Contents
Preface xvii
Acknowledgements xxiii
1 Measurement, Dimensions and Units 1
1.1 Introduction 1
1.2 The International System of Units (SI) 3
1.3 ‘Mass-to-Charge Ratio’ in Mass Spectrometry 6
1.4 Achievable Precision in Measurement of SI Base Quantities 9
1.5 Molecular Mass Limit for Trace Quantitation by Mass Spectrometry 11
1.6 Summary of Key Concepts 14
2 Tools of the Trade I. The Classical Tools 17
2.1 Introduction 17
2.2 Analytical and Internal Standards: Reference Materials 18
2.2.1 Analytical Standard (Reference Standard) and its Traceability 18
2.2.2 Certified (Standard) Reference Materials (CRMs) 20
2.2.3 Surrogate Internal Standard (SIS) 21
2.2.4 Volumetric Internal Standard (VIS) 25
2.3 The Analytical Balance 25
2.3.1 Balance Calibration 27
2.3.2 Sources of Uncertainty in Weighing 27
2.3.3 Weighing the Analytical Standard 31
2.4 Measurement and Dispensing of Volume 32
2.4.1 Standard Volumetric Flasks 32
2.4.2 Pipets 33
2.4.2a Classical Pipets 33
2.4.2b Micropipets 33
2.4.3 Loop Injectors for High Performance Liquid Chromatography (HPLC) 35
2.4.4 Syringes 38
2.5 Preparation of Solutions for Calibration 39
2.5.1 Matrix-Free Calibration Solutions 40
2.5.2 Matrix Matched Calibrators 40
2.5.3 Quality Control (QC) Samples 41
2.5.3a QCs in Method Development and Validation 41
2.5.3b QCs in Sample Analysis 41
2.6 Introduction to Calibration Methods for Quantitative Analysis 42
2.6.1 Calibration Using an External Standard 42
2.6.2 Calibration for the Method of Standard Additions 44
2.6.3 Calibration Using a Surrogate Internal Standard 44
2.6.4 Curves used in Conjunction with ‘Continuing Calibration Verification Standards’ 46
2.7 Summary of Key Concepts 47
3 Tools of the Trade II. Theory of Chromatography 51
3.1 Introduction 51
3.2 General Principles of Chemical Separations 53
3.3 Summary of Important Concepts 55
3.4 Plate Theory of Chromatography 58
3.4.1 Elution Equation for the Plate Theory 59
3.4.2 Retention Volume and Time 61
3.4.3 The Separation Ratio (Selectivity Factor) for Two Solutes 62
3.4.4 Capacity Factor (Ratio) of a Solute 62
3.4.5 Column Efficiency and Height Equivalent of the Theoretical Plate 63
3.4.6 Chromatographic Resolution 64
3.4.7 Effective Plate Number 65
3.4.8 Maximum Sample Injection Volume for a Specific Column 65
3.4.9 Peak Capacity of a Column 66
3.4.10 Gaussian Form of the Plate Theory Elution Equation 67
3.5 Nonequilibrium Effects in Chromatography: the van Deemter Equation 69
3.5.1 Multipath Dispersion 70
3.5.2 Longitudinal Diffusion 71
3.5.3 Resistance to Mass Transfer in the Mobile and Stationary Phases 71
3.5.4 Optimization to Maximize Column Efficiency 72
3.5.5 Relationships for Estimating Optimized Conditions 74
3.5.6 Numerical Estimates for Optimized Parameters 76
3.5.7 Ultra-Small Stationary Phase Particles 77
3.5.8 Monolithic Columns 80
3.5.9 Ultra High Flow Rate Liquid Chromatography 81
3.5.10 Packed Microcolumns 81
3.5.10a The Knox Equation 84
3.5.10b Chromatographic Dilution 87
3.5.10c Flow Impedance Parameter and Separation Impedance 87
3.5.11 Gas Chromatography 88
3.5.11a Effect of Gas Compressibility on Elution Equation for Packed Columns 88
3.5.11b Open Tubular Columns and the Golay Equation 89
3.5.12 Peak Asymmetry 90
3.6 Gradient Elution 92
3.7 Capillary Electrophoresis and Capillary Electrochromatography 97
Appendix 3.1 Derivation of the Plate Theory Equation for Chromatographic Elution 102
Appendix 3.2 Transformation of the Plate Theory Elution Equation from Poisson to Gaussian Form 103
