Recent Applications of Mass Spectrometry in the Study of  Grape and Wine Polyphenols

Flamini, Riccardo

doi:https://doi.org/10.1155/2013/813563

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Review Article | Open Access

Volume 2013 | Article ID 813563 | https://doi.org/10.1155/2013/813563

Recent Applications of Mass Spectrometry in the Study of Grape and Wine Polyphenols

Riccardo Flamini¹

Academic Editor: T. Stafilov, M. Valko, D.-A. Guo

Received24 Sept 2012

Accepted12 Oct 2012

Published27 Jan 2013

Abstract

Polyphenols are the principal compounds associated with health benefic 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. The soft 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. The 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.

1. Principal Polyphenols of Grape and Wine

Polyphenols are the principal compounds associated to health benefic 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. This phenomenon was called “the French paradox” and was related to the beneficial effects of red wine consumption [1]. In general; polyphenols have antioxidant activities. Their 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 flavan-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. These 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 identified in grape [5, 6]. The structures of flavonols are reported in Figure 2(b). The main biological activity of quercetin is to block human platelets aggregation, and it seems that it inhibits carcinogens and the cancer cell growth in human tumors (see [4] and references cited herein).

(a)

(b)

Stilbene compounds are the principal phytoalexins of grape: they include cis- and trans-resveratrol (3,5,4′-trihydroxystilbene) and their glucoside derivatives (cis- and trans-piceid), piceatannol (3,4,3′,5′-tetrahydroxy-trans-stilbene), and stilbene oligomers (viniferins). A number of studies evidenced anticancer, cardioprotection, anti-inflammatory and antioxidant activities, and platelet aggregation inhibition of trans-resveratrol [7–13]. Grapevine synthesizes viniferins in different parts of the plant (roots, clusters, and stems) in particular ε-viniferin, two δ-viniferin glucosides, and pallidol [14, 15]. Moreover, viniferins can arise from the oligomerisation of trans-resveratrol in grape tissues as active defense of the plant against exogenous attacks or could be produced from resveratrol by extracellular enzymes released from the pathogen in an attempt to eliminate undesirable toxic compounds [16, 17]. Structures of the principal vine viniferins are showed in Figure 3.

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. Often, also Mv-3-O-(6-O-caffeoyl)monoglucoside is present. More recently, pelargonidin (Pg) 3-O-monoglucoside was found in grape [24]. Often 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)).

The anthocyanin profile 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 [26–28].

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 profile 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 after 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 flavan-3-ols, procyanidins, and tannins shifts 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, 32–35].

More than hundred structures belonging the pigment families of anthocyanins, pyranoanthocyanins, direct flavanol-anthocyanin condensation products and acetaldehyde-mediated flavanol-anthocyanin condensation products (anthocyanin linked to flavan-3-ol either directly or by ethyl bridge), were identified in red wines. Structures of principal anthocyanin-derivatives are showed in Figure 4, [30].

2. Liquid Chromatography/Mass Spectrometry Analysis of Nonanthocyanic Polyphenols in Grape and Wine

Liquid Chromatography/Mass Spectrometry (LC/MS) coupled with Multiple Mass Spectrometry (MS/MS and ) 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 [37–40]. In general, these methods require minor sample purification, and MS/MS allows characterization of both aglycone and sugar moiety.

A study of flavonols 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 flavonols, such as kaempferol and laricitrin 3-galactosides, kaempferol-3-glucuronide, and quercetin and syringetin 3-(6-acetyl)glucoside, were identified [5]. Table 1 reports the flavonols identified in Petit Verdot grape skins extract. Extraction was performed by using a methanol/H₂O/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. After sample loading, the cartridge was washed with HCl 0.1 M solution, and the flavonol fraction containing neutral and acidic polyphenols was recovered with methanol. LC analysis was performed by using a reverse-phase C₁₈ 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.

