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 polyhydroxyalkaloids 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 mixture (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 discussed 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 available 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 classical 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 are sufﬁciently volatile to pass through GC columns at temperatures of up to 400 °C, the upper limit of ‘high temperature’ capillary columns. In natural product research, GC-MS is most frequently used to analyse essential oils. Legumes are not rich in essential oils but a number of genera do contain alkaloids based on the quinolizidine ring system that are sufﬁciently volatile to be analysed by GC-MS, and these have been studied inten- sively by this hyphenated technique. The range of compounds that can be analysed by GC-MS can be extended by employing chemical derivatisation to increase vola- tility. A frequently employed derivatisation method is to silylate groups having exchangeable protons, such as OH, NH and NH2. Performing GC-MS analyses of derivatised compounds only becomes advantageous over analysing the compounds in their native state by LC when the superior chromatographic resolving power and retention time consistency of GC is required to attempt separation of epimeric or isomeric forms. Examples of this approach in legumes are the analyses by GC-MS of two groups of hydroxylated polar nitrogen compounds: ‘polyhydroxyalkaloids’ and hydroxypipecolic acids.

3.1. Quinolizidine alkaloids

In legumes, GC-MS has been most readily applied to studies of quinolizidine alkaloids—the main class of alkaloids produced by the family (Kinghorn and Smo- lenski, 1981; Wink, 1993). More than 200 of these alkaloids are known (Ohmiya et al., 1995) and they can be broadly classified by the number and arrangement of their composite six-membered ring systems. Even before the advent of GC-MS a substantial data set had been accumulated on the occurrence of quinolizidine alkaloids from the time- and material-consuming process of purifying individual alkaloids and determining their structures by classical techniques or, less reliably, by thin layer chromatography or even spot tests (Mears and Mabry, 1971). These early data, when compared with Hutchinson’s classification (Hutchinson, 1964), indicated that the compounds were confined to subfamily Papil- ionoideae, being found in many members of tribes in the so-called ‘genistoid alliance’ and some members of the ‘primitive’ tribe Sophoreae (Mears and Mabry, 1971). The use of GC-MS in quinolizidine alkaloid analysis was developed in the late 1970s–early 1980s, first using packed column systems (Kinghorn and Balandrin, 1984) and then using capillary GC-MS (e.g. Wink et al., 1980). With subsequent development and wider availability of bench-top systems, GC-MS has become the G.C. Kite et al. / Biochemical Systematics and Ecology 31 (2003) 813–843 817 analytical method of choice, particularly in ecological and taxonomic studies. The suitability of GC-MS for the study of quinolizidine alkaloids in systematic work arises because most are sufficiently volatile and thermostable under GC conditions to permit analysis without chemical modification, although some hydroxylated quinolizidine alkaloids are better analysed as their trimethylsilyl derivatives (Veitch et al., 1997). Furthermore, the EI mass spectra of quinolizidine alkaloids have been well characterised and fragment ions have been identified that are indicative of certain features of the carbon skeleton (Ohmiya et al., 1995). Lists of mass spectra have also been compiled and published, often with relative retention or Kovat’s indices (e.g. Wink, 1993), and these can enable identification of the most common quinolizidine alkaloids (Fig. 1). GC-MS has enabled data on the quinolizidine alkaloids of less readily available taxa to be obtained by analysing crudely purified extracts made from small fragments of herbarium specimens. Some of the more recent work exemplifying this approach is that of Greinwald et al. (1993, 1995b, 1995c, 1996). These workers wanted to test the hypothesis, generated from a cladistic analysis of morphological characters (Crisp and Weston, 1987), that Brongniartieae sensu Polhill (1981) should be expanded to include the Templetonia group of legumes, placed in Bossiaeeae by Polhill (1981). Analysis of herbarium material of the two genera in Brongniartieae sensu Polhill (1981), Brongniartia and Harpalyce, and genera in the Templetonia group revealed that all contained quinolizidine alkaloids. As the remainder of Bos- siaeeae are not known to synthesize quinolizidine alkaloids, this data supported the transfer of the Templetonia group from Bossiaeeae to Brongniartieae (Polhill, 1994). In another study, GC-MS failed to reveal quinolizidine alkaloids in Hypocalyptus

Fig. 1. GC-MS analysis of quinolizidine alkaloids from Calia secundiflora (syn. Sophora secundiflora). Compounds identified by comparison of mass spectra and retention indices with published data (Wink, 1993). 818 G.C. Kite et al. / Biochemical Systematics and Ecology 31 (2003) 813Ð843

(van Wyk and Schutte, 1995), which was placed in the alkaloid-containing tribe Crotalarieae. Later the genus was moved to its own tribe Hypocalypteae unrelated to Crotalarieae (Schutte and van Wyk, 1998). GC-MS has been instrumental in compiling the extensive knowledge of the distribution of quinolizidine alkaloids that is now available (Table 1). The significance of these data is becoming more apparent with the publication of cladistic analyses of DNA sequence data from papilionoid genera. Currently, the most complete molecular phylogeny of the ‘genistoid alliance’ is that of Crisp et al. (2000) who analysed ITS1 and ITS2 sequences from several representatives of all the genistoid tribes. This analysis produced two sister clades (Brongniartieae and the ‘core genistoids’) containing all the quinolizidine alkaloid-containing genistoid genera sampled except Calia secundiflora (syn. Sophora secundiflora), which was placed as sister to these groups. Genera in the ‘genistoid alliance’ apparently not synthesising quinolizidine alkaloids, that is those in the Australian tribes Bossiaeeae and Mirbelieae, were excluded from the core genistoid clade, as was Hypocalyptus. Furthermore, the analysis placed Sophora (in part) and Maackia, two quinolizidine alkaloid-producing genera of Sophoreae, near the base of the core genistoid clade, whereas Baphia, a mem- ber of Sophoreae lacking quinolizidine alkaloids, was excluded. Poecilanthe, a genus with uncertain relationships based on morphological characters and classified vari- ously in Dalbergieae, Millettieae or Robinieae by different authors, was placed at the base of the Brongniartieae clade in the analysis of Crisp et al. (2000). Previously

Table 1 Alphabetical listing of legume genera reported to contain quinolizidine alkaloids

Acosmium Adenocarpus Ammodendron Ammopiptanthus Anagyris Anarthrophyllum Argyrocytisus Argyrolobium Aspalathus Baptisia Bolusanthus Bowdichia Brongniartia Cadia Calia Calicotome Calpurnia Camoensia Chamaecytisus Clathrotropis Cyclolobium∗ Cytisophyllum Cytisus Dichilus Dicraeopetalum Diplotropis Echinospartum Euchresta Genista Gonocytisus Haplormosia Harpalyce Hesperolaburnum Hovea Laburnum Lamprolobium Lebeckia Liparia Lotononis Lupinus Maackia Melolobium Ormosia Pearsonia Pericopsis Petteria Piptanthus Plagiocarpus Platycelyphium Podalyria Poecilanthe Polhillia Priestleya Rafnia Retama Robynsiophyton Rothia Sakoanala Sophora (part) Spartidium Spartium Stauracanthus Templetonia Thermopsis Ulex Virgilia Wiborgia

