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An Introduction to Mass Spectrometry in the Health Sciences

Tony Mallet

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An Introduction to Mass Spectrometry in the Health SciencesTony Mallet

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Published by IM Publications LLP 6 Charlton Mill, Charlton, Chichester, West Sussex PO18 0HY, UK. T: +44-1243-811334, F: +44-1243-811711, E: [email protected], www.impublications.com

Edition: 1.0

ISBN: 978-1-906715-19-9

British Library Cataloguing in Publication Data. A catalogue record for this book is available from the British Library.

© 2014 IM Publications LLP

All rights reserved. Apart from any fair dealing for the purposes of research or private study, or criticism or review, as permitted under the UK’s Copyright, Designs and Patents Act 1988, this publication may be reproduced, stored or transmitted, in any form or by any means, only with the prior permission in writing of the publishers, or in the case of reprographic reproduction, in accordance with the terms of licences issued by the Copyright Licensing Agency. Enquiries concerning reproduction outside those terms should be sent to the publishers at the address above.

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ContentsIntroduction ..............................................................................................................................................7Web site ....................................................................................................................................................................................................9

1. The essentials of a mass spectrometer and the results of analysis .............................................. 10Introduction ...........................................................................................................................................................................................10Ion source ...............................................................................................................................................................................................11Analyser ..................................................................................................................................................................................................20Tandem mass spectrometry ..............................................................................................................................................................25Ion detection .........................................................................................................................................................................................29Functions of an analyser and detector ..........................................................................................................................................30Summary .................................................................................................................................................................................................33

2. The mass spectrum .......................................................................................................................... 35Continuum and stick mass spectra ..................................................................................................................................................35EI spectra .................................................................................................................................................................................................35API spectra .............................................................................................................................................................................................36Stable isotopes ......................................................................................................................................................................................37Identifying the molecular ion ...........................................................................................................................................................39Elemental composition .......................................................................................................................................................................42Ion fragmentation .................................................................................................................................................................................42Scanned and selected ion experiments ........................................................................................................................................44Tandem mass spectrometry (MS/MS) ............................................................................................................................................45Summary .................................................................................................................................................................................................49

3. Sample inlet systems ........................................................................................................................ 52Introduction ...........................................................................................................................................................................................52Chromatographic separation ...........................................................................................................................................................52Gas chromatography ...........................................................................................................................................................................53Liquid chromatography: HPLC/UHPLC ........................................................................................................................................55Optimising separation in chromatography ..................................................................................................................................58Nano-flow LC/MS .................................................................................................................................................................................61Sample preparation prior to chromatography ...........................................................................................................................62

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FIA: Flow injection analysis ................................................................................................................................................................65Ionisation in the open laboratory—ambient mass spectrometry ........................................................................................66Summary .................................................................................................................................................................................................66

4. The use of mass spectrometry for qualitative analysis ................................................................. 69Structure from an EI spectrum .........................................................................................................................................................69Structural study with electrospray ionisation and MS/MS ......................................................................................................70Peptide characterisation and sequencing ....................................................................................................................................75MS/MS and sequence determination ............................................................................................................................................78Metabolite identification ...................................................................................................................................................................80Summary .................................................................................................................................................................................................81

5. Quantitative analysis ....................................................................................................................... 83Introduction ...........................................................................................................................................................................................83Ionisation ................................................................................................................................................................................................84Ion analysis .............................................................................................................................................................................................85Ion detection and recording ............................................................................................................................................................87Optimising mass spectrometer response and specificity........................................................................................................89Internal standards for quantitative analysis .................................................................................................................................90Matrix effects .........................................................................................................................................................................................96Validation and statistical treatment of quantitative data .........................................................................................................96Sample preparation for quantitative analysis...........................................................................................................................100Summary ..............................................................................................................................................................................................103

6. Mass spectrometry in the clinical laboratory: chromatographic sample input ....................... 106Vitamin D metabolite analysis .......................................................................................................................................................107Plasma estriol analysis ......................................................................................................................................................................110Urinary catecholamine analysis .....................................................................................................................................................112Testosterone analysis .......................................................................................................................................................................113Quantitative analysis of hepcidin .................................................................................................................................................114Summary ..............................................................................................................................................................................................117

