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E-raamat: Targeted Biomarker Quantitation by LC-MS

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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(118)
1 Overview of Targeted Quantitation of Biomarkers and Its Applications
3(14)
Naidong Weng
1.1 Introduction
3(1)
1.2 Biomarker Definition
4(1)
1.3 Current Challenges of a Biomarker
5(1)
1.4 Biomarker Validation Process
6(1)
1.5 Current Regulatory Requirement for Target Biomarker Quantitation
6(1)
1.6 Challenges of Biomarker Quantitation
7(1)
1.7 Current Technologies for Biomarker Quantitation
8(1)
1.7.1 LC-MS
8(1)
1.7.2 GC-MS
8(1)
1.7.3 Ligand-Binding Assay
9(1)
1.7.4 Flow Cytometry
9(1)
1.7.5 Quantitative PCR (qPCR)
9(1)
1.8 Current Biomarker Quantitation Applications
9(3)
1.8.1 Protein Biomarkers
9(1)
1.8.2 Peptide Biomarkers
10(1)
1.8.3 RNA Biomarkers
11(1)
1.8.4 Nucleotide Biomarkers
11(1)
1.8.5 Small Molecule Biomarkers
11(1)
1.9 Conclusion and Future Perspective
12(1)
References
13(4)
2 Translational Application of Biomarkers
17(18)
Ray Bakhtiar
2.1 Introduction
17(1)
2.2 Translational Medicine
17(1)
2.3 Biomarkers
18(1)
2.4 Biomarker Categories
18(3)
2.5 Neurobiological Disorders
21(1)
2.6 Cardiovascular Disorders
22(1)
2.7 Chronic Obstructive Pulmonary Disease
23(1)
2.8 Oncology
24(2)
2.9 Biomarker Measurements and Regulatory Considerations
26(1)
2.10 Conclusions
27(2)
References
29(6)
3 Current Regulatory Guidance Pertaining Biomarker Assay Establishment and Industrial Practice of Fit-for-Purpose and Tiered Approach
35(10)
Naidong Weng
3.1 Introduction
35(1)
3.2 Current Regulatory Guidance and Interpretation
36(1)
3.3 Current Industrial Discussion and Recommendations
37(2)
3.4 Considerations for Assay Validation and Sample Analysis
39(2)
3.4.1 Sensitivity
40(1)
3.4.2 Specificity and Selectivity
40(1)
3.4.3 Matrix Effects and Sample Variables
40(1)
3.4.3.1 Authentic Analyte/Authentic Matrix Approach
40(1)
3.4.3.2 Surrogate Analyte/Authentic Matrix Approach
40(1)
3.4.3.3 Authentic Analyte/Surrogate Matrix Approach
40(1)
3.4.4 Accuracy/Precision
40(1)
3.4.5 Stability
41(1)
3.4.6 Sample Analysis Consideration
41(1)
3.5 Examples of Fit-for-Purpose and Tiered Approach
41(1)
3.5.1 Relative Quantification of Glyco-isoforms of Intact Apolipoprotein C3 in Human Plasma by LC-HRMS
41(1)
3.5.2 Quantification of 4beta-Hydroxycholesterol Endogenous Biomarker for CYP3A4 Activity in Plasma Samples
41(1)
3.5.3 Quantitation of Leukotriene B4 in Human Sputum as a Biomarker Using UPLC-MS/MS
42(1)
3.6 Conclusion
42(1)
References
42(3)
4 Modern Liquid Chromatography and Mass Spectrometry for Targeted Biomarker Quantitation
45(20)
Wenying Jian
4.1 Introduction
45(1)
4.2 Liquid Chromatography
45(6)
4.2.1 Importance of Separation
45(2)
4.2.2 Basic Principle of LC
47(1)
