Recent Applications of Mass Spectrometry in the Study of Grape andWine Polyphenols

Polyphenols are the principal compounds associated with health bene�c effects of wine consumption and in general are characterized by antioxidant activities. Mass spectrometry is shown to play a very important role in the research of polyphenols in grape and wine and for the quality control of products. e so ionization of LC/MS makes these techniques suitable to study the structures of polyphenols and anthocyanins in grape extracts and to characterize polyphenolic derivatives formed in wines and correlated to the sensorial characteristics of the product. e coupling of the several MS techniques presented here is shown to be highly effective in structural characterization of the large number of low and high molecular weight polyphenols in grape and wine and also can be highly effective in the study of grape metabolomics.


Principal Polyphenols of Grape and Wine
Polyphenols are the principal compounds associated to health bene�c effects of wine consumption.A French epidemiological study performed in the end of 1970s reported that in France, despite the high consumption of foods rich in saturated fatty acids, the incidence of mortality from cardiovascular diseases was lower than that in other comparable countries.is phenomenon was called "the French paradox� and was related to the bene�cial effects of red wine consumption [1].In general; polyphenols have antioxidant activities.eir activity as peroxyl radical scavengers and in the formation of complexes with metals (Cu, Fe, etc.) has been shown by in vitro studies.Moreover, the ability of polyphenols to cross the intestinal wall of mammals confers their biological properties.
Flavan-3-ols are one of the principal classes of grape polyphenols which include (+)-catechin and (−)-epicatechin, and their oligomers called procyanidins, proanthocyanidins, and prodelphinidins.B-type and A-type procyanidins and proanthocyanidins (the latter are condensed tannins) are present in the grape skin and seeds; tannins are mainly present in seeds, and prodelphinidins are polymeric tannins composed of gallocatechin units (structures in Figure 1).
During winemaking the condensed (or nonhydrolyzable) tannins are transferred to the wine and contribute strongly to the sensorial characteristic of the product.In the mouth, the formation of complexes between tannins and saliva proteins confers to the wine the sensorial characteristic of astringency: bitterness and astringency of wine is linked to tannins structure, in particular galloylation degree (DG) and polymerization degree (DP) of �avan-3-ols [2,3].Grape tannins are used as active ingredients in medicinal products characterized by antioxidant plasma activity and for the treatment of circulatory disorders (capillary fragility, microangiopathy of the retina, reducing of platelet aggregation, decreasing of the susceptibility of healthy cells towards toxic and carcinogenic agents, and antioxidant activity toward human low density lipoprotein) (see [4], and references cited herein).
Flavonols are another important class of grape polyphenols.ese compounds are mainly present in the skin of berry; the principal are quercetin, kaempferol, and myricetin present in glycoside forms such as glucoside, glucuronide, and rutin.Recently, also isorhamnetin, laricitrin, and syringetin were identi�ed in grape [5,6].e structures of �avonols are reported in Figure 2(b).e main biological activity of quercetin is to block human platelets aggregation, and it seems that it inhibits carcinogens and the cancer F 2: (a) e principal monomer anthocyanins of grape: the glucose residue can be linked to an acetyl, coumaroyl, or caffeoyl group.(b) e principal �avonols of grape.cell growth in human tumors (see [4] and references cited herein).
Anthocyanins are the compounds responsible for the red color of grapes and wines.Principal anthocyanins of Vitis vinifera varieties are delphinidin (Dp), cyanidin (Cy), petunidin (Pt), peonidin (Pn), and malvidin (Mv), present in the skins as 3-O-monoglucoside, 3-O-acetylmonoglucoside, and 3-O-(6-O-p-coumaroyl)monoglucoside.Oen, also Mv-3-O-(6-O-caffeoyl)monoglucoside is present.More recently, pelargonidin (Pg) 3-O-monoglucoside was found in grape [24].Oen the not V. vinifera (hybrid) red grapes also contain diglucoside anthocyanins with the second glucose molecule linked to the C-5 hydroxyl group (structures showed in Figure 2(a)).e anthocyanin pro�le is also determined for the study of grape chemotaxonomy; for example, the presence of 3,5-O-diglucoside anthocyanins is used to distinguish between V. vinifera and hybrid grape varieties, the former being characterized by low presence or practical absence of these compounds.Moreover, grape anthocyanins are antioxidant and natural colorants used in the nutraceutical, food, and pharmaceutical industries [26][27][28].
During the wine aging, anthocyanins are undergone reactions with other matrix compounds, and new molecules with different chromatic characteristics, with respect to their precursors, are formed [29].As a consequence, the anthocyanic pro�le of wine changes dramatically during aging; for example, the LC-chromatogram of a 4-month aged wine recorded at 520 nm shows as main signals the grape anthocyanins, and aer 2-year aging, these signals disappear completely, and a broad peak due to the new anthocyanin derivatives overlaps the latter part of the chromatogram [30,31].Reaction of anthocyanins with �avan-3-ols, procyanidins, and tannins shis the wine from purple-red to brick-red hue, and the formation of pyranoanthocyanins, stable structures formed by reaction between anthocyanins and acetaldehyde, pyruvic acid, vinylphenol, vinylcatechol, vinylguaiacol, or vinyl(epi)catechin, toward orange hue [20,21,[32][33][34][35].