Appendix 3.3 A Brief Introduction to Snyder’s Theory of Gradient Elution 104
List of Symbols Used in Chapter 3 105
4 Tools of the Trade III. Separation Practicalities 109
4.1 Introduction 109
4.2 The Analyte and the Matrix 110
4.3 Extraction and Clean-Up: Sample Preparation Methods 111
4.3.1 Liquid–Liquid Extraction (LLE) 112
4.3.1a Solid-Supported Liquid–Liquid Extraction (SLE) 114
4.3.1b Single Drop Microextraction (SDME) 114
4.3.1c Dispersive Liquid–Liquid Microextraction (DLLE) 115
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4.3.1d Flow Injection Liquid–Liquid Extraction 115
4.3.1e Membrane Extraction 115
4.3.1f Protein Precipitation from Biological Fluids 117
4.3.2 Liquid Extraction of Analytes from Solid Matrices 117
4.3.2a Soxhlet Extraction 117
4.3.2b Pressurized Solvent Extraction 117
4.3.2c Sonication Assisted Liquid Extraction (SAE) 119
4.3.2d Microwave Assisted Extraction (MAE) 120
4.3.2e Supercritical Fluid Extraction (SFE) 121
4.3.3 Solid Phase Extraction from Liquids and Gases 124
4.3.3a Flash Chromatography 124
4.3.3b Purge-and-Trap Analysis for Volatile Organic Compounds 124
4.3.3c Solid Phase Extraction (SPE) 125
4.3.3d Turbulent Flow Chromatography 128
4.3.3e Molecularly Imprinted Polymers (MIPs) 130
4.3.3f Solid Phase Microextraction (SPME) 132
4.3.3g Stir-Bar Sorptive Extraction (SBSE) 133
4.4 Chromatographic Practicalities 133
4.4.1 Stationary Phases for SPE and Liquid Chromatography 133
4.4.1a Alumina and Silica Particles 134
4.4.1b Derivatization of Silica for Normal and Reverse Phase Chromatography 136
4.4.1c Ion Exchange Media 137
4.4.1d Chiral Separations 137
4.4.1e Affinity Media 143
4.4.2 Mobile Phases Used in SPE and Liquid Chromatography 144
4.4.2a Solvent Polarity and Elution Strength 145
4.4.2b Reverse Phase Chromatography 146
4.4.2c Hydrophilic Interaction Chromatography (HILIC) 146
4.4.3 Mobile and Stationary Phases for Gas Chromatography 147
4.4.3a GC Mobile Phase 147
4.4.3b Temperature Programming 149
4.4.3c GC Stationary Phases 150
4.4.4 Sample Injection Devices for Chromatography 152
4.4.4a Automated Loop Injectors for HPLC 152
4.4.4b GC Injectors 152
4.4.5 Pumps for HPLC 154
4.4.6 Capillary Electrophoresis and Electrochromatography 157
4.4.7 Micro Total Analysis Systems (Lab-on-a-Chip) 160
4.4.8 General Comments on Detectors for Chromatography 163
4.5 Summary of Key Concepts 166
Appendix 4.1 Responses of Chromatographic Detectors: Concentration vs Mass–Flux
Dependence 169
5 Tools of the Trade IV. Interfaces and Ion Sources for Chromatography–Mass Spectrometry 173
5.1 Introduction 174
5.1.1 Matrix Effects 175
5.2 Ion Sources that can Require a Discrete Interface Between Chromatograph and Source 176
5.2.1 Electron Ionization and Chemical Ionization 176
5.2.1a Discrete Chromatograph-Ion Source Interfaces 180
5.2.1b Chemical Derivatization for EI and CI 182
5.2.2 Matrix Assisted Laser Desorption/Ionization (MALDI) 184
5.2.3 ‘Lab-on-a-Chip’ 195
5.3 Ion Sources not Requiring a Discrete Interface 196
5.3.1 Flow Fast Atom Bombardment (Flow-FAB) 196
5.3.2 Thermospray Ionization 197
5.3.3 Atmospheric Pressure Ionization (API) 198
5.3.3a Coupling of API Sources to Mass Spectrometers 199
5.3.4 Atmospheric Pressure Chemical Ionization (APCI) 203
5.3.5 Atmospheric Pressure Photoionization (APPI) 206
5.3.6 Electrospray Ionization (ESI) 211
5.3.6a Ionization Suppression/Enhancement: Matrix Effects 221
5.3.6b ESI-MS: Concentration or Mass Flow Dependent? 230
