 252.2.4.1 Post-approval Changes to Drug Substance, Chemistry, and Manufacturing Controls 262.2.4.2 Post-approval Changes to Drug Product Chemistry, Manufacturing, and Controls 262.2.4.3 Changes to the Clinical Use of Drug Products 262.2.5 Other Considerations for Marketed Products 272.2.6 Drug Substance and Drug Product Impurity Assessment 272.2.6.1 Synthetic Impurities 282.2.6.2 Degradation Products 282.2.7 Hazard Assessment 292.2.8 Risk Characterization 322.2.8.1 Acceptable Intakes Based on Compound-specific Risk Assessments 322.2.8.2 Acceptable Intakes for Class 2 and Class 3 Compounds 332.2.8.3 Multiple Impurities 342.2.8.4 Exceptions and Flexibility in Approaches 352.2.9 Control Strategy 352.2.9.1 Considerations for Control Approaches 372.2.9.2 Considerations for Periodic Testing 372.2.9.3 Control of Degradation Products 382.2.10 Lifecycle Management 382.2.11 Documentation 382.2.11.1 Clinical Trail Applications 382.2.11.2 Common Technical Document (Marketing Application) 392.2.12 Other Aspects 392.2.12.1 Relationship Between ICH M7 and ICH Q3A 392.3 Conclusions 402.4 Commentary on ICH M7 Questions and Answers 402.4.1 Section 1 – Introduction 412.4.1.1 Question 1.1 412.4.1.2 Question 1.2 422.4.1.3 Question 1.3 422.4.1.4 Question 1.4 422.4.2 Section 2 – Scope 432.4.2.1 Question 2.1 432.4.3 Section 3 – General Principles 432.4.3.1 Question 3.1 442.4.3.2 Question 3.2 442.4.4 Section 4 – Considerations for Marketed Products 442.4.4.1 Question 4.1 452.4.5 Section 5 – Drug Substance and Drug Product Impurity Assessment 452.4.6 Section 6 – Hazard Assessment Elements 452.4.6.1 Question 6.1 452.4.6.2 Question 6.2 462.4.6.3 Question 6.3 472.4.6.4 Question 6.4 482.4.7 Section 7 – Risk Characterization 482.4.7.1 Question 7.1 482.4.7.2 Question 7.2 492.4.7.3 Question 7.3 492.4.7.4 Question 7.4 502.4.7.5 Question 7.5 512.4.8 Section 9 – Documentation 53References 553 Control Strategies for Mutagenic Impurities 57Andrew Teasdale, Michael Burns, and Michael Urquhart3.1 Introduction 573.2 Assessment Process 583.2.1 General 583.2.2 Step 1 – Evaluation of Drug Substance and Drug Product Processes for Sources of Potentially Mutagenic Impurities 603.2.3 Step 2 – Structural Assessment 613.2.4 Step 3 – Classification 613.2.5 Step 4 – Assessment of Risk of Potential Carryover of Impurities 633.2.6 Overall Quantification of Risk 633.2.6.1 Predicted Purge Factor 643.2.6.2 Required Purge Factor 653.2.6.3 Purge Ratio 663.2.6.4 High Predicted Purge 673.2.6.5 Moderate Predicted Purge 673.2.6.6 Low Predicted Purge 673.2.6.7 ICH M7 Control Option 1, 2, or 3 673.2.6.8 Step 5 – Further Evaluation 673.2.6.9 Safety Testing 673.2.7 Quantification of Level Present 683.3 Step 6 – Overall Risk Assessment 693.4 Further Evaluation of Risk – Purge (Spiking) Studies 703.5 Conclusion 703.6 Case Studies 713.6.1 Case Study 1 – GW641597X 713.6.1.1 Ethyl Bromoisobutyrate 2 733.6.1.2 Hydroxylamine 743.6.1.3 Alkyl Chloride 8 753.6.1.4 Additional Evidence for the Purging of Ethyl Bromoisobutyrate and Alkyl Chloride 8 763.6.2 Proposed ICH M7-aligned Potential Mutagenic Control Regulatory Discussion 783.6.3 Case Study 2 – Candesartan 78References 84Section 2 In Silico Assessment of Mutagenicity 874 Use of Structure–Activity Relationship (SAR) Evaluation as a Critical Tool in the Evaluation of the Genotoxic