List of Contributors xvi Preface xx List of Abbreviations xxii Part I Current Sample Preparation Techniques in LC‐MS Bioanalysis 1 1 Basic Sample Preparation Techniques in LC‐MS Bioanalysis: Protein Precipitation, Liquid–Liquid Extraction, and Solid‐Phase Extraction 3 Wenkui Li, Wenying Jian, and Yunlin Fu 1.1 Introduction 3 1.2 Physicochemical Properties of Drugs and Their Metabolites 4 1.3 Pre‐analytical Variables of Analyte(s) of Interest in Biological Matrix 5 1.4 Most Commonly Used Sample Preparation Methods in LC‐MS Bioanalysis 7 References 27 2 Online Extraction and Column Switching Techniques in LC‐MS Bioanalysis 31 Yan Mao and Mike (Qingtao) Huang 2.1 Introduction 31 2.2 System Configuration 32 2.3 Commonly Used Online Extraction Techniques 33 2.4 Considerations for Utilizing Online Extraction Techniques 40 2.5 Summary 41 References 42 3 Equilibrium Dialysis, Ultracentrifugation, and Ultrafiltration in LC‐MS Bioanalysis 45 Theo de Boer and Henri Meijering 3.1 Introduction 45 3.2 Challenges and Considerations 46 3.3 Experimental Procedures 46 3.4 Summary 49 References 51 4 Phospholipid Depletion Techniques in LC‐MS Bioanalysis 52 Stacy Brown and Jennifer Carmical 4.1 Introduction 52 4.2 Impact of Phospholipids on Bioanalytical Methods 52 4.3 Investigating Matrix Effects Associated with Phospholipids 55 4.4 Minimizing Matrix Effects Associated with Phospholipids 56 4.5 Removing Phospholipids Prior to LC‐MS Analysis 57 4.6 Example Methods that Demonstrate Successful Phospholipid Removal 60 4.7 Conclusions 63 Acknowledgement 64 References 64 5 Salting‐out Assisted Liquid–Liquid Extraction (SALLE) in LC‐MS Bioanalysis 68 Jun Zhang and Xin Xiong 5.1 Introduction 68 5.2 Considerations in Developing a SALLE Method 68 5.3 Combination of SALLE with Other Extraction Techniques 72 5.4 Matrix Effect in SALLE 72 5.5 Miniaturization and Automatization 73 5.6 Summary 73 References 73 6 Supported Liquid Extraction (SLE) in LC‐MS Bioanalysis 76 Zhongzhe Cheng and Hongliang Jiang 6.1 Introduction 76 6.2 Principle of SLE 76 6.3 Advantages and Limitation of SLE in Quantitative LC‐MS Bioanalysis 77 6.4 Key Consideration in Developing Robust SLE‐LC‐MS Bioanalytical Method 79 6.5 Representative Protocols 80 6.6 Summary 81 References 82 7 Immunocapture in LC‐MS Bioanalysis 85 Ang Liu 7.1 Introduction 85 7.2 Experimental Workflow and Optimization 85 7.3 Considerations on the Selection of Capture Reagents and the Limitations 86 7.4 Platforms for Immunocapture 89 7.5 Internal Standard Selection 89 7.6 Performance Evaluation 90 7.7 Applications and Representative Protocols 91 7.8 Validation Criteria and Regulatory Considerations 94 7.9 Summary 95 References 95 8 Microextraction Techniques in LC‐MS Bioanalysis 98 Marcio Rodrigues, Ana Fortuna, Amilcar Falcao, and Gilberto Alves 8.1 Introduction 98 8.2 Solid‐Phase Microextraction 99 8.3 Liquid‐Phase Microextraction 108 8.4 Summary 113 Acknowledgements 113 References 113 9 Microsampling Applications with LC‐MS Bioanalysis 117 Chester L. Bowen and Matthew Barfield 9.1 Introduction 117 9.2 Plasma Microsampling Considerations 118 9.3 Dried Blood (Matrix) Spot (DBS) Considerations 121 9.4 Volumetric Absorptive Microsampling (VAMS) 123 9.5 Emerging Techniques 125 9.6 Summary 126 Acknowledgements 126 References 126 10 Nanomaterials for Sample Preparation in LC‐MS Bioanalysis 128 Rodrigo A. Gonzalez‐Fuenzalida, Neus Jornet‐Martinez, Rosa Herraez‐Hernandez, and Pilar Campins‐Falco 10.1 Introduction 128 10.2 Carbon Nanomaterials 128 10.3 Metallic NPs 133 10.4 Nanoporous Materials 135 10.5 Future Perspectives 136 Acknowledgements 136 References 137 11 Sample Preparation via Molecularly Imprinted Polymers (MIPs) in LC‐MS Bioanalysis 139 Myriam Diaz‐Alvarez and Antonio Martin‐Esteban 11.1 Introduction 139 11.2 Preparation of MIPs 139 11.3 MIPs for Sample Preparation in Bioanalysis 142 11.4 Fragment Imprinting 147 11.5 Summary 147 References 147 12 Stir‐bar Sorptive Extraction for Sample