Quantitative Biological and Clinical Mass Spectrometry

Anthony I. Mallet has over 45 years of experience in the application of mass spectrometry to natural substances and clinical pharmacology. He has been employed in the University sector since 1967, has an Emeritus Professorship from King's College, London, and held a visiting chair in the University of Greenwich until 2013.

Acknowledgements x

Introduction 1

References 6

1 The Instrument: Ion Creation 7

1.1 Introduction 7

1.2 Sample handling 8

1.3 Vacuum ion sources 9

1.3.1 Electron ionisation 9

1.3.2 Chemical ionisation 11

1.3.3 Negative ion chemical ionization electron capture ionisation 12

1.3.4 Matrix‐assisted laser desorption ionisation 12

1.4 Atmospheric pressure ion sources 13

1.4.1 Electrospray ionisation methods for liquid samples 14

1.4.2 Atmospheric pressure chemical ionisation 19

1.4.3 Atmospheric pressure photoionisation 21

1.5 Ambient ionisation methods 23

References 23

2 The Instrument: Ion Analysis and Detection 25

2.1 The analyser 25

2.1.1 Quadrupole analyser 26

2.1.2 Ion trap 28

2.1.3 Orbitrap™ 31

2.1.4 MALDI‐TOF analyser 32

2.2 Tandem mass spectrometry 33

2.2.1 QqQ triple quadrupole analysers 36

2.2.2 Q‐TOF tandem mass spectrometry 36

2.2.3 MS/MS with an LIT analyser 38

2.2.4 Quadrupole with Orbitrap 38

2.3 The detector 40

2.3.1 Electron multiplier detectors 40

2.3.2 Fourier transform detection 42

2.4 Control and data handling 43

2.5 Ambient ionisation 45

2.6 Summary 46

References 48

3 The Mass Spectrum 49

3.1 Spectral output 49

3.2 Electron ionisation/chemical ionisation spectra 52

3.2.1 Radical cations from electron ionisation 52

3.2.2 Molecular weight nomenclature 53

3.3 Stable isotopes and accurate m/z determinations 54

3.3.1 Assignment of the molecular ion 54

3.3.2 Elemental composition 56

3.4 Chemical ionisation 57

3.4.1 Chemical ionisation with isobutane 57

3.4.2 Electron capture negative ion chemical ionisation 58

3.5 Atmospheric‐pressure spray ionisation 59

3.5.1 Electrospray ionisation 59

3.6 Tandem mass spectra, MS/MS 61

3.6.1 Fragmentation in the source 61

3.6.2 MS/MS analysis with multiple analysers 62

3.7 Manipulating chromatographic data output 64

3.7.1 Averaging spectra over eluting chromatogram 65

3.7.2 Background signal removal 65

3.7.3 SRM/MRM data presentation 66

3.8 Fragmentation of even‐ and odd‐electron ions 66

3.9 Spectra of peptides proteins and other biopolymers 66

3.10 Summary 70

References 70

4 Sample Handling Prior to Ionisation 72

4.1 Gas chromatography 73

4.2 Liquid chromatography: HPLC/UHPLC 75

4.2.1 Reversed‐phase HPLC 75

4.2.2 Normal‐phase HPLC 76

4.2.3 HILIC 76

4.2.4 Ion‐exchange HPLC 77

4.2.5 UHPLC 77

4.2.6 Effect of LC flow 77

4.3 Alternative sample purification methods 78

4.3.1 SPE cartridges 79

4.3.2 Supported liquid extraction cartridges 79

4.3.3 Protein crash cartridges 80

4.3.4 Less common chromatographic separation methods 80

4.4 Theory of chromatography relevant to clinical MS ion sources 82

4.4.1 Optimising separation and MS conditions 82

4.5 Avoiding chromatography: flow injection analysis 86

4.6 Summary 86

References 87

5 Establishing Optimum Specificity 88

5.1 Structure from the molecular ion or its derivative 88

5.1.1 Which is the molecular ion? 88

5.1.2 Examine the stable isotope ion patterns 89

5.1.3 What is the true molecular weight? 89

5.2 Structure from fragmentation 91

5.2.1 Simple rules for interpreting a spectrum 91

5.3 Spectra of peptides and proteins 92

5.3.1 ESI spectra of biopolymers 92

5.4 Example of the deduction of the identity of an unknown 94

5.4.1 ESI analysis of supposed fake material 94

5.4.2 MS/MS of proposed protonated molecular ion at 279 95

5.4.3 Examination of the stable isotope patterns to eliminate further possibilities 95

5.5 Potential problems with MS/MS for quantitative analysis 97

5.5.1 Crosstalk in MRM analyses 98

5.5.2 Mobile protons 98

5.6 Conclusions 101

References 102

6 Quantitative Analysis with Mass Spectrometry 103

6.1 Introduction 103

6.2 Calibration with internal standards 104

6.2.1 Analogue internal standards 104

6.2.2 Stable isotope internal standards 106

6.3 Creation of a calibration curve 107

6.4 Assay validation 110

6.4.1 Regulatory authorities 110

6.4.2 Errors 112

6.4.3 Parameters that need to be published for a valid assay 112

6.5 Matrix interference 114

6.6 Immediate calibrations 115

6.7 Selected or multiple ion recording 117

6.8 Summary 119

References 119

7 Examples of Quantitative Analysis: Combined Chromatography and Mass Spectrometry 121

7.1 Vitamin D metabolite analysis 122

7.2 Testosterone/ epitestosterone 126

7.3 Oxygenated neural sterols 129

7.4 Cholic acids 131

7.5 Phospholipids 131

7.6 8‐iso‐Prostaglandin F2α 133

7.7 Metanephrine and normetanephrine 134

7.8 Isotopic internal calibration assay for clozapine and norclozapine 135

7.9 Glycolipids and carbohydrates 137

7.10 Matrix‐assisted laser desorption ionisation analysis of simple carbohydrates 139

7.11 LC– MS/MS ceramides in Fabry disease 139

7.12 N‐Tetrasaccharides from protein glycosylation defects 140

7.13 Peptides 141

7.14 Hepcidin 141

7.15 Thyroglobulin 144

7.16 Quantitative proteomics 146

7.17 Summary 148

References 148

8 Rapid Clinical Analysis: Direct Sample Application to the Mass Spectrometer Source 153

8.1 Flow injection analysis 153

8.2 Dried blood spots and neonate inborn errors of metabolism analysis 154

8.3 Haemoglobin analyses 157

8.4 Application of ambient ionisation methods 163

8.4.1 Ambient spray ionisation 163

8.4.2 Ionisation with energetic beams 166

8.4.3 MALDI‐TOF and identification of microorganisms 168

8.4.4 Rapid evaporative ionisation mass spectrometry 170

8.5 Conclusions 172

References 173

A: Simple Mass Spectrometry Fragmentation Mechanisms 176

B: Some Simple Derivatisation Methods 179

C: Acronyms and Glossary of Common Terms 180

D: Simple Statistics 200

E: Helpful Web Links 202

Bibliography 204

Index 206