Liquid Chromatography-Mass Spectrometry: An Introduction (Analytical Techniques in the Sciences)

LIQUID CHROMATOGRAPHY–MASS SPECTROMETRY: AN INTRODUCTION......Page 3
Contents......Page 7
Series Preface......Page 11
Preface......Page 13
Acknowledgements......Page 15
Abbreviations, Acronyms and Symbols......Page 17
About the Author......Page 21
1 Introduction......Page 23
1.1 What are the Advantages of Linking High Performance Liquid Chromatography with Mass Spectrometry?......Page 24
1.2 What Capabilities are Required of the Combination?......Page 25
1.3 What Problems, if Any, Have to be Addressed to Allow the LC–MS Combination to Function, and Function Effectively?......Page 26
References......Page 27
2.1 Introduction......Page 29
2.2.1 Pump......Page 32
2.2.2 Sample Introduction (Injector)......Page 33
2.2.3 Mobile Phase......Page 34
2.2.4 Stationary Phase......Page 36
2.2.5 Detectors......Page 38
2.3 Chromatographic Properties......Page 40
2.4 Identification Using High Performance Liquid Chromatography......Page 43
2.5 Quantitation Using High Performance Liquid Chromatography......Page 45
2.6 The Need for High Performance Liquid Chromatography–Mass Spectrometry......Page 52
References......Page 53
3.1 Introduction......Page 55
3.2.2 Chemical Ionization......Page 58
3.2.3 Fast-Atom Bombardment......Page 60
3.2.4 Matrix-Assisted Laser Desorption Ionization......Page 61
3.3 Ion Separation......Page 62
3.3.1 The Quadrupole Mass Analyser......Page 63
3.3.3 The Double-Focusing and Tri-Sector Mass Analysers......Page 64
3.3.4 The Time-of-Flight Mass Analyser......Page 66
3.4 Tandem Mass Spectrometry (MS–MS)......Page 68
3.4.1 Instrumentation......Page 69
3.4.2 Techniques......Page 73
3.5.1 Identification......Page 75
3.5.2 Quantitation......Page 76
3.6.1 The Total-Ion-Current Trace......Page 80
3.6.2 Qualitative Analysis......Page 82
3.6.3 Quantitative Analysis......Page 90
3.6.4 The Use of Tandem Mass Spectrometry......Page 93
References......Page 95
4.1 Introduction......Page 97
4.2 The Moving-Belt Interface......Page 99
4.3 The Direct-Liquid-Introduction Interface......Page 104
4.4 The Continuous-Flow/Frit (Dynamic) Fast-Atom-Bombardment Interface......Page 107
4.5 The Particle-Beam Interface......Page 111
4.6 The Thermospray Interface......Page 116
4.7 The Electrospray Interface......Page 120
4.7.1 The Mechanism of Electrospray Ionization......Page 122
4.7.2 Sample Types......Page 127
4.7.3 The Appearance of the Electrospray Spectrum......Page 128
4.7.4 Structural Information from Electrospray Ionization......Page 139
4.8 The Atmospheric-Pressure Chemical Ionization Interface......Page 144
4.8.1 The Mechanism of Atmospheric-Pressure Chemical Ionization......Page 145
References......Page 148
5 Applications of High Performance Liquid Chromatography–Mass Spectrometry......Page 151
5.1 Method Development......Page 153
5.1.1 The Use of Experimental Design for Method Development......Page 155
5.1.2 The Choice of Electrospray or APCI......Page 156
5.2.1 Electrospray Spectra of Co-Eluting Components......Page 163
5.2.2 The Use of Selected-Ion Monitoring to Examine the Number of Terminal Galactose Moieties on a Glycoprotein......Page 165
5.2.3 The Effect of Mobile-Phase Additives and Cone-Voltage......Page 169
5.3.1 Amino Acid Sequencing of Proteins......Page 171
5.3.2 The Use of Enzymes for Amino Acid Sequencing......Page 172
5.3.4 Confirmation of Amino Acid Sequence Using the Analysis of LC–MS Data from an Enzyme Digest of a Protein......Page 174
5.3.5 Determination of the Amino Acid Sequence of a Novel Protein Using LC–MS Data from an Enzyme Digest......Page 182
5.3.6 Amino Acid Sequencing of Polypeptides Generated by Enzyme Digestion Using MS–MS......Page 188
5.3.7 The Location of Post-Translational Modifications Using LC–MS Data from an Enzyme Digest......Page 192
5.3.8 The Location of Post-Translational Modifications Using MS–MS......Page 195
5.3.9 The Analysis of Polysaccharides Present in Glycosylated Proteins......Page 199
5.3.10 Location of the Position of Attachment of a Glycan on the Polypeptide Backbone of a Glycoprotein......Page 203
5.4.1 The Use of Fast-LC–MS in Combinatorial Chemistry......Page 207
5.5.1 Method Development for Structural Studies......Page 211
5.5.2 The Use of Target-Compound Analysis and LC–MS–MS for the Identification of Drug Metabolites......Page 214
5.5.3 The Use of High-Accuracy Mass Measurements in Combination with LC–MS for the Structure Determination of Drug Metabolites......Page 221
5.5.4 The Use of Cone-Voltage Fragmentation in Conjunction with High-Accuracy Mass Measurements and LC–MS for Metabolite Identification......Page 225
5.5.5 The Use of LC–MS(n) for the Identification of Drug Metabolites......Page 229
5.6.1 Requirements of a Quantitative Method Involving LC–MS......Page 233
5.6.3 Matrix Effects in LC–MS......Page 235
5.6.4 The Method of Standard Additions to Overcome Matrix Effects......Page 240
5.6.5 The Quantitative Determination of DNA Oxidation Products......Page 244
5.6.6 The Use of MS–MS for Quantitative Determinations......Page 246
References......Page 255
Responses to Self-Assessment Questions......Page 257
Bibliography......Page 263
Glossary of Terms......Page 269
SI Units and Physical Constants......Page 279
Periodic Table......Page 283
Index......Page 285