The Use of Hyphenated Techniques in Comparative Phytochemical Studies of Legumes G.C

Biochemical Systematics and Ecology 31 (2003) 813–843 www.elsevier.com/locate/biochemsyseco The use of hyphenated techniques in comparative phytochemical studies of legumes G.C. Kite ∗, N.C. Veitch, R.J. Grayer, M.S.J. Simmonds Royal Botanic Gardens Kew, Richmond, Surrey TW9 3AB, UK Received 10 December 2002; accepted 28 February 2003 Abstract The coupling of instruments performing chromatographic separations to those providing structural data has had an enormous impact in analytical chemistry. These ‘hyphenated techniques’ are enabling compounds to be detected in plant extracts more effectively than ever before. At the same time, the rapid development of DNA sequencing technology and cladistic data analysis have provided taxonomists with the means to produce testable systematic hypotheses. These parallel developments in analytical chemistry and systematics have transformed the often criticised discipline of chemotaxonomy into modern integrated studies in comparative phytochemistry that aim to test cladistic hypotheses or gain insights into the biochemical evolution of plants. In this paper the key developments in the main hyphenated techniques, gas chromatography- mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS), are considered together with some of the challenges facing the further development of capillary electrophoresis-mass spectrometry (CE-MS) and liquid chromatography-nuclear magnetic resonance spectroscopy (LC-NMR). The application of GC-MS and LC-MS in comparative phytochemical studies in legumes is reviewed both from selected research in the literature and from the authors’ own experiences, with an emphasis on nitrogen-containing and phenolic compounds. The use of GC-MS has provided an extensive data set on the occurrence of quinolizidine alkaloids in legumes and this character is now assuming taxonomic significance at a high level. GC-MS also provides the means to separate the numerous isomeric forms of poly- hydroxyalkaloids and hydroxypipecolic acids as their volatile trimethylsilyl derivatives and surveys of these compounds are supporting systematic work at lower taxonomic levels. LC- MS is enabling the metabolic profiles of intact flavonoid glycosides to be obtained from small fragments of material while recent methods to analyse non-protein amino acids by LC-MS ∗ Corresponding author. Tel.: +1-44-208-332-5368; fax: +1-44-208-332-5310. E-mail address: [email protected] (G.C. Kite). 0305-1978/03/$ - see front matter 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0305-1978(03)00086-3 814 G.C. Kite et al. / Biochemical Systematics and Ecology 31 (2003) 813–843 without derivatisation hold much promise in surveys of these important taxonomic characters. Tandem mass spectrometry (MS/MS) provides a rapid means of sequencing peptides and so, as we enter the era of proteomics, LC-MS/MS is likely to play a central role in the analysis of legume proteins. 2003 Elsevier Science Ltd. All rights reserved. Keywords: Gas chromatography; Liquid chromatography; Mass spectrometry; Nuclear magnetic resonance spectroscopy; Hyphenated techniques; Leguminosae; Quinolizidine alkaloids; Non-protein amino acids; Flavonoids 1. Introduction Use of the term ‘hyphenated technique’ became popular in analytical chemistry during the 1980s as instruments employed in separation science were coupled directly to those used to elucidate the structures of organic compounds (Wilkins, 1983). The term has been applied most frequently to the coupling of mass spectrometry (MS) to gas chromatography (GC), high performance liquid chromatography (LC) or capillary electrophoresis (CE), and the combination of LC with nuclear magnetic resonance spectroscopy (NMR). However, many other techniques are ‘hyphenated’ such as the use of a photodiode array detector (PDA) with LC since this detector was the design solution to achieving on-line UV-visible spectrophotometry (UV). The distinction, perhaps, is that the evolution of the former techniques also involved the ‘hyphenation’ of scientists from different disciplines. The impact of hyphenated techniques in analytical chemistry has been enormous as they provide the analyst with structural information on the components present in complex mixtures (Stobiecki, 2001). This information may be sufficient to identify non-novel components or, in the case of LC-NMR, full structural elucidation of an unknown component is potentially possible without having to undergo the time- consuming process of isolating it. Hyphenated techniques are particularly adept at targeted analyses; i.e. determining whether a specific component is present in a mix- ture (Hopfgartner et al., 1999). They are, therefore, ideally suited to studies in systematic phytochemistry in which