More than hundred structures belonging the pigment families of anthocyanins, pyranoanthocyanins, direct �avanol-anthocyanin condensation products and acetaldehydemediated �avanol-anthocyanin condensation products (anthocyanin linked to �avan-3-ol either directly or by ethyl bridge), were identi�ed in red wines.Structures of principal anthocyanin-derivatives are showed in Figure 4, [30].  is the most effective tool for the structural characterization of low molecular weight (MW) polyphenols in grape extracts and wine.It is also widely used to characterize high-MW compounds, such as procyanidins, proanthocyanidins, prodelphinidins, and tannins [37][38][39][40].In general, these methods require minor sample puri�cation, and MS/MS allows characterization of both aglycone and sugar moiety.
A study of �avonols in different V. vinifera red grape extracts showed, in addition to myricetin and quercetin 3-O-glucosides and 3-glucuronides, and to kaempferol and isorhamnetin 3-O-glucosides, the presence of laricitrin and syringetin 3-glucosides.Also, minor �avonols, such as kaempferol and laricitrin 3-galactosides, kaempferol-3-glucuronide, and quercetin and syringetin 3-(6-acetyl)glucoside, were identi�ed [5].Table 1 reports the �avonols identi�ed in Petit Verdot grape skins extract.Extraction was performed by using a methanol/H 2 O/formic acid 50 : 48.5 : 1.5 (v/v/v), and the analytes were separated from anthocyanins by performing solid-phase extraction (SPE) using a combined reverse-phase and cationic-exchanger commercial cartridge.Aer sample loading, the cartridge was washed with HCl 0.1 M solution, and the �avonol fraction containing neutral and acidic polyphenols was recovered with methanol.LC analysis was performed by using a reverse-phase C 18 column with elution gradient with water/acetonitrile/formic acid 87 : 3 : 10 v/v/v and 40 : 50 : 10 v/v/v.Flavonols were detected by performing analysis with the mass spectrometer operating in positive ion mode.
Extraction of proanthocyanidins (PAs) and tannins from seeds and skins can be performed from the powder produced by grinding frozen material using a methanol/H 2 O 25 : 75 (v/v) solution (e.g., three consecutive extractions for 15 min under stirring at room temperature using ultrasounds [44,45] or with an acetone/H 2 O 60 : 40 (v/v) solution [3].Aer removing of organic solvent, the aqueous residue has to be washed with hexane in order to eliminate lipophilic substances.e extract can be then fractionated on a column for gel �ltration of natural products Sephadex LH-20 by eluting different fractions with ethanol and acetone aqueous solutions [44,45].Alternatively, puri�cation of aqueous extract can be done by performing chromatography on a methacrylic size-exclusion resin and elution from the column of two fractions with ethanol/H 2 O/TFA 55 : 45 : 0.02 v/v/v and acetone/H 2 O 30 : 70 v/v [3].e two solutions are pooled, concentrated under vacuum, and freeze dried.Further puri�cation of the residue can be performed on divinylbenzene-polystyrene resin: �avan-3-ol monomers are recovered with water and ether, then PAs with polymerization degree of 3 units (DP3) with methanol.Finally, the fraction containing DP10 is recovered with acetone/H 2 O 60 : 40 v/v.Seed extract, or grape �uice, can be also puri�ed by SPE using a reverse-phase C 18 cartridge.Extract is suspended in water, and the solution is passed through the cartridge previously conditioned by passage of methanol and water, and aer sample loading, the cartridge is rinsed with water, and the fraction containing PAs is eluted with acetone/H 2 O/acetic acid 70 : 29.5 : 0.5 v/v/v [46,47].
Extraction of tannins from skins is performed by preliminary removing the low MW phenolics (in particular anthocyanins) immerging the skins in a 12% v/v ethanol solution for 72  LC/ESI-MS analysis of PAs is usually performed by reverse-phase chromatography, even if normal phase chromatography using silica columns provided a satisfactory separation of oligomers based on their MW [46,47].e LC/ESI-MS positive-ion chromatogram of a grape seeds extract (analysis performed by a reverse-phase column and gradient elution with a binary solvent composed of aqueous F 8: Fragmentation pathways of monomer catechin in positive-ion mode: retro-Diels-Alder �ssion (RDA), heterocyclic ring �ssion (HRF), benzofuran forming �ssion (�FF), and loss of water molecule [41].0.1% formic acid and acetonitrile/0.1% formic acid) shows the signals of protonated catechin and epicatechin at  291, protonated catechin/epicatechin gallate at  443, protonated catechin/epicatechin dimer at  579, and protonated catechin/epicatechin gallate dimers and trimers at  731,  883 and  867, respectively [48].
PAs were also characterized by direct-infusion ESI-MS without performing chromatographic separation.Negativeion analysis of the extract dissolved in methanol/acetonitrile showed the highest intensity of ions including multiplied charged species [50].Moreover, simpler mass spectra were recorded due to the absence of intense adduct ion species and to the production of more multiply charged ions with respect to the positive ionization mode.e [M−H] − and [M−2H]   Neutral loss of 152 Da (corresponding to 3,4-dihydroxy-hydroxystyrene) induces formation of two fragments at  285 and 289 generated by cleavage of the A-type inter�avanic linkage [51].