Data from Mears and Mabry (1971); Kinghorn et al. (1982); Kinghorn and Balandrin (1984); Torrenegra et al. (1989), van Wyk and Verdoorn (1990), van Wyk et al. (1993); Wink (1993), Greinwald et al. (1993; 1995 a,b,c; 1996), Kass and Wink (1995); Ohmiya et al. (1995), van Wyk and Schutte (1995); Tosun and Akyuz (1999) and Kite (in prep., indicated by an asterisk). Cladrastis is not considered to be a quinolizidine alkaloid accumulating genus (see text). G.C. Kite et al. / Biochemical Systematics and Ecology 31 (2003) 813Ð843 819

GC-MS had been used to show that Poecilanthe contained quinolizidine alkaloids and the particular combination of structures present suggested a similarity with members of Brongniartieae (Greinwald et al., 1995a). On the basis of the molecular and chemical evidence, together with past suggestions, Crisp et al. (2000) formerly transferred Poecilanthe to Brongniartieae. A subsequent analysis of basal papilionoid legumes using sequences of the chloroplast intron trnL (Pennington et al., 2001) included more members of Sophoreae but fewer genistoids. This analysis generated a monophyletic clade in which Brongniart- ieae (only represented by Poecilanthe) was included with the core genistoids (Fig. 2). The clade also included Acosmium, Ormosia, Bowdichia, Sophora (in part), Pla- tycelyphium, Bolusanthus and Dicraeopetalum from Sophoreae, all of which are reported to contain quinolizidine alkaloids (Asres et al., 1986; Kinghorn et al., 1988; Torrenegra et al., 1989; van Wyk et al., 1993; Asres et al., 1997; Veitch et al., 1997). Also included in the clade, and sister to Poecilanthe,wasCyclolobium from

Fig. 2. Occurrence of quinolizidine alkaloids plotted onto a cladistic analysis of trnL intron DNA sequence data (simpliﬁed from Pennington et al., 2001) 820 G.C. Kite et al. / Biochemical Systematics and Ecology 31 (2003) 813Ð843

Millettieae. Analysis of herbarium material of Cyclolobium by GC-MS has revealed that it contains quinolizidine alkaloids, so supporting its placement in the core genistoids (Kite, in prep.). Thus, in these analyses, the presence of quinolizidine alkaloids appears to define a monophyletic clade or paraphyletic sister clades. Excluding reports of quinolizidine alkaloids in Oxytropis, Lotus and Dalbergia [see reviews by Wink (1993) and Ohmiya et al. (1995) for references], which are presumed anomalous, the only reports of quinolizidine alkaloids in taxa placed by Crisp et al. (2000) or Pennington et al. (2001) outside the core genistoid clade(s) are for Cladrastis, Calia secundiflora, Sophora pachycarpa [see reviews by Mears and Mabry (1971); Wink (1993) and Ohmiya et al. (1995) for alkaloid references] and Styphnolobium japonicum (as Sophora japonica; Kass and Wink, 1995). The position of Sophora pachycarpa in the analysis of Pennington et al. (2001) is now known to be due to an erroneous DNA sequence for this taxon and GC-MS analysis has been used to re-investigate the quinolizidine alkaloid status of the other taxa (Kite and Pennington, 2003). This study failed to detect quinolizidine alkaloids in cultivated specimens of Styphnolob- ium japonicum, S. affine and three species of Cladrastis but confirmed their presence in Calia secundiflora (Fig. 1). The absence of quinolizidine alkaloids in Cladrastis and Styphnolobium would concur with their position outside of the core genistoid clade(s) in either or both the analyses of Crisp et al. (2000) and Pennington et al. (2001), although it is difficult to prove conclusively that they lack the ability to synthesise quinolizidine alkaloids and continued investigation is required. Crisp et al. (2000) and Pennington et al. (2001) also found that Calia secundiflora was the sister taxon to the core genistoid clade(s), although it was part of a polytomy in the latter analysis, so the presence of quinolizidine alkaloids in this taxon does not con- flict with this placement. However, another recent molecular analysis using rbcL sequences (Kajita et al., 2001) positioned Calia in a clade with Styphnolobium and Cladrastis, again excluding them from the genistoid clade. This analysis also excluded Bolusanthus from the genistoid clade. Several quinolizidine alkaloids have been isolated from B. speciosus (Asres et al., 1986) creating another anomaly between chemical and molecular data that requires continued investigation. Further systematic work employing GC-MS analyses of quinolizidine alkaloids is likely to concentrate on the occurrence of particular patterns of alkaloids at a lower taxonomic levels. For example the presence of a-pyridone alkaloids in Argyrolobium, Dichilus, Melolobium and Polhillia (van Wyk and Verdoorn, 1990) supported their transfer from Crotalarieae to Genisteae, since a-pyridone alkaloids were typically absent from other genera in Crotalarieae (van Wyk and Schutte, 1995). At the species level the variability of quinolizidine alkaloid profiles within a species or even within individuals (e.g. Ricker et al., 1999) may diminish their usefulness as systematic characters. For example, an extensive GC-MS survey of quinolizidine alkaloids in Lupinus (Wink et al., 1995) found some correlation with molecular phylogenies but trends were quantitative rather than on the basis of discrete presence or absence of various types (Kass and Wink, 1997; Ainouche and Bayer, 1999). Alternative techniques, such as LC-MS, could also be employed to study non-volatile derivatives, such as quinolizidine alkaloid esters and glycosides (Abdel-Halim et al., 1999). G.C. Kite et al. / Biochemical Systematics and Ecology 31 (2003) 813Ð843 821

3.2. Polyhydroxyalkaloids

Polyhydroxyalkaloids are an unnatural assembly of compounds composed of one or two saturated heterocyclic ring systems bearing several hydroxyl groups (Watson et al., 2001). The different locations and conﬁgurations of the hydroxyl groups give rise to numerous isomers and epimers, the majority of which can be separated by GC-MS analysis of their trimethylsilyl derivatives. In Leguminosae, different types of polyhydroxyalkaloids are currently known from only a few genera in Sophoreae, Millettieae and Galegeae. Early work with GC-MS showed the presence of castanospermine-type polyhydroxyalkaloids, previously only known from the Australian genus Castanospermum (Hohenschutz et al., 1981), in herbarium specimens of the South American genus Alexa (Nash et al., 1988). This relationship was supported by other non-chemical characters and has now been conﬁrmed in molecular phylogenies (Pennington et al., 2001). Since then, however, GC-MS has been used largely to support intensive isolation work from a few legume taxa rather than in systematic studies (Asano et al., 2001). Nevertheless, since two of the ‘hotspots’ of polyhydroxyalkaloid occurrences, namely DMDP in Lonchocarpus of Millettieae and swainsonine in Astragalus of Galegeae, are within two of the largest and most intrac- table systematic complexes, GC-MS analysis of polyhydroxyalkaloids could have a role to play in future systematic studies in these areas.

