Characterisation of phenylethanoid glycosides by multiple-stage mass spectrometry.

RATIONALE: Although phenylethanoid glycosides (PhGs) occur widely in plants, their characterisation by liquid chromatography/mass spectrometry (LC/MS) is less well studied than other phenolic glycosides such as flavonoid glycosides. The multiple-stage mass spectrometry (MSn ) experiments required to improve the annotation of common verbascoside-type PhGs are described here. METHODS: Deprotonated, ammoniated and sodiated molecules of nine PhGs were subjected to low-energy collision-induced dissociation (CID) in a hybrid ion trap/orbitrap mass spectrometer. Most experiments were recorded at nominal mass using the linear ion trap analyser for wider applicability in the plant metabolomics community. Data interpretation was supported by high-resolution orbitrap scanning of product ions. Comparative data was acquired on the same instrument by performing higher-energy collisional dissociation (HCD) in the C-trap. RESULTS: Low-energy CID-MS2 of the deprotonated and ammoniated molecules generated diagnostic product ions from which the molecular masses of the phenolic acid and phenylethanoid moieties, respectively, could be determined. The sugar at C-3' of the core glucose was preferentially lost from the sodiated molecule following CID-MS2 , while CID-MSn produced a sodiated product ion from ring cleavage of the core glucose bearing the sugar at C-6'. Evidence of a disaccharide substitution came from a sodiated disaccharide residue in CID-MSn spectra. CONCLUSIONS: The consistency of PhG dissociation following low-energy CID-MSn of various ions is sufficient to enable annotation of verbascoside-type PhGs in LC/MS analyses of crude plant extracts. This can be achieved on a low-resolution instrument capable of MSn .

Mechanisms in mutualisms: a chemically mediated thrips pollination strategy in common elder.

MAIN CONCLUSION: This study provides first evidence of a thrips species pollinating Sambucus nigra and describes how interactions are driven by plant biochemical signalling and moderated by temporal changes in floral chemistry. The concept of flower-feeding thrips as pollinating insects in temperate regions is rarely considered as they are more frequently regarded to be destructive florivores feeding on pollen and surrounding plant tissue. Combining laboratory and field-based studies we examined interactions between Sambucus nigra (elderflower) and Thrips major within their native range to ascertain the role of thrips in the pollination of this species and to determine if floral chemicals mediated flower visits. If thrips provide a pollination service to S. nigra, then this will likely manifest in traits that attract the pollinating taxa at temporally critical points in floral development. T. major were highly abundant in inflorescences of S. nigra, entering flowers when stigmas were pollen-receptive and anthers were immature. When thrips were excluded from the inflorescences, fruit-set failed. Linalool was the major component of the inflorescence headspace with peak abundance coinciding with the highest number of adult thrips visiting flowers. Thrips were absent in buds and their numbers declined again in senescing flowers inversely correlating with the concentration of cyanogenic glycosides recorded in the floral tissue. Our data show that S. nigra floral chemistry mediates the behaviour of pollen-feeding thrips by attracting adults in high numbers to the flowers at pre-anthesis stage, while producing deterrent compounds prior to fruit development. Taking an integrative approach to studying thrips behaviour and floral biology we provide a new insight into the previously ambiguously defined pollination strategies of S. nigra and provide evidence suggesting that the relationship between T. major and S. nigra is mutualistic.

Medicinally Used Asarum Species: High-Resolution LC-MS Analysis of Aristolochic Acid Analogs and In vitro Toxicity Screening in HK-2 Cells.

Species of Asarum are used in traditional Chinese medicine and, similar to members of the genus Aristolochia, they contain aristolochic acid analogs (AAAs). These compounds are known for their nephrotoxic and carcinogenic effects. So far, the phytochemistry and nephrotoxicity of species of Asarum is not well studied. A high-resolution LC-MS-based metabolomic approach was used to study the phytochemical variation in medicinally used Asarum species. The cytotoxicity of the samples was assessed using human kidney (HK-2) cells. The majority of samples contained potentially nephrotoxic AAAs, including 9-methoxy aristolactam (AL) IV, AL I, and AL IV. These compounds were present in methanol as well as water extracts. AAAs were detected in all parts of the plant. The majority of the extracts were not cytotoxic to HK-2 cells at the doses tested. However, other mechanisms relating to aristolochic acid nephropathy and cancer development, such as DNA adduct formation may occur. The results of this study provide a model for assessing lesser-known plant species for toxicity.