In another study, the presence of galloylated A-type procyanidins in grape seeds was evidenced [52].Procyanidins were extracted from seeds with methanol and fractionated using graded methanol/chloroform precipitation in order to obtain the oligomers with lower MW.ESI-MS spectra recorded in positive mode showed the presence of [M+H] + ions of A-type and B-type nongalloylated and monogalloylated procyanidins with DP 2-5, and of digalloylated oligomers with DP 2-3.A-type procyanidins occurred with abundance 60%-80% with respect to the corresponding type-B species, the abundance of monogalloylated dimers was 20% of the abundance of the corresponding nongalloylated ones.For higher DP, the abundance of nongalloylated oligomers was higher of the corresponding galloylated oligomers.e type-A inter�avanic linkages were present in the terminal units, whereas the type-B inter�avanic linkages were extension units.
Figure 7 shows the positive-ion MS 3 fragmentations of the most intense trimeric procyanidins signals in the spectra at  867 [36].e schemes of positive fragmentation patterns for monomer catechin and of a B-type trimer at  883 are reported in Figures 8 and 9, respectively [41].Table  F 10: Fractionation of polyphenols in red wine (PA, proanthocyanidins) [43].
reports the positive-ion LC/ESI-MS  product ions of �avan-3-ol monomers and PAs dimers, trimers, and oligomers.A recent study reports the identi�cation of fourteen �avan-3-ol monoglycosides in Merlot grape seeds and wine extracts [53].Analyses were performed using an ESI-�uadrupole time of �ight mass spectrometry system operating in negative-ion mode.Compounds were identi�ed on the basis of their exact masses and speci�c fragmentation patterns and as aglycones showed (+)-catechin, (−)-epicatechin, (−)-epigallocatechin, and epicatechin gallate monomeric units.
Several methods of sample preparation were proposed to perform analysis of polyphenols in wine.e nonanthocyanic polyphenols identi�ed in four di�erent wine varieties (Tempranillo, Graciano, Cabernet Sauvignon, and Merlot) by performing LC/ESI-MS analysis are reported in Table 4. e analytes were extracted from 50 mL of wine previously reduced to 15 mL under vacuum in order to eliminate ethanol.Two consecutive extractions were performed by using diethyl ether and ethyl acetate.e two organic phases were combined, the solvent removed, and the residue was dissolved in a methanol/H 2 O solution.LC analyses were performed using a reverse-phase C 18 column performing the gradient elution with a binary solvent composed of H 2 O/acetic acid 98 : 2 v/v and H 2 O/acetonitrile/acetic acid 78 : 20 : 2 v/v/v [54].e compounds were identi�ed on the basis of their fragment ions and maximum absorption wavelengths recorded in the mass and UV-Vis spectra, respectively.Table 4 reports a number of compounds included in di�erent classes of phenols, such as �avonols, hydroxycinnamoyltartaric acids, stilbene compounds (cisand trans-resveratrol, piceid), phenolic acids, �avan-3-ols, and dimeric (B1, B3, B4, and B5) and trimeric (C1, T2 and T3) procyanidins.
Two di�erent puri�cation methods by using sizeexclusion and reverse-phase chromatography were used to perform sample preparation for the analysis of PCs and PAs in wine.A volume of dealcoholized wine was passed through a size-exclusion resin, the stationary phase was washed with water, and the fraction containing simple polyphenols was eluted with ethanol/H 2 O/TFA 55 : 45 : 0.005 v/v/v.e fraction containing dimers and trimers was recovered with acetone/H 2 O 60 : 40 v/v [55].Sample preparation by reverse-phase chromatography was instead performed using    a C 18 cartridge.A volume of dealcoholized wine was loaded onto the cartridge, and PAs were recovered with 10 mL of acetone/H 2 O/acetic acid 70 : 29.5 : 0.5 v/v/v solution [46].e �ow diagram in Figure 10 shows a method for fractionation of wine polyphenols by reverse-phase chromatography [43].e more complex polyphenols recovered in the fractions 8-10 were characterized by LC/ESI-MS and MS  negative-ion mode analysis and reported in Table 5, [57].

LC/MS of Grape Anthocyanins
Liquid chromatography mass spectrometry (LC/MS) coupled with multiple mass spectrometry (MS/MS and MS  ) has been widely used for structural characterization of grape anthocyanins [59] and to study the structure of new anthocyanin derivatives formed during wine aging [22,23,30,36,[60][61][62][63].LC analysis of anthocyanins is usually performed by a reverse-phase C 18 column by performing gradient elution of compounds using a binary solvent composed of H 2 O/formic acid 90 : 10 v/v and methanol/H 2 O/formic acid 50 : 40 : 10 v/v/v.e analytes are detected by recording the chromatogram at 520 nm.Due to the lack of standards commercially available, the identi�cation of compounds is usually performed on the basis of their column elution sequence.