4.2.3 Major Modes of LC Used for Targeted Biomarker Quantitation
47(2)
4.2.4 Modern LC Technologies
49(1)
4.2.4.1 HPLC and UHPLC
49(1)
4.2.4.2 Miniaturized Column LC
50(1)
4.2.4.3 2D-LC
51(1)
4.3 Mass Spectrometry
51(7)
4.3.1 Major Types of MS Used for Targeted Biomarker Quantitation
51(3)
4.3.2 Ionization Techniques
54(1)
4.3.3 Ion Mobility
54(1)
4.3.4 Fragmentation Mode
55(1)
4.3.5 Emerging MS Techniques
56(1)
4.3.5.1 MS Imaging
56(1)
4.3.5.2 Other Surface Analysis MS Techniques
58(1)
4.4 Summary and Future Perspectives
58(1)
References
59(6)
5 Comparison Between LC-MS and Ligand-Binding Assay Approaches for Biomarker Quantification
65(14)
QingQing Wang
Lili Guo
Ian A. Blair
5.1 General Considerations: LBAs or LC-MS Assays
65(1)
5.2 General Quantification Approaches
66(1)
5.3 Analytical Issues Specifically Related to LBAs
67(1)
5.3.1 There Is No Sample Pretreatment in Most LBAs
67(1)
5.3.2 It Is Hard to Distinguish Biomarkers and Their Variants by LBAs
68(1)
5.4 Analytical Features Specifically Related to LC-MS Methods
68(4)
5.4.1 Proper Sample Preparation Generates Better Data
69(1)
5.4.2 Biomarkers and Their Variants Can Be Distinguished
69(2)
5.4.3 Stable Isotope-Labeled Internal Standard Used for Assuring the Assay Accuracy
71(1)
5.5 Case Studies: Comparison Between ELISA and LC-MS
72(2)
5.5.1 Steroid Analysis
72(2)
5.5.2 Apolipoprotein Al
74(1)
5.6 Summary and Future Perspective
74(1)
References
74(5)
6 Sample Preparation Methods for Targeted Biomarker Quantification by LC-MS
79(28)
Shichen Shen
Bo An
Jun Qu
6.1 Introduction
79(1)
6.2 Sample Preparation Strategies for Small Molecule Biomarkers
79(7)
6.2.1 Primary Issues to Address for Sample Preparation
80(1)
6.2.1.1 Matrix Effects
80(1)
6.2.1.2 Sensitivity and Selectivity
81(1)
6.2.1.3 Selection of Calibration Methods
82(1)
6.2.2 Sample Preparation Techniques
82(1)
6.2.2.1 Dilute-and-Shoot
82(1)
6.2.2.2 Protein Precipitation (PPT)
82(1)
6.2.2.3 Liquid-Liquid Extraction (LLE)
82(1)
6.2.2.4 Solid-Phase Extraction (SPE)
84(2)
6.3 Sample Preparation Strategies for Macromolecule Biomarkers
86(8)
6.3.1 Considerations for Sample Preparation
86(1)
6.3.1.1 Matrix Effects
86(1)
6.3.1.2 Recovery of the Signature Peptide from the Target Analyte
86(1)
6.3.1.3 Selection of Calibration Methods
88(1)
6.3.1.4 Sensitivity and Selectivity
89(1)
6.3.2 Methods for Protein Extraction
89(1)
6.3.3 Methods for Protein and Peptide Enrichment
89(1)
6.3.3.1 Immunoaffinity Capture (IC)
90(1)
6.3.3.2 Sample Fractionation
90(1)
6.3.3.3 Depletion of High Abundance Proteins (HAPs)
91(1)
6.3.4 Methods for Protein Denaturation, Reduction, and Alkylation
92(1)
6.3.5 Methods for Proteolytic Digestion
93(1)
6.4 Conclusive Remarks
94(1)
References
95(12)
7 Overcome the Endogenous Levels in Biomarker Quantitation Using LC-MS
107(12)
Guowen Liu
7.1 Introduction
107(1)
