Targeted Biomarker Quantitation by LC-MS: (Wiley Series on Pharmaceutical Science and Biotechnology: Practices, Applications and Methods)

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.

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

About the Author :
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.