Variation of theanine, phenolic, and methylxanthine compounds in 21 cultivars of Camellia sinensis harvested in different seasons.

This is the first study to use chemometric methods to differentiate among 21 cultivars of Camellia sinensis from China and between leaves harvested at different times of the year using 30 compounds implicated in the taste and quality of tea. Unique patterns of catechin derivatives were observed among cultivars and across harvest seasons. C. sinensis var. pubilimba (You 510) differed from the cultivars of C. sinensis var. sinensis, with higher levels of theobromine, (+)-catechin, gallocatechin, gallocatechin gallate and theasinensin B, and lower levels of (-)-epicatechin, (-)-epigallocatechin (EGC) and (-)-epigallocatechin gallate (EGCG), respectively. Three cultivars of C. sinensis var. sinensis, Fuyun 7, Qiancha 7 and Zijuan contained significantly more caffeoylquinic acids than others cultivars. A Linear Discriminant Analysis model based on the abundance of 12 compounds was able to discriminate amongst all 21 tea cultivars. Harvest time impacted the abundance of EGC, theanine and afzelechin gallate.

LC-MS- and (1)H NMR-Based Metabolomic Analysis and in Vitro Toxicological Assessment of 43 Aristolochia Species.

Species of Aristolochia are used as herbal medicines worldwide. They cause aristolochic acid nephropathy (AAN), a devastating disease associated with kidney failure and renal cancer. Aristolochic acids I and II (1 and 2) are considered to be responsible for these nephrotoxic and carcinogenic effects. A wide range of other aristolochic acid analogues (AAAs) exist, and their implication in AAN may have been overlooked. An LC-MS- and (1)H NMR-based metabolomic analysis was carried out on 43 medicinally used Aristolochia species. The cytotoxicity and genotoxicity of 28 Aristolochia extracts were measured in human kidney (HK-2) cells. Compounds 1 and 2 were found to be the most common AAAs. However, AA IV (3), aristolactam I (4), and aristolactam BI (5) were also widespread. No correlation was found between the amounts of 1 or 2 and extract cytotoxicity against HK-2 cells. The genotoxicity and cytotoxicity of the extracts could be linked to their contents of 5, AA D (8), and AA IIIa (10). These results undermine the assumption that 1 and 2 are exclusively responsible for the toxicity of Aristolochia species. Other analogues are likely to contribute to their toxicity and need to be considered as nephrotoxic agents. These findings facilitate understanding of the nephrotoxic mechanisms of Aristolochia and have significance for the regulation of herbal medicines.

Phenolic profile characterization of Chemlali olive stones by liquid chromatography-ion trap mass spectrometry.

Aqueous methanol extracts of Chemlali olive stones were analyzed by reverse phase high-performance liquid chromatography (HPLC) with diode array detection and mass spectrometry [LC-MS/MS]. Oleoside, oleoside 11-methyl ester, nuezhenide, oleoside 11-methyloleoside, nuezhenide 11-methyloleoside, oleuropein, and glycosides of tryosol and hydroxytyrosol glycosides were identified in stones of Chemali olives. The antioxidant activity observed for the extract of the olive stones (IC50 = 13.84 µg/mL, TEAC = 0.436 mM) may be due to the high content of phenolic compounds, of which the main compounds are nuezhenide (325.78 mg/100g), methoxy derivative of nuezhenide (132.46 mg/100g), and nuezhenide-11-methyloleoside (82.91 mg/100g). These results suggest the use of olive stones as sources of natural antioxidants.

Is aristolochic acid nephropathy a widespread problem in developing countries? A case study of Aristolochia indica L. in Bangladesh using an ethnobotanical-phytochemical approach.