7. Mass spectrometry in the clinical laboratory: direct sample input and ambient mass spectrometry ....................................................................................................................................... 118MCAD determination in neonates ..............................................................................................................................................118

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Haemoglobin analyses .....................................................................................................................................................................119Ambient ion sources ........................................................................................................................................................................121REIMS and detection of cancerous tissue..................................................................................................................................124Automation of instrumentation ....................................................................................................................................................125Clinical microbiology .......................................................................................................................................................................125Summary ..............................................................................................................................................................................................126

8. Bibliography ................................................................................................................................... 128Further reading, general texts .......................................................................................................................................................128Mass spectrometry strategies in metabolomics ......................................................................................................................130The scientific literature ....................................................................................................................................................................131

9. References ...................................................................................................................................... 132

10. A model experiment in quantitative mass spectrometry based analysis............................... 142Objective of the experiment .........................................................................................................................................................142Analogue internal standard ...........................................................................................................................................................142Instrument and ESI MS and MS/MS .............................................................................................................................................143Liquid chromatography ..................................................................................................................................................................143Sample purification...........................................................................................................................................................................143Solutions of vincristine and vinblastine ......................................................................................................................................144Linearity and sensitivity; calibration ............................................................................................................................................144Accuracy and precision ...................................................................................................................................................................144Recovery ..............................................................................................................................................................................................145Matrix effects ......................................................................................................................................................................................145Conclusion ...........................................................................................................................................................................................145

11. A list of common terms ............................................................................................................... 146Acronyms .............................................................................................................................................................................................146Common terms ..................................................................................................................................................................................149

12. Simple mass spectrometry fragmentation mechanisms .......................................................... 180EI starting with a radical cation. Simple fission .........................................................................................................................180EI radical cation, rearrangement ...................................................................................................................................................180ESI protonated ion, loss of stable neutral ..................................................................................................................................181

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ESI even electron cations can also undergo rearrangements ..............................................................................................181

13. Surrogate analyte method for endogenous analytes............................................................... 182

14. Using this book ............................................................................................................................ 184Hyperlinks ...........................................................................................................................................................................................184Enhanced navigation ........................................................................................................................................................................184

Acknowledgements

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AcknowledgementsThe author thanks past and present colleagues and instrument manufacturers for many helpful discussions, suggestions for illustrations and content and especially to Professor Alison Ashcroft, Dr Michael Wright, Professor Mike Morris and Professor Neil Dalton for their encouragement while writing this book and, finally, to my wife for her patient exposure to the mysteries of mass spectrometry. Also to Ian Michael for many discussions on the merits and presentation of an ebook publication.

Introduction

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IntroductionMass spectrometry (MS) has for a century been an analytical method of choice for structural and elemental analysis on account of its sensitivity and specificity. It was limited in its early applications to the analysis of gases and volatile organic molecules and elements, by the apparent difficulty in ionising them, by the size and high cost of the early instruments and by the complexity of their operation. The advent of novel ionisation methods, ever increasingly powerful computer systems, advanced applications of ion optics and vacuum engineering and electronics has in the past 30 years made the technique applicable to the quantitative and qualitative analysis of a large range of organic and bio-organic molecules including endogenous and drug metabolites, proteins and other bio-polymers. The instruments are now smaller, totally controlled through a computer system and the latter can be made to output data in formats appropriate to the area of application. Up to now the most common uses of mass spectrometry in clinical settings has been to provide a back up in confirmation of more routine methods, in clinical pharmacology research, in metabolic studies, most successfully in diagnosis of inborn errors of metabolism, and in proteomic analyses. Many of these projects have been sited in University and pharmaceutical industry laboratories and in contract mass spectrometry enterprises.