3.3. Hydroxypipecolic acids

The compound code-named BR-1 isolated from Baphia racemosa was once classed as a polyhydroxyalkaloid (Manning et al., 1985). BR1 is now considered to be the only known naturally-occurring trihydroxylated pipecolic acid and it had not been reported from any other source until recently when it was revealed in the mono- typic Baphiopsis by GC-MS analyses of trimethylsilylated extracts (Kite, 2003). This study used ammonia chemical ionisation (CI) to confirm the molecular weights of the trimethylsilylated components detected, as molecular ions were absent from the quadrupole EI spectra (Fig. 3). Baphia and Baphiopsis were not considered related from morphology (even though their names suggest otherwise), being classified in the papilionoid tribes Sophoreae and Swartzieae, respectively (Polhill, 1994), but analyses of trnL DNA sequences (Ireland et al., 2000; Pennington et al., 2001) placed them as sister taxa. The occurrence of rare compounds in taxa considered related by other means provides strong support of systematic affinity and such studies show the value of hyphenated analytical techniques in the systematic study of chemical characters. More widely distributed in legumes are monohydroxypipecolic acids and, to a lesser extent, dihydroxypipecolic acids. Early work on the taxonomic distribution of these compounds was hindered by the difficulty of separating the epimeric and isomeric forms by traditional techniques (Romeo et al., 1983). In order to undertake a large survey of hydroxypipecolic acids in Inga, a GC-MS method was developed to separate, as their trimethylsilyl ethers, all eight common hydroxypipecolic acids in one analysis (Kite and Hughes, 1997). When applied to a survey of 92 species of 822 G.C. Kite et al. / Biochemical Systematics and Ecology 31 (2003) 813Ð843

Fig. 3. Use of GC-MS to detect the trihydroxypipecolic acid BR1 in an extract of Baphiopsis parviﬂora. The total ion chromatogram of trimethylsilyl derivatives (top) reveals a peak (arrowed) showing the same retention time and EI mass spectrum (middle) as a derivatised standard of BR1. The ammonia CI spectrum (bottom) conﬁrms the molecular weight of the derivatised compound.

Inga, this GC-MS method revealed several patterns of hydroxypipecolic acid accumulation which often supported concepts of closely related species groups, but at a higher taxonomic level particular patterns cut across the supposed morphological development in the genus (Kite, 1997). In Inga, the use of hydroxypipecolic acids as systematic characters at the species level may be limited by the occurrence of chemotypes in some species, such as Inga umbellifera. In another study using GC-MS (Kite and Wieringa, 2003), the presence or absence of hydroxypipecolic acids among species in the former genus Monopetalanthus was found to support the recent segregation of species into Aphanocalyx, Bikinia and Tetraberlinia (Wieringa, 1999). The species transferred to Aphanocalyx were found to lack these compounds, like species traditionally placed in that genus, while the species reclassiﬁed into the new genus Bikinia synthesised hydroxypipecolic acids and hydroxyprolines except for three species considered most derived on the basis of morphology. Tetraberlinia longiracemosa (formerly Monopetalanthus longiracemosus) also contained hydroxypipecolic acids as did all other species of Tetraberlinia. Future analyses by GC-MS are likely to reveal that mono- and dihydroxypipecolic acids are more widespread in legumes than was previously thought and therefore G.C. Kite et al. / Biochemical Systematics and Ecology 31 (2003) 813Ð843 823 will probably not be useful as characters in cladistic analyses of the whole family (Chappill, 1995). They may, however, provide useful characters at lower levels depending on the degree of variability shown.

4. Key developments in LC-MS

Before considering the potential of LC-MS in comparative studies of legume chemistry it is useful to review the key developments that have now made LC-MS a sensitive, robust and more widely used technique. Several interfaces were developed during the 1980s to transfer analyte molecules in solution, eluting from a liquid chromatograph, into gas phase ions suitable for mass analysis. Among those that found use in phytochemical laboratories during the evolution of LC-MS were the thermospray, particle beam and continuous-ﬂow fast atom bombardment interfaces. Each of these interfaces had its advantages and disad- vantages, but all have been superseded in the past decade by electrospray (ES) and atmospheric pressure chemical ionisation (APCI) (Herderich et al., 1997).

4.1. Electrospray and atmospheric pressure chemical ionisation

ES and APCI are now free of most major technical problems that bedevilled alternative ionisation methods (Oehme, 1999), and the suitability and simplicity of ES for the analysis of large biomolecules, as well as many micromolecules, has meant it has become the interface of choice in most laboratories. In ES the solvent flow from the LC sprays at atmospheric pressure from the end of a metal capillary as a result of applying a high voltage to the end of the capillary. This electrostatic dispersion of liquids into charged droplets was first described by John Zeleny in the early 1900s, but it was not until the 1960s that ES was pioneered by Malcolm Dole and colleagues (Dole et al., 1968) who realised that evaporation of the charged solvent droplets would lead to their repeated disintegration and ulti- mately to the ejection of gas-phase ions (Kebarle and Ho, 1997). Dole’s group in collaboration with the Bendix Corporation electrosprayed polystyrene molecules (Dole et al., 1968), but it was not until the 1980s that the first ES mass spectrometer was developed to analyse micromolecules (Yamashita and Fenn, 1984). Later ES was shown to produce multiple-charged ions of proteins, such that their mass to charge ratio fell within the scanning range of the quadrupole mass filter enabling their molecular weights to be calculated (Meng et al., 1988). The demand for such a technique meant that commercial ES interfaces followed soon after. In the complementary APCI interface, a spray of solvent is produced by a nebulis- ing gas and the solvent is evaporated as the droplets pass down a hot vaporiser tube. At the exit of the tube, gas phase chemical ionisation occurs as solvent vapour and analytes pass a point charge. APCI is useful for examining less polar and more volatile micromolecules. 824 G.C. Kite et al. / Biochemical Systematics and Ecology 31 (2003) 813Ð843