7.2 How Does Matrix Effect Affect Quantitation?
108(1)
7.3 Commonly Used Strategies
109(5)
7.3.1 Authentic Analyte in Authentic Matrix (Standard Addition)
109(1)
7.3.2 Surrogate Analyte in Authentic Matrix
109(3)
7.3.3 Authentic Analyte in Surrogate Matrix
112(2)
7.4 Discussions and Future Perspectives
114(1)
References
115(4)
Part II Challenges and Approaches 119(76)
8 Sample Collection for Targeted Biomarker Quantitation by LC-MS
121(16)
Yuzhong Deng
Xiaorong Liang
8.1 Introduction
121(1)
8.2 Timing of Biomarker Sample Collection
121(1)
8.3 Matrix Type
122(2)
8.3.1 Serum or Plasma
122(1)
8.3.2 Urine
123(1)
8.3.3 Tissue
123(1)
8.4 Collection Methods
124(4)
8.4.1 Plasma Sample Collection
124(1)
8.4.1.1 Anticoagulants
124(1)
8.4.1.2 Stabilizing Agents
125(1)
8.4.1.3 Temperature and Timing before Initial Processing
126(1)
8.4.1.4 Endogenous Degradation
126(1)
8.4.2 Urine Sample Collection
127(1)
8.4.3 Tissue Sample Collection
128(1)
8.5 Sample Storage Stability
128(1)
8.5.1 Storage of Blood-Derived Fluids and Urine Samples
128(1)
8.5.2 Storage of Tissue Samples
129(1)
8.5.3 Freeze/Thaw Effect
129(1)
8.6 Summary
129(1)
References
130(7)
9 Nonspecific Binding in LC-MS Bioanalysis
137(12)
Aimin Tan
John C. Fanaras
9.1 Introduction
137(1)
9.2 Identification and Evaluation of NSB
137(3)
9.2.1 Common Scenarios and Indicators for Potential NSB Issues
137(1)
9.2.2 Confirmation/Identification and Evaluation of NSB
138(1)
9.2.3 NSB versus Stability Issue
139(1)
9.3 Causes for NSB
140(1)
9.4 Overcoming NSB Challenges
140(4)
9.4.1 Solubilization of Compounds
140(1)
9.4.2 Overview of Measures for Overcoming NSB Challenges
141(2)
9.4.3 Application Examples
143(1)
9.5 Conclusion
144(2)
References
146(3)
10 Strategies for Improving Sensitivity for Targeted Quantitation by LC-MS
149(22)
Long Yuan
Qin C. Ji
10.1 Introduction
149(1)
10.2 Sample Preparation Strategies for Improving Sensitivity
150(6)
10.2.1 Protein Precipitation
151(1)
10.2.2 Liquid-Liquid Extraction
152(1)
10.2.3 Solid-Phase Extraction
153(1)
10.2.4 Immunoaffinity Extraction
154(1)
10.2.5 Chemical Derivatization
155(1)
10.2.6 Online Sample Preparation
155(1)
10.3 LC Separation Strategies for Improving Sensitivity
156(4)
10.3.1 Optimization of Mobile Phase
156(1)
10.3.2 2D-LC
157(1)
10.3.3 Low-Flow LC
157(3)
10.4 MS Detection Strategies for Improving Sensitivity
160(3)
10.4.1 SRM
160(2)
10.4.2 High-Resolution Mass Spectrometry (HRMS)
162(1)
10.4.3 IMS
163(1)
10.5 Conclusions
163(1)
References
163(8)