ETHNOPHARMACOLOGICAL RELEVANCE: Species of Aristolochia are associated with aristolochic acid nephropathy (AAN), a renal interstitial fibrosis and upper urinary tract cancer (UUC). Aristolochic acid nephropathy has been reported in ten countries but its true incidence is unknown and most likely underestimated. By combining an ethnobotanical and phytochemical approach we provide evidence for the risk of AAN occurring in Bangladesh. More specifically, we assess the intra-specific variation of aristolochic acid analogues in medicinally used Aristolochia indica samples from Bangladesh. MATERIALS AND METHODS: Ethnobotanical information was collected from 16 kavirajes (traditional healers) in different study locations in Bangladesh. Plant samples were obtained from native habitats, botanical gardens, herbal markets and pharmaceutical companies. The samples were extracted using 70% methanol and were analysed using LC-DAD-MS and (1)H-NMR. RESULTS: Roots as well as leaves are commonly used for symptoms such as snake bites and sexual problems. Among the informants knowledge about toxicity or side effects is very limited and Aristolochia indica is often administered in very high doses. Replacement of Aristolochia indica with other medicinal plants such as Rauvolfia serpentina (L.) Benth. ex Kurz was common. Aristolochia indica samples contained a variety of aristolochic acid analogues such as aristolochic acid I, aristolochic acid II, cepharadione A and related compounds. CONCLUSIONS: AAN cases are likely to occur in Bangladesh and more awareness needs to be raised about the health risks associated with the use of Aristolochia indica and other species of Aristolochia as herbal medicines.

Acyl spermidines in inflorescence extracts of elder (Sambucus nigra L., Adoxaceae) and elderflower drinks.

LC-UV-MS analyses of inflorescence extracts of Sambucus nigra L. (elder, Adoxaceae) revealed the presence of numerous acyl spermidines, with isomers of N,N-diferuloylspermidine and N-acetyl-N,N-diferuloylspermidine being most abundant. Pollen was the main source of the acyl spermidines in the inflorescence. Three of the major acyl spermidines were isolated and their structures determined by NMR spectroscopy as N5,N¹°-di-(E,E)-feruloylspermidine and the new compounds N¹-acetyl-N5,N¹°-di-(Z,E)-feruloylspermidine and N¹-acetyl-N5,N¹°-di-(E,E)-feruloylspermidine. An isomer of N,N,N-triferuloylspermidine was also obtained and identified as N¹,N5,N¹°-tri-(E,E,E)-feruloylspermidine. In addition to stereoisomers of the isolated acyl spermidines, other acyl spermidines detected by the positive ion LC-UV-MS were isomers of N-caffeoyl-N,N-diferuloylspermidine, N-coumaroyl-N,N-diferuloylspermidine, N-caffeoyl-N-feruloylspermidine, N-coumaroyl-N-feruloylspermidine, N-acetyl-N-caffeoyl-N-feruloylspermidine, and N-acetyl-N-coumaroyl-N-feruloylspermidine. Analysis of commercial elderflower drinks showed that acyl spermidines were persistent in these processed elderflower products. Examination of inflorescence extracts from Sambucus canadensis L. (American elder) revealed the presence of acyl spermidines that were different from those of S. nigra.

Generic detection of basic taxoids in wood of European Yew (Taxus baccata) by liquid chromatography-ion trap mass spectrometry.

The occurrence of the cardiotoxin taxine (comprising taxine B and several other basic taxoids) in leaves of Taxus baccata L. (European yew) is well known and has led to public concerns about the safety of eating or drinking from utensils crafted from the wood of this poisonous species. The occurrence of basic taxoids in the heartwood of T. baccata had not been examined in detail, although the bark is known to contain 2'ß-deacetoxyaustrospicatine. Initial examination of heartwood extracts for 2'ß-deacetoxyaustrospicatine by liquid chromatography-mass spectrometry (LC-MS) revealed the presence of this basic taxoid at about 0.0007% dry weight, using a standard isolated from bark. Analyses for taxine B, however, proved negative at the extract concentration analysed. Observing other basic taxoids within the heartwood extracts was facilitated by developing generic LC-MS methods that utilised a fragment arising from the N-containing acyl group of basic taxoids as a reporter ion. Of the various MS strategies available on a hybrid ion trap-orbitrap instrument that allowed observation of this reporter ion, combining all-ion collisions with high resolution ion filtering by the orbitrap was most effective, both in terms of the number of basic taxoids detected and sensitivity. Numerous basic taxoids, in addition to 2'ß-deacetoxyaustrospicatine, were revealed by this method in heartwood extracts of T. baccata. Red wine readily extracted the basic taxoids from heartwood while coffee extracted them less efficiently. Contamination with basic taxoids could also be detected in soft cheese that had been spread onto wood. The generic LC-MS method for detecting basic taxoids complements specific methods for detecting taxine B when investigating yew poisoning cases in which the analysis of complex extracts may be required or taxine B has not been detected.

