Sample Preparation in LC-MS Bioanalysis
Wiley Series on Pharmaceutical Science and Biotechnology: Practices, Applications and Methods Series

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 used 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, microsampling, 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, derivation 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.

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

WENKUI LI is a Senior Fellow at the Novartis Institutes for BioMedical Research Pharmacokinetic Sciences. He serves on the Editorial Board of Biomedical Chromatography.

WENYING JIAN is a Senior Principal Scientist in Janssen Research and Development at the Johnson & Johnson Company. She serves on the Editorial Board of Journal of Pharmacological and Toxicological Methods.

YUNLIN FU is an Investigator at the Novartis Institutes for BioMedical Research Pharmacokinetic Sciences.