Usually, LC/MS analysis of resveratrol (3,5,4′-trihydroxystilbene) and piceatannol (3,4,3′,5′-tetrahydroxy trans-stilbene) in grape is performed in negative-ion mode. Chromatographic separation can be performed by using a reverse-phase C₁₈ column and elution gradient program with a binary solvent composed of water/0.1% formic acid and methanol (e.g., 33% methanol for 40 min, 33%→100% methanol in 15 min, 100% methanol for 5 min) [42].

Recently, the mechanisms of the fragmentation of trans-resveratrol and piceatannol were studied by and deuterium exchange experiments and performing accurate mass measurements [25]. The product ion spectra of trans-resveratrol [M−H]⁻ ion at 227 and of piceatannol at 243 are reported in Figure 5. Fragmentation patterns of the two compounds are reported in Figure 6. Fragmentations were confirmed by deuterium labeling experiments by dissolving the standard compounds in deuterated methanol: the deprotonated molecules of trans-resveratrol and piceatannol were shifted at 229 and 246, respectively, proving the occurrence of OH hydrogen exchanges.

(a)

(b)

Extraction of proanthocyanidins (PAs) and tannins from seeds and skins can be performed from the powder produced by grinding frozen material using a methanol/H₂O 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/H₂O 60 : 40 (v/v) solution [3]. After removing of organic solvent, the aqueous residue has to be washed with hexane in order to eliminate lipophilic substances. The extract can be then fractionated on a column for gel filtration of natural products Sephadex LH-20 by eluting different fractions with ethanol and acetone aqueous solutions [44, 45]. Alternatively, purification 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/H₂O/TFA 55 : 45 : 0.02 v/v/v and acetone/H₂O 30 : 70 v/v [3]. The two solutions are pooled, concentrated under vacuum, and freeze dried. Further purification of the residue can be performed on divinylbenzene-polystyrene resin: flavan-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/H₂O 60 : 40 v/v. Seed extract, or grape juice, can be also purified by SPE using a reverse-phase C₁₈ cartridge. Extract is suspended in water, and the solution is passed through the cartridge previously conditioned by passage of methanol and water, and after sample loading, the cartridge is rinsed with water, and the fraction containing PAs is eluted with acetone/H₂O/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 h at 4°C. Then, skins are ground in methanol, and the solution is kept in immersion for other two hours at 4°C. Solid parts are extracted again with acetone/H₂O 60 : 40 v/v at 4°C overnight. The two extracts are concentrated under vacuum and fractionated separately on a size-exclusion resin. After removing of sugars and phenolic acids by washing the column with ethanol/H₂O/TFA 55 : 45 : 0.02 (v/v/v) and acetone/H₂O 30 : 70 (v/v), the fraction containing PAs with DP 12–20 is recovered with acetone/H₂O 60 : 40 v/v [3].

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]. The 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 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. Negative-ion 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. The [M−H]⁻ and [M−2H]²⁻ species of PAs with DP3 and DP9 are reported in Table 2. Abundant [M−H]⁻ singly charged ions separated by 288 Da in the ranges 289–2017 and 441–1881, were observed, corresponding to procyanidins (PCs) with DP 1–7 and procyanidin monogallates (PC1Gs) with DP 1–6, respectively. PAs with DP9 show the additional larger [M−H]⁻ ions derived from PC1G with DP7, PC2Gs (procyanidin digallates) with DP6 and DP7, and PC3Gs (trigallates) with DP4 and DP5.

