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Targeted Biomarker Quantitation by LC-MS

Naidong Weng (Editor), Wenying Jian (Editor)
ISBN: 978-1-119-10306-6
464 pages
July 2017
Targeted Biomarker Quantitation by LC-MS (1119103061) cover image

Description

The first book to offer a blueprint for overcoming the challenges to successfully quantifying biomarkers in living organisms 

The demand among scientists and clinicians for targeted quantitation experiments has experienced explosive growth in recent years. While there are a few books dedicated to bioanalysis and biomarkers in general, until now there were none devoted exclusively to addressing critical issues surrounding this area of intense research. Target Biomarker Quantitation by LC-MS provides a detailed blueprint for quantifying biomarkers in biological systems. It uses numerous real-world cases to exemplify key concepts, all of which were carefully selected and presented so as to allow the concepts they embody to be easily expanded to future applications, including new biomarker development. 

Target Biomarker Quantitation by LC-MS primarily focuses on the assay establishment for biomarker quantitation—a critical issue rarely treated in depth. It offers comprehensive coverage of three core areas of biomarker assay establishment: the relationship between the measured biomarkers and their intended usage; contemporary regulatory requirements for biomarker assays (a thorough understanding of which is essential to producing a successful and defendable submission); and the technical challenges of analyzing biomarkers produced inside a living organism or cell.

  • Covers the theory of and applications for state-of-the-art mass spectrometry and chromatography and their applications in biomarker analysis
  • Features real-life examples illustrating the challenges involved in target biomarker quantitation and the innovative approaches which have been used to overcome those challenges
  • Addresses potential obstacles to obtain effective biomarker level and data interpretation, such as specificity establishment and sample collection
  • Outlines a tiered approach and fit-for-purpose assay protocol for target biomarker quantitation
  • Highlights the current state of the biomarker regulatory environment and protocol standards

Target Biomarker Quantitation by LC-MS is a valuable resource for bioanalytical scientists, drug metabolism and pharmacokinetics scientists, clinical scientists, analytical chemists, and others for whom biomarker quantitation is an important tool of the trade. It also functions as an excellent text for graduate courses in pharmaceutical, biochemistry and chemistry.

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Table of Contents

List of Contributors xv

Preface xix

Abbreviations xxiii

Part I Overview 1

1 Overview of Targeted Quantitation of Biomarkers and Its Applications 3
Naidong Weng

1.1 Introduction 3

1.2 Biomarker Definition 4

1.3 Current Challenges of a Biomarker 5

1.4 Biomarker Validation Process 6

1.5 Current Regulatory Requirement for Target Biomarker Quantitation 6

1.6 Challenges of Biomarker Quantitation 7

1.7 Current Technologies for Biomarker Quantitation 8

1.7.1 LC–MS 8

1.7.2 GC–MS 8

1.7.3 Ligand ]Binding Assay 9

1.7.4 Flow Cytometry 9

1.7.5 Quantitative PCR (qPCR) 9

1.8 Current Biomarker Quantitation Applications 9

1.8.1 Protein Biomarkers 9

1.8.2 Peptide Biomarkers 10

1.8.3 RNA Biomarkers 11

1.8.4 Nucleotide Biomarkers 11

1.8.5 Small Molecule Biomarkers 11

1.9 Conclusion and Future Perspective 12

References 13

2 Translational Application of Biomarkers 17
Ray Bakhtiar

2.1 Introduction 17

2.2 Translational Medicine 17

2.3 Biomarkers 18

2.4 Biomarker Categories 18

2.5 Neurobiological Disorders 21

2.6 Cardiovascular Disorders 22

2.7 Chronic Obstructive Pulmonary Disease 23

2.8 Oncology 24

2.9 Biomarker Measurements and Regulatory Considerations 26

2.10 Conclusions 27

References 29

3 Current Regulatory Guidance Pertaining Biomarker Assay Establishment and Industrial Practice of Fit ]for ]Purpose and Tiered Approach 35
Naidong Weng

