A basic overview of mass spectrometry relevant to life and health science applications, illustrated throughout with relevant case studies

This introductory text provides information and assistance to new users of mass spectrometry (MS) working in clinical or biochemical fields who are faced with implementing and designing quantitative mass spectrometric assays for a variety of classes of molecules of biological interest. It presents a detailed discussion on how to optimize measurement parameters for a candidate reference quantitative analysis, including calibration procedures, sensitivity, reproducibility, speed of assay and compliance with regulatory authorities.

Quantitative Biological and Clinical Mass Spectrometry uses examples where development has not been immediately successful but where unforeseen problems have arisen and describes the strategies used to solve these. Advances in addressing the very large numbers of clinical samples that arise on routine screening programs such as those involved in inborn errors of metabolism studies are discussed. Direct mass spectrometric based analyses applicable to point of care testing (POCT) situations are also covered. The book concludes with a short section on possible novel developments, bibliography, references, and a glossary of terms.

* Shows how the presence of false results can be detected and understood

* Describes the 'parts' of modern instruments from sample introduction through ionization, mass analysis and detection, and the variety of techniques of tandem mass spectrometry

* Discusses the requirement for specificity in an assay method

* Fully illustrated throughout

* Highly relevant to all key areas of mass spectrometric analysis

Quantitative Biological and Clinical Mass Spectrometry appeals to those newly exposed to the use of combined chromatography and mass spectrometry for analysis of biological material and to scientists experienced in automated clinical analysis using immunoassays or who are new to 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.

Inhalt

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 Matrixassisted 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 MALDITOF analyser 32

2.2 Tandem mass spectrometry 33

2.2.1 QqQ triple quadrupole analysers 36

2.2.2 QTOF 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 Atmosphericpressure 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 oddelectron 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 Reversedphase HPLC 75

4.2.2 Normalphase HPLC 76

4.2.3 HILIC 76

4.2.4 Ionexchange 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 o...