Search

The aim of this study was to present integrated mass spectrometric methods for the structural characterization and identification of flavonoid glycoconjugates. During the liquid chromatography/mass spectrometry analyses, TriVersa NanoMate chip-based system with nanoelectrospray ionization and fraction collection was combined to a quadrupole time-of-flight mass spectrometer. In the extract samples prepared from green leaves of wheat plantlets, 41 flavonoid derivatives were recognized. Part of the target natural products had the full structure being characterized after the registration of mass spectra, where m/z values for protonated [M + H](+) and deprotonated molecules [M - H](-) were annotated. MS(2) and pseudo-MS(3) experiments were performed for [M + H](+) or [M - H](-) and aglycone ions (Y0(+/-)-type), respectively. It should be underlined that pseudo-MS(3) mass spectra were registered for aglycone product ions in the mass spectra of O-glycosides present in the extract samples. In many cases, only tentative structural identification of aglycones was possible, mainly because of the presence of numerous C-monoglycoside or C-diglycoside in the samples. Acylation of the sugar moiety and/or methylation of the aglycone in the flavonoid glycosides under study was observed. The existence of isobaric and/or isomeric compounds was demonstrated in the extract studied. The collision-induced dissociation mass spectra registered for C,O-diglycosides and C,C-diglycosides did not permit to draw complete structural conclusions about the compounds studied. For the investigated class of natural products, unambiguous classification of sugar moieties linked to the aglycones from the recorded mass spectra was not possible. Registration of the positive and negative ion mass spectra did not lead to the precise conclusion about the glycosylation position at C-6 or C-8, and O-4' or O-7 atoms. It was possible, on the basis of the collected MS(2) spectra, to differentiate between O-glycosides and C-glycosides present in the samples analyzed.

Introduction – Flavonoids are important plant compounds occurring in tissues mostly in the form of glycoconjugates. Most frequently the sugar moiety is comprised of mono- or oligosaccharides consisting of common sugars like glucose, rhamnose or galactose. In some plant species the glycosidic moiety contains glucuronic acid and may be acylated by phenylpropenoic acids. Methodology – Flavonoid glyconjugates were extracted from leaves of Medicago truncatula ecotype R108 and submitted to analysis using high-performance liquid chromatography combined with high-resolution tandem (quadrupole-time of flight, QToF) mass spectrometry. Results – The studied leaf extracts contained 26 different flavonoid glycosides among which 22 compounds were flavone (apigenin, luteolin, chrysoeriol and tricin) glucuronides and 13 were acylated with aromatic acids (p-coumaric, ferulic or sinapic). The fragmentation pathways observed in positive and negative ion mass spectra differed substantially between each other and from these of flavonoid glycosides which did not contain acidic sugars. The application of high-resolution MS techniques allowed unequivocal differentiation between ions with the same nominal m/z values containing different substituents (e.g. ferulic acid or glucuronic acid). Eleven of the identified flavonoids have not been reported previously in this species. Perspectives – The presented unique fragmentation pathways of flavonoid glucuronates enable detection of these compounds in tissue extracts from different plant species. Copyright © 2009 John Wiley & Sons, Ltd.

Anthocyanins are secondary plant metabolites ubiquitous in the plant kingdom. They have different biological activities, so monitoring their content in plant tissue or in feed/food derived from plants may be an important task in different projects from various fields of molecular biology and biotechnology. Profiling of secondary metabolites with high-performance liquid chromatography/mass spectrometry (HPLC/MS) systems is time-consuming, especially when many samples have to be checked within a defined time frame with a reasonable number of repetitions according to the metabolomic standards. Even application of the advanced ultra-performance liquid chromatography (UPLC)/MS or equivalent systems would require a long time for analysis of numerous samples. We demonstrate the applicability of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the assessment of level (concentration) of anthocyanins in leaf tissues of four Arabidopsis thaliana ecotypes grown at normal (20 degrees C/16 degrees C day/night) and decreased (4 degrees C) temperature. The quantitative results were obtained for anthocyanins with MALDI-TOF MS using ferulic acid as a matrix. The amounts of anthocyanins in leaves of A. thaliana varied from 0.3-2.5 microg per gram of leaves for ecotypes Col-0 and C24, respectively, and contents of these markedly increased in plants grown in the cold. The applied analytical method exhibited better repeatability of measurements than obtained with an HPLC/ion trap MS system.