3.1 Introduction 35

3.2 Current Regulatory Guidance and Interpretation 36

3.3 Current Industrial Discussion and Recommendations 37

3.4 Considerations for Assay Validation and Sample Analysis 39

3.4.1 Sensitivity 40

3.4.2 Specificity and Selectivity 40

3.4.3 Matrix Effects and Sample Variables 40

3.4.3.1 Authentic Analyte/Authentic Matrix Approach 40

3.4.3.2 Surrogate Analyte/Authentic Matrix Approach 40

3.4.3.3 Authentic Analyte/Surrogate Matrix Approach 40

3.4.4 Accuracy/Precision 40

3.4.5 Stability 41

3.4.6 Sample Analysis Consideration 41

3.5 Examples of Fit ]for ]Purpose and Tiered Approach 41

3.5.1 Relative Quantification of Glyco ]isoforms of Intact Apolipoprotein C3 in Human Plasma by LC ]HRMS 41

3.5.2 Quantification of 4β ]Hydroxycholesterol Endogenous Biomarker for CYP3A4 Activity in Plasma Samples 41

3.5.3 Quantitation of Leukotriene B4 in Human Sputum as a Biomarker Using UPLC–MS/MS 42

3.6 Conclusion 42

References 42

4 Modern Liquid Chromatography and Mass Spectrometry for Targeted Biomarker Quantitation 45
Wenying Jian

4.1 Introduction 45

4.2 Liquid Chromatography 45

4.2.1 Importance of Separation 45

4.2.2 Basic Principle of LC 47

4.2.3 Major Modes of LC Used for Targeted Biomarker Quantitation 47

4.2.4 Modern LC Technologies 49

4.2.4.1 HPLC and UHPLC 49

4.2.4.2 Miniaturized Column LC 50

4.2.4.3 2D ]LC 51

4.3 Mass Spectrometry 51

4.3.1 Major Types of MS Used for Targeted Biomarker Quantitation 51

4.3.2 Ionization Techniques 54

4.3.3 Ion Mobility 54

4.3.4 Fragmentation Mode 55

4.3.5 Emerging MS Techniques 56

4.3.5.1 MS Imaging 56

4.3.5.2 Other Surface Analysis MS Techniques 58

4.4 Summary and Future Perspectives 58

References 59

5 Comparison Between LC–MS and Ligand ]Binding Assay Approaches for Biomarker Quantification 65
QingQing Wang, Lili Guo, and Ian A. Blair

