Preface

Biomarker has been increasingly playing a significant role in drug discovery and development. Its application ranges from target and candidate selection and refinement in discovery to safety and efficacy evaluation in drug development and to patient stratification and market differentiation at late phase development and post market. With the assistance of ever-improving mass spectrometry in conjunction with liquid chromatography, assays for new and novel biomarkers, some of which at very low levels, are being developed and validated for being applied in abovementioned areas. At mean time, regulatory bodies such as the FDA have increased their drug approval evaluation using information driven by biomarkers. Yet, assay establishment for biomarkers remains to be a daunting task, partially due to the fact that biomarkers inherently are endogenous analytes, and therefore the existing assay validation guidance, albeit very useful for drug candidates, is less straightforward for the biomarkers, partially due to the lack of thorough understanding of the relationship between a target biomarker and its usage in decision making.

A comprehensive bioanalytical overview on quantitative liquid chromatography with mass spectrometric detection (LC-MS) analysis of biomarkers appropriate to the drug discovery/development stage and usage of biomarkers as discussed in this book is timely needed in the industry. For a given biomarker that can play pivotal role in drug discovery and development go-no-go decision, the bioanalytical assay needs to be appropriately established to meet both the regulatory and scientific rigor. It is the hope that this book will illustrate this concept using real-life examples.

This book contains 26 chapters that are divided into three parts. Part 1 (Chapters 1-7) provides an overview of quantitative bioanalysis of biomarkers using LC-MS. Chapter 1 provides an overall introduction of targeted quantitation of biomarkers and its application, and Chapter 2 describes the role of biomarker in drug discovery and development, with emphasis on clinical application. In Chapter 3, a thorough review of current regulatory landscape on biomarker quantitation and industrial practices on biomarker method validation/qualification strategy is discussed. Important considerations in biomarker analysis method development and assay establishment are discussed in detail in Chapters 4-6. Chapter 4 introduces modern LC-MS on bioanalysis with emphasis on state-of-art mass spectrometry and liquid chromatography for targeted biomarker quantitation. Chapter 5 compares the advantages and disadvantages of LC-MS-based biomarker quantitation with traditional ligand binding assays. Sample preparation is probably the most important parameter for a successful biomarker assay establishment. Review and contrast of the sample preparation strategies and challenges for biomarkers and the different sample preparation procedures for various types of biomarkers (small molecules, peptides, and proteins) are the subject of Chapter 6. Several practical and successful approaches are discussed in Chapter 7, in particular, the surrogate matrix versus surrogate analyte approaches. Preanalytical consideration is pivotally important for the successful biomarker assay establishment.

Part 2 (Chapters 8-12) presents the challenges of bioanalysis quantitation and approaches to overcome those challenges. As biomarkers are endogenous compounds, they present unique challenges to appropriately establish assay parameters that are usually prescribed to drug candidates. Study design to maximize the opportunity to observe biomarker changes including sample collection, stability, circadian rhythm, etc. is extensively discussed in Chapter 8. Biomarker analyte loss due to nonspecific adsorption to the container is a frequently observed phenomenon, and Chapter 9 is specifically designed to address this issue. Another fundamental challenge for biomarker measurement is the lack of assay sensitivity. Strategies for improving sensitivity using novel strategy such as immunoaffinity extraction, derivatization, etc. are the subjects for Chapter 10. Strategy to address assay specificity, fundamentally a difficult task due to the endogenous nature of the biomarkers, is covered in Chapter 11. One of the aims of this book is also to leverage technologies that are already used in other disciplines such as proteomics. Chapter 12 certainly bridges this by presenting novel quantitative strategy of using heavy and light labeled derivatives for the relative quantitation of biomarkers.

Part 3 (Chapters 13-26) focuses on in-depth discussion of different types of biomarkers and demonstrates case studies for their targeted quantitation using LC-MS. The first four chapters focus on biomarkers related to amino acids, peptides, proteins, and glycoproteins (Chapters 13-16). Quantitative measurement of amino acids in biological matrices can yield important information into disease identification and monitoring, treatment efficacy, and overall improvement of human health. Chapter 13 offers guidance to ensure optimal assay characteristics such as accuracy, precision, and selectivity for LC-MS quantitation of amino acid biomarkers. In Chapter 14, targeted quantification of peptide biomarkers and a case study of amyloid peptides are presented. Aspects on important issues related to biomarker assays such as adsorption, solubility, and stability are discussed in detail. Chapter 15 focuses on proteins, one of the most important types of biomarkers, and analytical approaches using signature peptide following enzymatic digestion of the proteins (bottom-up) and direct analysis of whole (intact) proteins without enzymatic digestion (top-down) are the subjects of this chapter. Key aspects in assay establishment such as sample preparation, use of internal standards, assay sensitivity and throughput, etc. are also discussed. Chapter 16 reviews recent technological advances related to glycoproteomics and glycomics biomarker analyses. Glycosylation is one of the most common protein modifications that could alter protein function and lead to various physiological and pathological consequences.

Part 3 continues with review and case studies of another class of important biomarkers related to lipid structures such as lipids (Chapter 17), hormones (Chapter 18), sterols (Chapter 19), bile acids (Chapter 20), and vitamins (Chapter 21). Chapter 17 focuses on targeted LC-MS methods for measuring class I lipids-the fatty acyls in biological samples-and the various methodologies presented in this chapter demonstrate the recent advancement in the field of lipidomics and allow for more efficient quantitation and monitoring of lipid metabolites. Chapter 18 describes the targeted LC-MS quantification of androgens and estrogens for biomarker development for hormone-dependent tumors such as that of the breast and prostate. Chapter 19 mainly focuses on the current discussions of some of the glucocorticoids and sterols as biomarkers and their corresponding bioanalysis by LC-MS, in particular 4ß-hydroxycholesterol, a potential P450 3A4/5 endogenous biomarker. Since bile acids play a role for the development and for the therapy of certain metabolic diseases, Chapter 20 discusses the importance of having access to an appropriate analytical platform to adequately quantify cholesterol and bile acids species. Chapter 21 reviews vitamins as biomarkers for vitamin status and deficiency. It focuses on vitamin D, not only because its deficiency is a worldwide problem but also because that there are multiple competing quantification methodologies besides LC-MS. The diversity in assay methods as well as high variability in measurements has caused controversy and confusion in clinical testing.

Part 3 concludes with review and case studies of other important biomarkers with diversified structures such as acyl-coenzyme A thioesters (Chapter 22), neurotransmitters (Chapter 23), carbohydrates (Chapter 24), nucleotides/nucleosides (Chapter 25), and oligonucleotides (Chapter 26). Chapter 22 discusses the structure and function of acyl-coenzyme A thioesters, provides an overview of the LC-MS-based methods of quantifying acyl-coenzyme A thioesters, and gives specific examples of the analysis of acyl-coenzyme A thioesters as biomarkers for current drug targets, metabolic diseases, and drug metabolism. Chapter 23 discusses the recent LC-MS methodologies developed for the measurement of neurotransmitters, which combine optimized sample preparation, chemical derivatization, and chromatographic conditions. They enable more sensitive and specific measurement of neurotransmitters in low concentration ranges, with reproducibility, high throughput, and short run time. Chapter 24 discusses the critical role of targeted LC-MS methods for quantitative analysis of carbohydrates from biological fluids. Optimal assay conditions require careful consideration of sample extraction, chromatography, and mass spectrometric detection. Chapter 25 is an informative source for LC-MS assays for the measurement of nucleoside/nucleotide biomarkers in biological samples and how to overcome challenges related to the determination of nucleosides/nucleotides due to their low...