4.2. Collision-induced decomposition

ES and APCI interfaces are ‘soft ionisation’ techniques in which compounds survive ionisation relatively intact, often as a protonated molecule ([M+H]+) or adduct (e.g. [M+Na]+) or, in negative source polarity, as a deprotonated molecule ([M-H]Ϫ). This is particularly true for ES and so the resulting mass spectrum provides molecular weight information, but generally lacks the high degree of structural information present in the fragmentation pattern seen in, for example, an EI mass spectrum. With APCI more fragment ions are often observed in the spectrum due to the harsher vaporisation and ionisation process. In order to obtain fragmentation of ions produced by ES (or APCI) they are accelerated inside the mass spectrometer so as to collide with molecules of the bath gas, usually helium. Such collision-induced decomposition (CID) of ions was observed in the early days of EI-MS and was the subject of fundamental studies from the 1960s, but it was of little value in analytical work and usually considered a nuisance (Jennings, 2000). This changed with the advent of soft ionisation techniques and CID became an integral part of analytical mass spectrometry. CID can be performed on all the ions emerging from the source, but this produces mixed CID spectra when more than one compound enters the source at the same time, as frequently occurs in LC-MS. To obtain pure CID spectra, the ion of interest (the precursor ion) needs to be isolated. Initially the quadrupole mass filter was the ideal instrument to do this since its rf voltage can be ‘parked’ at a given value to only allow the selected precursor ion through. This ion can then be accelerated into the bath gas in a collision cell (also of quadrupole design) and the products can be recorded using a third quadrupole operated in normal scanning mode. Tandem mass spectrometry (MS/MS) in space therefore requires three quadrupoles. The first analytical triple quadrupole (QqQ) instrument was developed in 1978 (Yost and Enke, 1978) followed by the first commercial instruments in 1982, but these were very expensive.

4.3. Tandem mass spectrometry in time

The high cost of triple quadrupoles caused a resurgence of interest in the quadrupole ion trap (QIT) used in GC-MS since it had the potential to perform MS/MS in time within one analyser. Ions of a given mass to charge ratio can be isolated within a QIT and then excited such that they collide with bath gas and the resulting product ions are trapped and scanned out to the detector. Indeed, rather than being scanned out, the cycle of ion isolation and fragmentation can be repeated a number of times to achieve multistage MS (MSn). The first ES-QIT mass spectrometer was con- structed in 1990 followed by the first bench-top system in 1992 and the first commercial LC-MS instrument in 1995 (Bier and Schwartz, 1997). An important feature of these instruments is that they can record MS/MS spectra automatically during an LC-MS analysis without prior manual programming of the m/z values of ions to be selected for MS/MS (Siethoff et al., 1999). A survey scan monitors the ions being generated by the source and then the most intense ion is automatically selected and G.C. Kite et al. / Biochemical Systematics and Ecology 31 (2003) 813Ð843 825 analysed in a subsequent MS/MS scan. Even more powerfully, if the next survey scan reveals that the most intense ion is at the same m/z value as in the previous survey scan, then the second most intense ion will be analysed in the subsequent MS/MS scan. This data-dependent analysis continues to successively lower intensity ions until the m/z value of the most intense ion in the survey scan changes to a value that has not been analysed within a user-set time period. By this means, CID spectra of co-eluting compounds are obtained in a single LC-MS analysis, which may, therefore, generate hundreds of useful spectra.

4.4. Current developments

The major disadvantage with triple quadrupole and quadrupole ion trap systems is that they are low-resolution instruments, delivering an accuracy of 0.1 m/z units in the range m/z 50–2000 in the case of the QIT. Recording MS/MS spectra with a QIT is also a relatively slow process and advances in automatic control of instruments have been such that a QIT may not record spectra quickly enough to obtain all possible spectra from an eluting LC peak, particularly one containing several compounds. Thus the most recent commercial drive is to introduce the faster and higher resolution time-of-ﬂight analysers into LC-MS. One such instrument, men- tioned earlier in the discussion of mass analysers, replaces the third quadrupole of the triple quadrupole instrument with a TOF analyser. Ion mobility mass spectrometers are also being used to transfer ions successively to a TOF analyser to produce an extremely fast tandem mass spectrometer (Steiner et al., 2001).

5. Potential of LC-MS in legume systematics

In applying LC-MS to a phytochemical problem in legume systematics, or indeed any similar analytical problem, the prime consideration is the ability of the source to ionise the compound or class of compounds under study. Students new to LC- MS often have the misguided impression, gained from experience with GC-EI-MS, that the mass spectrometer will detect all compounds eluting from the column. While it is true that the mass analyser will detect all appropriately charged ions within its scan range, ES and APCI sources do not ionise all the compounds entering them. Ideally, efficient ionisation should occur in the mobile phase used for chromatography. If not then post column modification of mobile phase may be possible to permit or promote ionisation. However, some compounds remain that are difficult to ionise, so ES-MS and APCI-MS are by no means universal detection techniques. Further challenges in LC-MS are to achieve efficient chromatographic separation of compounds using column and mobile phase conditions that are not only compatible with ionisation but also compatible with the mass spectrometer hardware and data acquisition. Mobile phases must not contain a high level of non-volatile components otherwise contamination of the source will occur. Consequently, phosphate buffers commonly used in LC cannot be employed in LC-MS, although recently introduced ‘Z-spray’ sources show greater tolerance to non-volatile buffers. Corros- 826 G.C. Kite et al. / Biochemical Systematics and Ecology 31 (2003) 813Ð843 ive mobile phases should also be avoided. Bleed from the column or mobile phase ionisation products may generate intense signals at m/z values within the desired scanning range, which can cause problems with data acquisition. With quadrupole and TOF analysers these ions will merely create a high baseline that may be rectified by data manipulation, but with a QIT analyser they will exclude sample ions from the trap and so reduce sensitivity. A final general consideration in employing LC-MS is the predictability of the ionisation products generated in an analysis. It would be desirable if the molecular weights of ‘unknowns’ could be assigned confidently from the ionisation products observed. However, our experience shows that it cannot be taken for granted that all examples of a particular class of compound will be ionised in the same manner. For example, a compound prone to dehydration may not survive ionisation intact + + and produce a [(M H)-H2O] ion. The molecular weight of such a compound could easily be assigned incorrectly if this compound were an unknown in the analysis. Flavonoids, alkaloids and non-protein amino acids are among the classes of compounds that have been most frequently used in traditional chemotaxonomic studies in Leguminosae, and fortunately these are easily and in most cases predictably ionised by ES and APCI. Terpenoids may also be useful, taxonomically, but these can be more problematic for LC-MS. The generalities of LC-MS analysis for these compound classes is discussed, briefly, below, although as yet there are few published systematic studies using LC-MS to survey these compounds in legume taxa.