Potential of Impurities 89Catrin Hasselgren and Glenn Myatt4.1 Introduction 894.2 (Q)SAR Assessment 904.2.1 Looking-up Experimental Data 904.2.2 (Q)SAR Methodologies 914.2.2.1 Overview 914.2.2.2 OECD Validation Principles 914.2.3 Expert Rule-Based Methodology 924.2.4 Statistical-Based Methodology 954.2.5 Applying (Q)SAR Models 974.2.6 Expert Review 984.2.6.1 Overview 984.2.6.2 Refuting a Statistical-Based Prediction 1004.2.6.3 Mechanistic Assessment 1014.2.6.4 Assessing Lack of Chemical Reactivity 1014.2.7 Class Assignment 1034.2.7.1 Overview 1034.2.8 Documentation 1094.3 Discussion 1094.4 Conclusions 110Acknowledgments 111References 1115 Evolution of Quantitative Structure–Activity Relationships ((Q)SAR) for Mutagenicity 115James Harvey and David Elder5.1 Introduction 1155.2 Pre ICH M7 Guideline 1165.3 Post ICH M7 1175.3.1 Evolution of (Q)SAR Platforms 1175.3.2 Robust Negative In Silico (Q)SAR Predictions 1185.3.3 Development of Composite (Q)SAR Models 1195.3.4 Expansion of Training Data Sets to Enhance the Predictive Power of (Q)SAR Tools 1205.3.5 Focused Data Sharing Initiatives on Specific Chemical Classes 1205.3.5.1 Understanding In Vitro Mechanisms Leading to Mutagenicity 1215.3.5.2 Shared Data, Shared Progress 1225.3.6 Novel Data Mining Approaches 1255.3.6.1 Case Study: Primary Aromatic Amines (PAAs) 1255.3.6.2 Case Study: Aromatic N-oxides 1255.4 Expert Knowledge 1275.5 Future Direction 129References 131Section 3 Toxicological Perspective on Mutagenic Impurities 1376 Toxicity Testing to Understand the Mutagenicity of Pharmaceutical Impurities 139Andrew Teasdale, John Nicolette, Joel P. Bercu, James Harvey, Stephen Dertinger, Michael O’Donovan, and Christine Mee6.1 Introduction 1396.2 In Vitro Genotoxicity Tests 1416.2.1 Background 1416.2.2 Bacterial Reverse Mutation or “Ames” Test 1426.2.3 Modifications to the Standard Ames Test 1456.2.3.1 Six-well Ames Assay 1466.2.4 Test Strategy 1466.3 In Vivo Mutation Assays 1486.3.1 In Vivo Pig-a Gene Mutation Assay 1486.3.2 Rodent Micronucleus Test 1526.3.3 Rodent “Comet” Assay 1556.3.4 Transgenic Rodent (TGR) Mutation Assay 1556.4 Conclusions 158Glossary 159References 1607 Compound-and Class-Specific Limits for Common Impurities in Pharmaceuticals 165Joel P. Bercu, Melisa J. Masuda-Herrera, Alejandra Trejo-Martin, David J. Snodin, Kevin P. Cross, George E. Johnson, James Harvey, Robert S. Foster, David J. Ponting, and Richard V. Williams7.1 Introduction 1657.2 Monograph Development 1677.2.1 Exposure to the General Population 1677.2.2 Mutagenicity/Genotoxicity 1707.2.3 Noncarcinogenic Effects 1707.2.4 Carcinogenic Effects 1707.2.5 Mode of Action (MOA) and Assessment of Human Relevance 1717.2.6 Toxicokinetics 1717.2.7 Regulatory/Published Limits 1717.3 Derivation of the Compound-specific Limit 1717.3.1 PoD Selection 1727.3.2 Limited Data Sets 1727.3.3 PDE Development 1727.3.4 AI Development 1727.3.5 Class-specific Limit 1737.3.6 Less than Lifetime (LTL) AIs 1737.4 Examples of Published Compound-specific Limits 1737.4.1 Mutagenic Carcinogens 1737.4.2 Nonmutagenic Carcinogens 1767.4.3 Mutagenic Noncarcinogens 1767.4.4 Nonmutagenic Compounds 1767.4.5 Mutagenic In vitro but not In vivo 1767.4.6 Route of Administration-specific Limits 1777.5 Class-specific Limits 1777.5.1 Alkyl Chlorides 1777.5.2 Alkyl Bromides 1787.5.3 