5.3.7 Atmospheric Pressure Desorption Methods 236
5.4 Source–Analyzer Interfaces Based on Ion Mobility 237
5.5 Summary of Key Concepts 238
5.1 Appendix 5.1: Methods of Sample Preparation for Analysis by MALDI 242
6 Tools of the Trade V. Mass Analyzers for Quantitation: Separation of Ions by m/z Values 245
6.1 Introduction 245
6.2 Mass Analyzer Operation Modes and Tandem Mass Spectrometry 248
6.2.1 The Selectivity–Sensitivity Compromise 249
6.2.2 Tandem Mass Spectrometry (MS/MS) 251
6.2.3 Figures of Merit for Mass Analyzers 255
6.2.3a Accessible m/z Range 255
6.2.3b Resolving Power 256
6.2.3c Accuracy and Precision of Mass Measurement 257
6.2.3d Transmission Efficiency 258
6.2.3e Duty Cycle 259
6.2.3f Data Acquisition Rate 259
6.2.3g Dynamic Range (Range of Reliable Response) 261
6.2.3h Versatility for Tandem Mass Spectrometry 261
6.2.3i Ease of Use 261
6.2.3j Capital and Maintenance Costs 261
6.3 Motion of Ions in Electric and Magnetic Fields 261
6.3.1 Introduction to Interactions of Electric and Magnetic Fields with Ions 263
6.3.2 Ion Optics and Lenses: Instrument Tuning 265
6.4 Mass Analyzers 266
6.4.1 Calibration of the m/z Axis (‘Mass Calibration’) 266
6.4.2 Quadrupole Mass Filters 267
6.4.2a RF-Only Quadrupoles 276
6.4.3 Triple Quadrupole Instruments 277
6.4.4 Magnetic Sector Analyzers 280
6.4.5 Quadrupole Ion Traps 284
6.4.5a Three-Dimensional (Paul) Traps 285
6.4.5b Two-Dimensional (Linear) Traps 301
6.4.6 The QqQtrap Analyzer 309
6.4.7 Time of Flight and QqTOF Analyzers 311
6.4.8 FTICR and Orbitrap Analyzers 320
6.5 Activation and Dissociation of Ions 320
6.6 Vacuum Systems 326
6.6.1 Pumping Speed, Conductance and Gas Flow 327
6.6.2 Vacuum Pumps 329
6.6.2a Rotary Vane Pumps 329
6.6.2b Diffusion Pumps 330
6.6.2c Turbomolecular Pumps 330
6.6.2d Differential Pumping 332
6.6.3 Vacuum Gauges 334
6.6.3a Capacitance Manometer 334
6.6.3b Pirani Gauge 334
6.6.3c Thermocouple Gauge 335
6.6.3d Ionization Gauge 335
6.7 Summary of Key Concepts 336
Appendix 6.1 Interaction of Electric and Magnetic Fields with Charged Particles 339
Appendix 6.2 Leak Detection 340
Appendix 6.3 List of Symbols Used in Chapter 6 341
7 Tools of the Trade VI. Ion Detection and Data Processing 345
7.1 Introduction 345
7.1.1 Signal:Noise vs Signal:Background 347
7.1.1a Shot Noise in the Ion Beam 350
7.1.1b Data Smoothing Before Integration 352
7.1.1c Integration and Experimental Determination of Signal:Background 353
7.2 Faraday Cup Detectors 353
7.3 Electron Multipliers 354
7.3.1 Discrete Dynode Secondary Electron Multipliers 354
7.3.1a Off-Axis Conversion Dynodes 357
7.3.2 Channel Electron Multipliers 359
7.3.2a Single Channel Electron Multipliers 359
7.3.2b Channel Electron Multiplier Arrays 362
7.4 Post-Detector Electronics 365
7.4.1 Analog Signal Processing 365
7.4.2 Digital Electronics for Ion Counting Detectors 366
7.4.3 Computer-Based Data Systems 367
7.5 Summary of Key Concepts 368
8 Tools of the Trade VII: Statistics of Calibration, Measurement and Sampling 373
8.1 Introduction 374
8.1.1 Systematic and Random Errors: Accuracy and Precision 375
8.2 Univariate Data: Tools and Tests for Determining Accuracy and Precision 377
8.2.1 Mean, Median, Standard Deviation, Standard Error 377
8.2.2 Significant Figures and Propagation of Error 379
8.2.3 Normal (Gaussian) Distribution 382
8.2.4 Hypothesis Tests: False Positives and False Negatives 385
8.2.5 Student’s t-Test and Fisher F-Test for Comparison of Means and Variances: Applications