A study showed that� nonacidi�ed methanol is a solvent suitable for extraction of anthocyanins from grape skins reducing the risk of hydrolysis of acetylated compounds [64].Alternatively, extraction by methanol/H 2 O/formic acid solution (50 : 48.5 : 1.5 v/v/v) was proposed [65].Anthocyanins can be then puri�ed by passing through a C 18 cartridge: aer sample loading, the nonanthocyanic phenols are eluted from the cartridge with ethyl acetate, then anthocyanins are recovered with methanol.A fast direct-injection ESI-MS/MS analysis in positive-ion mode provides structural characterization and semiquantitative data of the anthocyanins in the extract [66].As may be seen in the ESI-direct injection spectrum of Clinton grape skins extract in Figure 11, all anthocyanins show evident signal of the M + ion.
MS/MS and collision-induced-dissociation (CID) provide characterization of compounds.Experiments are performed by applying a supplementary radio frequency �eld to the endcaps of the ion trap (1-15 V) in order to make the selected ions collide with He.Precursor ions and the fragments recorded for the sample in Figure 11      Phloroglucinol F 16: Fragmentation scheme proposed for two anthocyanin dimers.�CR: heterocyclic ring �ssion and RDA: retro-Diels-Alder �ssion [56]. in Table 6.A list of other monomer anthocyanins identi�ed in extracts of other grape varieties is reported in Table 7, [66].
In general, MS  is highly effective in differentiation also of isobaric anthocyanins.Of course, the collision energy applied affects the relative abundance of diagnostic fragments.In the case of Mv-3,5-O-diglucoside and Mv-3-O-(6-caffeoyl)monoglucoside, to distinguish between two compounds by performing MS  experiments is not possible because they have identical molecular mass and aglycone.For identi�cation of two compounds, dissolution of the extract in deuterated water was performed to observe the different mass shis in agreement with the different number of exchangeable acidic proton present in each molecule [66].Direct-ESI/MS also provided semiquantitative data of anthocyanins in the extract.Quan-ti�cation of compounds was performed on the calibration curves of Mv-3-O-glucoside for monoglucosides (M + at  493) and of Mv-3,5-O-diglucoside for diglucosides (M + at  655) being standard commercially available compounds.An Mv-3-O-glucoside 40-ppm solution in water/acetonitrile 95 : 5 v/v was used to optimize the ESI parameters in order to maximize the signals [36,66].
Also, the capabilities of quadrupole-time-of-�ight (Q-TOF) MS coupled with LC-Chip were used to distinguish between Mv-3,5-O-diglucoside and Mv-3-O-(6-O-caffeoyl)monoglucoside in Clinton extract, and the method was compared with direct-infusion ESI/IT-MS [49].LC-Chip analyses were performed using a chromatographic system composed of an enrichment column Zorbax 300 SB-C 18 (40 nL, 5 m) and analytical column Zorbax 300 SB-C 18 (75 m × 43 mm, 5 m).Elution of compounds from the column was performed by a binary solvent mixture composed of aqueous 0.1% formic acid and methanolic 0.1% formic acid working at a �ow rate of 400 nL/min.Before analysis, the grape skins extract was diluted with a loading solution (aqueous 5% methanol containing 0.1% formic acid), and 1 L of the sample was injected.LC-Chip/ESI-QTOF MS provided the complete anthocyanin �ngerprint of the sample in less than 5 minutes with practically no solvent consumption.Neither MS/MS was necessary for identi�cation of isobaric compounds, nor deuterium exchange experiments to distinguish between compounds having the same aglycone.e fast separation bypassed also the problem of Pt   in the analysis of Clinton grape skins extract are showed in Figure 12.
Reverse-phase LC-Chip allowed the separation of all the isobaric pairs of compounds with an elution sequence from the column linked to the polarity of compounds: �rst elute the more polar diglucosides, followed by monoglucosides, and �nally the less polar acylated monoglucosides (acetates and pcoumarates, resp.).�e anthocyanins identi�ed in the Clinton extract by LC-Chip/Q-TOF and direct-ESI/IT-MS analysis are reported in Table 8.
To distinguish between isobaric pairs of anthocyanins, also accurate mass measurements were performed.Figure 13 shows the Q-TOF signals recorded aer LC-Chip separation of isobaric pairs at nominal mass  611 (Cy-diglucoside and Dp-p-coumaroylmonoglucoside),  625 (Pn-diglucoside and Pt-p-coumaroylmonoglucoside), and  655 (Mv-diglucoside and Mv-caffeoylmonoglucoside). For these ions, the Q-TOF system used did not provide sufficient resolution to distinguish between Cy-3,5-O-diglucoside (C 27    F 20: Mass spectra of a Cabernet Sauvignon wine recorded by application of spray capillary voltage 3 kV (a) and 0.5 kV (b).* : main anthocyanin signals [70].
Concentration of anthocyanins in the extract was determined on the calibration curve calculated by spiking the sample with Mv-3,5-O-diglucoside standard at three different concentrations.e resulting calibration curve is showed in Figure 15.
Quantitative percentage data of LC-Chip/ESI-QTOF-MS and ESI/IT-MS analysis of Clinton extract are reported in    for Pt-  8), inferring that probably protonated �avonols present in the extract were not separated from the chromatographic chip used.
In grape skins, also the oligomeric anthocyanins listed in Table 9 were identi�ed [56,59].Figure 16 shows the fragmentation scheme proposed for two anthocyanin dimers.