N-Nitrosamines 1787.5.3.1 Regulatory Limits for N-Nitrosamines 1787.5.3.2 Additional Proposed Limits for N-Nitrosamines 1807.5.3.3 N-Nitrosamine Exposure in the General Population 1817.5.3.4 Developing a Class-specific Limit for N-Nitrosamines 1827.5.4 Arylboronic Acids and Esters 1937.6 EMS Case Study and Updated Toxicity Analysis 1967.6.1 Potential for Human Exposure 1967.6.2 Mutagenicity/Genotoxicity 1967.6.3 Noncarcinogenic Effects 1987.6.4 Carcinogenicity 1997.6.5 Regulatory and/or Published Limits 1997.6.6 Permitted Daily Exposure 1997.7 Extractables and Leachables 2027.8 Lhasa AI/PDE Database for Impurities 2037.9 Conclusions and Future Directions 203Acknowledgments 204References 2048 Genotoxic Threshold Mechanisms and Points of Departure 213George E. Johnson, Shareen H. Doak, Gareth J.S. Jenkins, and Adam D. Thomas8.1 Introduction to Genotoxic Dose Responses 2138.1.1 The Linear Default Position for Genotoxic Carcinogens 2138.1.2 Theoretical Evidence for Rejecting the Linear Approach 2148.1.3 In Vitro Experimental Evidence for Threshold Mechanism 2158.1.4 In Vivo Evidence for Genotoxic Thresholds 2188.2 Threshold Mechanisms 2218.2.1 Statistical Assessment of Dose Response Data Sets 2248.2.2 Extrapolation from One Chemical to Another 2248.2.3 Extrapolation of Threshold Mechanisms and PoDs to Populations 2258.3 Conclusions 227References 227Section 4 Quality Perspective on Genotoxic Impurities 2339 Mutagenic Impurities – Assessment of Fate and Control Options 235Michael W. Urquhart, Andrew Teasdale, and Michael Burns9.1 Introduction/Background 2359.2 Reactivity 2369.2.1 Reactivity Classification 2389.3 Solubility – Isolated Stages 2389.4 Recrystallization 2399.4.1 Solubility – Liquid/Liquid Partitioning 2399.5 Volatility 2419.6 Chromatography 2419.7 Other Techniques 2429.7.1 Activated Charcoal 2429.7.2 Scavenger Resins 2429.8 Overall Quantification of Risk 2439.9 Alignment to ICH M7 – Control Options 2449.10 Control Option Selection 2479.10.1 Predicted Purge Factor 2489.10.2 Required Purge Factor 2499.10.3 Purge Ratio 2499.10.4 High Predicted Purge 2509.10.5 Moderate Predicted Purge 2509.10.6 Low Predicted Purge 2509.10.7 ICH M7 Control Option 1, 2, or 3 2519.10.8 Representative Data to be Supplied in Regulatory Submission Under an ICH M7 Control Strategy 2519.10.9 Summary of PMI Purging Across the Synthetic Route 2519.10.10 Details of Individual Impurity Purging Through the Subsequent Downstream Chemistry 2539.10.11 Development of a Knowledge Base Expert In Silico System 2549.10.12 Experimental Work to Assess Reactivity 2579.11 Utilizing Mirabilis for a Purge Calculation 2599.11.1 Utility of In Silico Predictions 2609.11.1.1 Case Study – Camicinal [38] 260References 26610 N-Nitrosamines 269Andrew Teasdale, Justin Moser, J. Gair Ford, and Jason Creasey10.1 Background 26910.2 Generation of N-Nitrosamines 27010.3 Article 31 27310.4 Further Issues – Cross Contamination and Ranitidine 27510.4.1 Article 5(3) and Associated Q&A Document 27610.5 How to Assess the Risk Posed in Pharmaceuticals 27810.5.1 Drug Substance 27810.5.1.1 Where do Nitrites Come Within Drug Substance Come From? 27810.5.1.2 What Other Sources Are There? 