to Two Data Sets 387
8.2.6 Statistical Testing of More Than Two Data Sets: Bartlett Test and ANOVA 394
8.2.7 Multiple-Range Test and Huber–Davies Test for Outliers 398
8.3 Bivariate Data: Tools and Tests for Regression and Correlation 398
8.3.1 Correlation Analysis 401
8.3.2 Simple Linear Regression for Homoscedastic Data 401
8.3.3 Test for Goodness of Fit: Irreproducibility, Heteroscedacity and Nonlinearity 407
8.3.4 Inversion of the Calibration Equation 410
8.3.5 Weighted Linear Regression 411
8.3.6 Regression for Nonlinear Data: the Quadratic Fitting Function 415
8.3.7 Summarized Procedure to Determine Best-Fit Calibration Curve 416
8.3.8 Nonlinear Least-Squares Regression 418
8.4 Limits of Detection and Quantitation 418
8.4.1 Limit of Detection 419
8.4.2 Limits of Quantitation 427
8.5 Calibration and Measurement: Systematic and Random Errors 428
8.5.1 Analyses Without Internal Standards 428
8.5.1a External Linear Calibration With a Zero Intercept 428
8.5.1b Method of Standard Additions 430
8.5.1c External Linear Calibration With a Nonzero Intercept 432
8.5.1d Systematic Errors in the Method of Standard Additions: Youden Plots 434
8.5.1e Strategies When Analytical Standards are not Available 435
8.5.2 Analyses Using Internal Standards 436
8.5.2a Volumetric Internal Standards for GC Analyses 437
8.5.2b Use of Surrogate Internal Standards 439
8.5.2c Cross-Contributions Between Analyte and Internal Standard – a Need for
Nonlinear Regression 444
8.6 Statistics of Sampling of Heterogeneous Matrices 448
8.7 Summary of Key Concepts 453
Appendix 8.1 A Brief Statistics Glossary 455
Appendix 8.2 Symbols Used in Discussion of Calibration Methods 458
9 Method Development and Fitness for Purpose 461
9.1 Introduction 461
9.2 Fitness for Purpose and Managing Uncertainty 461
9.3 Issues Between Analyst and Client: Examining What’s at Stake 463
9.3.1 Define the Analytical Problem 463
9.3.2 Consider the Needs of all Interested Parties 463
9.3.2a Client’s Perspective vs Analyst’s Perspective 463
9.3.3 Define Uncertainty Tolerance 465
9.3.3a Setting Targets for Quantitation 466
9.3.3b Setting Targets for Identification 466
9.3.4 Balancing Thoroughness vs Time and Resources 472
9.3.5 The Analyst as Expert Witness 474
9.4 Formulating a Strategy 474
9.4.1 Defining the Scope and Method Requirements 474
9.4.2 Considering Prevailing Guidelines or Regulatory Requirements 476
9.4.2a Good Laboratory Practice 476
9.4.2b Laboratory Accreditation 478
9.4.2c Method Development and Validation Without Established
Guidance 478
9.4.3 The Analyte 478
9.4.3a Structures and Chemical Properties – MS Considerations 478
9.4.3b Structures and Chemical Properties – Analytical Considerations 479
9.4.4 The Reference Standard 479
9.4.4a Availability and Source 479
9.4.4b Receipt and Documentation 480
9.4.4c Certificate of Analysis 481
9.4.4d Assigned Purity 481
9.4.4e Chiral Purity 482
9.4.4f Storage and Stability 482
9.4.5 The Surrogate Internal Standard (SIS) 482
9.4.5a General Considerations 482
9.4.5b Stable Isotope Internal Standards 482
9.4.5c Analog or Homolog Internal Standards 484
9.4.5d SIS for Multi-analyte Assays 484
9.4.6 The Analytical Sample 484
9.4.6a Sample Availability vs Method Requirements 485
9.4.6b Documentation of Sample Receipt, Storage and Use 485
9.4.7 The Control Matrix 486
9.4.7a Obtaining a Suitable Control Matrix 486
9.4.7b Using the Control Matrix to Assess Selectivity 486
9.4.7c Surrogate Matrices 489
9.4.8 Evaluate Analytical Options 489