5.1 General Considerations: LBAs or LC–MS Assays 65

5.2 General Quantification Approaches 66

5.3 Analytical Issues Specifically Related to LBAs 67

5.3.1 There Is No Sample Pretreatment in Most LBAs 67

5.3.2 It Is Hard to Distinguish Biomarkers and Their Variants by LBAs 68

5.4 Analytical Features Specifically Related to LC–MS Methods 68

5.4.1 Proper Sample Preparation Generates Better Data 69

5.4.2 Biomarkers and Their Variants Can Be Distinguished 69

5.4.3 Stable Isotope ]Labeled Internal Standard Used for Assuring the Assay Accuracy 71

5.5 Case Studies: Comparison Between ELISA and LC–MS 72

5.5.1 Steroid Analysis 72

5.5.2 Apolipoprotein A1 74

5.6 Summary and Future Perspective 74

References 74

6 Sample Preparation Methods for Targeted Biomarker Quantification by LC ]MS 79
Shichen Shen, Bo An, and Jun Qu

6.1 Introduction 79

6.2 Sample Preparation Strategies for Small Molecule Biomarkers 79

6.2.1 Primary Issues to Address for Sample Preparation 80

6.2.1.1 Matrix Effects 80

6.2.1.2 Sensitivity and Selectivity 81

6.2.1.3 Selection of Calibration Methods 82

6.2.2 Sample Preparation Techniques 82

6.2.2.1 Dilute ]and ]Shoot 82

6.2.2.2 Protein Precipitation (PPT) 82

6.2.2.3 Liquid–Liquid Extraction (LLE) 82

6.2.2.4 Solid ]Phase Extraction (SPE) 84

6.3 Sample Preparation Strategies for Macromolecule Biomarkers 86

6.3.1 Considerations for Sample Preparation 86

6.3.1.1 Matrix Effects 86

6.3.1.2 Recovery of the Signature Peptide from the Target Analyte 86

6.3.1.3 Selection of Calibration Methods 88

6.3.1.4 Sensitivity and Selectivity 89

6.3.2 Methods for Protein Extraction 89

6.3.3 Methods for Protein and Peptide Enrichment 89

6.3.3.1 Immunoaffinity Capture (IC) 90

6.3.3.2 Sample Fractionation 90

6.3.3.3 Depletion of High Abundance Proteins (HAPs) 91

6.3.4 Methods for Protein Denaturation, Reduction, and Alkylation 92

6.3.5 Methods for Proteolytic Digestion 93

6.4 Conclusive

Remarks 94

References 95

7 Overcome the Endogenous Levels in Biomarker Quantitation Using LC–MS 107
Guowen Liu

7.1 Introduction 107

7.2 How Does Matrix Effect Affect Quantitation? 108

7.3 Commonly Used Strategies 109

7.3.1 Authentic Analyte in Authentic Matrix (Standard Addition) 109

7.3.2 Surrogate Analyte in Authentic Matrix 109

7.3.3 Authentic Analyte in Surrogate Matrix 112

7.4 Discussions and Future Perspectives 114

References 115

Part II Challenges and Approaches 119

8 Sample Collection for Targeted Biomarker Quantitation by LC–MS 121
Yuzhong Deng and Xiaorong Liang

8.1 Introduction 121

8.2 Timing of Biomarker Sample Collection 121

8.3 Matrix Type 122

8.3.1 Serum or Plasma 122

8.3.2 Urine 123

8.3.3 Tissue 123

8.4 Collection Methods 124

8.4.1 Plasma Sample Collection 124

8.4.1.1 Anticoagulants 124

8.4.1.2 Stabilizing Agents 125

8.4.1.3 Temperature and Timing before Initial Processing 126

8.4.1.4 Endogenous Degradation 126

8.4.2 Urine Sample Collection 127

8.4.3 Tissue Sample Collection 128

8.5 Sample Storage Stability 128

8.5.1 Storage of Blood ]Derived Fluids and Urine Samples 128

8.5.2 Storage of Tissue Samples 129

8.5.3 Freeze/Thaw Effect 129

8.6 Summary 129

References 130

9 Nonspecific Binding in LC–MS Bioanalysis 137
Aimin Tan and John C. Fanaras

9.1 Introduction 137

9.2 Identification and Evaluation of NSB 137

9.2.1 Common Scenarios and Indicators for Potential NSB Issues 137

9.2.2 Confirmation/Identification and Evaluation of NSB 138

9.2.3 NSB versus Stability Issue 139

9.3 Causes for NSB 140

9.4 Overcoming NSB Challenges 140

9.4.1 Solubilization of Compounds 140

9.4.2 Overview of Measures for Overcoming NSB Challenges 141

9.4.3 Application Examples 143

9.5 Conclusion 144

References 146

10 Strategies for Improving Sensitivity for Targeted Quantitation by LC–MS 149
Long Yuan and Qin C. Ji

10.1 Introduction 149

10.2 Sample Preparation Strategies for Improving Sensitivity 150

10.2.1 Protein Precipitation 151

10.2.2 Liquid–Liquid Extraction 152

10.2.3 Solid ]Phase Extraction 153

10.2.4 Immunoaffinity Extraction 154

10.2.5 Chemical Derivatization 155

10.2.6 Online Sample Preparation 155

10.3 LC Separation Strategies for Improving Sensitivity 156

10.3.1 Optimization of Mobile Phase 156

10.3.2 2D ]LC 157

10.3.3 Low ]Flow LC 157

10.4 MS Detection Strategies for Improving Sensitivity 160

10.4.1 SRM 160

10.4.2 High ]Resolution Mass Spectrometry (HRMS) 162

10.4.3 IMS 163

10.5 Conclusions 163

References 163

11 Strategies to Improve Specificity for Targeted Biomarker Quantitation by LC–MS 171
Yuan ]Qing Xia and Jeffrey D. Miller