27810.5.1.3 Other Factors Associated with Drug Substance Synthesis 28010.5.2 Process to Assess Drug Substance-Related Risk 28010.5.3 Drug Product-Related Risk 28210.5.3.1 Related Risks of Contamination and Formation in Drug Products 28210.5.4 Container Closure Systems 28910.5.5 Elastomeric Components 29110.5.6 Nitrosamine Impurities in Biologics 29310.5.6.1 Active Substance 29310.5.6.2 The Water Used in Formulation Is Depleted in Nitrosating Agents 29510.5.6.3 Bioconjugated or Chemically Modified Products 29510.5.6.4 Excipients 29610.6 Regulatory Guidance Pursuant to N-Nitrosamines and its Implications 29710.6.1 Article 31 Process and Outcomes 29710.6.1.1 Article 31 Request 29710.6.2 Sartans Lessons Learnt Report 29810.6.2.1 Reflection on the Initial Section of the EMA Report 29910.6.3 Article 5(3) Report 29910.6.3.1 Quality 29910.6.3.2 Consideration for Analytical Method Development to Identify and Quantify N-Nitrosamines in Drug Substances and Medicinal Products 30010.6.3.3 Safety 30110.6.3.4 Conclusions 30510.6.4 EMA Question and Answer Document [6] 30510.6.4.1 Further Revision of the EMA Question and Answer Document 31010.6.5 FDA Guideline 31010.6.5.1 Introduction and Background 31010.6.5.2 Recommendations 31010.6.5.3 Acceptable Intakes (section III.A) 31310.6.5.4 Quality/Chemistry and Controls 31410.7 Way Forward 315Acknowledgments 316References 31711 Conditions Potentially Leading to the Formation of Mutagenic Impurities 321Lucie Lovelle, Andrew Teasdale, Ian Ashworth, Adrian Clarke, and Alan Steven11.1 Problematic Reagent Combinations per Structural Alert 32311.1.1 N-Nitroso Compounds (COC) 32311.1.1.1 Amines and Nitrosating Agents [10] 32311.1.1.2 Amine Derivatives and Nitrosating Agents 32411.1.1.3 Other 32411.1.2 Alkyl-azoxy Compounds (COC) 32511.1.2.1 Reduction [52–54] 32511.1.2.2 Oxidation 32511.1.2.3 Others 32511.1.3 Other N-O Compounds 32611.1.3.1 Reduction of Nitro Groups 32611.1.3.2 Oxidation of Amines and Hydroxylamines 32611.1.4 Nitration 32611.1.5 Other N-N Compounds [59, 60] 32611.1.6 Aflatoxin-like Compounds [62] (COC) 32711.1.7 Dioxin-like Compounds (Including Polychlorinated Biphenyls = PCBs) [63] 32711.1.8 Alkyl and Acyl Halides 32711.1.8.1 ROH + HCl → RCl + H2O 32711.1.8.2 Ether Opening with Halides 32811.1.9 Methyl Sulfoxides and Pummerer Rearrangement 32811.1.10 Acyl Chlorides Formation [82] 32911.1.11 Halogenation of Unsaturated Compounds 32911.1.12 Ammonium Salts (Hofmann Elimination) 32911.1.12.1 Alkyl Sulfonates [90] 32911.1.13 Epoxides and Aziridines [95–97] 33011.2 Miscellaneous 33111.2.1 B and P Based Compounds 33111.2.2 Formation of N-Methylol 33111.2.3 Acetamide 33211.2.4 Quinones and Quinone Derivatives 33211.2.5 Anilines [100] 33211.2.6 Michael Acceptors 33311.2.7 Others 33311.3 Mechanism and Processing Factors Affecting the Formation of N-nitrosamines 33311.3.1 Introduction 33311.3.2 Mechanisms of Amine Nitrosation 33311.3.2.1 Nitrosation of Secondary Amines 33311.3.2.2 Aqueous Nitrosation 33411.3.2.3 Nitrosation in Organic Solvents 33611.3.3 Nitrosation of Tertiary Amines 33711.3.3.1 Nitrosation of Quaternary Amines 33711.3.3.2 Nitrosation of Amine Oxides 33811.3.4 Sources of Nitrosating Agents 33811.3.4.1 Process Water 33811.3.4.2 Nitric Acid 33911.3.4.3 Atmospheric Sources 33911.3.4.4 Excipients Used in Drug Product Manufacture 34011.3.4.5 Nitrocellulose 34011.3.4.6 Nitrosating Agent Scavengers 34011.3.4.7 Removal of Nitrosamines 34111.4 Formation, Fate, and