Amides and an alkaloid from Portulaca oleracea.

A total of 16 phenolic compounds, including one new and five known N-cinnamoyl phenylethylamides, one new pyrrole alkaloid named portulacaldehyde, five phenylpropanoid acids and amides, and derivatives of benzaldehyde and benzoic acid, were isolated and identified from a polar fraction of an extract of Portulaca oleracea. Their structures were determined through spectroscopic analyses.

Flavonol glycosides acylated with 3-hydroxy-3-methylglutaric acid as systematic characters in Rosa.

LC-UV-MS/MS analysis of leaf extracts from 146 accessions of 71 species of Rosa revealed that some taxa accumulated flavonol O-glycosides acylated with 3-hydroxy-3-methylglutaric acid, which are relatively uncommon in plants. The structures of two previously unrecorded examples isolated from Rosa spinosissima L. (syn. Rosa pimpinellifolia L.) were elucidated using spectroscopic and chemical methods as the 3-O-α-L-rhamnopyranosyl-(1→2)-[6-O-(3-hydroxy-3-methylglutaryl)-ß-D-galactopyranosides] of kaempferol (3,5,7,4'-tetrahydroxyflavone) and quercetin (3,5,7,3',4'-pentahydroxyflavone). The corresponding 3-O-[6-O-(3-hydroxy-3-methylglutaryl)-ß-D-galactopyranoside] of quercetin was also present in R. spinosissima, but at lower levels, together with 17 other flavonol O-glycosides for which structures were assigned using LC-UV-MS/MS. The distribution of flavonol 3-hydroxy-3-methylglutarylgalactosides in Rosa was limited to some species of subgenus Rosa section Pimpinellifoliae and Rosa roxburghii Sw. of the monotypic subgenus Platyrhodon, indicating that this character could be of value in phylogenetic analyses of the genus.

Inhibition of human calcineurin and yeast calcineurin-dependent gene expression by Jasminum humile leaf and root extracts.

ETHNOPHARMACOLOGICAL RELEVANCE: The leaves of Jasminum humile are used to treat skin disorders in a way which resembles the use of modern topical anti-inflammatory drugs. Ethanolic extracts of the roots and leaves were shown to inhibit calcineurin which is a regulator of inflammatory gene expression. MATERIALS AND METHODS: A novel yeast calcineurin reporter gene assay suitable for a 96 well plate format was developed to test for inhibition of calcineurin-dependent gene expression. Calmodulin/calcineurin phosphatase assays were then used to further elucidate the mode of action of the extracts. RESULTS AND CONCLUSIONS: Jasminum humile root and leaf extract exhibited calcineurin inhibition activity that was shown to be mediated through a direct interaction with calcineurin enzyme. The activity is sufficient to block calcineurin-dependent gene expression in a yeast model. The activity of the plant supports its traditional use in the treatment of inflammatory skin disorders. The specially adapted yeast reporter assay was found to be a highly effective way of detecting calcineurin inhibitors in plant extracts.

Applications of phytochemical and in vitro techniques for reducing over-harvesting of medicinal and pesticidal plants and generating income for the rural poor.

Plants provide medicine and pest control resources for millions of poor people world-wide. Widespread harvesting of medicinal and pesticidal plants puts pressure on natural populations, thus severely compromising their contribution to the income and well-being of traders and consumers. The development of in vitro propagation techniques appropriate for developing countries will provide a robust platform for effective propagation and cultivation of endangered plants. This review focuses on advances in the application of phytochemical and in vitro tools to identify and rapidly propagate medicinal and pesticidal plants. Problems of over-harvesting can be alleviated and ex situ cultivation in agroforestry systems can be facilitated through improving seed germination, in vitro cloning and the use of mycorrhizal fungi. We also present a case for effective use of phytochemical analyses for the accurate identification of elite materials from wild stands and validation of the desired quality in order to counter loss of efficacy in the long run through selection, propagation or ex situ management in agroforestry systems. Future prospects are discussed in the context of medicinal activity screening, sustainable propagation, on-farm planting, management and utilization.