5.1. Flavonoids

Acceptable chromatographic separation of flavonoids and other phenolic compounds can be achieved on standard C18 reverse phase columns using a gradient of aqueous methanol or acetonitrile under acidic conditions. Both ES and APCI sources ionise flavonoids in these mobile phases and acceptable ionisation can be achieved in both positive and negative modes to yield [M+H]+ and [M-H]Ϫ ions, respectively, although limits of detection can be 10–100 times greater than with a UV monitor. Production of an ion corresponding to the protonated or deprotonated flavonoid molecule (depending on source polarity) is generally predictable. One common excep- tion, though, occurs with malonylated flavonoids which decarboxylate in the APCI source; ES yields, unambiguously, the true protonated molecule (Lin et al., 2000). Inclusion of an acid, usually acetic acid, in the mobile phase is essential to promote ionisation of flavonoids with ES but its presence has little effect on flavonoid ionisation using APCI. Thus if an LC separation is better performed under neutral conditions, then post column acidification is necessary when using ES. Legumes can be very amenable to systematic studies of flavonoids by LC-MS as for many species it is possible to analyse crude aqueous methanol extracts of leaves or seeds without further purification to obtain ‘metabolomic’ profiles of the flavonoids produced. Lin et al. (2000) used this approach to analyse the flavonoids of Tri- folium pratense. ES is particularly appropriate for profiling flavonoids as, with appropriate tuning of the source, the compounds can be ionised with minimal fragmentation. Thus coeluting flavonoids of differing masses are readily detected in G.C. Kite et al. / Biochemical Systematics and Ecology 31 (2003) 813Ð843 827 a three-dimensional plot of the chromatographic analysis where m/z value is the third dimension (Fig. 4). With APCI, flavonoids tend to show some degree of in-source fragmentation which can confuse the detection of co-eluting compounds (Grayer et al., 2000). Analysis by LC-MS has potential in screening legumes for unusual flavonoids, such as the various isoflavonoids and neoflavonoids that have been detected in members of the Papilionoideae (Aoki et al., 2000). The general distribution of these may be of higher level taxonomic interest when plotted against molecular phylogenies, or the distribution of unusual structures may support lower level taxonomic relationships. Developing traditional methods to screen for a range of possible isoflavonoid and neoflavonoids in small samples of material is difficult (Harborne, 1971), but it should be possible by LC-MS once a suitable library of MS/MS spectra has been compiled to facilitate identification of the compounds. Another area where LC-MS may improve systematic flavonoid research is in the analysis of flavonoid glycosides. Flavonoids generally occur as glycosides but, in the past, surveys of flavonoids were undertaken on hydrolysed extracts since, with planar chromatography, profiles of flavonoid aglycones were more accurately compared than intact glycosides. LC-MS has more potential to exploit the taxonomic variability that may be shown by the intact glycosides. The CID spectra of protonated flavonoid glycosides generally show the successive losses of sugar moieties to leave a protonated aglycone fragment. Thus information can be obtained on the number

Fig. 4. Three-dimensional plot of an LC-ES-MS analysis of flavonoids in a crude 80% methanol extract of leaves of Ateleia cubensis. ES protonates flavonoid glycosides with minimal fragmentation, thus co- eluting flavonoids with different masses are readily revealed. Numbers above peaks are the m/z values of the [M+H]+ ions of the flavonoids. 828 G.C. Kite et al. / Biochemical Systematics and Ecology 31 (2003) 813Ð843 and type (hexose, pentose, deoxyhexose, etc.) of sugars or other compounds involved in the glycoside and MS/MS analyses of all major flavonoid glycosides can be performed automatically with some instruments, even if compounds co-elute (Fig. 5). Fur- thermore, the CID spectrum of the protonated aglycone fragment can be used to achieve an identification by comparison with library spectra (Ma et al., 1997). For on-line identification of the aglycone moiety, recording a UV spectrum, using LC- UV-MS, is useful and sometimes essential for aglycones with similar CID spectra. With ES, obtaining a CID spectrum of the aglycone of glycosides generally requires a MS/MS/MS experiment, but with APCI an aglycone fragment is usually produced in the ionisation process and can be subjected directly to MS/MS. Thus LC-APCI-

Fig. 5. Positive ion MS/MS spectra of the protonated molecules of two of the co-eluting ﬂavonoid glycosides shown in Fig. 4 ([M+H]+ at m/z 917 and 887) recorded automatically in a single LC-MS/MS analysis by a quadrupole ion trap mass spectrometer. Losses of 146 are due to rhamnosyl moieties while losses of 308 and 338 are indicative of cinnamoylhexosyl and feruloylhexosyl moieties, respectively. A combined loss of rhamnosyl and hexosyl moieties could also result in the loss of 308, but the lack of an intermediate ion makes this less likely. MS/MS/MS analysis of the aglycone fragments at m/z 287 in a subsequent analysis revealed that the ﬂavonoids are kaempferol glycosides. G.C. Kite et al. / Biochemical Systematics and Ecology 31 (2003) 813Ð843 829

MS provides a more efficient approach to obtaining information on aglycone moieties than ES. LC-UV-MS can be used to demonstrate, with acceptable confidence, the existence of the same flavonoid glycosides in related taxa and sufficient spectral data may be provided to assign the structures of common monoglycosides and diglycosides by comparison of the mass and UV spectra and retention times with standards. The complete structures of more complex glycosides can only be obtained by NMR, either off-line, after isolating the compound, or possibly by LC-NMR. However, the CID spectra of flavonoid glycosides may contain more information than simply an indication of the number and masses of the glycosidic moieties involved. For example, based on the relative abundance of ions formed by the fragmentation of glycosidic bonds in positive ion MS/MS, four pairs of isomeric flavonoid O-diglycosides have been differentiated (Ma et al., 2001). It has also been shown that flavonol 3-O-rhamnosyl(1→6)glucosides can be distinguished from 3-O-rhamnosyl (1→2)glucosides using negative ion MS/MS since the CID spectra of the deprotonated molecules and aglycone ions differ (Grayer et al., 2000). Positional isomers of flavone C-glycosides, such as vitexin and isovitexin, have also been distinguished by various CID spectra (Li et al., 1992; Grayer et al., 2000; Waridel et al., 2001) and even quercetin 3-O-glucoside and quercetin 3-O-galactoside can be distinguished from consistent differences in the intensities of certain ions in the negative CID spectra of the aglycone fragment (Fig. 6). Further careful comparison of the MS/MS spectra of positional isomers of flavonoid glycosides and glycosides involving isomeric sugars may aid the on-line characterisation of common and relatively simple flavonoid glycosides. In this respect differences in the CID spectra of flavonoid glycosides complexed with metal ions holds promise (Satterfield and Brodbelt, 2001) but it is doubtful that more elaborate glycosides will be amenable to such studies due to the number of possible positional isomers that will require analysis, although some generalised rules may become apparent. A hindrance to this area of research is that the fragmentation behaviour of flavonoids varies among the types of mass analyser used (Waridel et al., 2001).

