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

Authors: Riccardo Flamini
Journal: ISRN Spectroscopy
 (2013)

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## 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.


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## Body

## 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 Figure1). 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).Figure 1
B-type and A-type flavan-3-ol dimers and trimers present in the grape seeds.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) The principal monomer anthocyanins of grape: the glucose residue can be linked to an acetyl, coumaroyl, or caffeoyl group. (b) The principal flavonols of grape.
(a)
(b)Stilbene compounds are the principal phytoalexins of grape: they includecis- 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 oftrans-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.trans-resveratrol (1) and principal viniferins of the vine: Z-ε-viniferin (2), E-ε-viniferin (3), E-ω-viniferin (4), Z-ω-viniferin (5), pallidol (6), E-ampelopsin D (7), E-quadrangularin A (8), α-viniferin (9), E-cis-miyabenol C (10), Z-miyabenol C (11), E-miyabenol C (12), isohopeaphenol (13), ampelopsin H (14), vaticanol C isomer (15), and hopeaphenol (16).Anthocyanins are the compounds responsible for the red color of grapes and wines. Principal anthocyanins ofVitis 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 betweenV. 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 Figure4, [30].Figure 4
Above: compounds formed in wines during aging: (a) structure with direct linkage between anthocyanin and flavan-3-ol proposed by Somers [18] and (b) anthocyanin-flavan-3-ol structure by ethyl bridge proposed by Timberlake and Bridle [19]. Below: structures of C-4 substituted anthocyanins identified in aged red wines formed by reaction with pyruvic acid, vinylphenol, vinylcatechol, vinylguaiacol, and vinyl(epi)catechin [20–23].