Purge of Impurities Arising from the Hydrogenation of Nitroarenes to Anilines 34111.4.1 Primary Reaction Mechanism 34111.4.2 Mass and Heat Transfer Effects 34211.4.3 Condensation Chemistry 34411.4.4 Factors Affecting Aryl Hydroxylamine Accumulation 34611.4.5 Aryl Hydroxylamine Control 34711.4.5.1 Use of Cocatalysts 34711.4.5.2 Physical Adsorption 34811.4.5.3 Kinetic Understanding Around Formation and Consumption 34911.4.5.4 Holistic Control of Impurity Profile 34911.4.6 Controlling Residual Nitroarene 35111.4.7 Specific Considerations of Alkyl Nitro Reductions 35311.4.8 Closing Comments on Hydrogenation of Nitroarenes to Anilines 35311.5 Mechanism and Processing Parameters Affecting the Formation of Sulfonate Esters – Summary of the PQRI Studies 35311.5.1 Introduction 35311.5.2 Reaction Mechanism 35511.5.3 Experimental Results 35711.5.3.1 Experimental Results from Study of the Ethyl Methanesulfonate (EMS) System 35711.5.3.2 Other Methanesulfonic Acid Systems 35911.5.3.3 Experimental Results from Study of the Isopropyl Methanesulfonate (IMS) System 36011.5.4 Experimental Results from Study of Toluenesulfonic (Tosic) Acid Systems 36111.5.4.1 Experimental Results from Study of the Ethyl Tosylate (ETS) System 36211.5.4.2 Kinetic Modeling 36311.5.4.3 Key Learnings and Their Implications for Process Design 36511.5.4.4 Processing Rules 36611.5.5 What About Viracept™? 36611.5.6 What About Other Sources of Sulfonate Esters? 36711.5.7 Potential for Ester Formation in the Solid Phase 36811.5.8 Conclusions 369References 36912 Strategic Approaches to the Chromatographic Analysis of Mutagenic Impurities 381Frank David, Gerd Vanhoenacker, Koen Sandra, Pat Sandra, Tony Bristow, and Mark Harrison12.1 Introduction 38112.2 Method Development and Validation 38412.3 Analytical Equipment for Mutagenic Impurity Analysis 38512.4 Alkyl Halides and Aryl Halides 38812.4.1 Method Selection 38812.4.2 Typical Conditions Used for Alkyl-and Aryl Halide Analysis by SHS-GC-MS and SPME-GC-MS 39012.4.2.1 Sample Preparation 39012.4.2.2 GC-MS Parameters 39112.4.3 Typical Results Obtained for Alkyl-and Aryl Halide Analysis by SHS-GC-MS and SPME-GC-MS 39112.5 Sulfonates 39312.5.1 Method Selection 39312.5.2 Typical Conditions Used for Sulfonate Analysis by Derivatization SHS-GC-MS 39412.5.2.1 Sample Preparation 39512.5.2.2 Synthesis of Deuterated Internal Standards 39512.5.2.3 GC-MS Parameters 39512.5.3 Typical Results Obtained Using Derivatization – SHS – GC-MS 39512.5.4 Confirmation Analysis by PTV-GC-MS 39612.6 S-and N-mustards 39812.6.1 Method Selection 39812.6.2 Typical Analytical Conditions for the Analysis of N-mustards by Derivatization – SPME-GC-MS 39912.6.2.1 Sample Preparation 39912.6.3 Typical Results for N-mustards by Derivatization – SPME-GC-MS 39912.7 Michael Reaction Acceptors 40012.7.1 Method Selection 40012.7.2 Typical Analytical Conditions for Michael Reaction Acceptors 40012.7.2.1 Sample Preparation 40112.7.2.2 Parameters for SHS-GC-MS 40112.7.2.3 Parameters for Liquid Injection and GC-MS with Back-flush 40212.7.3 Typical Results Obtained for Trace Analysis of Michael Reaction Acceptors 40212.7.3.1 SHS with PTV 40212.7.3.2 Liquid Injection GC-MS 40312.8 Epoxides 40412.8.1 Method Selection 40412.8.2 Typical Analytical Conditions for the Analysis of Volatile Epoxides by