5.2. Alkaloids

Most quinolizidine alkaloids, the major class of alkaloids found in legumes, are best analysed by GC-MS, as indicated earlier. However, LC-MS will be useful for the analysis of less volatile quinolizidine alkaloids and their glycosides and for other types of alkaloids that occur in the family, such as those characteristic of Erythrina, Physostigma and Erythrophleum. Alkaloids are very efﬁciently ionised by positive APCI and ES generating [M+H]+ ions or, in the case of quaternary alkaloids, the pre-formed M+ ion is revealed. LC-UV of alkaloids has usually been achieved on reverse phase columns in the presence of an ion-pairing salt. Volatile ion-pair reagents have been substituted to transfer the method to LC-MS (Fabre et al., 2000) but the use of such reagents may be unnecessary with modern C18 columns on which acceptable chromatographic separation of many alkaloids can be achieved merely by using ammonium acetate buffer, usually at basic pH to increase retention 830 G.C. Kite et al. / Biochemical Systematics and Ecology 31 (2003) 813Ð843

Fig. 6. Use of negative ion APCI-MS3 analyses in a quadrupole ion trap to distinguish quercetin 3-O- glucoside from quercetin 3-O-galactoside. Dissociation of the deprotonated molecule at m/z 463 in the 2 = MS analysis yields an ‘aglycone’ ion at m/z 301 (quercetin Mr 302) for both glycosides. These ions, however, are not equivalent as there are consistent differences in the relative intensities of ions at m/z analyses (two CID spectra through (301בand 255 in their CID spectra, obtained by MS3 (m/z 463 271 an LC peak are shown for each glycoside).

(Verpoorte and Niessen, 1994). With recently-introduced ether-linked phenyl reverse phase columns, speciﬁcally designed to retain polar aromatic compounds, suitable chromatography of aromatic alkaloids can be obtained simply with a gradient of aqueous methanol (Fig. 7). G.C. Kite et al. / Biochemical Systematics and Ecology 31 (2003) 813Ð843 831

Fig. 7. LC-APCI-MS analysis of a crude methanol extract from seeds of Physostigma venenosum showing acceptable chromatography of alkaloids on a hydrophilic endcapped, ether-linked phenyl phase column using a simple aqueous methanol mobile phase gradient. Numbers above the peaks are the m/z values of the [M+H]+ ions observed as the base ion in the mass spectra. The CID spectrum (recorded by MS-MS) of eseridine is shown as an example.

5.3. Non-protein amino acids and polyhydroxyalkaloids

The majority of non-protein amino acids, including hydroxypipecolic acids, can be protonated or deprotonated with the appropriate source polarity using ES and APCI. The basic polyhydroxyalkaloids protonate readily, but some can also be deprotonated, albeit at very low efficiency, using negative APCI. The advantage of using negative source polarity is that the CID spectra of deprotonated isomers or epimers may show more differences than the spectra of the protonated forms, which therefore assists identification. This has been demonstrated for both hydroxypipecolic acids from legumes (Kite, 1999) and polyhydroxyalkaloids (Egan et al., 2000). MS-compatible LC of non-protein amino acids and polyhydroxyalkaloids, without employing derivatisation, is problematical due to their very polar nature and charged state. Their low molecular weights also require the mass spectrometer to be scanned down into the cluster ions that generally form from solvents used in the analysis and this can lead to data analysis problems or reduced sensitivity. The standard methods for LC analysis of derivatised amino acids (Bidlingmeyer et al., 1984) can be modified to perform LC-MS of derivatised non-protein amino acids and polyhydroxyalkaloids; for example, 9-fluorenylmethyloxycarbonyl derivatives of catecholamines have been analysed by LC-MS (Chen et al., 1999). With unknowns, however, this creates possible ambiguity in determining the molecular weight of the underivatised form, since ions resulting from the loss of the derivatising groups are 832 G.C. Kite et al. / Biochemical Systematics and Ecology 31 (2003) 813Ð843 not readily identified in CID spectra (Chen et al., 1999). Reagent peaks and multiple derivatisation products also complicate the analysis. Underivatised amino acids have been analysed by LC-MS using a normal phase cyano column (Casetta et al., 2000), but superior retention and chromatographic separation has been achieved by employing perfluorinated acids as volatile ion-pair reagents (Chaimbault et al., 1999). Recently, heptafluorobutyric acid was used as a volatile ion pair reagent in a LC-MS study of the non-protein amino acids in the papilionoid legume genus Bocoa (Kite and Ireland, 2002). In some species of Bocoa the analyses revealed compounds, such as 2,4-methanoproline and 2,4-methanoglut- amic acid, that are characteristic of species of Ateleia and Cyathostegia (Fig. 8). Two species of Bocoa, however, contained hydroxypipecolic acids. These chemical data supported DNA sequence evidence, indicating that Bocoa is polyphyletic and that some species were closely allied to Ateleia and Cyathostegia, a relationship that had not been suggested previously (Ireland et al., 2000). GC-MS of tert-butyldimethylsilyl derivatives of non-protein amino acids has been used in systematic surveys of legumes; for example, in a survey of glutamic acid and phenylalanine based non-protein amino acids in Caesalpinia sensu lato (Kite and Lewis, 1994). This method also suffers from the production of partial derivatives, and compounds with more than four derivatisable groups are not analysed satisfac- torily. Thus LC-MS of underivatised non-protein amino acids is likely to be the method of choice in future systematic studies.

Fig. 8. LC-APCI-MS analysis of underivatised non-protein amino acids in an extract from Ateleia herbert-smithii using heptaﬂuorobutyric acid as an ion pair reagent. Numbers near peaks are the m/z values of the protonated molecules observed as the base ion in the mass spectra. G.C. Kite et al. / Biochemical Systematics and Ecology 31 (2003) 813Ð843 833