In a recent work anthocyanins of 21 hybrid red grape varieties produced by crossing of different Vitis vinifera, riparia, labrusca, Lincecumii, and rupestris species were studied [58].In general, hybrid grapes are characterized by peculiar contents of anthocyanins, oen qualitatively and quantitatively different-and superior-to the V. vinifera varieties [66,[71][72][73][74][75][76][77].Also due to the increasing industrial demand for natural colorants, the knowledge of their composition may be useful for industrial purposes [78,79].
e study was performed by using an ultraperformance liquid chromatography and triple quadrupole mass spectrometry system (UPLC/MS).Precursor-ion analysis of the anthocyanidin and monoglucoside-anthocyanin fragments produced by CID was performed.Twenty-four compounds were identi�ed using two different experimental conditions: precursor-ions scan of the aglycone fragments produced at a collision energy of 4 eV and precursors scan of monoglucoside anthocyanin fragments produced at collision energy 25 eV.Analysis of precursor ions of these fragments was possible because usually no fragmentation occurs in the glucose and acyl group linkage, nor formation of acylglucoside anthocyanins was observed in neutral-loss analysis [80].e samples were subdivided into two groups on the basis of their anthocyanin pro�les which were characterized by the substantial presence or scarce presence of diglucoside compounds, respectively.Analysis of precursor ions showed to be a highly selective method: by monitoring each aglycone, the signals of all corresponding derivatives are detected.is approach can be used for selective study of particular anthocyanidin derivatives in the sample; for example, by monitoring the product ion at  331 (corresponding to Mv), the signals of precursors at  493 (the monoglucoside derivative),  535 (acetyl monoglucoside),  639 (pcoumaroylmonoglucoside),  655 (diglucoside), and  801 (p-coumaroyl diglucoside) were detected.In addition, precursor-ion analysis enhances the signal-to-noise ratio, allowing more sensitivity in analysis of anthocyanins in complex matrices [80].
Figure 17 shows the TIC and precursor-ion chromatograms of aglycone fragments from analysis of a hybrid grape sample (Bacò 30 -12).ree precursor ions for Dp, Pn, and, Cy anthocyanidin fragments and �ve precursor ions for Mv and Pt were found, leading to identi�cation of the 20 anthocyanins (13 monoglucosides and 7 diglucosides) reported in Table 10.

LC/MS of Anthocyanin Derivatives in Wine
LC/MS analysis of anthocyanin derivatives in wine can be performed by direct injection of the sample without prior sample preparation, and several methods with different chromatographic conditions were proposed by this approach.Table 12 shows the compounds identi�ed in Graciano, Tempranillo, Cabernet Sauvignon, and Primitivo wines.Alternatively, a previous sample puri�cation can be performed on a C 18 cartridge recovering anthocyanins with methanol [81].
Several methods for isolation and fractionation of oligomeric pigments in the analysis of pyranoanthocyanins and anthocyanin derivatives in wine were proposed [22,33].Anthocyanin-�avanol derivatives can be characterized by MS/MS experiments (Table 12).For example, Figure 18 shows the fragmentation schemes proposed for (epi)catechin-Mv-3-glu (M + at  781) and for Mv-3-glu-(epi)catechin with A-type linkage (M + at  783).
A list of anthocyanin derivatives identi�ed in wines at different aging stages is reported in Table 13.As may be seen, ethyl-bridge derivatives, pyranoanthocyanins, and pigments formed by anthocyanin-�avanol linkage are included.Some of these compounds are already present in wine in the �rst aging stage and disappear in the time, and others are formed with long time aging.Also, oligomeric pigments F-A-A + type (F, �avanol� A, anthocyanin) were identi�ed in wines and characterized by ESI/MS  .Table 14 reports the compounds identi�ed in Tempranillo aged wines.Possible structures proposed for the M + species at  1273 are shown in Figure 19, [60].
A fast and selective method for screening of the anthocyanic composition of wine was recently proposed [70].Analysis of wine extract was performed by direct-infusion ESI-MS/MS operating in positive-ion mode with application of a low spray capillary voltage (0.5 kV).e high selectivity of the method towards anthocyanins is due to the following: by operating far from ESI conditions, any process related to electrospray ionization occurs (these processes taking place with capillary voltages up 3 kV [83]), and only the species already present in ionic form in the sprayed solution can be detected [84].In ESI conditions, simultaneous detection of anthocyanins, their derivatives, and protonated ions of other wine components can lead to a quite complex panorama, with possible occurrence of matrix effects, favouring the detection of nonanthocyanic compounds.Operating with a low spraying capillary voltage, the protonation of these molecules is strongly reduced, or avoided, so allowing to obtain directly a �ngerprint of the cations already present in the sample.As a matter of fact, by decreasing the spray capillary voltage, a dramatic increase of selectivity toward anthocyanins was observed and their peaks become the most abundant (Figure 20).In these conditions, the electrospray phenomena are practically inhibited and the solution spray is generated only by pneumatic effects, so that the method was called direct-infusion pneumatic spray (DIPS) mass spectrometry.e previous spectrum was obtained by application of an usual capillary voltage of 3 kV and the most abundant signals at  977, 949, 739, and 711 are of unknown compounds.By decreasing the spray capillary voltage to 0.5 kV, signals of the main anthocyanins in the sample at  331, 479, 493, 535, and 609 increase (marked peaks in the spectrum in the following).
e anthocyanic pro�les of several red wines at different aging stages were studied by DIPS, and a great number of anthocyanins and anthocyanin-derivatives were detected.Sample preparation for analysis was performed by extraction of anthocyanins using a reverse-phase C 18 cartridge and recovering the analytes with methanol.In order to verify the structural assignment of the ionic species detected in the DIPS spectra, MS/MS was performed.e product ions found con�rmed the presence of 15 anthocyanin-�avanol derivatives, including 11 pyranoanthocyanins, 3 vitisin A-type compounds (at  531, 533, and 561, resp.) and malvin-vinyl(epi)catechin ( 805) (in particular in aged wines), and other several anthocyanin derivatives.e list of compounds identi�ed is reported in Table 15.