Identification of common glycosyl groups of flavonoid O-glycosides by serial mass spectrometry of sodiated species.

Flavonoid O-glycosides are a ubiquitous and important group of plant natural products in which a wide variety of sugars are O-linked to an aglycone. Determining the identity of the sugars, and the manner in which they are linked, by mass spectrometry alone is challenging. To improve the identification of common O-linked di- and trisaccharides when analysing mixtures of flavonoid O-glycosides by liquid chromatography/mass spectrometry (LC/MS), the fragmentation of electrosprayed sodium adducts in an ion trap mass spectrometer was investigated. The sodium adducts [M + Na](+) of kaempferol 3-O-glycosides generated sodiated glycosyl groups by the neutral loss of kaempferol. The product ion spectra of these sodiated glycosyl groups differed between four isomeric kaempferol 3-O-rhamnosylhexosides and four isomeric kaempferol 3-O-glucosylhexosides in which the primary hexose was either glucose or galactose and bore the terminal glucose or rhamnose at either C-2 or C-6. Fragmentation of sodiated glycosyl groups from linear O-triglucosides and branched O-glucosyl-(1 → 2)-[rhamnosyl-(1 → 6)]-hexosides produced sodiated disaccharide residues, and the product ion spectra of these ions assisted the identification of the complete sugar. The product ion spectra of the sodiated glycosyl groups were consistent among flavonoid O-glycosides differing in the position at which the sugar was O-linked to the aglycone, and the nature of the aglycone. The abundance of sodiated species was enhanced by application of a pre-trap collision voltage, without the need to dope with salt, allowing automated LC/MS methods to be used to identify the glycosyl groups of common flavonoid O-glycosides, such as rutinosides, robinobiosides, neohesperidosides, gentiobiosides and sophorosides.

Flavonoid glycosides of the black locust tree, Robinia pseudoacacia (Leguminosae).

Four flavone glycosides isolated from extracts of the leaves of Robinia pseudoacacia (Leguminosae) were characterised by spectroscopic and chemical methods as the 7-O-beta-d-glucuronopyranosyl-(1-->2)[alpha-l-rhamnopyranosyl-(1-->6)]-beta-d-glucopyranosides of acacetin (5,7-dihydroxy-4'-methoxyflavone), apigenin (5,7,4'-trihydroxyflavone), diosmetin (5,7,3'-trihydroxy-4'-methoxyflavone) and luteolin (5,7,3',4'-tetrahydroxyflavone). Assignment of glycosidic (1)H and (13)C resonances in their NMR spectra was facilitated by (2)J(HC) correlations detected using the H2BC (heteronuclear two-bond correlation) pulse sequence. Spectroscopic analysis of two known triglycosides, acacetin 7-O-beta-d-glucopyranosyl-(1-->2)[alpha-l-rhamnopyranosyl-(1-->6)]-beta-d-glucopyranoside (previously unrecorded from this species) and acacetin 7-O-beta-d-xylopyranosyl-(1-->2)[alpha-l-rhamnopyranosyl-(1-->6)]-beta-d-glucopyranoside ('acacetin trioside'), enabled inconsistencies in the literature relating to these structures to be resolved. Comparison of the flavonoid chemistry of leaves and flowers of R. pseudoacacia using LC-UV and LC-MS showed that flavone 7-O-glycosides, particularly of acacetin, predominated in the former, whereas the latter comprised mainly flavonol 3,7-di-O-glycosides, including several examples new to this species. Tissue dependent differences in flavonoid chemistry were also evident from the glycosylation patterns of the compounds.

Assigning glucose or galactose as the primary glycosidic sugar in 3-O-mono-, di- and triglycosides of kaempferol using negative ion electrospray and serial mass spectrometry.