SHS-GC-MS 40612.8.2.1 Sample Preparation 40612.8.2.2 SHS-GC-MS Parameters 40612.8.3 Typical Results Obtained for Volatile Epoxides Using SHS-GC-MS 40712.9 Haloalcohols 40712.9.1 Method Selection 40712.9.2 Analytical Conditions for Trace Analysis of Halo-alcohols by Derivatization and Liquid Injection - 2DGC-MS 40912.9.2.1 Sample Preparation 40912.9.2.2 2D-GC-MS Parameters 41012.9.3 Typical Results for Analysis of Halo-alcohols by Derivatization and Liquid Injection - 2DGC-MS 41012.10 Aziridines 41112.10.1 Method Selection 41112.10.2 Typical Analytical Conditions for RPLC-MS and HILIC-MS Analysis of Aziridines 41212.10.2.1 Sample Preparation 41212.10.2.2 RPLC-MS Method Parameters 41312.10.2.3 HILIC-MS Method Parameters 41312.10.3 Typical Results Obtained for Aziridine Analysis Using RPLC and HILIC 41312.11 Arylamines and Amino Pyridines 41412.11.1 Method Selection 41412.11.2 Typical Analytical Conditions for Arylamines and Aminopyridines by RPLC-MSD 41512.11.2.1 Sample Preparation 41512.11.2.2 HPLC-MS Parameters 41612.11.3 Typical Results for Arylamines and Aminopyridines by RPLC-MSD 41712.12 Hydrazines and Hydroxylamine 41912.12.1 Method Selection 41912.12.2 Analytical Conditions for the Analysis of Hydrazines Using Derivatization and HPLC-MS 42012.12.2.1 Sample Preparation 42112.12.2.2 HPLC-MS Parameters 42112.12.3 Typical Results Obtained for Hydrazines Using Derivatization LC-MS 42112.13 Aldehydes and Ketones 42312.13.1 Method Selection 42312.13.2 Typical Analytical Conditions for Analysis of Aldehydes and Ketones by DNPH Derivatization, Followed by LC-MS Analysis 42312.13.2.1 Sample Preparation 42412.13.2.2 Derivatization Reagent Solution 42512.13.2.3 HPLC-MS Parameters 42512.13.3 Typical Results Obtained for Aldehyde Analysis by DNPH Derivatization – LC-MS 42612.14 Nitrosamines 42612.14.1 Method Selection 42612.14.2 Sample preparation for SHS-GC-MS Analysis (according to ref [85]) 42812.14.2.1 SHS-GC-MS Analysis [85] Sample Preparation 42812.14.2.2 GC-MS (HRAM-MS) Conditions 42812.14.2.3 UHPLC-MS Analysis 42912.14.2.4 Sample Preparation for Hydrophilic Samples (e.g. Metformin) 42912.14.2.5 Sample Preparation for Hydrophobic Matrices 43012.14.2.6 UHPLC Conditions 43012.14.2.7 HRAM-MS and MS/MS Conditions 43012.14.3 Typical Results Obtained for Volatile N-nitrosamines Using SHS-GC-MS 43012.14.4 Typical Results Obtained for N-nitrosamines Using LC-MS 43112.15 Nontarget Analysis of PMI/MIs 43412.16 Conclusions 435Acknowledgements 436References 43613 Analysis of Mutagenic Impurities by Nuclear Magnetic Resonance (NMR) Spectroscopy 439Andrew R. Phillips and Stephen Coombes13.1 Introduction to NMR 43913.2 Why Is NMR an Insensitive Technique? 43913.2.1 Nuclear Spin 43913.2.2 Boltzmann Distribution 44013.3 How Could NMR Be Used for Trace Analysis? 44013.3.1 Generating an NMR Spectrum 44013.3.2 Chemical Shift 44213.3.3 Scalar Coupling 44313.3.4 The Quantitative Nature of NMR 44413.3.5 Relaxation 44513.3.6 Summary 44613.4 What Can Be Done to Maximize Sensitivity? 