11 Strategies to Improve Specificity for Targeted Biomarker Quantitation by LC-MS
171(12)
Yuan-Qing Xia
Jeffrey D. Miller
11.1 Introduction
171(1)
11.2 Differential Mobility Spectrometry
171(4)
11.3 High-Resolution Mass Spectrometry
175(5)
11.4 Conclusions
180(1)
References
180(3)
12 Biomarker Quantitation Using Relative Approaches
183(12)
Shane M. Lamos
Katrina E. Wiesner
12.1 Introduction
183(1)
12.2 Relative Quantitation Isotope Labeling Approaches
183(8)
12.2.1 Enzymatic Isotopic Incorporation
183(2)
12.2.2 Metabolic Isotopic Incorporation
185(2)
12.2.3 Chemical Labeling (Nonisobaric)
187(1)
12.2.4 Chemical Labeling (Isobaric)
188(3)
12.3 Conclusions
191(1)
References
192(3)
Part III Applications 195(230)
13 Targeted Quantification of Amino Acid Biomarkers Using LC-MS
197(14)
Barry R. Jones
Raymond F. Biondolillo
John E. Buckholz
13.1 Introduction
197(1)
13.2 Amino Acids as Biomarkers
198(3)
13.2.1 Biomarker of Heart Failure
199(1)
13.2.2 Citrulline as Biomarker of Intestinal Failure
199(1)
13.2.3 Oncological Biomarkers
200(1)
13.2.4 Branched-Chain Amino Acids in Diabetes and Cancer
200(1)
13.2.5 Inborn Errors of Metabolism
200(1)
13.2.6 Biomarker of Phenylketonuria (PKU)
201(1)
13.2.7 Amino Acid Supplementation
201(1)
13.3 Methods of Measurement
201(2)
13.3.1 LC-MS Considerations for Measurement of 2-Hydroxyglutarate
202(1)
13.4 Accuracy, Precision, Selectivity, and Stability Considerations
203(4)
13.4.1 Accuracy
203(1)
13.4.1.1 Accuracy: Surrogate Matrix
203(1)
13.4.1.2 Accuracy: Surrogate Analyte
205(1)
13.4.1.3 Surrogate Matrix/Analyte Considerations for Multiplexed Amino Acid Assays
205(1)
13.4.2 Precision
206(1)
13.4.3 Selectivity
206(1)
13.4.4 Stability
207(1)
13.5 Assay Design
207(1)
13.6 Conclusion
207(1)
References
208(3)
14 Targeted Quantification of Peptide Biomarkers: A Case Study of Amyloid Peptides
211(16)
Lieve Dillen
Marc De Meulder
Tom Verhaeghe
14.1 Overview
211(1)
14.2 Challenges and Approaches
212(4)
14.2.1 Multiply Charged Ions: SRM Versus HRMS
212(2)
14.2.2 Adsorption-Solubility-Stability Aspects
214(1)
14.2.3 Blank Matrix-Internal Standard-Surrogate Analytes
214(1)
14.2.4 Extraction-Sample Pretreatment
215(1)
14.3 Application to the Quantification of Alzheimer's Disease Biomarkers
216(6)
14.3.1 Introduction: Amyloid Peptides in CSF as Biomarkers for Alzheimer's Disease
216(1)
14.3.2 LC-MS/MS Method for Analysis of Amyloid Peptides in CSF in Support of Preclinical Development
216(1)
14.3.3 LC-MS/MS Method for Analysis of Amyloid Peptides in CSF in Support of Clinical Development
217(2)
