Targeted Biomarker Quantitation by LC-MS
Wiley Series on Pharmaceutical Science
1. Edition September 2017
464 Pages, Hardcover
Wiley & Sons Ltd
ISBN:
978-1-119-10306-6
John Wiley & Sons
Further versions
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.
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 4ß?]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 4ß?]OHC as a P450 3A4/5 Endogenous Biomarker 307
19.2.2 Quantitation of 4ß?]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 ß?]Hydroxycortisol (6ß?]HC) as Biomarkers and Their Quantitation 312
19.3.1 Cortisol and 6ß?]HC as Biomarkers 312
19.3.2 Measurement of Cortisol and 6ß?]HC 313
19.3.2.1 Measurement of Cortisol in Serum 313
19.3.2.2 Measurement of Cortisol and 6ß?]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
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 4ß?]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 4ß?]OHC as a P450 3A4/5 Endogenous Biomarker 307
19.2.2 Quantitation of 4ß?]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 ß?]Hydroxycortisol (6ß?]HC) as Biomarkers and Their Quantitation 312
19.3.1 Cortisol and 6ß?]HC as Biomarkers 312
19.3.2 Measurement of Cortisol and 6ß?]HC 313
19.3.2.1 Measurement of Cortisol in Serum 313
19.3.2.2 Measurement of Cortisol and 6ß?]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
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.
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.
