Elucidations on the structures of some putative flavonoids identified in postharvest withered grapes (Vitis vinifera L.) by quadrupole time-of-flight mass spectrometry.

Grape dehydration is an oenological process used for production of high-quality reinforced and sweet wines. High-resolution mass spectrometry (ultrahigh-performance liquid chromatography/quadrupole time-of-flight [UHPLC/QTOF]) was used to deepen the characterization of some flavonoids previously proposed in Corvina and Raboso Piave withered grapes. By performing a targeted data analysis workflow and orthogonal identification approaches (tandem mass spectrometry [MS/MS]), in silico fragmentation, and calculation of putative retention time), elucidation on the structures of six compounds previously proposed was achieved (taxifolin-pentoside, two tetrahydroxyflavanone-hexoside derivatives, a tetrahydroxy-dimethoxyflavanone-hexoside derivative, a pentahydroxyflavone, and peonidin-O-pentoside); and the structures of four putative new grape flavonoids were characterized (dihydromyricetin-O-hexoside, taxifolin-di-O-hexoside, isorhamnetin, and a pinoquercetin isomer). Findings enlarge the panorama of flavonoids in grape and of their possible biosynthetic pathways.

Insights on the stilbenes in Raboso Piave grape (Vitis vinifera L.) as a consequence of postharvest vs on-vine dehydration.

BACKGROUND: Grape withering is a process used to produce reinforced wines and raisins. Dehydration is usually carried out postharvest by keeping ripe grapes in special warehouses in controlled conditions of temperature, relative humidity (RH) and air flow. Alternatively, grape clusters can be left on the vines after the canes have been pruned. In general, dehydration increases stilbenes in grape, but there are few studies on the effects of on-vine withering. The stilbene profiles of Raboso Piave grape during postharvest and on-vine dehydration were studied here. RESULTS: High-resolution mass spectrometry (MS) was used to identify 19 stilbenes, including resveratrol monomers, dimers (viniferins), oligomers and glucoside derivatives. The two dehydration methods generally had different effects on the above nutraceuticals in grape. The samples kept in warehouses revealed significant increases in Z-ω-viniferin, E-ϵ-viniferin, δ-viniferin and another resveratrol dimer which were not observed in the plants. Trans-Resveratrol increased significantly only in samples dehydrated in the warehouse at 21 °C and 60-70% RH. CONCLUSION: The findings increase knowledge of stilbene composition in grapes subjected to withering on-vine. The choice of dehydration method affects the contents of these nutraceuticals in the grape and consequently in wines. Reasonably, it could also affect other secondary metabolites important for wine quality. © 2017 Society of Chemical Industry.

Association between taste receptor (TAS) genes and the perception of wine characteristics.

Several studies have suggested a possible relationship between polymorphic variants of the taste receptors genes and the acceptance, liking and intake of food and beverages. In the last decade investigators have attempted to link the individual ability to taste 6-n-propylthiouracil (PROP) and the sensations, such as astringency and bitterness, elicited by wine or its components, but with contradictory results. We have used the genotype instead of the phenotype (responsiveness to PROP or other tastants), to test the possible relation between genetic variability and the perception of wine characteristic in 528 subjects from Italy and the Czech Republic. We observed several interesting associations, among which the association between several TAS2R38 gene single nucleotide polymorphisms (P = 0.002) and the TAS2R16-rs6466849 polymorphism with wine sourness P = 0.0003). These associations were consistent in both populations, even though the country of origin was an important factor in the two models, thus indicating therefore that genetics alongside cultural factors also play a significant role in the individual liking of wine.

Putative identification of new p-coumaroyl glycoside flavonoids in grape by ultra-high performance liquid chromatography/high-resolution mass spectrometry.