In 2008 M. Vogeser and C. Seger [1] wrote a review of the state of use of mass spectrometry in clinical settings. Since then several of the hoped-for improvements they suggested have taken place and more clinically oriented laboratories and institutions are taking up mass spectrometry for quantitative as well as qualitative analysis to help solve diagnostic problems. This account is aimed to help newcomers to understand the science behind the operation of modern mass spectrometry and combined chromatography-mass spectrometry systems and to highlight some of the most common misunderstandings and problems that can occur.

1M. Vogeser and C. Seger, “A decade of HPLC-MS/MS in the routine clinical laboratory—goals for further

developments”, Clin. Biochem. 41, 649–662 (2008). doi: 10.1016/j.clinbiochem.2008.02.017

http://dx.doi.org/10.1016/j.clinbiochem.2008.02.017

Introduction

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While much biological mass spectrometry development has been led by the pharmaceutical industry, application areas include forensics, metabolomics, proteomics and sports medicine in all of which the ability of the technique to provide structural information can be crucial. A principal reason for choosing a quantitative analysis by mass spectrometry over the more common methods in use in clinical laboratories is that the former can provide greater specificity and frequently, a clearer marker for the occurrence of interferences from extraneous signals. Routine mass spectrometric analyses are now in use or being developed for several common endogenous plasma molecules and are in use also to provide a reference standard analytical procedure for routine immuno-assays. Recent development work has been on the production of rapid, sensitive and stable instruments and automated sample introduction methods.

The development of a near “black box” instrument while making operation more straightforward, has the disadvantage of masking the occasions when inaccurate data is output and may not be recognised as such. Any analytical technique in which a sample is placed in an instrument has a potential to interfere with the operation of the device and in the case of mass spectrometry where small apertures and high voltages are present such problems are always a possibility.

The acronym POCT “point of care testing” is increasingly being quoted in connection with mass spectrometric methods of analysis. The common instrumental arrangements of a chromatographic separation linked to a mass spectrometer do not make for a comfortable patient bedside test. However, several direct simple sample ionisation methods are being developed for use in the open atmosphere and these are being seriously considered for such situations. Recent descriptions for very large scale surveys are becoming more common e.g. Reference [2].

2T.P. Mechtler, S. Stary, T.F. Metz, V.R. De Jesús, S. Greber-Platzer, A. Pollak, K.R. Herkner, B. Streubel and D.C.

Kasper, “Neonatal screening for lysosomal storage disorders: feasibility and incidence from a nationwide study in Austria”, Lancet 379, 335–341 (2012). doi: 10.1016/s0140-6736(11)61266-x

http://dx.doi.org/10.1016/s0140-6736%2811%2961266-x

http://dx.doi.org/10.1016/s0140-6736%2811%2961266-x

IntroductionWeb site

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This book is designed to explain the science behind the operation of a mass spectrometer, to suggest methods for the production of reliable data and to flag cases where the production of false data can occur. No “recipes” are given for specific analyte methods; these are plentiful in the scientific literature. The intention has been to prepare the reader to examine critically the published methods and to be aware of the major pitfalls in designing and implementing an analytical procedure.

The first five chapters describe the operation of a mass spectrometer system and its use for qualitative and quantitative analysis while the final chapters describe some successful examples of application and explain how the methods chosen were designed to improve sensitivity, specificity and speed of analyses. Possible future applications are also discussed. A list of acronyms and a short section on the meaning of common terms is included at the end of the volume. Links to references, web sites and other useful information are included.

Web site

Readers may like to visit the book’s website (http://www.impublications.com/mshealthsci/) for updates and the opportunity to ask questions and clarifications of the author. Please use the password “xxxxx” to access the site.

http://www.impublications.com/mshealthsci/

The essentials of a mass spectrometer and the results of analysisIntroduction

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1. The essentials of a mass spectrometer and the results of analysis

Introduction

The “parts” of a mass spectrometer shown in Figure 1.1 consist of a sample introduction method, a source of ions, an analyser where the ions are separated and their mass-to-charge ratios (m/z) are measured and a detector. In addition a computerised operating and data system are used to control the instrument and collect, manipulate and store the data. The analyser and detector and some ion sources need to be maintained under a vacuum and efficient pumping is absolutely essential.

