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 xIntroduction 1References 61 The Instrument: Ion Creation 71.1 Introduction 71.2 Sample handling 81.3 Vacuum ion sources 91.3.1 Electron ionisation 91.3.2 Chemical ionisation 111.3.3 Negative ion chemical ionization electron capture ionisation 121.3.4 Matrix‐assisted laser desorption ionisation 121.4 Atmospheric pressure ion sources 131.4.1 Electrospray ionisation methods for liquid samples 141.4.2 Atmospheric pressure chemical ionisation 191.4.3 Atmospheric pressure photoionisation 211.5 Ambient ionisation methods 23References 232 The Instrument: Ion Analysis and Detection 252.1 The analyser 252.1.1 Quadrupole analyser 262.1.2 Ion trap 282.1.3 Orbitrap™ 312.1.4 MALDI‐TOF analyser 322.2 Tandem mass spectrometry 332.2.1 QqQ triple quadrupole analysers 362.2.2 Q‐TOF tandem mass spectrometry 362.2.3 MS/MS with an LIT analyser 382.2.4 Quadrupole with Orbitrap 382.3 The detector 402.3.1 Electron multiplier detectors 402.3.2 Fourier transform detection 422.4 Control and data handling 432.5 Ambient ionisation 452.6 Summary 46References 483 The Mass Spectrum 493.1 Spectral output 493.2 Electron ionisation/chemical ionisation spectra 523.2.1 Radical cations from electron ionisation 523.2.2 Molecular weight nomenclature 533.3 Stable isotopes and accurate m/z determinations 543.3.1 Assignment of the molecular ion 543.3.2 Elemental composition 563.4 Chemical ionisation 573.4.1 Chemical ionisation with isobutane 573.4.2 Electron capture negative ion chemical ionisation 583.5 Atmospheric‐pressure spray ionisation 593.5.1 Electrospray ionisation 593.6 Tandem mass spectra, MS/MS 613.6.1 Fragmentation in the source 613.6.2 MS/MS analysis with multiple analysers 623.7 Manipulating chromatographic data output 643.7.1 Averaging spectra over eluting chromatogram 653.7.2 Background signal removal 653.7.3 SRM/MRM data presentation 663.8 Fragmentation of even‐ and odd‐electron ions 663.9 Spectra of peptides proteins and other biopolymers 663.10 Summary 70References 704 Sample Handling Prior to Ionisation 724.1 Gas chromatography 734.2 Liquid chromatography: HPLC/UHPLC 754.2.1 Reversed‐phase HPLC 754.2.2 Normal‐phase HPLC 764.2.3 HILIC 764.2.4 Ion‐exchange HPLC 774.2.5 UHPLC 774.2.6 Effect of LC flow 774.3 Alternative sample purification methods 784.3.1 SPE cartridges 794.3.2 Supported liquid extraction cartridges 794.3.3 Protein crash cartridges 804.3.4 Less common chromatographic separation methods 804.4 Theory of chromatography relevant to clinical MS ion sources 824.4.1 Optimising separation and MS conditions 824.5 Avoiding chromatography: flow injection analysis 864.6 Summary 86References 875 Establishing Optimum Specificity 885.1 Structure from the molecular ion or its derivative 885.1.1 Which is the molecular ion? 885.1.2 Examine the stable isotope ion patterns 895.1.3 What is the true molecular weight? 895.2 Structure from fragmentation 915.2.1 Simple rules for interpreting a spectrum 915.3 Spectra of peptides and proteins 925.3.1 ESI spectra of biopolymers 925.4 Example of the deduction of the identity of an unknown 945.4.1 ESI analysis of supposed fake material 945.4.2 MS/MS of proposed protonated molecular ion at 279 955.4.3 Examination of the stable isotope patterns to eliminate further possibilities 955.5 Potential problems with MS/MS for quantitative analysis 975.5.1 Crosstalk in MRM analyses 985.5.2 Mobile protons 985.6 Conclusions 101References 1026 Quantitative Analysis with Mass Spectrometry 1036.1 Introduction 1036.2 Calibration with internal standards 1046.2.1 Analogue internal standards 1046.2.2 Stable isotope internal standards 1066.3 Creation of a calibration curve 1076.4 Assay validation 1106.4.1 Regulatory authorities 1106.4.2 Errors 1126.4.3 Parameters that need to be published for a valid assay 1126.5 Matrix interference 1146.6 Immediate calibrations 1156.7 Selected or multiple ion recording 1176.8 Summary 119References 1197 Examples of Quantitative Analysis: Combined Chromatography and Mass Spectrometry 1217.1 Vitamin D metabolite analysis 1227.2 Testosterone/ epitestosterone 1267.3 Oxygenated neural sterols 1297.4 Cholic acids 1317.5 Phospholipids 1317.6 8‐iso‐Prostaglandin F2α 1337.7 Metanephrine and normetanephrine 1347.8 Isotopic internal calibration assay for clozapine and norclozapine 1357.9 Glycolipids and carbohydrates 1377.10 Matrix‐assisted laser desorption ionisation analysis of simple carbohydrates 1397.11 LC– MS/MS ceramides in Fabry disease 1397.12 N‐Tetrasaccharides from protein glycosylation defects 1407.13 Peptides 1417.14 Hepcidin 1417.15 Thyroglobulin 1447.16 Quantitative proteomics 1467.17 Summary 148References 1488 Rapid Clinical Analysis: Direct Sample Application to the Mass Spectrometer Source 1538.1 Flow injection analysis 1538.2 Dried blood spots and neonate inborn errors of metabolism analysis 1548.3 Haemoglobin analyses 1578.4 Application of ambient ionisation methods 1638.4.1 Ambient spray ionisation 1638.4.2 Ionisation with energetic beams 1668.4.3 MALDI‐TOF and identification of microorganisms 1688.4.4 Rapid evaporative ionisation mass spectrometry 1708.5 Conclusions 172References 173A: Simple Mass Spectrometry Fragmentation Mechanisms 176B: Some Simple Derivatisation Methods 179C: Acronyms and Glossary of Common Terms 180D: Simple Statistics 200E: Helpful Web Links 202Bibliography 204Index 206