11.1 Introduction 171

11.2 Differential Mobility Spectrometry 171

11.3 High ]Resolution Mass Spectrometry 175

11.4 Conclusions 180

References 180

12 Biomarker Quantitation Using Relative Approaches 183
Shane M. Lamos and Katrina E. Wiesner

12.1 Introduction 183

12.2 Relative Quantitation Isotope Labeling Approaches 183

12.2.1 Enzymatic Isotopic Incorporation 183

12.2.2 Metabolic Isotopic Incorporation 185

12.2.3 Chemical Labeling (Nonisobaric) 187

12.2.4 Chemical Labeling (Isobaric) 188

12.3 Conclusions 191

References 192

Part III Applications 195

13 Targeted Quantification of Amino Acid Biomarkers Using LC ]MS 197
Barry R. Jones, Raymond F. Biondolillo, and John E. Buckholz

13.1 Introduction 197

13.2 Amino Acids as Biomarkers 198

13.2.1 Biomarker of Heart Failure 199

13.2.2 Citrulline as Biomarker of Intestinal Failure 199

13.2.3 Oncological Biomarkers 200

13.2.4 Branched ]Chain Amino Acids in Diabetes and Cancer 200

13.2.5 Inborn Errors of Metabolism 200

13.2.6 Biomarker of Phenylketonuria (PKU) 201

13.2.7 Amino Acid Supplementation 201

13.3 Methods of Measurement 201

13.3.1 LC ]MS Considerations for Measurement of 2 ]Hydroxyglutarate 202

13.4 Accuracy, Precision, Selectivity, and Stability Considerations 203

13.4.1 Accuracy 203

13.4.1.1 Accuracy: Surrogate Matrix 203

13.4.1.2 Accuracy: Surrogate Analyte 205

13.4.1.3 Surrogate Matrix/Analyte Considerations for Multiplexed Amino Acid Assays 205

13.4.2 Precision 206

13.4.3 Selectivity 206

13.4.4 Stability 207

13.5 Assay Design 207

13.6 Conclusion 207

References 208

14 Targeted Quantification of Peptide Biomarkers: A Case Study of Amyloid Peptides 211
Lieve Dillen, Marc De Meulder, and Tom Verhaeghe

14.1 Overview 211

14.2 Challenges and Approaches 212

14.2.1 Multiply Charged Ions: SRM Versus HRMS 212

14.2.2 Adsorption–Solubility–Stability Aspects 214

14.2.3 Blank Matrix–Internal Standard–Surrogate Analytes 214

14.2.4 Extraction–Sample Pretreatment 215

14.3 Application to the Quantification of Alzheimer’s Disease Biomarkers 216

14.3.1 Introduction: Amyloid Peptides in CSF as Biomarkers for Alzheimer’s Disease 216

14.3.2 LC ]MS/MS Method for Analysis of Amyloid Peptides in CSF in Support of Preclinical Development 216

14.3.3 LC ]MS/MS Method for Analysis of Amyloid Peptides in CSF in Support of Clinical Development 217

14.3.4 Comparison of Immunoassay and UHPLC ]MS/MS: Are the Results Comparable? 219

14.4 Conclusion 222

References 222

15 Targeted Protein Biomarker Quantitation by LC ]MS 227
Yongle Pang, Chuan Shi, and Wenying Jian

15.1 Introduction 227

15.2 Sample Preparation for Targeted Protein Biomarker Quantitation 231

15.2.1 Protein Precipitation 232

15.2.2 Solid Phase Extraction 232

15.2.3 Abundant Protein Depletion 232

15.2.4 Affinity Enrichment 233

15.3 “Bottom ]Up” Approach for Targeted Protein Biomarker Quantitation Using LC ]MS 233