44613.4.1 System Performance 44713.4.1.1 Field Strength 44713.4.2 Probe Performance 44713.4.2.1 Probe Design 44713.4.2.2 Probe Diameter 44813.4.2.3 Cryogenically Cooled Probes 44813.4.3 Substrate Concentration 44913.4.4 Molecular Weight Ratio 45113.4.5 Acquisition Time and Signal Averaging 45113.4.6 Number of Protons and Linewidth 45313.4.7 Resolution 45513.4.8 Dynamic Range 45513.4.8.1 Selective Excitation 45813.4.8.2 Shaped Pulses 45813.4.8.3 Quantification Using Selective Pulses 46013.4.8.4 Excitation Sculpting 46113.4.9 Limit Tests 46113.4.9.1 Method Development 46213.4.9.2 Validation 46313.4.9.3 Unresolved Signals 46313.4.9.4 Rapid Analysis 46413.4.10 Expanded Use of MI NMR Methodology 46413.4.11 Summary 46413.5 Case Studies 46413.5.1 Case Study 1 – An Aldehyde Functionalized MI 46413.5.2 Case Study 2 – Use of 19F NMR 46613.5.3 Case Study 3 – Epoxide and Chlorohydrin MIs 46813.5.4 Case Study 4 – Sulfonate Esters 46913.5.5 Case Study 5 – Limit Test for Poorly Resolved Signals 47013.5.6 Case Study 6 – Using NMR MI Methodology for Cleaning Validation 47213.6 Conclusion 473References 47514 Addressing the Complex Problem of Degradation-Derived Mutagenic Impurities in Drug Substances and Products 477Steven W. Baertschi and Andrew Teasdale14.1 Introduction 47714.1.1 Background 47714.2 Working Definitions 47814.3 Challenges Associated with the Assessment of Risk Posed by (Potentially) Mutagenic Degradation Products 47914.4 Risk Assessment Process for Mutagenic Degradants 47914.4.1 Stability-Related MRA Process Overview 47914.4.2 Stress Studies 48014.4.3 Accelerated Stability Studies 48014.4.4 Long-term ICH Stability Studies 48114.4.5 Deciding Which Products to Include in the MRA 48114.4.6 In Silico Tools for the Prediction of Potential Degradation Products 48214.5 Using Stress Testing to Select Degradation Products for Identification 48214.5.1 Approach 1: Criteria for Structure Identification After Observation in Accelerated and Long-term Stability Studies 48314.5.2 Approach 2: Criteria for Structure Identification Through Use of an Algorithm in Stress Testing Studies 48314.5.3 Approach 3: Structure Identification Through Use of Kinetic Equivalence and Scaled ICH Q3B Thresholds 48514.5.3.1 Kinetic Equivalence 48514.5.3.2 Scaled ICH Q3B Thresholds 48614.6 Development Timeline Considerations 48714.6.1 Drug Discovery Stage 48714.6.2 Preclinical to Phases 1/2 48714.6.3 Phase 3 to New Drug Application (NDA) Regulatory Submission 48814.6.4 Post-marketing/Line Extensions 48814.7 Developing Control Strategies for (Potential) Mutagenic Degradation Products 48814.7.1 Determining Relevancy of Potential Degradation Products and Developing Control Strategies for Actual Degradation Products 48814.7.2 Accelerated Stability (40 °C/75% RH Six months) or Kinetic Equivalent 48914.7.3 Photostability Studies 48914.7.4 Degradation Chemistry Knowledge 49014.8 Risk Assessment Process Illustrated 49114.8.1 Case Study #1: Molecule A 49114.8.2 Case Study #2: Galunisertib 49214.8.3 Case Study #3: Naloxegol 49414.8.4 Case Study #4: Selumetinib Side Chain 49614.9 Significance of the Risk of Forming Mutagenic Degradation Products 49814.9.1 Frequency of Alerting Structures in Degradation Products 49814.10 Degradation Reactions Leading to Alerting Structures in Degradation Products 49914.10.1 Frequency of Alerting Structures Giving Rise to Ames Positive Tests 50314.10.2 Mutagenic Degradation Products: Overall Predicted Frequency 50314.11 N-Nitrosamines: Special Considerations 50314.11.1 Evaluation of Potential Formation of N-Nitrosamines in Drug Product 50414.12 Conclusions 506References 507Index 513