9.4.8a Reviewing The Literature and Previous Methodologies 489
9.4.8b Review Existing Laboratory SOPs 490
9.5 Method Development 490
9.5.1 Instrument Qualification, Calibration and Maintenance 490
9.5.1a Equipment Qualification 491
9.5.1b Mass Spectrometer Calibration 494
9.5.1c Maintenance 494
9.5.1d Validation of Computerized Laboratory Instruments and Equipment 494
9.5.2 Instrument Optimization 496
9.5.3 LC–MS/MS Optimization for MRM Analysis Using QqQ 497
9.5.3a API Interface and Q1 Optimization 498
9.5.3b Selecting Product Ions and CID Conditions 502
9.5.3c Final MRM Optimization 503
9.5.4 Preparation of Stock and Spiking Solutions 504
9.5.4a Primary Stock Solutions 504
9.5.4b Sub-Stocks and Spiking Solutions 506
9.5.4c Miscellaneous Considerations 506
9.5.5 Chromatography Method Development 507
9.5.5a General Considerations 507
9.5.5b Sample Throughput and Selectivity 507
9.5.5c Multi-dimensional Separations 508
9.5.5d Miscellaneous Components 510
9.5.6 Sample Preparation – Extraction and Clean-Up 511
9.5.6a Evaluating Options 512
9.5.6b Use of Blanks During Method Development and Analysis 513
9.5.6c Using Spikes and ‘Recovery Samples’ 513
9.5.7 Evaluating Sensitivity and Linearity in Matrix 515
9.6 Matrix Effects 517
9.6.1 Evaluating Ion Suppression in Method Development 518
9.6.2 Addressing Ion Suppression/Enhancement 519
9.6.3 Interferences 520
9.7 Contamination and Carryover 522
9.7.1 Laboratory Contamination 522
9.7.1a Common Causes and Impact 522
9.7.1b Monitoring Contamination in the Laboratory 522
9.7.2 Carryover 523
9.7.2a Evaluating Carryover During Method Development 524
9.7.2b Addressing Carryover in the Method 525
9.8 Establishing the Final Method 527
9.8.1 Curve Preparation 527
9.8.1a Calibration Solutions 527
9.8.1b Matrix Matched Calibrators 528
9.8.2 Preparation of QCs 530
9.8.3 System Suitability 531
9.8.4 Testing Method Robustness and Ruggedness 533
9.8.5 Establishing Final Stepwise Procedure 535
9.8.6 Final Method Qualification/Assessment 536
10 Method Validation and Sample Analysis in a Controlled Laboratory Environment 539
10.1 Introduction 539
10.2 Method Validation 540
10.2.1 Figures of Merit for Full Validation 540
10.2.2 Selectivity 540
10.2.3 Sensitivity, Range of Reliable Response and Linearity 542
10.2.4 Accuracy and Precision 543
10.2.5 Reproducibility 543
10.2.6 Recovery 544
10.2.7 Stability in Solution 544
10.2.8 Stability in Matrix 545
10.2.8a In-Process Stability (Room Temperature Stability) 545
10.2.8b Freeze–Thaw Stability 546
10.2.8c Long Term Storage Stability (Sample Stability) 546
10.2.9 Other Special Validation Requirements 547
10.2.9a Extract and Re-Injection Stability 547
10.2.9b Assessing Carryover and Potential for Laboratory
Contamination 548
10.2.9c Incurred Sample Re-Analysis and Stability 548
10.2.9d Integrity of Dilution 549
10.2.10 Abbreviated and Partial Validation 549
10.2.11 Cross Validations 550
10.3 Conduct of the Validaton 551
10.3.1 Validation Plan or Protocol 551
10.3.2 Validation Sample Analysis 552
10.3.2a Data Review 552
10.3.2b Addressing Validation Run Failures 553
10.3.3 Documentation of Supporting Data 553
10.3.3a Assay Procedure 553
10.3.3b Standards and Stock Solutions 553
10.3.3c Preparation of Calibrators and QCs 553
10.3.3d Acceptance Criteria 553
10.3.3e Sample Analysis 554
10.3.3f Run Summary Sheets 555
10.3.3g Chromatograms 555
10.3.3h Communications 555
10.3.4 The Validation Report and Scientific Review 555