15.3.1 Surrogate Peptide Selection 233

15.3.2 Sample Pretreatment Prior to Proteolytic Digestion 234

15.3.3 Proteolytic Digestion 234

15.3.4 LC ]MS Analysis 235

15.4 “Top Down” Approach for Targeted Protein Biomarker Quantitation Using LC ]MS 235

15.5 Key Considerations in Targeted Protein Biomarker Quantitation Using LC ]MS 236

15.5.1 Preanalytical Considerations 236

15.5.2 Internal Standard 236

15.5.3 Reference Standard 237

15.5.4 Improving Sensitivity of the Assay 238

15.5.5 Improving Throughput of the Assay 238

15.5.6 Correlating MS Data with LBA Data 239

15.6 Summary and Future Perspectives 239

References 240

16 Glycoprotein Biomarkers 245
Shuwei Li, Stefani N. Thomas, and Shuang Yang

16.1 Introduction 245

16.2 Technologies for Glycoprotein Analysis 246

16.2.1 Glycoprotein Enrichment 246

16.2.1.1 Techniques for the Enrichment of Glycoproteins 246

16.2.1.2 Hybrid Chemical Metabolic Labeling 248

16.2.2 Glycan Analysis 251

16.2.2.1 In ]Solution Glycan Analysis 251

16.2.2.2 Solid ]Phase Glycan Analysis 252

16.2.3 Automated Platform for Processing Clinical Specimens 252

16.2.4 MS Analysis of Glycoproteins 254

16.2.4.1 Bottom ]Up Approaches 254

16.2.4.2 Top ]Down Approaches 254

16.2.4.3 MS/MS Fragmentation Methods for Glycopeptides 254

16.3 Glycoprotein Biomarker Quantification Using LC ]MS 255

16.3.1 Quantification by Stable Isotope Labeling 255

16.3.2 Metabolic Labeling Strategies 255

16.3.3 Label ]Free Glycoprotein Quantification 257

16.3.4 Methods for Targeted Quantification Using LC ]MS/MS 259

16.4 Protein Biomarkers for Clinical Applications 259

16.4.1 FDA ]Approved Glycoprotein Biomarkers 259

16.4.2 Classes of Biomarkers 260

16.4.3 New Glycoprotein Biomarker Discovery 260

16.5 Summary and Future Direction 264

References 265

17 Targeted Lipid Biomarker Quantitation Using Liquid Chromatography–Mass Spectrometry (LC–MS) 273
Ashkan Salamatipour, Ian A. Blair, and Clementina Mesaros