RATIONALE: Grape polyphenols are antioxidant compounds, markers in vine chemotaxonomy, and involved in color stabilization of red wines. Sugar acylation usually confers higher stability on glycoside derivatives and this effect is enhanced by an aromatic substituent such as p-coumaric acid. Until now, only p-coumaroyl anthocyanins have been found in grape. METHODS: A method of 'suspect screening analysis' by ultra-high-performance liquid chromatography/high-resolution mass spectrometry (UHPLC/QTOFMS) has recently been developed to study grape metabolomics. In the present study, this approach was used to identify new polyphenols in grape by accurate mass measurement, MS/MS fragmentation, and study of correlations between fragments observed and putative structures. RESULTS: Three putative p-coumaroyl flavonoids were identified in Raboso Piave grape extract: a dihydrokaempferide-3-O-p-coumaroylhexoside-like flavanone, isorhamnetin-3-O-p-coumaroylglucoside, and a chrysoeriol-p-coumaroylhexoside-like flavone. Accurate MS provided structural characterization of functional groups, and literature data indicates their probable position in the molecule. A fragmentation scheme is proposed for each compound. CONCLUSIONS: Compounds were identified by overlapping various analytical methods according to recommendations in the MS-based metabolomics literature. Stereochemistry and the definitive position of substituents in the molecule can only be confirmed by isolation and characterization or synthesis of each compound. These findings suggest addressing research of acylated polyphenol glycosides to other grape varieties.

Profiling of grape monoterpene glycosides (aroma precursors) by ultra-high performance-liquid chromatography-high resolution mass spectrometry (UHPLC/QTOF).

A 'suspect screening analysis' method for grape metabolomics by ultra-high performance-liquid chromatography (UHPLC) and high-resolution quadrupole-time of flight (QTOF) mass spectrometry was recently developed. This method was applied to study grape monoterpene glycosides, the main grape aroma precursors. Since standard compounds were not available, they were tentatively identified by overlapping various analytical approaches, in agreement with the indications recommended in mass spectrometry (MS)-based metabolomics. Accurate mass and isotopic pattern, MS/MS fragmentation, correlation between fragments observed and putative structures and between liquid chromatography coupled with mass spectrometry (LC/MS) and gas chromatography/mass spectrometry signals were studied. Seventeen monoterpene glycosides were identified without performing the hydrolytic artifacts commonly used to study these compounds which may affect sample profile. This is the first time that a detailed study of these aroma precursors has been carried out by direct LC/MS analysis.

Study of isobaric grape seed proanthocyanidins by MALDI-TOF MS.

Positive matrix-assisted laser desorption ionization (MALDI) spectra of grape seed raw extracts show the signals of putative proanthocyanidin (PA) oligomers, including those of several sodium and potassium adducts with the same nominal molecular weight. As a result, the MALDI time-of-flight profiles are characterized by several isobaric signals from different PAs. The presence of isobaric PA adducts was studied by sodium and potassium exchange experiments on the seed extracts of six grape varieties. This approach was effective in differentiating PAs with isobaric signals, and 15 different PAs were identified in the MALDI spectra of all samples.

Identification of saffron aroma compound ß-isophorone (3,5,5-trimethyl-3-cyclohexen-1-one) in some V. vinifera grape varieties.

Carotenoid-derived aroma compounds play an important role in the composition of aroma and grapes, and consequently of wine. The volatile composition of forty-five grape varieties harvested in 2011 and 2012 was investigated by solid phase extraction of samples and gas chromatography-mass spectrometry (GC-MS) analysis. In grape extracts of cultivars Barbera, Refosco dal Peducolo Rosso, Ribolla gialla and Rossese, the C9-norisoprenoid compound ß-isophorone (3,5,5-trimethyl-3-cyclohexen-1-one) was found and quantified. This carotenoid-derived compound is characterised by a saffron aroma and is here reported in grape for the first time.

Seed oil triglyceride profiling of thirty-two hybrid grape varieties.