Flavonoid conjugates constitute several classes of plant phenolic secondary metabolites including many isomeric compounds differing in the hydroxylation pattern and substitution of their rings with different groups such as alkyls, acyls or sugars. These compounds occur in plant tissues mainly as glycosides and in many cases it is necessary to have reliable and detailed information concerning the structure of these natural products. Our results were obtained using leaf extracts of Arabidopsis thaliana and Lupinus angustifolius in which different glycosides of flavones, flavonols and isoflavones are present. Analysis of collision-induced dissociation (CID)/MS/MS spectra of protonated [M + H](+), sodiated [M + Na](+) or deprotonated [M - H](-) molecules recorded during HPLC runs may bring needed information in this respect. However, registration of mass spectra of [M + Na](+) ions with a good efficiency is possible only after post-column addition of a sodium acetate solution to the LC column eluate. The retention of sodium cation on the saccharidic parts of the molecule is observed after the CID fragmentation. In many cases, the location of this cation on the glycan attached to C-3 hydroxyl group of flavonol led to assignment of its structure. Additionally, the determination of the structure of the aglycone and of the sequence of the glycan part was made possible through the CID data obtained from the [M + H](+) and [M - H](-) ions. CID spectra show a different order of sugar elimination from hydroxyl groups at C-3 and C-7 in flavonol glycosides isolated from A. thaliana leaves and give sufficient information to discriminate flavonoid O-diglycosides from flavonoid di-O-glycosides.

The profiles of isoflavone conjugates in extracts obtained from different parts of Lupinus exaltatus Zucc. grown in Mexico were compared using HPLC-UV and HPLC-ESI/MSn. Collision-induced dissociation–MSn experiments were performed using an ion trap analyser during HPLC-ESI/MS analyses. Nineteen isoflavone conjugates were identified in samples obtained from air-dried roots, leaves, stems and inflorescences of lupin plants. It was possible to determine the structures of the studied compounds on the basis of the MS recorded. The compounds identified were di- and mono-glucosides of genistein and 2′-hydroxygenistein with a different pattern of C- and O-glycosylation. Some glucosides were acylated with malonic acid. It was not possible to establish the glycosylation sites on the basis of MS alone; however, it was possible to differentiate isoflavone C- and O-glucosides. The highest levels of isoflavones and their conjugates were detected in roots and the lowest in stems. Free aglycones were identified in roots and inflorescences but they were not found in stems and leaves. Copyright © 2006 John Wiley & Sons, Ltd.

Extracts from plantlets of different species of the genus Drosera, grown as in vitro cultures, were evaluated for the level of phenolic secondary metabolites from the group of naphthoquinones and flavonols. The profiles of natural products in the extracts obtained from different species were monitored by HPLC with UV detection at 260 and 330 nm. On the basis of the data obtained, Drosera binata, the species with the highest amount of plumbagin, was selected for further studies. The most effective method of extraction of quinones was established and the composition of phenolic secondary metabolites in the tissues was determined. For the identification of phenolic compounds, HPLC-UV and HPLC-ESI/MS were applied. Copyright © 2005 John Wiley & Sons, Ltd.

Mass spectrometric fragmentation patterns of herbacetin 3-O-glucopyranoside-8-O-glucuronopyranoside (1), gossypetin 3-O-glucopyranoside-8-O-glucuronopyranoside (2) and takakin 7-O-glucopyranoside-8-O-glucuronopyranoside (3) were elucidated from mass spectra obtained with electrospray ionisation. The usefulness of the fragmentation patterns observed in the positive and negative mode for structural elucidation of the studied compounds is discussed. The fragmentation of 3 was substantially different from 1 and 2, especially in the negative mode. In order to explain these differences, theoretical calculations were performed. Copyright © 2003 John Wiley & Sons, Ltd.