17.1 Introduction of Lipids 273

17.2 LC–MS Analysis of Lipids 276

17.3 Examples of LC–MS Analysis of Lipids 278

17.3.1 Omega ]6 ]Derived Eicosanoids 278

17.3.2 Docosahexaenoic Acid (DHA) 279

17.3.3 N ]Acylethanolamines (NAEs) and Eicosanoids 281

17.3.4 Arachidonic Acid (AA) 282

17.4 Summary and Future Directions 283

References 283

18 Targeted LC–MS Quantification of Androgens and Estrogens for Biomarker Development 289
Daniel Tamae

18.1 Introduction 289

18.1.1 History of Estrogen and Androgen Quantification 289

18.1.2 Androgen Biosynthesis and Metabolism 290

18.1.3 Estrogen Biosynthesis and Metabolism 290

18.2 Current Considerations in Biomarker Validation 292

18.3 Current Considerations in LC–MS Method Development 293

18.3.1 Chromatography 293

18.3.2 Direct Detection Methods 293

18.3.3 Derivatization Strategies 294

18.3.4 Stable Isotope Standards 295

18.3.5 Hydrolysis of Conjugated Steroids 296

18.4 Clinical Application of LC–MS Quantification of Estrogens and Androgens 296

18.4.1 Reference Ranges of Estrogens and Androgens 296

18.4.2 Estrogens in Postmenopausal Women and Low Androgens in Aging Men 297

18.4.3 Estrogens and Breast Cancer 297

18.4.4 Androgens and Prostate Cancer 298

18.5 Conclusion and Perspective 301

References 301

19 Steroid Biomarkers 307
Mike (Qingtao) Huang, Shefali Patel, and Zhongping (John) Lin

19.1 Introduction 307

19.2 Sterols as Endogenous Biomarkers and Their Quantitation 307

19.2.1 4β ]OHC as a P450 3A4/5 Endogenous Biomarker 307

19.2.2 Quantitation of 4β ]OHC in Human and Animal Species 310

19.2.3 24S ]OHC and 27 ]OHC as Biomarkers 311

19.2.4 Quantitation of 24S ]OHC and 27 ]OHC 312

19.3 Cortisol and 6 β ]Hydroxycortisol (6β ]HC) as Biomarkers and Their Quantitation 312

19.3.1 Cortisol and 6β ]HC as Biomarkers 312

19.3.2 Measurement of Cortisol and 6β ]HC 313

19.3.2.1 Measurement of Cortisol in Serum 313

19.3.2.2 Measurement of Cortisol and 6β ]HC in Urine 314

19.3.2.3 Measurement of Cortisol in Saliva and Hair 315

19.4 Summary 316

References 316

20 Bile Acids as Biomarkers 321
Clara John, Philipp Werner, Joerg Heeren, and Markus Fischer

20.1 Introduction 321

20.2 Analytical Platform for Bile Acids 323

20.3 Summary 327

References 327

21 Biomarkers for Vitamin Status and Deficiency: LC ]MS Based Approach 331
Stanley (Weihua) Zhang and Jonathan Crowther

21.1 Introduction to Vitamin and Vitamin Deficiency 331

21.2 Detection of Vitamin D by LC ]MS/MS and Comparison with Other Methods 332

21.2.1 Vitamin D and Vitamin D Deficiency 332

21.2.2 Target the Right Metabolites 332

21.2.3 Analytical Challenges 332

21.2.4 History of Vitamin D Quantification Assays 333

21.2.5 Quantification of 25(OH)D by LC ]MS/MS 334

21.2.5.1 Considerations in Assay Development and Validation 334

21.2.5.2 Sample Preparation 335

21.2.5.3 LC ]MS/MS 335

21.2.5.4 Method Comparison and Standardization 336

21.3 Other Vitamin Biomarkers 338

21.3.1 Retinol: Biomarkers of Vitamin A Status and Deficiency 338

21.3.2 Folic Acid: Biomarkers for Vitamin B9 Dietary Intake 339

21.3.3 Vitamin C: An Appropriate Biomarker of Vitamin C Intake 340

21.4 Conclusions and Perspectives 340

References 341

22 Quantitation of Acyl ]Coenzyme A Thioesters as Metabolic Biomarkers 347
Nathaniel Snyder

22.1 Introduction 347

22.2 Structure and Function of Acyl ]CoAs 347

22.3 Detection and Quantitation of Acyl ]CoAs 349

22.4 Acyl ]CoA Analysis for Current Drug Targets 352

22.5 Acyl ]CoAs as Biomarkers in Metabolic Disease 352

22.6 The Involvement of Acyl ]CoAs in Drug Metabolism 353

References 353

23 Neurotransmitter Biomarkers 357
Guodong Zhang

23.1 Introduction 357

23.2 Chromatographic Platforms of Biological Measurement for Neurotransmitters 358