Triglyceride profile of seed oil samples from 32 hybrid grape varieties not studied before was investigated. A new method for the analysis of triacylglycerols (TAGs) has been developed based on the direct infusion in the electrospray ionization (ESI) source and employing tetrahydrofuran/methanol/water (85:10:5 v|v|v) as solvent; the formation of [M + Na](+) ions in high yield has been observed. TAGs were identified by ESI-tandem mass spectrometry analysis, and the matrix-assisted-laser-desorption-ionization and time-of-flight profile of samples was determined. Six were the principal TAGs identified in seed oil: trilinolein (LLL) was the most abundant (43%), followed by dilinoleoyl-oleoylglycerol (LOL, 23%), and dilinoleoyl-palmitoylglycerol (LPL, 15%). Compounds present in lower concentration were LSL and LOO (11%), LOP (6%), and LSP (2%). Compared with seed oils produced from V. Vinifera grapes, some significant differences in the relative abundances of TAGs were found, in particular hybrid grape seed oils showed higher LOL and lower LPL content, respectively. Among the samples studied, a particularly high content of LLL (rich in unsaturated fatty acids) was found in seed oils from two red varieties.

A fast and selective method for anthocyanin profiling of red wines by direct-infusion pneumatic spray mass spectrometry.

RATIONALE: Anthocyanins confer the color to red wines. During wine aging, new anthocyanin derivatives with peculiar chromatic characteristics are formed. A fast and selective method for screening the anthocyanin composition of wine has been developed. METHODS: Direct-infusion electrospray ionization tandem mass spectrometry (ESI-MS/MS) analysis of wine extracts has been performed by operating in positive ion mode with a low spray capillary voltage (0.5 kV) to detect only the species already present in ionic form. The method has been called direct-infusion pneumatic spray (DIPS) mass spectrometry. The anthocyanin profiles of thirteen red wines at different aging stages were studied. Using malvidin-3,5-diglucoside as internal standard, the indices of wine color and of wine color evolution were estimated. RESULTS: By decreasing the spray capillary voltage a dramatic increase in selectivity toward anthocyanins has been observed and the related peaks are the most abundant in the spectra. Several anthocyanins and anthocyanin derivatives (pyranoanthocyanins and derivatives formed by reaction with flavan-3-ols) have been identified in the wines. The younger wines showed higher total anthocyanin index. The aged wines showed color evolution index higher than 20% while it was lower in the younger samples. CONCLUSIONS: The anthocyanin fingerprint is effective for estimating the aging state of wine. The color evolution index can be correlated to barrel conditions during wine aging and used to distinguish between aged and younger wines. The study of pyranoanthocyanins and anthocyanin-flavanol derivatives can be useful to predict wine color stability.

Chemical characterization and enological potential of Raboso varieties by study of secondary grape metabolites.

DNA studies supported by matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis of seed proteins showed that the Vitis vinifera red grape variety Raboso Veronese is the progeny of a spontaneous cross between Raboso Piave (red) and Marzemina Bianca (white) varieties. In the present work, the main secondary grape metabolites of Raboso varieties were studied, and their enological potential was evaluated and compared with that of other red varieties reported in the literature. In general, Raboso grapes had high flavonoid contents and high percentages of polyphenols extractable in winemaking and substantial contents of norisoprenoid aroma precursors. Raboso Veronese stood out for its high content of cyanidin and had higher (+)-catechin and (-)-epicatechin contents than Raboso Piave and abundant quercetin glucoside, indicating substantial plant biosynthesis toward compounds dihydroxylated in the B-ring. Study of secondary grape metabolites is confirmed as an effective tool in differentiating similar varieties, in particular on the basis of polyphenol profiles.

Parental relationships among three grape varieties studied by MALDI of grape seed protein profiles.