5.4. Terpenoids

Given the variety of terpenoids that have been isolated from legumes, their distribution may be of considerable systematic value. LC-MS of non-glycosylated terpenoids will be more difficult than for phenolic and nitrogen-containing compounds. Some of the problems may be anticipated from attempts to analyse steroids by LC-APCI- MS and LC-ESI-MS (Ma and Kim, 1997) in which multiple ionisation products (extensive fragmention and adduct formation) and variable sensitivity were observed, and both of these may depend on the mobile phase used. For these reasons, LC- MS has been applied more extensively to the analysis of terpenoid glycosides, in particular saponins. Saponins, when analysed by positive ion ES, usually yield the sodium adduct because of their strong alkali cation affinity (Fang et al., 1998; Cui et al., 1999) and sensitivity can be improved by cationization with Ag+ (Cui et al., 2001). However, saponins are probably better analysed in negative mode as the [M-H]- ion is formed more clearly (Fang et al., 1998). With negative LC-ES-MS, the efficiency of depro- tonation may be improved by post column addition of ammonium hydroxide solution (Martinet et al., 2001). MS/MS analysis of the [M-H]Ϫ ion of saponins generally shows the successive losses of sugar moieties to yield an aglycone ion, although multistage MS appears necessary to reveal the full sequence (Cui et al., 2000). In contrast, the positive ion CID spectra of cationized saponins may be complicated by fragments resulting from cross-ring cleavage reactions and there may be neutral loss of the aglycone moiety, or aglycone plus sugars, to leave a cationized sugar fragment (Fang et al., 1998; Cui et al., 2001). Thus negative ion LC-MS of saponins produces results that are more readily interpreted. In legumes, LC-MS has been used to analyse the saponins of Glycine max (Fuzzati et al., 1997) and Vigna mungo (Lee et al., 1999) and GC-MS has been used to analyse the aglycones of hydrolysed saponins in, for example, Swartzia schomburgkii (Abdel-Kader et al., 2000) and Glycyrrhiza glabra (Hayashi et al., 1993). The potential of systematic surveys of saponins is indicated from a study of six species of Glycyrrhiza in which rbcL sequence analysis revealed two groups that also differed in the major saponin produced, as revealed by LC-UV (Hayashi et al., 2000).

6. Other hyphenated techniques

Two hyphenated techniques that promise much potential, but have yet to be adopted widely due to various technical problems, are the coupling of CE to MS and the combination of LC and NMR. CE-MS offers the possibility of achieving the chromatographic resolution obtained with GC but with analytes in solution while LC-NMR offers the attraction of on-line structural elucidation of components in mixtures. 834 G.C. Kite et al. / Biochemical Systematics and Ecology 31 (2003) 813Ð843

6.1. Capillary electrophoresis-mass spectrometry Electrophoresis on solid supports, such as paper or agarose, has been employed for over a century to separate charged molecules and has been widely employed in the study of legume non-protein amino acids (e.g. Evans and Bell, 1978). The use of high voltage electrophoresis in capillaries was developed in its modern form by Jorgensen and Lukacs (1981) following the pioneering work of Hjerteń (1967). Sep- arations are created by the electrophoretic mobility of the analyte in the presence of electroosmotic flow and high separation efficiencies can be achieved, as well as low sample consumption. The development of CE-MS was stimulated by early reports of ES with the first report of CE-ES-MS appearing in 1987 (Olivares et al., 1987). For the analysis of charged molecules, the features of CE and ES offer a near ideal marriage with, in the simplest form of CE-MS, the high voltage applied to the electrospray needle creating both the electrospray effect and closing the CE circuit. Thus CE-MS/MS is an ideal technique for protein sequence determination through the analysis of the complex peptide mixtures that result from enzyme digests. For small charged molecules, such as alkaloids, the possibilities for CE-MS are beginning to be recognised (Cherkaoui et al., 1999). For uncharged molecules, though, the modified CE technique of capillary electrokinetic chromatography is necessary to achieve analysis and this creates incompatibilities with ES that are hindering development in this area (Luedtke and Unger, 1999). 6.2. Liquid chromatography-nuclear magnetic resonance spectroscopy LC-NMR is arguably the most powerful of the hyphenated techniques currently available to researchers in phytochemistry (Hostettmann and Wolfender, 2001; Wol- fender et al., 2001). In principle it can provide the molecular structures of the components of an extract or fraction in a single online experiment. The potential of this method was first recognised in the 1970s, although it was not until the early 1990s that the technical problems associated with its implementation were overcome satis- factorily. Of all the hyphenated techniques described in this review, LC-NMR is inherently the least sensitive, and thus high-field NMR spectrometers (500 MHz and above) are necessary to achieve useful results with the amount of material that can be applied to an HPLC column. Recent advances in NMR probe technology have also contributed to improved sensitivity. Another major difficulty that must be overcome is the suppression of solvent signals from the mobile phase without loss of those due to the compounds present. This is particularly important when gradient LC methods are used as the signals from more than one solvent must be suppressed, and their chemical shift values vary with composition. New solvent suppression techniques have therefore been developed to meet these requirements (Dalvit et al., 1999). The chemical shift values for the signals of the analytes may also be solvent dependent. This means that spectral matching for compound identification against data acquired in normal NMR experiments is compromised. Conversely analyte signals lost because of solvent suppression in one system (e.g. MeOH-H2O) may be recovered by repeating the LC-NMR analysis with a different solvent (e.g.

MeCN-H2O). G.C. Kite et al. / Biochemical Systematics and Ecology 31 (2003) 813Ð843 835

Two methods of data collection are commonly used in LC-NMR. In both cases the analytes eluted from a LC column pass into a NMR microflow probe that typically has an active volume of 60 µl and a diameter of 3 mm (this should be compared with the conventional NMR sample size of 500–600 µl and a sample tube of 5 mm diameter). In the on-flow mode, 1H-NMR spectra are acquired continuously throughout the separation, whereas in the stop-flow mode the separation can be halted when a component of interest reaches the probe and several NMR datasets can be acquired over a longer time period. These may include 2D experiments such as 1H-1HCOSY and inverse-detected heteronuclear correlation experiments such as HMQC/HSQC and HMBC. For compounds with a relative molecular mass of 400 or below, detection limits with the microflow probe are typically 3 µg and 100 ng of analyte in on- flow and stop-flow modes, respectively. One limitation of both approaches therefore is that direct observation 1D 13C-NMR spectra cannot be obtained for comparison with existing literature data. Applications of LC-NMR to phytochemical problems first appeared in the literature in the mid 1990s and the number of papers published continues to increase (Wolfender et al., 2001). The technique has been most successful when applied to plant extracts rich in natural products of relatively low molecular mass or those containing aglycones rather than glycosides. As yet, Leguminosae are poorly represented in terms of published LC-NMR applications, with only a single report available that describes the interconversion of diastereoisomeric diterpenes from the root bark of Bobgunnia madagascariensis (Schaller et al., 2001). However, studies in other plant families emphasise the value of LC-NMR as a technique for obtaining detailed chemical profiles of species for taxonomic work. For example, the major metabolites of several Gentianaceae taxa have been identified using LC-NMR in a combination of on-flow and stop-flow modes (Wolfender et al., 1997). The compound classes analysed included flavones, xanthones and secoiridoids. In another successful application, Vogler et al. (1998) used LC-NMR in the on-flow mode to identify nine anti-bacterial sesquiterpene lactones from a partially purified extract of Vernonia fastigiata (Asteraceae) without the need for isolation of the individual compounds. Similar studies could yield valuable data in systematic surveys of chemi- cals in Leguminosae and in particular those whose distribution is restricted, such as isoflavonoids in the subfamily Papilionoideae.