the occurrences of specific compounds or types of compounds are surveyed in taxonomic groups to test or sometimes suggest taxonomic relationships in combination with other character evidence, such as DNA sequence or morphological data. The main focus of this paper is to examine the use of hyphenated mass spectrometry techniques in systematic phytochemistry, with particular reference to legumes. The development of the mass analysers used in hyphenated techniques is dis- cussed in relation to GC-MS and the contribution that this more established technique has made in comparative legume chemistry is reviewed. However, it is LC-MS that is likely to have the greatest application in systematic phytochemistry as it progresses from being an expensive ‘state-of-the-art’ technique to one that is more widely avail- able in research laboratories (Niessen, 1999). The future application of LC-MS in the analysis of compounds that may be of systematic value in legumes is therefore G.C. Kite et al. / Biochemical Systematics and Ecology 31 (2003) 813–843 815 reviewed together with the key developments that have made LC-MS an accepted technique. The challenges facing CE-MS and LC-NMR, two hyphenated techniques that still require further development, are also considered briefly. 2. Development of mass analysers Throughout most of the past 100 years or so since its development, mass spectrometry lay in the realm of specialised laboratories and used large, complex and expensive magnetic deflection and double-focussing instruments. The 1980s, however, saw the beginning of a period of rapid change and now, during the centenary of its inception, mass spectrometry has become a widely used research tool. Progress has come through the commercialisation of different types of mass analysers, increasingly sophisticated computer control and, most significantly, the coupling of mass analysers to chromatographic techniques. Transferring an analyte from a gaseous chromatographic mobile phase to the high vacuum of a mass spectrometer presents fewer technical challenges than transferring an analyte from a liquid mobile phase. Thus, new mass analysers were introduced into GC-MS before LC-MS, although they are essentially the same for both. Direct coupling of GC to magnetic sector instruments was achieved about 50 years ago, but today such GC-MS systems are rare and the most widely used analyser for GC-MS is the quadrupole mass filter. Developed by Paul in the 1950s, this mass analyser uses a quadrupolar electrical field, comprising radiofrequency and direct current components, generated by four rods to scan out ions onto a detector (Dawson, 1976). ‘Quadrupoles’ are only capable of low-resolution mass analysis but the ion intensities in the mass spectra obtained from electron ionisation (EI) are similar to those recorded on high resolution sector instruments. Although the first commercial quadrupole GC-MS was introduced in 1968, it was not until 1983 that the wider availability of GC-MS was catalysed with the introduction of an alternative and lower cost mass analyser, the quadrupole ion trap (Stafford et al., 1984). The ion trap, first proposed by Paul and Steinwedel (1953), traps and analyses ions using a three-dimensional quadrupolar radiofrequency electric field (March, 2000). Ion traps are sensitive but ion intensities in their EI spectra differ slightly from quadrupoles. They do, however, have the advantage of performing lower cost tandem mass spectrometry (see later) which is ideally suited to target analyte analyses in complex matr- ices. The most recent mass analyser to be employed in GC-MS is the time-of-flight (TOF) tube proposed by Stephens (1946). In TOFMS, ions of different mass-to- charge ratio are separated by differences in their velocities as they move in a straight path towards a collector (Standing, 2000). In theory, these mass analysers have unlimited mass range and so they are very suitable for biomolecular MS, particularly for determining the molecular weights of proteins. TOF analysers can also record mass spectra very rapidly and can deliver a mass accuracy approaching that of classi- cal double-focussing instruments; thus they are highly suited to hyphenated techniques. Coupling of TOFMS to GC was achieved back in the 1950s, but it was the 816 G.C. Kite et al. / Biochemical Systematics and Ecology 31 (2003) 813–843 construction in 1996–1997 of hybrid instruments, using a quadrupole mass filter to inject ions orthogonally into a TOF analyser (Morris et al., 1996; Shevchenko et al., 1997), that resulted in recently-introduced commercial systems. GC-TOFMS is likely to play an increasingly major role in future phytochemical studies. 3. Use of GC-MS in legume systematics The use of GC-MS is restricted to compounds that

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