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.

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 1.8 Current Biomarker Quantitation Applications 9 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 3.5 Examples of Fit‐for‐Purpose and Tiered Approach 41 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 4.3 Mass Spectrometry 51 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 5.4 Analytical Features Specifically Related to LC–MS Methods 68 5.5 Case Studies: Comparison Between ELISA and LC–MS 72 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 6.3 Sample Preparation Strategies for Macromolecule Biomarkers 86 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 7.4 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 8.4 Collection Methods 124 8.5 Sample Storage Stability 128 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 9.3 Causes for NSB 140 9.4 Overcoming NSB Challenges 140 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 10.3 LC Separation Strategies for Improving Sensitivity 156 10.4 MS Detection Strategies for Improving Sensitivity 160 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 12.3 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 13.3 Methods of Measurement 201 13.4 Accuracy, Precision, Selectivity, and Stability Considerations 203 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 14.3 Application to the Quantification of Alzheimer’s Disease Biomarkers 216 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 15.3 “Bottom‐Up” Approach for Targeted Protein Biomarker Quantitation Using LC‐MS 233 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 15.6 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 16.3 Glycoprotein Biomarker Quantification Using LC‐MS 255 16.4 Protein Biomarkers for Clinical Applications 259 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 17.4 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 18.2 Current Considerations in Biomarker Validation 292 18.3 Current Considerations in LC–MS Method Development 293 18.4 Clinical Application of LC–MS Quantification of Estrogens and Androgens 296 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 19.3 Cortisol and 6 β‐Hydroxycortisol (6β‐HC) as Biomarkers and Their Quantitation 312 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 21.3 Other Vitamin Biomarkers 338 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 23.3 Bioanalytical Methodologies 359 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 24.3 Bioanalytical Method Development for Carbohydrate Biomarkers 374 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 25.3 Bioanalytical Methodologies 391 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 26.5 Case Studies 415 References 418 Index 425

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. Targeted 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. Targeted 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 for 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 analysisFeatures real-life examples illustrating the challenges involved in targeted biomarker quantitation and the innovative approaches which have been used to overcome those challengesAddresses potential obstacles to obtain effective biomarker level and data interpretation, such as specificity establishment and sample collectionOutlines a tiered approach and fit-for-purpose assay protocol for targeted biomarker quantitationHighlights the current state of the biomarker regulatory environment and protocol standards Targeted 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.