7. The future for hyphenated techniques in legume systematics

The great majority of LC-MS systems currently in use are employed in the analysis of proteins and peptides, rather than small molecules. We are at the beginning of the era of ‘proteomics’ and LC-MS/MS will be at the centre of technological developments to enable large-scale protein analysis. Isozyme analysis has been a favourite chemotaxonomic tool in the past. In the future, isozyme fingerprints will probably be achieved by LC-MS/MS analysis and indeed this technology has the potential to allow the entire proteomes of species to be compared. It is beyond the scope of the present paper to cover these methods in detail and the reader is referred to recent 836 G.C. Kite et al. / Biochemical Systematics and Ecology 31 (2003) 813Ð843 reviews (e.g. Peng and Gygi, 2001), papers (e.g. Devreese et al., 2001) and practical manuals (e.g. Humphrey-Smith and Ward, 2000). At present, proteomes are compared by two-dimensional gel electrophoresis and individual spots are excised, digested in trypsin and the resulting peptides are analysed by LC-ES-MS/MS. This not only provides the molecular weights of the peptides but each can be sequenced by analysis of its CID spectrum or, more usually, identified by database searching. In legumes, this approach has been used to study the proteome of Melilotus alba and its symbiotic nitrogen-fixing bacterium Sinorhizobium meliloti (Natera et al., 2000). Two dimensional electrophoresis is a skilled, time-consuming and expensive technique, so the present move is to use the increasing chromatographic resolution and speed of LC-MS/MS systems, coupled with powerful bioinformatics software, to analyse trypsin digests of entire cell lysates in which the number of peptides has been reduced by simple processing. The logic behind this strategy is that the sequence of only one or two trypsin fragments are needed to identify a protein whose sequence is known. One such method of reducing the number of peptides in a whole cell digest is to tag cysteine residues so that peptides containing cysteine can be separated by affinity chromatography. Recently, using this technique, about 300 proteins were profiled from yeast in a single LC-MS/MS analysis (Ideker et al., 2001). In future studies of legume systematics, such techniques will allow the analysis of proteins controlling development and the expression of suites of morphological (and chemical) characters, as the emphasis of comparative plant biology moves from the study of systematic relationships to evolution and development.

8. Materials and methods

The following provides a brief account of the analyses undertaken during the preparation of this review to illustrate the use of hyphenated techniques in legume systematics.

8.1. Plant material

Plant material was obtained from specimens in the living (L), herbarium (H) or seed (S) collections of the Royal Botanic Gardens, Kew. The material studied and collection references are as follows: Ateleia cubensis Griseb., leaf (Rico et al 1128, H); A. herbert-smithii Pittier, leaf (1997-6547, L); Baphiopsis parviflora Baker, leaf (Gosline 246,H);Calia secundiflora (Ortega) Yakovlev, leaf (1990-899, L); Clad- rastis delavayi (Franch.) Prain (syn. C. sinensis Hemsl.), leaf (1910-65043, L); C. kentukea (Dum.-Cours.) Rudd, leaf (1920-10301, L); C. platycarpa Mak., leaf (1973- 15671, L); Cyclolobium nutans C.T. Rizzini and E.P.Heringer, fruit (B. Balansa 4425, H); Physostigma venenosum Balf., seed (J475, S); Styphnolobium affine (Torrey and A. Gray) Walp., leaf (1931-18102, L); S. japonicum (L.) Schott, leaf and seed (1972-10834, L). G.C. Kite et al. / Biochemical Systematics and Ecology 31 (2003) 813Ð843 837

8.2. Extract preparation

Quinolizidine alkaloids were extracted from fresh material with 0.5 M HCl. The extract was partitioned against dichloromethane under the initial acidic conditions and then under basic conditions (ammonia solution was used for basification). The organic phase from the basic partition was analysed for quinolizidine alkaloids. Non- protein amino acids and polyhydroxyalkaloids were extracted from dry material with 70% aqueous methanol and purified and trimethylsilylated, if required, as described by Kite and Hughes (1997). Physostigmine alkaloids and flavonoid glycosides were extracted with methanol and 80% aqueous methanol, respectively; analyses were performed on these crude extracts.

8.3. GC-MS and LC-MS analyses

GC-MS analyses were undertaken using a Perkin-Elmer ‘Autosystems XL’ gas chromatograph coupled to a Perkin-Elmer ‘TurboMass’ quadrupole mass spectrometer equipped with either an electron ionisation (70 eV) or chemical ionisation source (reagent gas ammonia). Chromatographic separation of quinolizidine alkaloids was performed on a 30 m × 0.25 mm (i.d.) × 0.25 µm DB1-MS (J and W Scientific) capillary column using an oven temperature program of 120–320 °Cat6°CminϪ1. Analysis of trimethylsilylated polyhydroxyalkaloids was performed on a 30 m × 0.25 mm (i.d.) × 0.25 µm PE5-MS capillary column (Perkin-Elmer Ltd) using an oven temperature program of 120–240 °Cat3°CminϪ1. For both analyses the helium carrier gas flow was 1 ml minϪ1. LC-MS/MS analyses were undertaken using a Thermo Finnigan ‘Surveyor’ high performance liquid chromatograph coupled to a Thermo Finnigan ‘LCQ Classic’ quadrupole ion trap mass spectrometer equipped with an ES or APCI source. Source conditions were as described by Egan et al. (2000). Chromatographic separations of flavonoid glycosides were performed on a 150 × 4.6 mm (i.d.), 5 µm Phenomenex Luna C18 column using a 1 ml minϪ1 solvent gradient of 20–100% aqueous methanol (containing 1% acetic acid) in 20 min. Physostigmine alkaloids were analysed using a 150 × 4.6 mm (i.d.), 5 µm Phenomenex Polar-RP column and eluting with water for 5 min followed by a gradient of 0–100% aqueous methanol over 25 min. Non- protein amino acids were separated on a 250 × 4.6 mm (i.d.), 5 µm Phenomenex Aqua-C18 column using a 1 ml minϪ1 gradient of methanol and 0.1% aqueous heptafluorobutyric acid as described by Kite and Ireland (2002). The flow to the APCI source was the full 1 ml min-1 from the column but the flow to the ES source was reduced to 200 µmminϪ1 by a flow splitter. CID spectra were recorded by MS/MS and used an ion isolation width of 5 m/z units and a scaled collision energy of 45% regardless of the source employed and its polarity.

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