The mass spectrometer is not a spectroscopic device but a true metric measurement technique by which the mass of an element, a molecule or a fragment of one can be determined. Unlike true spectroscopic detection methods such as ultraviolet (UV) or nuclear magnetic resonance (NMR) that are governed by the intrinsic properties of the molecules and in which a given molecule will behave identically in a variety of instruments, in mass spectrometry it is the interaction between the ionised molecule and the design of the instrument which is being determined. Variation in design and dimensions of the ion source and analyser between different manufacturers can lead to significant differences in behavior of analyte species. Even the same model of instrument may produce different results. The basis of its operation depends upon the production of an electrically charged species, an ion, which is made to move in electrical and magnetic fields. The physics of these induced movements, in a wide variety of configurations, produces equations of motion that include the term m/z, the ratio of mass to charge of the ion. We will see that the value of z is often 1 and hence the mass of

Figure 1.1. The “parts” of a mass spectrometer instrument.

The essentials of a mass spectrometer and the results of analysisIon source: the part of the instrument in which ions are formed

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the ion is easily obtained. Later on we will discuss cases where z is greater than unity and how the value can be obtained.

In this chapter we will limit the ion sources to those that can be easily linked to a chromatographic separation system; more direct techniques will be discussed later.

Ion source: the part of the instrument in which ions are formed

Electron ionisation (EI)

This method, originally used by Thomson and Aston in the early 20th century involves the introduction of the volatilised sample into a vacuum chamber where it is exposed to a beam of electrons from a heated filament. The technique illustrated in Figure 1.2 is well suited to a sample introduced from a gas-chromatography separation system as the analyte will be already in a vapour state and the mass spectrometer instrument can easily maintain a good vacuum with a constant flow of helium carrier gas from a GC capillary column.

When an energetic electron passes in close proximity to an atom or molecule it strips off one electron from the outer electronic shell of the target leaving a positive charge on a free radical:

M + ε → M+• + 2ε–

The resulting radical cation may still have enough excess energy for bond breaking and other reactions and hence a family of fragment ions is also produced. We will see later that this leads to a “spectrum” of ions of differing m/z where z is, in most cases, unity.

electrontrap

ion repeller

S N

filament

electronbeam

extraction grid

focussing lens

magnet

To MassSpectrometer

Figure 1.2. An EI source; the sample from a GC column eluent is introduced into the plane of the diagram to intersect with the electron beam. The whole source is inside a high vacuum. (Figure provided by Dr P. Gates, University of Bristol.)


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Chemical ionisation (CI)

A closely related method, that uses essentially the same arrangement, is to include a reagent gas in the EI source that is itself ionised by the electron beam. The design of the ion source is similar to the EI source above but all apertures are kept as small as possible so as to maintain an increased gas pressure and a line to introduce the reagent gas is provided. The reagent gas is ionised by an EI process and the radical cations so formed react with neutral species to form energetic cations. These then react with analyte molecules in a secondary process usually by proton transfer to make a new cationised analyte ion, Figure 1.3. Common reagent gases include methane, iso-butane and ammonia as shown below:

In the case of methane CI the initial step is the formation of the radical cation of methane; this is followed by a number of chemical reactions, including the reaction of this with more methane. The CH5

+ cation produced is a strong acid that can ionise analyte molecules. No radical cations are formed and thus less energy is present to induce fragmentation.

Electrospray ionisation (ESI)

EI and CI can only be used where it is possible to introduce a volatilised sample into a vacuum. The majority of biological molecules and biopolymers are therefore excluded from these methods. In the 1980’s the development of ionisation methods taking place at atmospheric pressure has led to the employment of mass spectrometry for virtually any type of molecule and the most widely used in liquid chromatography with mass spectrometry approach has been that of John Fenn [3], Figure 1.4. Electrospray ionisation takes place from a solution of the analyte molecule in appropriate solvent mixtures, Figure 1.4. The solvent is sprayed through a fine aperture in the presence of a high voltage.

Figure 1.3. Protonation by methane in a CI source.