23.2.1 Challenges for Neurotransmitter Measurement 358

23.2.2 LBA, LC, GC, and CE 358

23.2.3 LC–MS/MS 359

23.3 Bioanalytical Methodologies 359

23.3.1 Sample Preparation Strategies 359

23.3.2 Sensitivity and Chromatography Enhancement by Chemical Derivatization Using LC ]MS/MS 362

23.3.3 Chromatographic Strategies for LC ]MS/MS Assays 362

23.3.4 NTs Stability and Sample Collection 363

23.3.5 Case Studies 367

23.4 Conclusion 367

References 367

24 Targeted Quantification of Carbohydrate Biomarkers Using LC–MS 371
Cong Wei and Hong Gao

24.1 Introduction 371

24.2 Overview 371

24.2.1 Clinical Diagnostic Carbohydrate Biomarkers 371

24.2.2 Overview of Bioanalytical Analysis of Carbohydrate Biomarker 372

24.3 Bioanalytical Method Development for Carbohydrate Biomarkers 374

24.3.1 Sample Preparation 374

24.3.1.1 Sample Preparation by Solid ]Phase Extraction (SPE) 374

24.3.1.2 Sample Preparation by Liquid–Liquid Extraction (LLE) 376

24.3.1.3 Sample Preparation by Derivatization 378

24.3.1.4 Sample Preparation by Enzymatic Digestion or Chemical Reduction 378

24.3.2 Chromatography and Column Options 380

24.3.2.1 HILIC for LC–MS/MS Bioanalysis 381

24.3.2.2 Porous Graphic Hypercarb Chromatography for LC–MS/MS Bioanalysis 381

24.3.2.3 Reversed ]Phase Chromatography for LC–MS/MS Bioanalysis 382

24.3.2.4 Reversed ]Phase Ion ]Pair Chromatography for LC–MS Bioanalysis 382

24.3.3 LC–MS/MS Analysis 383

24.4 Conclusions 384

References 384

25 Nucleoside/Nucleotide Biomarkers 389
Guodong Zhang

25.1 Introduction 389

25.2 Chromatographic Platforms for Nucleosides/Nucleotides 390

25.2.1 Challenges for Nucleosides and Nucleotides Measurement 390

25.2.2 Conventional Immunoassays, CE, GC and HPLC 390

25.2.3 LC–MS/MS 391

25.3 Bioanalytical Methodologies 391

25.3.1 Sample Preparation Strategies 391

25.3.2 Chromatographic Strategies for LC–MS/MS Assays 394

25.4 Nucleoside/Nucleotide Biomarkers and Case Studies 398

25.5 Conclusion 399

References 402

26 LC–MS of RNA Biomarkers 407
Michael G. Bartlett, Babak Basiri, and Ning Li

26.1 Introduction 407

26.2 Role in Disease and Therapeutics 408

26.3 Role of Mass Spectrometry in RNA Biomarkers 409

26.4 LC–MS Approaches for RNA Determination 411

26.4.1 Sample Preparation 411

26.4.2 Ion ]Pair Chromatography 413

26.4.3 Capillary Chromatography 414

26.4.4 Liquid Chromatography–Inductively Coupled Plasma Mass Spectrometry 415

26.5 Case Studies 415

26.5.1 Single Nucleotide Polymorphisms as Biomarkers 415

26.5.2 Small Interfering RNA Determination 416

26.5.3 MicroRNA Determination 416

References 418

Index 425

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Author Information

Naidong Weng, PhD, is Scientific Director, Janssen Fellow, and Head of Bioanalytical Chemistry and Pharmacokinetics within Department of Pharmacokinetics, Dynamics and Metabolism at US East Coast, Janssen Research & Development, Johnson and Johnson. He has over 25 years of experiences on quantitative bioanalysis. His research interest includes using HILIC-MS/MS for quantitation of highly polar analytes as well as chiral analysis. He has published more than 110 journal papers and book chapters.

Wenying Jian, PhD, a Senior Principal Scientist of Bioanalytical Chemistry and Pharmacokinetics within Department of Pharmacokinetics, Dynamics and Metabolism at US East Coast, Janssen Research & Development, Johnson and Johnson. Her research experience and interest center on application of advanced LC-MS methodologies in detection, identification, and quantitation of endogenous molecules, drugs and their metabolites, including small and large molecules, and in complicated biological matrices. She has published more than 40 journal papers and book chapters.

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