Kaempferol 3-O-beta-glucopyranoside, kaempferol 3-O-beta-galactopyranoside and higher glycosides of these two flavonoids with alpha-rhamnose at C-2 and/or C-6 of the primary sugar were studied by negative ion electrospray ionisation and serial mass spectrometry in a three-dimensional (3D) ion trap mass spectrometer. Kaempferol 3-O-beta-glucopyranoside and kaempferol 3-O-alpha-rhamnopyranosyl(1-->6)-beta-glucopyranoside could be distinguished from their respective galactose analogues by differences in the ratio of the radical aglycone ion [Y(0) - H](*-) to the rearrangement aglycone ion Y(0) (-) following MS/MS of the deprotonated molecules. Kaempferol 3-O-rhamnopyranosyl(1-->2)-beta-glucopyranoside and kaempferol 3-O-alpha-rhamnopyranosyl(1-->2)[alpha-rhamnopyranosyl(1-->6)]-beta-glucopyranoside could be distinguished from their respective galactose analogues by differences in the product ion spectra of the [(M - H) - rhamnose](-) ion following serial mass spectrometry. In the triglycoside, it was deduced that this ion resulted from the loss of the rhamnose substituted at 2-OH of the primary sugar by observing that MS/MS of deprotonated kaempferol 3-O-beta-glucopyranosyl(1-->2)[alpha-rhamnopyranosyl(1-->6)]-beta-glucopyranoside showed the loss of glucose and not rhamnose. Thus the class of sugar (hexose, deoxyhexose, pentose) at C-2 and C-6 of the primary sugar can be determined. These observations aid the assignment of kaempferol 3-O-glycosides, having glucose or galactose as the primary glycosidic sugar, in LC/MS analyses of plant extracts, and this can be done with reference to only a few standards.

Flavonol tetraglycosides from fruits of Styphnolobium japonicum (Leguminosae) and the authentication of Fructus Sophorae and Flos Sophorae.

The dried fruits and seeds of Styphnolobium japonicum (L.) Schott (syn. Sophora japonica L.) are used in traditional Chinese medicine and known as Fructus Sophorae or Huai Jiao. The major flavonoids in these fruits and seeds were studied by LC-MS and other spectroscopic techniques to aid the chemical authentication of Fructus Sophorae. Among the flavonoids were two previously unreported kaempferol glycosides: kaempferol 3-O-beta-glucopyranosyl(1-->2)-beta-galactopyranoside-7-O-alpha-rhamnopyranoside and kaempferol 3-O-beta-xylopyranosyl(1-->3)-alpha-rhamnopyranosyl(1-->6)[beta-glucopyranosyl(1-->2)]-beta-glucopyranoside, the structures of which were determined by NMR. Two further tetraglycosides were identified for the first time in S. japonicum as kaempferol 3-O-beta-glucopyranosyl(1-->2)[alpha-rhamnopyranosyl(1-->6)]-beta-glucopyranoside-7-O-alpha-rhamnopyranoside and kaempferol 3-O-beta-glucopyranosyl(1-->2)[alpha-rhamnopyranosyl(1-->6)]-beta-galactopyranoside-7-O-alpha-rhamnopyranoside; the latter was the main flavonoid in mature seeds. The chromatographic profiles of 27 recorded flavonoids were relatively consistent among fruits of similar ages collected from five trees of S. japonicum, and those of maturing unripe and ripe fruits were similar to a market sample of Fructus Sophorae, and thus provide useful markers for authentication of this herbal ingredient. The flower buds (Huai Mi) and flowers (Huai Hua) of S. japonicum (collectively Flos Sophorae) contained rutin as the main flavonoid and lacked the flavone glycosides that were present in flower buds and flowers of Sophora flavescens Ait., reported to be occasional substitutes for Flos Sophorae. The single major flavonoid in fruits of S. flavescens was determined as 3'-hydroxydaidzein.

Flavonol tetraglycosides and other constituents from leaves of Styphnolobium japonicum (Leguminosae) and related taxa.