The fragmentation pathways of [M−H]^- and [M−3H]⁻ ions at 577, 575, 729, 727, and 441 are probably due to the cleavage of the interflavanic bond, Retro-Diels-Alder (RDA) fission on the C ring followed by the elimination of water with formation of [M−H−152]⁻ ( 713, 425, 865, and 577) and [M−H−152−H₂O]⁻ ( 695, 407, 847, and 559) ions, and [M−H–126]⁻ ions at 739 and 451 formed by elimination of a phloroglucinol molecule. The ion at 881 corresponds to a epicatechin-gallate dimer or a epicatechin-epicatechin-epigallocatechin trimer (two isobaric compounds). Doubly charged ions showed a series of abundant ions separated by 144 Da from 652.4 to 1948.8, which correspond to the [M−2H]²⁻ ions of PC1Gs with DP 4–13. Increase of the orifice voltage showed two different fragmentation patterns of trimeric species: formation of two ions at 575 and 573 from the ions at 863 (A-type) and formation of ions at 711 due to RDA fragmentation. 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 interflavanic linkage [51].

Operating in positive ionization mode provides also the signals of PCs and PAs protonated molecules: [M+H]⁺ ion of catechin dimers, trimers, and tetramers at 579, 867, and 1155, of their mono- and digalloyl derivatives at 731, 883, 1019, 1171, 1307, and 1459, of trimers and tetramers trigalloyl derivatives at 1323 and 1611, together with those of [M+H]⁺ ions of flavan-3-ols pentamers, hexamers, and heptamers ( 1443, 1731, 2019), their monogalloyl derivatives ( 1595, 1883, 2171), pentamers and hexamers digalloyl derivatives ( 1747 and 2035), and pentamers and hexamers trigalloyl derivatives ( 1899 and 2187) are observed [45].

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. The type-A interflavanic linkages were present in the terminal units, whereas the type-B interflavanic linkages were extension units.

Figure 7 shows the positive-ion MS³ fragmentations of the most intense trimeric procyanidins signals in the spectra at 867 [36]. The 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 3 reports the positive-ion LC/ESI- product ions of flavan-3-ol monomers and PAs dimers, trimers, and oligomers.

A recent study reports the identification of fourteen flavan-3-ol monoglycosides in Merlot grape seeds and wine extracts [53]. Analyses were performed using an ESI-quadrupole time of flight mass spectrometry system operating in negative-ion mode. Compounds were identified on the basis of their exact masses and specific 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. The nonanthocyanic polyphenols identified in four different wine varieties (Tempranillo, Graciano, Cabernet Sauvignon, and Merlot) by performing LC/ESI-MS analysis are reported in Table 4. The 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. The two organic phases were combined, the solvent removed, and the residue was dissolved in a methanol/H₂O solution. LC analyses were performed using a reverse-phase C₁₈ column performing the gradient elution with a binary solvent composed of H₂O/acetic acid 98 : 2 v/v and H₂O/acetonitrile/acetic acid 78 : 20 : 2 v/v/v [54]. The compounds were identified 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 different classes of phenols, such as flavonols, hydroxycinnamoyltartaric acids, stilbene compounds (cis- and trans-resveratrol, piceid), phenolic acids, flavan-3-ols, and dimeric (B1, B3, B4, and B5) and trimeric (C1, T2 and T3) procyanidins.

Two different purification methods by using size-exclusion 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/H₂O/TFA 55 : 45 : 0.005 v/v/v. The fraction containing dimers and trimers was recovered with acetone/H₂O 60 : 40 v/v [55]. Sample preparation by reverse-phase chromatography was instead performed using a C₁₈ cartridge. A volume of dealcoholized wine was loaded onto the cartridge, and PAs were recovered with 10 mL of acetone/H₂O/acetic acid 70 : 29.5 : 0.5 v/v/v solution [46].

The flow diagram in Figure 10 shows a method for fractionation of wine polyphenols by reverse-phase chromatography [43]. The more complex polyphenols recovered in the fractions 8–10 were characterized by LC/ESI-MS and negative-ion mode analysis and reported in Table 5, [57].