Preparation in LC‐MS Bioanalysis 152 Maria Kechagia, Maria Kissoudi, and Victoria F. Samanidou 12.1 Introduction 152 12.2 SBSE Principle 153 12.3 SBSE Steps 154 12.4 Derivatization 155 12.5 Coating Materials 156 12.6 Applications 158 12.7 Summary 161 References 161 13 Monolithic Spin Column Extraction in LC‐MS Bioanalysis 163 Akira Namera and Takeshi Saito 13.1 Introduction 163 13.2 History of Monoliths 163 13.3 The Use of Monolith as Sorbent in Solid‐Phase Extraction 164 13.4 Monolithic Spin Column for Sample Preparation 165 References 171 14 Aptamer‐based Sample Preparation in LC‐MS Bioanalysis 174 Fuyou Du, Zongyu Wei, Qiulian Zeng, and Guihua Ruan 14.1 Introduction 174 14.2 Aptamer‐based Sample Preparation 175 14.3 Representative Protocols 181 14.4 Summary 185 Acknowledgements 185 References 185 15 Sample Extraction via Electromembrane in LC‐MS Bioanalysis 188 Maria Ramos‐Payan, Rut Fernandez‐Torres, and Miguel Angel Bello‐Lopez 15.1 Introduction 188 15.2 Factors Affecting the Extraction Efficiency of EME 190 15.3 Recent Developments in EME 191 15.4 Bioanalytical Applications 193 15.5 Summary 197 References 197 Part II Matrix‐specific Sample Preparation Techniques in LC‐MS Bioanalysis 201 16 Tissue Sample Preparation in LC‐MS Bioanalysis 203 Hong Gao and John Williams 16.1 Introduction 203 16.2 Selection of Homogenization Method 203 16.3 Common Protocols 209 16.4 Protocols for Special Tissue Sample Preparation 210 16.5 Challenges Associated with Tissue Homogenization 212 16.6 Summary 214 References 214 17 Sample Preparation for LC‐MS Bioanalysis of Peripheral Blood Mononuclear Cells 217 Yunlin Fu and Wenkui Li 17.1 Introduction 217 17.2 Peripheral Blood Mononuclear Cells (PBMCs) 217 17.3 Sample Preparation Workflow for LC‐MS Bioanalysis of PBMC Samples 217 17.4 Representative Protocols 220 17.5 Summary 222 References 222 18 Sample Preparation for LC‐MS Bioanalysis of Urine, Cerebrospinal Fluid, Synovial Fluid, Sweat, Tears, and Aqueous Humor Samples 225 Allena J. Ji 18.1 Introduction 225 18.2 Sample Preparation Methods for Urine 225 18.3 Sample Preparation Methods for Cerebrospinal Fluid 230 18.4 Sample Preparation Methods for Synovial Fluid 231 18.5 Sample Preparation Methods for Sweat 231 18.6 Sample Preparation Methods for Tears 234 18.7 Sample Preparation Methods for Aqueous Humor 236 18.8 Summary 236 References 236 19 Sample Preparation for LC‐MS Bioanalysis of Liposomal Samples 238 Wenying Jian, Rao N.V.S. Mamidi, and Naidong Weng 19.1 Introduction 238 19.2 Major Types of Sample Extraction Techniques for Liposomal Samples 239 19.3 Key Considerations in Sample Preparation for Liposomal Samples 242 19.4 Typical Protocols 245 19.5 Summary 246 References 246 Part III Sample Preparation Techniques for LC‐MS Bioanalysis of Challenging Molecules 249 20 Key Pre‐analytical Considerations in LC‐MS Bioanalysis 251 Fumin Li, Bruce J. Hidy, and Rand G. Jenkins 20.1 Introduction 251 20.2 The Pre‐analytical Phase 251 20.3 Bioanalytical Evaluation‐planning 253 20.4 Common Pre‐analytical Issues in LC‐MS Bioanalysis 255 20.5 Summary 258 References 259 21 Derivatization in Sample Preparation for LC‐MS Bioanalysis 260 Dafang Zhong and Yunting Zhu 21.1 Introduction 260 21.2 Derivatization Strategies 260 21.3 Key Considerations for Derivatization 264 21.4 Application of Derivatization for Quantitative LC‐MS Bioanalysis 265 21.5 Summary 270 References 271 22 Sample Preparation for LC‐MS Bioanalysis of Lipids 275 Samuel Hofbauer, Ashkan Salamatipour, Ian A. Blair, and Clementina Mesaros 22.1 Introduction 275 22.2 Sample Preparation for LC‐MS Bioanalysis of Lipids 275 22.3 Case Studies of LC‐MS Bioanalysis of Lipids 280 22.4 Summary 281 References 282 23 Sample Preparation for LC‐MS Bioanalysis of Peptides 284 Long Yuan 23.1 Introduction 284 23.2 Properties of Peptides and Sample Pretreatment 285 23.3 Sample Preparation Strategies 287 23.4 Conclusions 296 Acknowledgements 297 References 297 24 Sample Preparation