Of course, the absolute intensity of anthocyanin signals is lower than that observed using spraying capillary voltage of 3 kV due to the lack of the ion focusing effect originating by the high voltage.is aspect re�ects negatively in higher values of limit of detection (LOD) and of limit of quanti�cation (LO�) but positively in higher speci�city of the data.DIPS �ngerprint of a several years aged wine shows profound transformations of anthocyanin composition, as shown in the spectrum in Figure 21.Signals of pyranoanthocyanin and anthocyanin-�avanol derivatives arise in two  ranges of the spectrum, the �rst at  579-771 and the second at  751-929, respectively.
For a semiquantitative analysis, Mv-3,5-diglucoside (compound usually not present in V. vinifera grapes and wines) was added to the sample as internal standard (IS), and two indexes, of wine color and of wine color evolution, were calculated.e �rst is a total anthocyanin index of the sample.It was calculated as the sum of intensity of all anthocyanin and anthocyanin derivative signals in the TIC and was expressed as mg/L of IS.As expected, higher values of this parameter were found for nonaged wines (between 40 and 100 mg/L), instead the aged samples had anthocyanin content between 4 and 80 mg/L.In particular, very low contents were found in the two oldest samples aged in barrels (between 3 and 10 mg/L), inferring that anthocyanins and derivatives are undergone to severe degradation processes in barrel ageing for long time.
During aging, the chemical composition of anthocyanins (i.e., color) changes as well.Index of wine color evolution was calculated as the ratio Σanthocyanin-derived signals intensity/total anthocyanin index and represents these chemical changes.All the nonaged wines had a value lower than 20%, while it was higher in all aged samples.As a consequence, a wine color evolution index of 20% was proposed as the limit for distinguishing between aged and nonaged wines.is index can be also correlated to the wine aging conditions, such as presence of oxidative or reductive environment (barrel or bottle), oxygen level in wine, and air exchange through the barrel staves.Moreover, because pyranoanthocyanins remain colored over a wide pH range and in the presence of sulphites [32,85], the study of the ratio between pyranoanthocyanins and the other anthocyanin-�avanol derivatives was proposed to predict the wine color stability.

Study of Grape Procyanidins by MALDI-TOF MS
Matrix-assisted laser desorption-ionization and time of �ight (MALDI-TOF) mass spectrometry is a technique in which an acidic solution containing an energy-absorbing molecule (matrix) is mixed with the analyte and a highly focused laser pulses are directed to the mixture [86].Molecules are desorbed, ionized, and accelerated by a high electrical potential, and the ions arrive to the detector in the order of their increasing  ratio.Due to robustness, tolerance to salt-and detergent-related impurities, and ability to be automated, MALDI-TOF is used to perform generation of mass map of proteins aer enzymatic digestion [87].cyano-4-hydroxycinnamic acid (CHCA) is the matrix commonly used for analysis of peptides and small proteins and sinapinic acid (SA) of higher molecular weight (MW) proteins (10-100 kDa).Advantages of MALDI-TOF are good mass accuracy (0.01%) and sensitivity to require very little sample for analysis.
MALDI-TOF has been used also for characterization of grape procyanidins [88][89][90][91][92]. LC/MS does not allow separation and identi�cation of oligomers higher than pentamers because the separation of a large number of diastereoisomers is not possible.By operating positive-ion MALDI-TOF in the re�ectron mode, �avan-3-ol oligomers and their galloylated derivatives in grape seeds extracts were studied [88].Oligomers were detected up to heptamer as sodium adducts [M+Na] + using 2,5-dihydroxybenzoic acid (DHB) as matrix with a resolution higher than 3000, allowing to separate individual ions of different isotope composition (e.g., the ion at  1177.46 was further resolved into a group of four peaks).In another study, to perform analysis of seed extracts in positive-ion re�ectron mode using trans-3-indoleacrylic acid (t-IAA) as matrix allowed the identi�cation of a series of compounds with MW 2 Da lower, which correspond to Atype polycatechins.Linear mode analysis provided detection of PAs oligomers sodium adducts up to nonamers [89].e lower sensitivity of the re�ectron mode for the large ions is reasonably due to their collisionally induced decomposition occurring in the �ight path [88,89].Masses of PAs determined in both re�ectron and liner modes are reported in Table 16.On the basis of the galloylated structures, the equation 290 + 288 + 2 + 2 used to predict the mass distribution of PGPF in grape seeds was calculated (290 is the MW of the terminal catechin/epicatechin unit, c degree of polymerization,  number of galloyl esters, 23 Na atomic mass) [89].Extraction of PAs from grape seeds for MALDI-TOF analysis was carried out with acetone/water, ethanol, or methanol/water mixtures, then a puri�cation by extraction with ethyl acetate or chloroform which can be performed [88,89,91,93].