14.3.4 Comparison of Immunoassay and UHPLC-MS/MS: Are the Results Comparable?
219(3)
14.4 Conclusion
222(1)
References
222(5)
15 Targeted Protein Biomarker Quantitation by LC-MS
227(18)
Yongle Pang
Chuan Shi
Wenying Jian
15.1 Introduction
227(4)
15.2 Sample Preparation for Targeted Protein Biomarker Quantitation
231(2)
15.2.1 Protein Precipitation
232(1)
15.2.2 Solid Phase Extraction
232(1)
15.2.3 Abundant Protein Depletion
232(1)
15.2.4 Affinity Enrichment
233(1)
15.3 "Bottom-Up" Approach for Targeted Protein Biomarker Quantitation Using LC-MS
233(2)
15.3.1 Surrogate Peptide Selection
233(1)
15.3.2 Sample Pretreatment Prior to Proteolytic Digestion
234(1)
15.3.3 Proteolytic Digestion
234(1)
15.3.4 LC-MS Analysis
235(1)
15.4 "Top Down" Approach for Targeted Protein Biomarker Quantitation Using LC-MS
235(1)
15.5 Key Considerations in Targeted Protein Biomarker Quantitation Using LC-MS
236(3)
15.5.1 Preanalytical Considerations
236(1)
15.5.2 Internal Standard
236(1)
15.5.3 Reference Standard
237(1)
15.5.4 Improving Sensitivity of the Assay
238(1)
15.5.5 Improving Throughput of the Assay
238(1)
15.5.6 Correlating MS Data with LBA Data
239(1)
15.6 Summary and Future Perspectives
239(1)
References
240(5)
16 Glycoprotein Biomarkers
245(28)
Shuwei Li
Stefani N. Thomas
Shuang Yang
16.1 Introduction
245(1)
16.2 Technologies for Glycoprotein Analysis
246(9)
16.2.1 Glycoprotein Enrichment
246(1)
16.2.1.1 Techniques for the Enrichment of Glycoproteins
246(1)
16.2.1.2 Hybrid Chemical Metabolic Labeling
248(3)
16.2.2 Glycan Analysis
251(1)
16.2.2.1 In-Solution Glycan Analysis
251(1)
16.2.2.2 Solid-Phase Glycan Analysis
252(1)
16.2.3 Automated Platform for Processing Clinical Specimens
252(2)
16.2.4 MS Analysis of Glycoproteins
254(1)
16.2.4.1 Bottom-Up Approaches
254(1)
16.2.4.2 Top-Down Approaches
254(1)
16.2.4.3 MS/MS Fragmentation Methods for Glycopeptides
254(1)
16.3 Glycoprotein Biomarker Quantification Using LC-MS
255(4)
16.3.1 Quantification by Stable Isotope Labeling
255(1)
16.3.2 Metabolic Labeling Strategies
255(2)
16.3.3 Label-Free Glycoprotein Quantification
257(2)
16.3.4 Methods for Targeted Quantification Using LC-MS/MS
259(1)
16.4 Protein Biomarkers for Clinical Applications
259(5)
16.4.1 FDA-Approved Glycoprotein Biomarkers
259(1)
16.4.2 Classes of Biomarkers
260(1)
16.4.3 New Glycoprotein Biomarker Discovery
260(4)
16.5 Summary and Future Direction
264(1)
References
265(8)
17 Targeted Lipid Biomarker Quantitation Using Liquid Chromatography-Mass Spectrometry (LC-MS)
273(16)
Ashkan Salamatipour
Ian A. Blair
Clementina Mesaros
17.1 Introduction of Lipids
273(3)
17.2 LC-MS Analysis of Lipids
276(2)
17.3 Examples of LC-MS Analysis of Lipids
278(5)
17.3.1 Omega-6-Derived Eicosanoids
278(1)
17.3.2 Docosahexaenoic Acid (DHA)
279(2)
17.3.3 N-Acylethanolamines (NAEs) and Eicosanoids
281(1)
17.3.4 Arachidonic Acid (AA)
282(1)
17.4 Summary and Future Directions
283(1)
References
283(6)
18 Targeted LC-MS Quantification of Androgens and Estrogens for Biomarker Development
289(18)
Daniel Tamae
18.1 Introduction
289(3)
