Contents
Series Preface ix
Preface xi
Acknowledgements xiii
Abbreviations, Acronyms and Symbols xv
About the Author xix
1 Introduction 1
1.1 What are the Advantages of Linking High Performance
Liquid Chromatography with Mass Spectrometry? 2
1.2 What Capabilities are Required of the Combination? 3
1.3 What Problems, if Any, Have to be Addressed to Allow
the LC–MS Combination to Function, and Function Effectively? 4
References 5
2 Liquid Chromatography 7
2.1 Introduction 7
2.2 High Performance Liquid Chromatography 10
2.2.1 Pump 10
2.2.2 Sample Introduction (Injector) 11
2.2.3 Mobile Phase 12
2.2.4 Stationary Phase 14
2.2.5 Detectors 16
2.3 Chromatographic Properties 18
2.4 Identification Using High Performance Liquid Chromatography 21
vi Liquid Chromatography–Mass Spectrometry
2.5 Quantitation Using High Performance Liquid Chromatography 23
2.6 The Need for High Performance Liquid Chromatography–
Mass Spectrometry 30
References 31
3 Mass Spectrometry 33
3.1 Introduction 33
3.2 Ionization Methods 36
3.2.1 Electron Ionization 36
3.2.2 Chemical Ionization 36
3.2.3 Fast-Atom Bombardment 38
3.2.4 Matrix-Assisted Laser Desorption Ionization 39
3.2.5 Negative Ionization 40
3.3 Ion Separation 40
3.3.1 The Quadrupole Mass Analyser 41
3.3.2 The (Quadrupole) Ion-Trap Mass Analyser 42
3.3.3 The Double-Focusing and Tri-Sector Mass Analysers 42
3.3.4 The Time-of-Flight Mass Analyser 44
3.4 Tandem Mass Spectrometry (MS–MS) 46
3.4.1 Instrumentation 47
3.4.2 Techniques 51
3.5 Data Acquisition 53
3.5.1 Identification 53
3.5.2 Quantitation 54
3.6 Processing of Mass Spectral Data 58
3.6.1 The Total-Ion-Current Trace 58
3.6.2 Qualitative Analysis 60
3.6.3 Quantitative Analysis 68
3.6.4 The Use of Tandem Mass Spectrometry 71
References 73
4 Interface Technology 75
4.1 Introduction 75
4.2 The Moving-Belt Interface 77
4.3 The Direct-Liquid-Introduction Interface 82
4.4 The Continuous-Flow/Frit
(Dynamic) Fast-Atom-Bombardment Interface 85
4.5 The Particle-Beam Interface 89
4.6 The Thermospray Interface 94
4.7 The Electrospray Interface 98
4.7.1 The Mechanism of Electrospray Ionization 100
4.7.2 Sample Types 105
Contents vii
4.7.3 The Appearance of the Electrospray Spectrum 106
4.7.4 Structural Information from Electrospray Ionization 117
4.8 The Atmospheric-Pressure Chemical Ionization Interface 122
4.8.1 The Mechanism of Atmospheric-Pressure Chemical
Ionization 123
References 126
5 Applications of High Performance Liquid Chromatography–
Mass Spectrometry 129
5.1 Method Development 131
5.1.1 The Use of Experimental Design for Method
Development 133
5.1.2 The Choice of Electrospray or APCI 134
5.2 The Molecular Weight Determination of Biopolymers 141
5.2.1 Electrospray Spectra of Co-Eluting Components 141
5.2.2 The Use of Selected-Ion Monitoring to Examine
the Number of Terminal Galactose Moieties on a
Glycoprotein 143
5.2.3 The Effect of Mobile-Phase Additives and
Cone-Voltage 147
5.3 Structure Determination of Biopolymers 149
5.3.1 Amino Acid Sequencing of Proteins 149
5.3.2 The Use of Enzymes for Amino Acid Sequencing 150
5.3.3 The Mass Spectral Fragmentation of Peptides 152
5.3.4 Confirmation of Amino Acid Sequence Using
the Analysis of LC–MS Data from an Enzyme
Digest of a Protein 152
5.3.5 Determination of the Amino Acid Sequence of a Novel
Protein Using LC–MS Data from an Enzyme Digest 160
5.3.6 Amino Acid Sequencing of Polypeptides
Generated by Enzyme Digestion Using MS–MS 166
5.3.7 The Location of Post-Translational Modifications
Using LC–MS Data from an Enzyme Digest 170
5.3.8 The Location of Post-Translational Modifications
Using MS–MS 173
5.3.9 The Analysis of Polysaccharides Present
in Glycosylated Proteins 177
5.3.10 Location of the Position of Attachment of
a Glycan on the Polypeptide Backbone of a Glycoprotein 181
5.4 Molecular Weight Determination of Small (<1000 Da)
Molecules 185
5.4.1 The Use of Fast-LC–MS in Combinatorial Chemistry 185
viii Liquid Chromatography–Mass Spectrometry
5.5 Structure Determination of Low-Molecular-Weight
Compounds 189
5.5.1 Method Development for Structural Studies 189
5.5.2 The Use of Target-Compound Analysis and
LC–MS–MS for the Identification of Drug Metabolites 192
5.5.3 The Use of High-Accuracy Mass Measurements
in Combination with LC–MS for the Structure
Determination of Drug Metabolites 199
5.5.4 The Use of Cone-Voltage Fragmentation in Conjunction
with High-Accuracy Mass Measurements and LC–MS
for Metabolite Identification 203
5.5.5 The Use of LC–MSn for the Identification
of Drug Metabolites 207
5.6 Quantitation 211
5.6.1 Requirements of a Quantitative Method
Involving LC–MS 211
5.6.2 Quantitative Standardization 213
5.6.3 Matrix Effects in LC–MS 213
5.6.4 The Method of Standard Additions to Overcome
Matrix Effects 218
5.6.5 The Quantitative Determination of DNA
Oxidation Products 222
5.6.6 The Use of MS–MS for Quantitative Determinations 224
References 233
Responses to Self-Assessment Questions 235
Bibliography 241
Glossary of Terms 247
SI Units and Physical Constants 257
Periodic Table 261
Index 263
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