Among the matrices tested for MALDI, DHB and t-IAA showed to be highly suited for PAs analysis.e experiments performed by Yang and Chien showed that DHB provides the broadest mass range and the least background noise.e dry grape seed extract was dissolved in acetone or methanol at 2 mg/mL, and a DHB matrix solution 20 mg/mL in tetrahydrofuran was prepared (the sample and matrix solutions were mixed at 1 : 1 v/v) [88].Sodium apparently arises from the seeds themselves, and only a minute amount of sodium was needed.e use of DHB and water-free solvents such as anhydrous tetrahydrofuran, acetone, or methanol for the sample and matrix preparation showed the best analytical conditions in re�ectron mode.
In general tannins containing A-type bonds showed lower signal intensity with respect to the corresponding compounds with B-type bonds (between 10% and 50%).By the formula in Table 16 and the data from the literature, PAs with DP from 2 up to 11 were identi�ed [52,57,89,92].
A study of PAs in grape seeds of 35 hybrid and V. vinifera grape varieties is now in progress.PAs are extracted with a methanol/water 70 : 30 v/v solution and analyzed by MALDI-TOF in positive-ion mode using matrix DHB.PAs showed [M+Na] + adducts as main signal, and some of [M+K] + and [M+H] + adducts (Table 17).Signals of tannins containing Btype bonds and one or more A-type bonds were found in the mass spectra (Figure 22).For example, the protonated trimer was present in different forms: with two B-type bonds and MW 867 Da, with one A-type and one B-type bond and MW 865 Da, and with two A-type bonds with MW 863 Da.

Polyphenols and Grape Metabolomics
"Metabolomics" is the comprehensive quantitative and qualitative study of all the metabolites within a cell, tissue, or organism.e major limitation of this approach is its current inability to exhaustively describe the whole "metabolome" pro�le.is is due to its chemical complexity and the dynamic range limitations of most instrumental approaches.Because a single analytical technique does not provide suf-�cient description of whole metabolome, more methods are needed for a comprehensive view.LC/MS, direct-injection MS, and electrospray ionization (ESI) are powerful tools that offer high selectivity and sensitivity, allowing detection of nonvolatile and labile components in the extract.e coupling of the techniques with GC/MS analysis of volatile compounds, in general, provides a sufficiently wide panorama of the sample metabolomics.e selection of the most suitable techniques is generally a compromise between speed, selectivity, and sensitivity.Due to the complexity of plant extracts, statistical multivariate analysis of data (principal component analysis and cluster analysis) has to be performed to ensure good analytical rigorousness and de�ne both similarities and differences among samples [94].
In general, an "untargeted" metabolomics approach provides sensitivity, resolution, and high-throughput capacity and identi�cation of thousands compounds in a single run [95].Several studies reported that by performing a "targeted" analysis of speci�c metabolites, large part of the molecular information regarding the metabolome of complex samples (e.g., the wine) is missed [96,97].On the other hand, other several studies performed in target analysis provided interesting results in the wine study [98][99][100].For example, in targeted analysis of red wines, anthocyanins and the pigments formed during wine ageing were reported as main biomarkers [95].
A middle-way method between these two approaches is the "suspects screening analysis." In this study the identi�cation of metabolites relies on available speci�c information on compounds such as their molecular formula and structure [101].is approach is applied in our laboratories to the study of metabolomics of grape varieties.Grape berries are powered using liquid nitrogen (in order to minimize possible artifacts) and extracted with methanol.An internal standard is added, and the extract is analyzed with a LC/QTOF system with nominal resolution 40.000 which provides accurate mass measurements.
A library called Grape Metabolomics is actually under construction.is database includes the information available in the literature and found in electronic databases on the potential grape metabolites.Partial con�rmation of the library hits was achieved by performing the identi�cation of metabolites in extracts of some grape varieties taken as models for the peculiar chemical characteristics previously studied (e.g., Raboso Piave for the study of anthocyanins and polyphenols and Moscato Bianco for aroma precursors) [102,103].Currently, this library contains around 1000 putative compounds of grape with MW between 100 and 1700 Da.When data processing of a sample provides identi�cation of a new compound with score su�ciently con�dent, it is added to the library.As a consequence, a further increase of the library will be possible.Compounds are identi�ed on the basis of accurate mass measurements and their isotope patterns, and the identi�cation is con�rmed by MS/MS (multiple mass spectrometry).
With this approach, between 260 and 450 signals were assigned to putative phenolic compounds in grapes (the number depending on the grape variety), mainly including nutraceutical and antioxidant compounds such as anthocyanins, �avones and �avanones, �avanols and procyanidins, phytoalexins, and phenolic acids.Average 30-60 hits had an identi�cation score higher than 99% and a hundred higher 95% (Table 18).For example, in analysis of Raboso Piave grape extract, 17 stilbenes and derivatives were identi�ed, among them trans-resveratrol, piceatannol, cis-and transpiceid, several viniferins, and resveratrol dimers, trimers, and tetramers.Moreover, tentative of identi�cation of a number of aroma precursors (mono-and diterpenols glycoside, norisoprenoids), primary metabolites and peptides, is now in progress.