Two flavonol tetraglycosides comprising a trisaccharide at C-3 and a monosaccharide at C-7 were isolated from the leaves of Styphnolobium japonicum (L.) Schott and characterised as the 3-O-alpha-rhamnopyranosyl(1-->2)[alpha-rhamnopyranosyl(1-->6)]-beta-glucopyranoside-7-O-alpha-rhamnopyranosides of quercetin and kaempferol. The 3-O-alpha-rhamnopyranosyl(1-->2)[alpha-rhamnopyranosyl(1-->6)]-beta-galactopyranoside-7-O-alpha-rhamnopyranoside of kaempferol, the 3-O-alpha-rhamnopyranosyl(1-->2)[alpha-rhamnopyranosyl(1-->6)]-beta-glucopyranosides of kaempferol and quercetin and the 3-O-alpha-rhamnopyranosyl(1-->2)[alpha-rhamnopyranosyl(1-->6)]-beta-galactopyranoside of kaempferol were also obtained from this species for the first time. Some or all of these flavonol tetra- and triglycosides were detected in 17 of 18 specimens of S. japonicum examined from living and herbarium material, although the most abundant flavonoid in the leaves was generally quercetin 3-O-alpha-rhamnopyranosyl(1-->6)-beta-glucopyranoside (rutin). The triglycosides, but not the tetraglycosides, were detected in herbarium specimens of Styphnolobium burseroides M. Sousa, Rudd & Medrano and Styphnolobium monteviridis M. Sousa & Rudd, but specimens of Styphnolobium affine (Torrey & A. Gray) Walp. contained a different profile of flavonol glycosides. The flavonol tetra- and triglycosides of S. japonicum were also present in leaves of Cladrastis kentukea (Dum. Cours.) Rudd, a representative of a genus placed close to Styphnolobium in current molecular phylogenies. An additional constituent obtained from leaves of Styphnolobium japonicum was identified as the maltol derivative, 3-hydroxy-2-methyl-4H-pyran-4-one 3-O-(4'-O-p-coumaroyl-6'-O-(3-hydroxy-3-methylglutaroyl))-beta-glucopyranoside.

Data-directed scan sequence for the general assignment of C-glycosylflavone O-glycosides in plant extracts by liquid chromatography-ion trap mass spectrometry.

An ion trap LC-MS/MS method is described for the analysis of C-glycosylflavone O-glycosides in crude methanolic extracts of plants. The method employs survey scans with and without the application of up-front collision induced dissociation (CID) to generate diagnostic ions for data-directed MS/MS. The spectra acquired allow assignment of the C-linked sugar to either the C-6 or C-8 position of the aglycone and provide data on the molecular mass of the compound, the number and type of O-linked sugars and the molecular mass of the flavone aglycone. These data for the majority of C-glycosylflavone O-glycosides in an extract are obtained automatically in one LC-MS/MS analysis without manual pre-programming. Key to the assignment of the C-6 or C-8 site of C-glycosylation is the generation, by up-front CID, of the (0,1)X+ product ion formed by internal cleavage of the C-linked sugar. MS/MS of this ion is found to have diagnostic value in addition to the (0,2)X+ product ion described by other authors. Ion trap MS/MS spectra of [M+H]+ of the 6,8-di-C-glycosylflavones schaftoside and isoschaftoside show an additional and previously unreported diagnostic product ion that is useful in determining the type of sugar at the C-6 position. The product ion spectra of protonated kaempferol 3-O-glucosylrhamnosides show similarities to the spectra of C-glycosylflavone O-glycosides; this is a potential source of confusion if the analysis of such glycosides is limited solely to MS/MS of [M+H]+.

Metabolomic analysis of saponins in crude extracts of Quillaja saponaria by liquid chromatography/mass spectrometry for product authentication.

Analysis of 50% aqueous methanolic extracts of bark of Quillaja saponaria Molina (quillaja) by liquid chromatography/mass spectrometry (LC/MS), using negative ion electrospray, revealed over 100 saponins. The majority could be assigned to known structures or generalised variations of these from the product ion spectra obtained by serial mass spectrometry in a quadrupole ion trap mass spectrometer. Ten saponins contained a fatty acid domain terminated with both a pentose and deoxyhexose unit, a feature thus far only reported in QS-III. Twenty saponins were based on a hydroxylated derivative of quillaic acid, whereas only six 22beta-hydroxyquillaic acid saponins have been described. The occurrence of pairs of saponins differing only by the presence of a rhamnose or xylose unit in the C-3-substituted saccharide was readily observed in two-dimensional mass maps, and these showed the presence of the unreported 'rhamnose partner' of QS-III. However, one sample labelled as Q. saponaria appeared to lack all saponins containing rhamnose in the C-3 saccharide. Methods to authenticate saponin extracts of quillaja by LC/MS are suggested based on the general metabolomic profile, the occurrence of specific major saponins covering known structural variations, or the presence of saponins containing the unusual fatty acid domain, revealed by neutral loss analysis.