18.1.1 History of Estrogen and Androgen Quantification
289(1)
18.1.2 Androgen Biosynthesis and Metabolism
290(1)
18.1.3 Estrogen Biosynthesis and Metabolism
290(2)
18.2 Current Considerations in Biomarker Validation
292(1)
18.3 Current Considerations in LC-MS Method Development
293(3)
18.3.1 Chromatography
293(1)
18.3.2 Direct Detection Methods
293(1)
18.3.3 Derivatization Strategies
294(1)
18.3.4 Stable Isotope Standards
295(1)
18.3.5 Hydrolysis of Conjugated Steroids
296(1)
18.4 Clinical Application of LC-MS Quantification of Estrogens and Androgens
296(5)
18.4.1 Reference Ranges of Estrogens and Androgens
296(1)
18.4.2 Estrogens in Postmenopausal Women and Low Androgens in Aging Men
297(1)
18.4.3 Estrogens and Breast Cancer
297(1)
18.4.4 Androgens and Prostate Cancer
298(3)
18.5 Conclusion and Perspective
301(1)
References
301(6)
19 Steroid Biomarkers
307(14)
Mike Huang
Shefali Patel
Zhongping Lin
19.1 Introduction
307(1)
19.2 Sterols as Endogenous Biomarkers and Their Quantitation
307(5)
19.2.1 4beta-OHC as a P450 3A4/5 Endogenous Biomarker
307(3)
19.2.2 Quantitation of 4beta-OHC in Human and Animal Species
310(1)
19.2.3 24S-OHC and 27-OHC as Biomarkers
311(1)
19.2.4 Quantitation of 24S-OHC and 27-OHC
312(1)
19.3 Cortisol and 6 beta-Hydroxycortisol (6beta-HC) as Biomarkers and Their Quantitation
312(4)
19.3.1 Cortisol and 6beta-HC as Biomarkers
312(1)
19.3.2 Measurement of Cortisol and 6beta-HC
313(1)
19.3.2.1 Measurement of Cortisol in Serum
313(1)
19.3.2.2 Measurement of Cortisol and 6beta-HC in Urine
314(1)
19.3.2.3 Measurement of Cortisol in Saliva and Hair
315(1)
19.4 Summary
316(1)
References
316(5)
20 Bile Acids as Biomarkers
321(10)
Clara John
Philipp Werner
Joerg Heeren
Markus Fischer
20.1 Introduction
321(2)
20.2 Analytical Platform for Bile Acids
323(4)
20.3 Summary
327(1)
References
327(4)
21 Biomarkers for Vitamin Status and Deficiency: LC-MS Based Approach
331(16)
Stanley Zhang
Jonathan Crowther
21.1 Introduction to Vitamin and Vitamin Deficiency
331(1)
21.2 Detection of Vitamin D by LC-MS/MS and Comparison with Other Methods
332(6)
21.2.1 Vitamin D and Vitamin D Deficiency
332(1)
21.2.2 Target the Right Metabolites
332(1)
21.2.3 Analytical Challenges
332(1)
21.2.4 Histgry of Vitamin D Quantification Assays
333(1)
21.2.5 Quantification of 25(OH)D by LC-MS/MS
334(1)
21.2.5.1 Considerations in Assay Development and Validation
334(1)
21.2.5.2 Sample Preparation
335(1)
21.2.5.3 LC-MS/MS
335(1)
21.2.5.4 Method Comparison and Standardization
336(2)
21.3 Other Vitamin Biomarkers
338(2)
21.3.1 Retinol: Biomarkers of Vitamin A Status and Deficiency
338(1)
21.3.2 Folic Acid: Biomarkers for Vitamin B9 Dietary Intake
339(1)
21.3.3 Vitamin C: An Appropriate Biomarker of Vitamin C Intake
340(1)
21.4 Conclusions and Perspectives
340(1)
References
341(6)
22 Quantitation of Acyl-Coenzyme A Thioesters as Metabolic Biomarkers
347(10)
Nathaniel Snyder
22.1 Introduction
347(1)
22.2 Structure and Function of Acyl-CoAs
347(2)
22.3 Detection and Quantitation of Acyl-CoAs
349(3)
22.4 Acyl-CoA Analysis for Current Drug Targets
352(1)