T 18: Phenolic compounds identi�ed by LC�QTOF mass spectrometry and �suspects screening� analysis using the library GrapeMetabolomics in the study of metabolomics of Raboso Piave grape extract.�e compounds identi�ed �ith identi�cation score (id.) higher than 95% are reported.

Phenolic compound
Id

Conclusions
Mass spectrometry plays a very important role for research and quality control in the viticulture and oenology �eld.e so ionization conditions of LC/MS and the minor sample puri�cation usually needed make these techniques more suitable to study the structures of polyphenols and anthocyanins in grape extracts and for the study of structures correlated to the color changing of red wines.ese methods also allow to characterize the high-MW compounds of grape, such as procyanidins, proanthocyanidins, prodelphinidins, and tannins.e important role of LC/MS in the structural study of polyphenols is also con�rmed by the considerable number of papers appeared in the literature in the last years.Complementary use of different MS techniques can be highly effective to characterize the large panorama of compounds of grape and wine.For example, the use of LC/MS, MALDI-TOF and MS/MS techniques allowed the characterization of procyanidin oligomers up to dodecamers; the coupling of LC/MS with MS/MS techniques is very effective in particular for characterization of glycoside compounds, and GC/MS and LC/MS analyses allow the characterization of hundreds volatile and nonvolatile compounds providing practically the whole metabolome of grape.Further development of these metabolomic approaches will provide effective tools for identi�cation of a high number of important compounds in grape with few analyses and minimal sample preparation, providing useful information on the compounds involved in the metabolisms of cells and tissues.

F 9 :
Positive-ion mode fragmentation pathways of B-type trimer  883: retro-Diels-Alder �ssion (RDA), heterocyclic ring �ssion (HRF), benzofuran forming �ssion (BFF), �uinone methide �ssion (QM), and loss of water molecule.QM CD↓ : the ion derived from the QM �ssion of ring-C/ring-D linkage bond by the loss of upper unit and QM FG↑ : the ion derived from the QM �ssion of ring-F/ring-G linkage bond by the loss of lower unit[41].
-3-O-(6-O-acetyl)monoglucoside and Dp-3,5-O-diglucoside quanti�cation of direct-infusion ESI/IT MS due to overlapping of their signals with matrix interferences.e high speci�city of LC-Chip/Q-TOF-MS was due to highly reproducible retention times (Chip-chromatography reduces the problems arising from dead volumes, eluent �ow constancy, and ESI condition stabilities), highly effective and reproducible collisional experiments, accurate mass measurements, and consequent elemental formula determination of the precursor and fragment ions.Total ion chromatogram (TIC) and extracted ion chromatogram (EIC) of the three isobaric compound pairs at  611, 625, and 655 identi�ed

T 10 :T 11 :
Precursors of t�e aglycone fragment ions identi�ed by �P�C�MS precursor-ion analysis in t�e study of 21 �ybrid grape varieties.Varieties Precursor ions of m/z 331 (Mv) Precursor ions of m/z 317 (Pt) Precursor ions of m/z 303 (Dp) Precursor ions of m/z 301 (Pn) Precursor ions of m/z 287 (Cy) m/z 331 (Mv) Precursor ions of m/z 317 (Pt) Precursor ions of m/z 303 (Dp) Precursor ions of m/z 301 (Pn) Precursor ions of m/z 287 (Cy) Precursors of t�e monoglucoside ant�ocyanin fragment ions identi�ed by UP�C�MS precursor-ion analysis in 21 �ybrid grape varieties.Varieties Precursor ions of m/z 493 (Mv) Precursor ions of m/z 479 (Pt) Precursor ions of m/z 465 (Dp) Precursor ions of m/z 463 (Pn) Precursor ions of m/z 449 (Cy)
are reported

Table 8 .
e two methods showed good agreement, except T 8: Anthocyanins identi�ed in Clinton grape skins extract by direct-ESI/IT-MS and LC-Chip/ESI-QTOF-MS analysis performed in positive-ion mode (LC-Chip retention times are reported).Data are expressed as relative percentage of M + signal of compound with respect to M + signal of Mv-3-O-monoglucoside at  611 and  493.
F 22: Signals of PAs in the MALDI-TOF spectrum of a Cabernet Sauvignon grape seeds extract.
T 14: Molecular and fragment ions of the �avanol-anthocyanin-anthocyanin (F-A-A + ) trimers identi�ed in Tempranillo aged wines.e fragment ions are reported in order of abundance.
[88] 16: Masses observed by MALDI-TOF analysis calculated using the equation 290 + 288c + 152g + 23 (290: MW of the terminal catechin unit; c: degree of polymerization; g: number of galloyl ester; 23: Na atomic mass).n.d.: not observed[88].Main PAs identi�ed in the MALDI-TOF positive-ion spectra of seeds extracts of 35 hybrid and V. vinifera grape varieties.Signals were divided in A and B series in reference to the inter-�avanic bond type: series B only B-type inter�avanic bonds, and also A-type bonds are included in series A. Signals of sodium and protonated ions are reported.G: galloyl ester.