for LC‐MS Bioanalysis of Proteins 304 Nico van de Merbel 24.1 Introduction 304 24.2 Intact Versus Digested Protein Analysis 305 24.3 Enzymatic Digestion 306 24.4 Protein Depletion 308 24.5 Protein Extraction (Before Digestion) 308 24.6 Peptide Extraction (After Digestion) 312 24.7 Combined Protein and Peptide Extraction 314 24.8 Summary 314 References 315 25 Sample Preparation for LC‐MS Bioanalysis of Oligonucleotides 319 Michael G. Bartlett, Jaeah Kim, Babak Basiri, and Ning Li 25.1 Introduction 319 25.2 Properties of Oligonucleotides and Associated Challenges in LC‐MS Bioanalysis 319 25.3 Classes of Oligonucleotides 322 25.4 Major Types of Sample Extraction Techniques 323 25.5 Key Considerations in Sample Preparation for LC‐MS Bioanalysis of Oligonucleotides 327 25.6 Representative Protocols 328 25.7 Summary 330 References 330 26 Sample Preparation for LC‐MS Bioanalysis of Antibody–Drug Conjugates 335 Cong Wei and Ragu Ramanathan 26.1 Introduction 335 26.2 Properties of ADC and Challenges for Sample Preparation 335 26.3 Sample Preparation Methods and Common Protocols 342 26.4 Future Perspective 347 Acknowledgements 348 References 348 Index 351

Revised and expanded handbook provides comprehensive introduction and complete instruction for sample preparation in vital category of bioanalysis Following in the footsteps of the previously published Handbook of LC-MS Bioanalysis, this book is a thorough and timely guide to all important sample preparation techniques used for quantitative Liquid Chromatography–Mass Spectrometry (LC-MS) bioanalysis of small and large molecules. LC-MS bioanalysis is a key element of pharmaceutical research and development, post-approval therapeutic drug monitoring, and many other studies in human healthcare. While advances are continually being made in key aspects of LC-MS bioanalysis such as sensitivity and throughput, the value of research/study mentioned above is still heavily dependent on the availability of high-quality data, for which sample preparation plays the critical role. Thus, this text provides researchers in industry, academia, and regulatory agencies with detailed sample preparation techniques and step-by-step protocols on proper extraction of various analyte(s) of interest from biological samples for LC-MS quantification, in accordance with current health authority regulations and industry best practices. The three sections of the book with a total of 26 chapters cover topics that include: Current basic sample preparation techniques (e.g., protein precipitation, liquid-liquid extraction, solid-phase extraction, salting-out assisted liquid-liquid extraction, ultracentrifugation and ultrafiltration, supported liquid extraction, and sample extraction via electromembranes)Sample preparation techniques for uncommon biological matrices (e.g., tissues, hair, skin, nails, bones, mononuclear cells, cerebrospinal fluid, aqueous humor)Crucial aspects of LC-MS bioanalytical method development (e.g., pre-analytical considerations, derivatization strategies, stability, non-specific binding) in addition to sample preparation techniques for challenging molecules (e.g., lipids, peptides, proteins, oligonucleotides, antibody-drug conjugates) Sample Preparation in LC-MS Bioanalysis will prove a practical and highly valuable addition to the reference shelves of scientists and related professionals in a variety of fields, including pharmaceutical and biomedical research, mass spectrometry, and analytical chemistry, as well as practitioners in clinical pharmacology, toxicology, and therapeutic drug monitoring.

"A welcome reference which provides a complete overview of current and new approaches in sample preparation for LC-MS...To have an exhaustive compilation of strategies with “practical tips and tricks” can only be inspiring for the readers helping him to make the best choice for his specific challenge." Gérard Hopfgartner, Analytical and Bioanalytical Chemistry, January 2020