Two Raboso cultivars, i.e. Raboso Veronese and Raboso Piave, are two black Vitis vinifera varieties. A genetic study suggested that Raboso Veronese is the progeny of a spontaneous cross between Raboso Piave and Marzemina Bianca cultivars. Parental relationships are usually investigated by genetic studies, which are effective to establish genetic links among different cultivars. Considering that proteome is the genome expression, in this article we evaluated the power of seed protein profiles obtained by matrix-assisted laser desorption/ionization (MALDI)/MS for parentage investigation. The three cultivars lead to very similar spectra with differences in the relative intensity of the most abundant species and the presence of very weak specific ions. In order to evaluate the analytical significance of these aspects, the variability due to instrumental factors and due to different harvesting areas and years of the same cultivars have been considered and measured by the calculation of discrepancy factor values. On one hand, the results obtained can be considered a valid confirmation of the genomic findings, whereas on the other hand, the results provide evidence for the ability of MALDI/MS to individuate minor differences in protein profiles of complex protein mixtures.

A solid-phase microextraction gas chromatography/ion trap tandem mass spectrometry method for simultaneous determination of 'foxy smelling compounds' and 3-alkyl-2-methoxypyrazines in grape juice.

2'-Aminoacetophenone (o-AAP) was identified as the main cause of the aging note called 'hybrid note' or 'foxy smell' which is typical of non-Vitis vinifera grapes. Together with methyl anthranilate (MA), this compound contributes to the typical foxy taint of wines made with non-Vitis vinifera grapes. 3-Alkyl-2-methoxypyrazines in grapes, with their herbaceous note and very low sensory thresholds, contribute to the aroma of several wines. In this study, a solid-phase microextraction gas chromatography/ion trap tandem mass spectrometry (SPME-GC/IT-MS/MS) method for simultaneous detection of o-AAP, MA and ethyl-, isopropyl-, sec-butyl- and isobutylmethoxypyrazine in grape juice was developed. The method was applied to the study of several grape juices: the time required for analysis was less than 24 min, and the method was considered to be suitable for analysis of 'foxy compounds' and methoxypyrazines in grape juice. The high levels of MA and o-AAP found in Clinton and Siebel grapes confirmed the 'hybrid' character of these varieties.

Changes in chemical composition of a red wine aged in acacia, cherry, chestnut, mulberry, and oak wood barrels.

Aging in wooden barrels is a process used to stabilize the color and enrich the sensorial characteristics of wine. Many compounds are released from wood into the wine; oxygen permeation through the wood favors formation of new anthocyanin and tannin derivatives. Recently, polyphenols and volatile compounds released from acacia, chestnut, cherry, mulberry, and oak wood used in making barrels for spirits and wine aging were studied. Here, changes in volatile and polyphenolic compositions of a red wine aged for 9 months in acacia, cherry, chestnut, mulberry, and oak barrels are studied. Mulberry showed significant decreases of fruity-note ethyl esters and ethylguaiacol and a great cession of ethylphenol (horsey-odor defect). Cherry promoted the highest polyphenol oxidation, making it less suitable for long aging. LC/ESI-MS(n) showed the relevant presence of cis- and trans-piceatannol in mulberry-aged wine, a phytoalexin with antileukemia and antimelanoma activities.

GC/MS-positive ion chemical ionization and MS/MS study of volatile benzene compounds in five different woods used in barrel making.

Extracts from acacia, chestnut, cherry, mulberry, and oak wood, used in making barrels for aging wine and spirits were studied by GC/MS positive ion chemical ionization (PICI). Wood chips were extracted by a 50% water/ethanol solution and a tartrate buffer pH 3.2-12% ethanol (model wine) solution. The principal compounds identified in extracts were guaiacol-containing aldehydes and alcohols, such as benzaldehyde and derivatives, vanillin and syringaldehyde, cinnamaldehyde and coniferaldehyde, eugenol and methoxyeugenol, guaiacol and methoxyguaiacol derivatives. PICI using methane as reagent gas produced a high yield of the protonated molecular ion of volatile phenols, compound identification was confirmed by collision-induced-dissociation (CID) experiments on [M + H](+) species. MS/MS fragmentation patterns were studied with standard compounds: guaiacol-containing molecules were characterized by neutral methyl and methanol losses, benzaldehyde derivatives by CO loss. Acacia wood extracts contained significant syringaldehyde and anisaldehyde, but no eugenol and methoxyeugenol. Significant syringaldehyde, eugenol and methoxyeugenol, and high vanillin were found in chestnut and oak wood extracts; low presence of volatile benzene compounds was found in mulberry wood extracts. Cherry wood extracts were characterized by the presence of several benzaldehyde derivatives and high trimethoxyphenol.