Plants respond to various biotic or abiotic stimuli with different mechanisms and the mobilization of phenolic compounds is one of them. In white lupin (Lupinus albus L.) plants, isoflavones and their glucosides were localized in cell walls where the high constitutive activity of β-glucosidase (EC 3·2·1·21) was also identified. The enzyme was partially purified from root cell walls. Its polymeric active form has 180 or 200 kDa as determined by non-denaturing electrophoresis and gel filtration, respectively, and the isoelectric point is at pH 6·9. The enzyme is an exoglucosidase, preferentially hydrolysing conjugates of phenolic compounds with β anomers of glucose. It is not active against purified lupin cell walls. The specific β-glucosidase activity varies in different tissues with the highest one in roots, and always higher in cell walls than in protoplast. The cell wall location of the enzyme was confirmed biochemically by its activity in intercellular washing fluids. Both aglycones and glycosides were also present in these fluids. The specific β-glucosidase activity correlated well with the isoflavonoid aglycone/glycoside ratios in various tissues: the higher β-glucosidase activity, the higher relative aglycone content. β-glucosidase activity was found to be inducible under conditions of yeast elicitor treatment. Induction of the enzyme was accompanied by changes in the isoflavone secretion and accumulation, suggesting the regulatory role of β-glucosidase activity in root exudation of isoflavonoids. No correlation however, was found between changes in β-glucosidase activity and the presence of isoflavonoids in root exudates of plants grown under various nitrogen nutrition regimes.

Various mass spectrometric techniques were used for the structural elucidation of flavonoid glycosides isolated from green parts of yellow lupin plants (Lupinus luteus). A methodological approach is proposed consisting of (1) extraction and purification of flavonoid glycosides from plant tissues, (2) registration of liquid secondary ion mass spectra of intact compounds and (3) gas chromatographic/mass spectrometric analyses of those compounds subjected to several types of chemical modification. On the basis of these data, it was possible to define the molecular mass of the glycosides, the structure of the aglycones, the configuration of sugar moieties and the position of glycosidic linkages between sugars in diglycosides or between aglycone and sugar moieties. Analysis of the mass spectrometric data permitted structural identification of four flavonoid diglycosides, where the aglycones had flavone, isoflavone and flavonol structures. They were recognized as: apigenin 7-O-(2″-O -rhamnopyranosyl)glucopyranoside (1), genistein 4′,7-O-diglucopyranoside (2), kaempferol 3-O -(6″-rhamnopyranosyl)glucopyranoside (3) and 3′- or 4′-methylquercetin 3-O-(6″-O -rhamnopyranosyl)glucopyranoside (4). Complete structural evaluation of flavonoid glycosides was possible with only submilligram quantities of samples needed to register various mass spectra. It was not possible, however, to define the anomeric configuration of the C-1 carbons in investigated glycosides. Copyright © 1999 John Wiley & Sons, Ltd.

Solid phase extraction on strong cation exchanger (benzene sulphonic groups) and reverse phase (octadecyl groups) cartridges connected in series was used for a one-step isolation of quinolizidine alkaloids and phenolic compounds present in extracts from lupin seedlings. Both classes of compounds were further analysed by gas chromatography-mass spectrometry. This technique was used to analyse changes in the alkaloid and isoflavonoid profiles during the defence reaction of lupin plants evoked by abiotic (CuCl2) and biotic (elicitor preparation from Colletotrichum lindemuthianumcell walls) factors. © 1997 John Wiley & Sons, Ltd.

Mass spectrometry is an important tool for the identification and structural determination of flavonoid glycosides. The advantages of mass spectrometry are high sensitivity and possibilities of hyphenation with liquid chromatographic methods for the analysis of mixtures of compounds. Different desorption ionization methods allow the analysis of underivatized glycosides. A review of mass spectrometric techniques applied to the identification and structural studies of flavonoid glycosides is presented.

Three flavonoid glycosides isolated from plants were identified using fast atom bombardment mass spectrometry and gas chromatography/mass spectrometry. In the latter studies, electron impact and ammonia chemical ionization were used.

The electron impact induced mass fragmentations of 16 new 2- and 4-alkylthio-5-chloro- and 2- and 4-alkylthio-5-bromouracils are presented. The elucidation of the fragmentation pathways is described. The fragmentation routes have been confirmed by accurate mass measurements and metastable transitions. It has been found that the position of uracil ring thiation may be deduced on the basis of the mass spectral fragmentation patterns.

The electron impact induced fragmentation of a large number of 2- and 4-S-(2-naphthoxy)-polymethyienethiouracils is discussed in detail, with the aid of exact mass measurements and observation of metastable decompositions. The 2- and 4-thiopyrimidine structures can be easily distinguished by their mass spectra.