3. LC/MS of Grape Anthocyanins

Liquid chromatography mass spectrometry (LC/MS) coupled with multiple mass spectrometry (MS/MS and ) 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, 60–63]. LC analysis of anthocyanins is usually performed by a reverse-phase C₁₈ column by performing gradient elution of compounds using a binary solvent composed of H₂O/formic acid 90 : 10 v/v and methanol/H₂O/formic acid 50 : 40 : 10 v/v/v. The analytes are detected by recording the chromatogram at 520 nm. Due to the lack of standards commercially available, the identification of compounds is usually performed on the basis of their column elution sequence.

A study showed that; nonacidified 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/H₂O/formic acid solution (50 : 48.5 : 1.5 v/v/v) was proposed [65]. Anthocyanins can be then purified by passing through a C₁₈ cartridge: after 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 field 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 are reported in Table 6. A list of other monomer anthocyanins identified in extracts of other grape varieties is reported in Table 7, [66].

In general, is highly effective in differentiation also of isobaric anthocyanins. The fragment ions [M−162]⁺, [M−324]⁺ (formed by two consecutive losses of sugar residue), [M−204]⁺, [M−308]⁺, and [M−470]⁺ (consecutive losses of p-coumaroylglucose and glucose) allow characterization of both monoglucoside and diglucoside compounds. 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 experiments is not possible because they have identical molecular mass and aglycone. For identification of two compounds, dissolution of the extract in deuterated water was performed to observe the different mass shifts 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. Quantification 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-flight (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-C₁₈ (40 nL, 5 m) and analytical column Zorbax 300 SB-C₁₈ (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 flow 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 fingerprint of the sample in less than 5 minutes with practically no solvent consumption. Neither MS/MS was necessary for identification of isobaric compounds, nor deuterium exchange experiments to distinguish between compounds having the same aglycone. The fast separation bypassed also the problem of Pt-3-O-(6-O-acetyl)monoglucoside and Dp-3,5-O-diglucoside quantification of direct-infusion ESI/IT MS due to overlapping of their signals with matrix interferences. The high specificity of LC-Chip/Q-TOF-MS was due to highly reproducible retention times (Chip-chromatography reduces the problems arising from dead volumes, eluent flow 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 identified in the analysis of Clinton grape skins extract are showed in Figure 12.

(a)

(b)

(c)

(d)

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: first elute the more polar diglucosides, followed by monoglucosides, and finally the less polar acylated monoglucosides (acetates and p-coumarates, resp.). The anthocyanins identified 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 after 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 (C₂₇H₃₁O₁₆, MW 611.1612) and Dp-3-O-(6-O-p-coumaroyl)monoglucoside (C₃₀H₂₇O₁₄, MW 611.1401), Pn-3,5-O-diglucoside (C₂₈H₃₃O₁₆, MW 625.1769) and Pt-3-O-(6-O-p-coumaroyl)monoglucoside (C₃₁H₂₉O₁₄, MW 625.1557), or Mv-3,5-O-diglucoside (C₂₉H₃₅O₁₇, MW 655.1874) and Mv-3-O-(6-O-caffeoyl)monoglucoside (C₃₂H₃₁O₁₅, MW 655.1663) because a resolution at least 30.000 is necessary. MS/MS allowed to confirm identification of the compounds with M⁺ at 611 and 625 (on the basis of their different aglycone ions), but only chip separation allowed differentiation of two isobaric compounds with the same aglycone moiety (nominal mass 655 Da).

(a)

(b)

(c)

(d)

(e)

(f)

Figure 14 shows the MS/MS mass spectra of the isobaric anthocyanin pairs Cy-diglucoside and Dp-p-coumaroylmonoglucoside (M⁺ at 611), Pn-diglucoside and Pt-p-coumaroylmonoglucoside (M⁺ at 625), and Mv-diglucoside and Mv-caffeoylmonoglucoside (M⁺ at 655). The spectra (a), (c), and (e) are characteristic of diglucoside compounds and show the ions formed by 162 Da loss corresponding to glucose residue at 449, 463, and 493 for Cy-diglucoside, Pn-diglucoside, and Mv-diglucoside, respectively, and the aglycone ions at 287, 301, and 331. The spectra (b), (d), and (f), characteristic of cinnamoylmonoglucoside derivatives, show the aglycones of Dp-p-coumaroylmonoglucoside, Pt-p-coumaroylmonoglucoside, and Mv-caffeoylmonoglucoside at 303, 317, and 331, respectively.

