Mass Spectrometry for Metabolite Identification

YUQIN WANG AND WILLIAM J. GRIFFITHS

The School of Pharmacy, University of London, 29–39 Brunswick Square, London WC1N 1AX, UK

1.1 Introduction

Mass spectrometry (MS) and nuclear magnetic resonance (NMR) constitute the two major pillars upon which the disciplines of metabolomics and metabolite profiling are built. Both these techniques have their advantages and disadvantages, but the fundamental difference in the nature of their spectroscopy means that they provide complementary information to the analytical scientist. NMR spectroscopy is discussed in detail in Chapter 2, while this chapter will concentrate on mass spectrometry and associated methodologies appropriate in metabolomics research. The principles of mass spectrometry will be described and examples of metabolite analysis given. As the range of metabolite structures present in biology (almost) exceeds the imagination, we will take many examples from the class of biomolecules that we have been most intimately involved with, i.e. sterols and steroids. However, many of the references quoted are equally applicable to other area of metabolite profiling and metabolomics.

1.2 Mass Spectrometry

1.2.1 Principles

Simplistically, a mass spectrometer consists of an ion source, a mass analyser, a detector and a data system (Figure 1.1). Sample molecules are admitted to the ion source, where they become ionised. The ions, which are now in the gas phase, are separated according to their mass-to-charge ratio (m/z) in the mass analyser and are then detected. The resulting signals are transmitted to the data system and a plot of ion abundance against m/z corresponds to a mass spectrum. In many cases, a separating inlet device precedes the ion source, so that complex mixtures can be separated prior to admission to the mass spectrometer. Today, the separating inlet device is usually either a capillary gas chromatography (GC) column or a high-performance liquid chromatography (HPLC) column, although capillary electrophoresis and thin-layer chromatography can be interfaced with mass spectrometry.

For metabolite analysis a number of different types of ionisation methods are used to generate gas-phase ions and these include: electron ionisation (EI), chemical ionisation (CI), electrospray (ES), atmospheric pressure chemical ionisation (APCI), atmospheric pressure photoionisation (APPI), and the recently introduced, desorption electrospray ionisation (DESI) technique. Other ionisation techniques used, but to a lesser extent, are liquid secondary ion mass spectrometry (LSIMS) and fast atom bombardment (FAB), or for more specific applications, matrix-assisted laser desorption/ionisation (MALDI) (see Chapter 9) and desorption ionisation on silicon (DIOS). The most widely used ionisation modes are discussed below, as are the chromatographic devices to which they are interfaced.

1.2.2 Ionisation

1.2.2.1 Electron Ionisation (EI) and Chemical Ionisation (CI)

Historically, the most important method of ionisation of small biomolecules (< ~500 Da) is EI. The effluent from a GC column is readily transferred to an EI source, thereby allowing the combination of the high separating power of a GC column with mass analysis. EI involves the bombardment of gas-phase sample molecules (M) with high-energy electrons (e-), usually of 70 eV energy; the result is the generation of [M]+• ions which are usually radical cations, and thermal energy free electrons (e-) (eqn 1).

[FORMULA NOT REPRODUCIBLE IN ASCII] (1)

In many cases the molecular ions, [M]+•, are unstable and fragment to generate more stable products (eqn 2).

[FORMULA NOT REPRODUCIBLE IN ASCII] (2)

Fragmentation upon EI can be seen as both advantageous and disadvantageous. On the plus side, the fragmentation pattern resulting from decomposition of a molecular ion can provide structural information, allowing its identification. On the negative side, however, fragmentation may be so extensive that the molecular ion may not be observed, and thus the molecular weight of the compound of interest not determined. EI can be used to generate either positive ions [M]+•or negative ions [M]+-. Negative ions are generated via an electron capture event, which involves the capture of secondary low-energy electrons generated by ionisation of a bath gas (e.g. Ar, N2) (eqn 3).

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[FORMULA NOT REPRODUCIBLE IN ASCII] (3b)

A prerequisite of EI is that the sample to be ionised must be in the gas phase; this is also true for GC and has led to the extensive development of derivatisation chemistry to allow the vaporisation of many small biomolecules without their decomposition.

CI is a close relative of EI. It differs in that analyte ionisation is achieved via proton attachment rather than electron ejection (positive ion). In CI the ion source contains a reagent gas, often methane, which becomes ionised by EI and acts as a proton donor to the analyte (eqn 4).

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[FORMULA NOT REPRODUCIBLE IN ASCII] (4b)

[FORMULA NOT REPRODUCIBLE IN ASCII] (4c)

The resulting ion, [M + H]+, is an even-electron protonated molecule, which is more stable than the equivalent odd-electron molecular ion, [M] +•, formed by EI, and thus fragments to only a minor extent.

Electron-capture negative ionisation (ECNI), also called electron-capture negative chemical ionisation (EC-NCI), exploits the electron capturing properties of groups with high electron affinities (eqn 5). The method often utilises fluori-nated agents in the preparation of volatile derivatives with high electron affinities. For example, trifluoroacetic, pentafluoropropionic or heptafluorobutyric anhydrides can be used to prepare acyl derivatives of amines and hydroxyl groups, perfluorinated alcohols can be used to generate esters of carboxylic acids, while carbonyl groups can be converted to oximes which can then be converted to...