10.3.4a Reference Standard and Solutions 556
10.3.4b QCs and Calibrators 556
10.3.4c Assay Procedures 556
10.3.4d Run Acceptance Criteria 556
10.3.4e Sample Analysis Tables 556
10.3.4f Failed Runs 557
10.3.4g Deviations From Method or SOPs 558
10.3.4h Chromatograms 558
10.3.4i Amendments 558
10.4 Examples of Methods and Validations Fit for Purpose 559
10.4.1 Bioanalytical Method Validation 559
10.4.1a Sensitivity and the Calibration/Standard Curve 560
10.4.1b Accuracy and Precision 561
10.4.1c Selectivity 562
10.4.1d Matrix Effects 562
10.4.1e Recovery 563
10.4.1f Reproducibility 563
10.4.1g Stability – Overview 563
10.4.1h Stock Solution Stability 563
10.4.1i Freeze and Thaw Stability 564
10.4.1j Short Term Temperature Stability 564
10.4.1k Long Term Stability 564
10.4.1l Incurred Sample Re-Analysis 565
10.4.2 Risk Assessment Methods 567
10.4.3 Enforcement Methods 567
10.4.3a FDA Enforcement Methods for Drug Residues in Animal Food Products 568
10.5 Validated Sample Analysis 570
10.5.1 Sample Batch Preparation and Extraction 570
10.5.2 Sample Analysis 570
10.5.2a Analytical Run Lists 570
10.5.2b Instrument Set-Up and System Suitability 571
10.5.2c Failed Runs and Re-Analysis 571
10.5.3 Data Review 572
10.5.3a Evaluating Chromatography and Instrument Response 572
10.5.3b Evaluating the Curve and QCs 575
10.5.3c Rejection of Data 576
10.5.4 Sample Re-Assays 577
10.5.5 Addressing Carryover During Sample Analysis 578
10.5.6 Investigation and Corrective Action 580
10.6 Documentation 582
10.6.1 Sample Tracking 582
10.6.2 Sample Analytical Report 583
10.7 Traceability 583
11 Examples from the Literature 585
11.1 Introduction 585
11.2 Food Contaminants 585
11.2.1 Acrylamide 586
11.2.2 Paralytic Shellfish Poisons 598
11.3 Anthropogenic Pollutants in Water 605
11.3.1 Disinfection By-Products 605
11.3.1a MX [3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone] 607
11.3.1b N-nitrosodialkylamines 612
11.3.2 Multi-residue Methods for Pharmaceutical Residues 616
11.3.2a Validated Method for Macrolide Antibiotics 617
11.3.2b Extraction/Clean-up for Multi-residue Analysis of Compounds of Widely
Different Polarity etc. 620
11.3.2c Screening Method for Multiclass Antibiotics 621
11.4 GC–MS Analyses of Persistent Environmental Pollutants 623
11.4.1 ‘Dioxin-like’ Compounds 623
11.4.2 Other Persistent Pollutants 636
11.5 Bioanalytical Applications 637
11.5.1 Drug Discovery Methods 638
11.5.1a Passive Permeability Test (Caco-2 Assays) 639
11.5.1b Drug–Drug Interaction Studies (CYP450 Assays) 641
11.5.1c Clearance Rate Tests (Metabolic Stability) 643
11.5.1d Pharmacokinetics and ADME Studies 646
11.5.1e Metabolite Identification and Quantitation 648
11.5.2 Drug Development Phase Validated Methods 654
11.5.2a Enantiomer-Specific Analyses 655
11.5.2b Methods for Two Concentration Ranges 660
11.6 Quantitative Proteomics 661
11.6.1 Identification of Proteins by Mass Spectrometry 664
11.6.2 Relative (Comparative) Quantitation 667
11.6.2a Isotope-Coded Affinity Tag (ICAT) 669
11.6.2b Isotope Tagging for Relative and Absolute Quantitation (ITRAQ) 669
11.6.2c Other Chemical Labeling Methods 671
11.6.2d Proteolytic Labeling Methods 672
11.6.2e Culture-Based Labeling Methods 673
11.6.3 Absolute Quantitation 675
11.6.3a AQUA 675
11.6.3b QCAT Peptides 676
11.6.3c Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) 677
11.7 Analysis of Endogenous Analytes 680
Epilog 683
References 685
Index 709
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