Fast determination of the total free resveratrol content in wine by direct-exposure-probe, positive-ion chemical ionization and collision-induced-dissociation mass spectrometry.

In the present work, a fast method has been developed for the determination of total free resveratrol in wine extracts by positive-ion chemical ionization using methane as reagent gas and direct introduction of the sample into the mass spectrometer. Quantitative analysis is performed using 4',5,7-trihydroxyflavone as internal standard, by recording the product ions at m/z 135 for resveratrol and m/z 153 for the internal standard, both generated by collision-induced dissociation experiments on the related [M+H](+) ions. The method showed sufficient repeatability and satisfactory linearity in the range of concentration studied. Alternatively, because of the marked effect of matrices on the compounds released in the vaporization step, quantitative analysis was performed by addition of standard resveratrol to the sample, and the total free resveratrol content extrapolated from the calibration curve. Calibration curves for three different wine extracts had correlation coefficients between 0.91 and 0.99, and total free resveratrol contents between 11.8 and 17.2 mg/L were found. This method enabled us to determine the resveratrol-potential of wine within a few minutes, thereby overcoming the time- and solvent-consuming procedures needed for chromatographic methods.

Glyoxal and methylglyoxal levels in diabetic patients: quantitative determination by a new GC/MS method.

Determination of glyoxal and methylglyoxal levels in plasma is of great interest, since it allows us to evaluate oxidation processes occurring in glycated proteins. A method based on a simple derivatization procedure followed by gas chromatography/mass spectrometry (GC/MS) analysis has been developed. Ten diabetic patients were evaluated before and after improvement of glycemic control. Fasting plasma glucose, hemoglobin A1c (HbA1c), advanced glycation end products (AGE), pentosidine, glyoxal and methylglyoxal levels were measured. The percentage decreases of the levels of fasting plasma glucose, HbA1c and AGE were larger than those of pentosidine, glyoxal and methylglyoxal. These results may be explained by considering the different position of these compounds in the Maillard reaction pathways: these two sets of metabolic parameters give different pictures of patients' metabolic control. The measurement of glyoxal and methylglyoxal may be particularly important in the evaluation of the possible effect of oxidative stress. Other metabolic pathways can contribute to glyoxal production, and the observed minor decrease in these compounds can be, in principle, ascribed to such effect. However, a similar behavior of pentosidine indicates that these alternative pathways can be only partially responsible for glyoxal and methylglyoxal production.

Glyoxal/glycolaldehyde: a redox system involved in malolactic fermentation of wine.

To verify the presence of glycolaldehyde in wine resulting from reduction of glyoxal after malolactic fermentation, sterilized solutions of synthetic cultures were inoculated with a lactic bacterium of the type Oenococcus oeni. Fermentation was also carried out on solutions with glyoxal added. The resulting glycolaldehyde concentrations turned out to be associated with the amounts of glyoxal present, and glyoxal was seen to decrease as glycolaldehyde increased. In addition, it was observed that glyoxal principally forms from breakdown of sugars and that reduction to glycolaldehyde is mainly promoted by bacterial activity. Finally, the ability of glycolaldehyde to induce browning of (+)-catechin in a model wine system was verified and turned out to be about 10 times higher than that of ascorbic acid.