(a)

(b)

(c)

(d)

(e)

(f)

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. The 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 Table 8. The two methods showed good agreement, except for Pt-3-O-(6-O-acetyl)monoglucoside ( 521) and Dp-3,5-O-diglucoside ( 627) for which the M⁺ signal area resulted overestimated in the ESI/IT-MS analysis. CID of the species at 627 in the ESI/MS spectrum, other than the ions at 465 and 303 corresponding to Dp-3,5-O-diglucoside, produced the ion at 317 as the most intense signal probably corresponding to petunidin or protonated isorhamnetin revealing the overlapping of at least two compounds. CID spectrum of the ion at 521, other than the aglycone base peak signal at 317, showed two intense signals at 522 and 488 with relative abundance of about 40% each. MS³ of the ion at 488 showed that at least two isobaric compounds were present in the extract.

TOF/MS was not suitable to distinguish between isobaric pairs of compounds such as protonated isorhamnetin glucoside and petunidin glucoside (C₂₂H₂₃O₁₂, exact mass 479.1190), and protonated kaempferol monoglucoside and cyanidin monoglucoside (C₂₁H₂₁O₁₁, exact mass 449.1084). Intensities of the signals at 449 and 479 recorded by direct-ESI/IT-MS and LC-Chip/ESI-QTOF-MS are similar (Table 8), inferring that probably protonated flavonols present in the extract were not separated from the chromatographic chip used.

In grape skins, also the oligomeric anthocyanins listed in Table 9 were identified [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, often qualitatively and quantitatively different—and superior—to the V. vinifera varieties [66, 71–77]. Also due to the increasing industrial demand for natural colorants, the knowledge of their composition may be useful for industrial purposes [78, 79].

The 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 identified 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]. The samples were subdivided into two groups on the basis of their anthocyanin profiles 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. This 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 (p-coumaroylmonoglucoside), 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). Three precursor ions for Dp, Pn, and, Cy anthocyanidin fragments and five precursor ions for Mv and Pt were found, leading to identification of the 20 anthocyanins (13 monoglucosides and 7 diglucosides) reported in Table 10.

(a)

(b)

(c)

(d)

(e)

(f)

The precursors identified by precursor-ion analysis of monoglucoside anthocyanins are reported in Table 11. Identification of diglucoside compounds found in precursor-ion analysis of aglycone fragments was confirmed, and the signals of four additional diglucoside compounds were also found [Mv-3-O-(6-O-acetyl)diglucoside at 697, Dp-3,5-diglucoside at 627, Pn-3-O-(6-O-p-coumaroyl)diglucoside at 771, and Cy-3-O-(6-O-p-coumaroyl)diglucoside at 757], leading to identification of total 11 diglucoside anthocyanins. Therefore, the coupling of UPLC and precursor-ion analysis with monitoring of monoglucosides resulted a highly specific method for selective detection of diglucoside compounds.

4. 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 identified in Graciano, Tempranillo, Cabernet Sauvignon, and Primitivo wines. Alternatively, a previous sample purification can be performed on a C₁₈ 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-flavanol 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 identified in wines at different aging stages is reported in Table 13. As may be seen, ethyl-bridge derivatives, pyranoanthocyanins, and pigments formed by anthocyanin-flavanol linkage are included. Some of these compounds are already present in wine in the first aging stage and disappear in the time, and others are formed with long time aging.

Also, oligomeric pigments F-A-A⁺ type (F, flavanol; A, anthocyanin) were identified in wines and characterized by ESI/. Table 14 reports the compounds identified in Tempranillo aged wines. Possible structures proposed for the M⁺ species at 1273 are shown in Figure 19, [60].