22.5 Acyl-CoAs as Biomarkers in Metabolic Disease
352(1)
22.6 The Involvement of Acyl-CoAs in Drug Metabolism
353(1)
References
353(4)
23 Neurotransmitter Biomarkers
357(14)
Guodong Zhang
23.1 Introduction
357(1)
23.2 Chromatographic Platforms of Biological Measurement for Neurotransmitters
358(1)
23.2.1 Challenges for Neurotransmitter Measurement
358(1)
23.2.2 LBA, LC, GC, and CE
358(1)
23.2.3 LC-MS/MS
359(1)
23.3 Bioanalytical Methodologies
359(8)
23.3.1 Sample Preparation Strategies
359(3)
23.3.2 Sensitivity and Chromatography Enhancement by Chemical Derivatization Using LC-MS/MS
362(1)
23.3.3 Chromatographic Strategies for LC-MS/MS Assays
362(1)
23.3.4 NTs Stability and Sample Collection
363(4)
23.3.5 Case Studies
367(1)
23.4 Conclusion
367(1)
References
367(4)
24 Targeted Quantification of Carbohydrate Biomarkers Using LC-MS
371(18)
Cong Wei
Hong Gao
24.1 Introduction
371(1)
24.2 Overview
371(3)
24.2.1 Clinical Diagnostic Carbohydrate Biomarkers
371(1)
24.2.2 Overview of Bioanalytical Analysis of Carbohydrate Biomarker
372(2)
24.3 Bioanalytical Method Development for Carbohydrate Biomarkers
374(10)
24.3.1 Sample Preparation
374(1)
24.3.1.1 Sample Preparation by Solid-Phase Extraction (SPE)
374(1)
24.3.1.2 Sample Preparation by Liquid-Liquid Extraction (LLE)
376(1)
24.3.1.3 Sample Preparation by Derivatization
378(1)
24.3.1.4 Sample Preparation by Enzymatic Digestion or Chemical Reduction
378(2)
24.3.2 Chromatography and Column Options
380(1)
24.3.2.1 HILIC for LC-MS/MS Bioanalysis
381(1)
24.3.2.2 Porous Graphic Hypercarb Chromatography for LC-MS/MS Bioanalysis
381(1)
24.3.2.3 Reversed-Phase Chromatography for LC-MS/MS Bioanalysis
382(1)
24.3.2.4 Reversed-Phase Ion-Pair Chromatography for LC-MS Bioanalysis
382(1)
24.3.3 LC-MS/MS Analysis
383(1)
24.4 Conclusions
384(1)
References
384(5)
25 Nucleoside/Nucleotide Biomarkers
389(18)
Guodong Zhang
25.1 Introduction
389(1)
25.2 Chromatographic Platforms for Nucleosides/Nucleotides
390(1)
25.2.1 Challenges for Nucleosides and Nucleotides Measurement
390(1)
25.2.2 Conventional Immunoassays, CE, GC and HPLC
390(1)
25.2.3 LC-MS/MS
391(1)
25.3 Bioanalytical Methodologies
391(7)
25.3.1 Sample Preparation Strategies
391(3)
25.3.2 Chromatographic Strategies for LC-MS/MS Assays
394(4)
25.4 Nucleoside/Nucleotide Biomarkers and Case Studies
398(1)
25.5 Conclusion
399(3)
References
402(5)
26 LC-MS of RNA Biomarkers
407(18)
Michael G. Bartlett
Babak Basiri
Ning Li
26.1 Introduction
407(1)
26.2 Role in Disease and Therapeutics
408(1)
26.3 Role of Mass Spectrometry in RNA Biomarkers
409(2)
26.4 LC-MS Approaches for RNA Determination
411(4)
26.4.1 Sample Preparation
411(2)
26.4.2 Ion-Pair Chromatography
413(1)
26.4.3 Capillary Chromatography
414(1)
26.4.4 Liquid Chromatography-Inductively Coupled Plasma Mass Spectrometry
415(1)
26.5 Case Studies
415(3)
26.5.1 Single Nucleotide Polymorphisms as Biomarkers
415(1)
26.5.2 Small Interfering RNA Determination
416(1)
26.5.3 MicroRNA Determination
416(2)
References
418(7)
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.
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