CH4 + ε– → CH4•+

CH4•+ + CH4 → CH5

+ + CH3•

CH5+ + M → MH+ + CH4

3J. Fenn, M. Mann, C. Meng, S. Wong and C. Whitehouse, “Electrospray ionization for mass spectrometry of

large biomolecules”, Science 246, 64–71 (1989). doi: 10.1126/science.2675315

http://dx.doi.org/10.1126/science.2675315


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This leads to a so-called Taylor cone (see Figure 1.6) from which an aerosol of fine charged droplets is formed. The addition of nitrogen gas and heat to assist in the evaporation of the solvent and to define a narrow stream of droplets leads to the formation of a succession of diminishing droplets and finally to solvent free, charged molecular ions.

The choice of solvent is important; it should be volatile and usually contains an acid or a base to assist in the addition or removal of a proton from the analyte molecule. Common solvent mixtures are aqueous acetic or formic acids with methanol or acetonitrile and these are also common RP-HPLC chromatography solvents. The atmospheric pressure nature of ESI makes the coupling to any liquid chromatography separation system a simple job. The ESI solvent should not contain an involatile buffer as the fine apertures in the source will otherwise

Figure 1.4. Electrospray ionisation and ion source overview. The sample is present in a stream of volatile solvent which is sprayed through a fine capillary held at high voltage, with a stream of nitrogen gas. (Reproduced with permission from the Lamond Laboratory, University of Dundee.)


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soon become blocked. In spite of biochemists’ fears it has almost always been possible to find a volatile replacement for most common buffer recipes. Table 1.1 suggests appropriate volatile ESI solvents and buffer solutions.

While a small ionised molecule can carry a single +ve or –ve charge, larger species in which charges can be physically separated provide a case where the z in m/z becomes greater than unity. Most common are larger molecules and polymeric materials such as peptides and proteins where z can be anything from 2 upwards. This provides a means of analysis for molecules with molecular weights greater than the transmission upper limit of the analyser.

API, LC/MS compatible solvents

Solvent Mol. wt. UV cut-offAcetonitrile 41.05 190Chloroform 119.4 245Dichloromethanea 84.93 235Ethanol 46.08 210Ethyl acetate 88.12 260Heptane 100.21 200Hexane 86.18 200Isopropanol 60.11 210Methanol 32.04 205n-Propanol 60.11 210Tetrahydrofurana 72.12 215Toluene 92.15 285Water 18.02 <190

aDichloromethane and THF are not compatible with PEEK HPLC tubing

Compatible volatile buffers

Buffer pKa RangeTrifluoroacetic acidb 0.5 3.8–5.8Formic acid 3.8 —Ammonium formate 3.8 2.8–4.8Acetic acid 4.8 —Ammonium acetate 4.8 3.8–5.84-Methylmorpholine 8.4 7.4–9.4Ammonium bicarbonate 6.3–10.3 6.8–11.3Ammonium acetate 9.2 8.2–10.2Ammonium formate 9.2 8.2–10.21-Methylpiperidine 10.1 10.0–12.0Triethylammonium acetate 11.0 10.0–12.0Pyrrolidine 11.3 10.3–12.3

bTrifluroacetic acid can affect negative ion sensitivity and its ion signal is very persistent; avoid in ESI if possible

Table 1.1. Suggested ESI volatile solvents and buffer compositions.


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A typical case is shown here, Figure 1.5. The measured m/z of the most intense peak arises from the formula [M + 5] / 5 where M is the molecular weight of insulin. We discuss the methods for deconvoluting these spectra later on.

The low energy of ion production will also result in charged species other than protons as well as solvent molecules being attached to the analyte molecule and a complex family of molecular ions may result. The presence of sodium is nearly impossible to prevent totally from everyday samples.

ESI and the next three techniques are referred to as Atmospheric Pressure Ionisation (API).

Figure 1.5. ESI spectrum of bovine insulin. The molecular weight of this compound is 5734 Da and the three principal signals here are all molecular ions and arise from ions with 6, 5 and 4 protons attached to the molecule.

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