(a)

(b)

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). The 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 fingerprint 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. The 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).

(a)

(b)

The anthocyanic profiles 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 C₁₈ 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. The product ions found confirmed the presence of 15 anthocyanin-flavanol 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. The list of compounds identified 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. This aspect reflects negatively in higher values of limit of detection (LOD) and of limit of quantification (LOQ) but positively in higher specificity of the data. DIPS fingerprint 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-flavanol derivatives arise in two ranges of the spectrum, the first 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. The first 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 anthocyanin-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. This 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-flavanol derivatives was proposed to predict the wine color stability.

5. Study of Grape Procyanidins by MALDI-TOF MS

Matrix-assisted laser desorption-ionization and time of flight (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 after 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 [88–92]. LC/MS does not allow separation and identification 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 reflectron mode, flavan-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 reflectron mode using trans-3-indoleacrylic acid (t-IAA) as matrix allowed the identification of a series of compounds with MW 2 Da lower, which correspond to A-type polycatechins. Linear mode analysis provided detection of PAs oligomers sodium adducts up to nonamers [89]. The lower sensitivity of the reflectron mode for the large ions is reasonably due to their collisionally induced decomposition occurring in the flight path [88, 89]. Masses of PAs determined in both reflectron and liner modes are reported in Table 16. On the basis of the galloylated structures, the equation 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 purification 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. The experiments performed by Yang and Chien showed that DHB provides the broadest mass range and the least background noise. The 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. The 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 reflectron 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 identified [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 B-type 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.

6. Polyphenols and Grape Metabolomics

“Metabolomics” is the comprehensive quantitative and qualitative study of all the metabolites within a cell, tissue, or organism. The major limitation of this approach is its current inability to exhaustively describe the whole “metabolome” profile. This is due to its chemical complexity and the dynamic range limitations of most instrumental approaches. Because a single analytical technique does not provide sufficient 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. The coupling of the techniques with GC/MS analysis of volatile compounds, in general, provides a sufficiently wide panorama of the sample metabolomics. The 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 define both similarities and differences among samples [94].

In general, an “untargeted” metabolomics approach provides sensitivity, resolution, and high-throughput capacity and identification of thousands compounds in a single run [95]. Several studies reported that by performing a “targeted” analysis of specific 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 [98–100]. 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 identification of metabolites relies on available specific information on compounds such as their molecular formula and structure [101]. This 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. This database includes the information available in the literature and found in electronic databases on the potential grape metabolites. Partial confirmation of the library hits was achieved by performing the identification 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 identification of a new compound with score sufficiently confident, it is added to the library. As a consequence, a further increase of the library will be possible. Compounds are identified on the basis of accurate mass measurements and their isotope patterns, and the identification is confirmed 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, flavones and flavanones, flavanols and procyanidins, phytoalexins, and phenolic acids. Average 30–60 hits had an identification 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 identified, among them trans-resveratrol, piceatannol, cis- and trans-piceid, several viniferins, and resveratrol dimers, trimers, and tetramers. Moreover, tentative of identification of a number of aroma precursors (mono- and diterpenols glycoside, norisoprenoids), primary metabolites and peptides, is now in progress.

7. Conclusions

Mass spectrometry plays a very important role for research and quality control in the viticulture and oenology field. The soft ionization conditions of LC/MS and the minor sample purification 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. These methods also allow to characterize the high-MW compounds of grape, such as procyanidins, proanthocyanidins, prodelphinidins, and tannins. The important role of LC/MS in the structural study of polyphenols is also confirmed 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 identification 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.

Acknowledgments

The author would like to thank Dr. Laura Molin and Dr. Roberta Seraglia for the MALDI-TOF data of PAs and for the spectrum of Cabernet Sauvignon grape seeds extract kindly provided.

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