## 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 andMSn) 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 differentV. 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.Table 1
LC retention times (RTs), maximum adsorption UV-Vis wavelengths, and mass spectra data of flavonols identified inPetit Verdot grape skins.
Flavonol
HPLC RT (min)
λ
max (nm)
[M − H]+ and product ion (m/z)
Myricetin-3-glucuronide
13.9
257(sh), 261, 301(sh), 353
495, 319
Myricetin-3-glucoside
14.5
257(sh), 262, 298(sh), 355
481, 319
Quercetin-3-glucuronide
18.0
257, 265(sh), 299(sh), 354
479, 303
Quercetin-3-glucoside
18.8
256, 265(sh), 295(sh), 354
465, 303
Laricitrin-3-glucoside
19.9
256, 265(sh), 301(sh), 357
495, 333
Kaempferol-3-glucoside
22.6
265, 298(sh), 320(sh), 348
449, 287
Isorhamnetin-3-glucoside
24.3
255, 265(sh), 297(sh), 354
479, 317
Syringetin-3-glucoside
24.9
255, 265(sh), 300(sh), 357
509, 347
Laricitrin-3-galactoside
19.4
256, 265(sh), 302(sh), 357
495, 333
Kaempferol-3-galactoside
21.1
266, 292(sh), 320(sh), 348
449, 287
Kaempferol-3-glucuronide
21.9
265, 290(sh), 320(sh), 348
463, 287
Quercetin-3-(6-acetyl)glucoside
22.9
257, 265(sh), 295(sh), 352
517, 303
Syringetin-3-(6-acetyl)glucoside
30.4
255, 265(sh), 298(sh), 358
517, 347Usually, LC/MS analysis of resveratrol (3,5,4′-trihydroxystilbene) and piceatannol (3,4,3′,5′-tetrahydroxytrans-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 oftrans-resveratrol and piceatannol were studied by MSn and deuterium exchange experiments and performing accurate mass measurements [25]. The product ion spectra of trans-resveratrol [M−H]− ion at m/z 227 and of piceatannol at m/z 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 m/z 229 and m/z 246, respectively, proving the occurrence of OH hydrogen exchanges.Product negative-ion spectrum of direct infusion ESI-generated [M−H]− species of trans-resveratrol (a) and piceatannol (b) [25].
(a)
(b)Figure 6
Collisionally induced fragmentation patterns of [M−H]− ions of trans-resveratrol at m/z 227 (compound 1) considering that the deprotonation reaction occurred on the phenol moiety (Scheme 1a), and of [M−H]− ions of trans-resveratrol (R = H, compound 1) and piceatannol at m/z 243 (R = OH, compound 2) considering that the deprotonation reaction occurred on the resorcinol moiety (Scheme 1b),  [25].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 m/z 291, protonated catechin/epicatechin gallate at m/z 443, protonated catechin/epicatechin dimer at m/z 579, and protonated catechin/epicatechin gallate dimers and trimers at m/z 731, m/z 883 and m/z 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 m/z 289–2017 and m/z 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.Table 2
[M − H]− and [M − 2H]2− ions of proanthocyanidins with degree of polymerization (DP) 3 and 9 (na: not assigned) [50].
Procyanidins (PCs)
Monogallates (PC1Gs)
Digallates (PC2Gs)
Trigallates (PC3Gs)
Tetragallates (PC4Gs)
Pentagallates (PC5Gs)
DP
Sdp3
Sdp9
Sdp3
Sdp9
Sdp3
Sdp9
Sdp3
Sdp9
Sdp3
Sdp9
Sdp3
Sdp9
[M − H]−
[M − H]−
[M − 2H]2−
[M − H]−
[M − H]−
[M − 2H]2−
[M − H]−
[M − H]−
[M − 2H]2−
[M − H]−
[M − H]−
[M − 2H]2−
[M − H]−
[M − H]−
[M − 2H]2−
[M − H]−
[M − H]−
[M − 2H]2−
1
289.2
289.4
441.2
441.4
2
577.4
577.4
729.4
729.4
881.4
881.4
3
865.4
865.4
1017.6
1017.4
1169.8
1169.8
4
1153.4
1153.4
1305.8
1305.8
652.4
1457.6
1457.4
1609.2
804.4
5
1441.8
1441.6
720.4
1593.4
1593.4
796.4
1745.4
1745.2
872.6
1897.8
948.8
1024.8
1100.2
6
1729.8
1729.2
Na
1881.8
1881.6
940.8
2034.0
Na
1092.6
Na
1243.8
7
2017.2
2017.2
1009.2
2169.8
1084.2
2322.2
1161.0
1236.8
1312.6
1388.4
8
Na
1228.6
Na
1380.6
Na
1532.2
9
1296.6
1373.2
1448.6
1524.6
1601.2
1676.4
10
Na
1516.8
Na
1669.2
Na
1821.8
11
1584.0
1661.0
1737.0
1813.2
1889.2
12
1805.4
Na
13
1948.8
2025.2The fragmentation pathways of [M−H]- and [M−3H]− ions at m/z 577, 575, 729, 727, and m/z 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]− (m/z 713, 425, 865, and 577) and [M−H−152−H2O]− (m/z 695, 407, 847, and 559) ions, and [M−H–126]− ions at m/z 739 and 451 formed by elimination of a phloroglucinol molecule. The ion at m/z 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 m/z 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 m/z 575 and 573 from the ions at m/z 863 (A-type) and formation of ions at m/z 711 due to RDA fragmentation. Neutral loss of 152 Da (corresponding to 3,4-dihydroxy-α-hydroxystyrene) induces formation of two fragments at m/z 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 m/z 579, 867, and 1155, of their mono- and digalloyl derivatives at m/z 731, 883, 1019, 1171, 1307, and 1459, of trimers and tetramers trigalloyl derivatives at m/z 1323 and 1611, together with those of [M+H]+ ions of flavan-3-ols pentamers, hexamers, and heptamers (m/z 1443, 1731, 2019), their monogalloyl derivatives (m/z 1595, 1883, 2171), pentamers and hexamers digalloyl derivatives (m/z 1747 and 2035), and pentamers and hexamers trigalloyl derivatives (m/z 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.Figure7 shows the positive-ion MS3 fragmentations of the most intense trimeric procyanidins signals in the spectra at m/z 867 [36]. The schemes of positive fragmentation patterns for monomer catechin and of a B-type trimer at m/z 883 are reported in Figures 8 and 9, respectively [41]. Table 3 reports the positive-ion LC/ESI-MSn product ions of flavan-3-ol monomers and PAs dimers, trimers, and oligomers.Table 3
LC/ESI-MSn product ions of flavan-3-ol monomers, dimers, and oligomers recorded in positive-ion mode. Neutral losses are shown in parentheses. C, EC, and GC: catechin, epicatechin, and gallocatechin, respectively. (E)C and (E)GC: (epi)catechin and (epi)gallocatechin, respectively. (E) indicates either catechin/epicatechin or gallocatechin/epigallocatechin.
Compound
[M + H]+
HRFC
RDAC
BFFC
BFFC/H2O
HRFC/H2O
C
291
165 (126)
139 (152)
169 (122), 123
151 (140), 123
147 (144)
EC
291
165 (126)
139 (152)
169 (122), 123
151 (140), 123
147 (144)
GC
307
181 (126)
139 (168)
169 (138), 139
151 (156), 139
163 (144)
[M + H]+
Q
M
CD
↑
Q
M
CD
↑
HRFC
RDAC
RDAF
HRFC
RDAF/HRFC
RDAF
H2O/BFFF
(E)GC-(4,8)-(E)C
595
305
469 (126)
427 (168)
443 (152)
317 (152)
329 (140)
317 (126)
(E)GC-(4,6)-(E)C
595
469 (126)
427 (168)
443 (152)
317 (152)
329 (140)
317 (126)
(E)C-(4,8)-(E)C
579
291
453 (126)
427 (152)
427 (152)
301 (152)
313 (140)
301 (126)
(E)C-(4,8)-(E)C
579
289
453 (126)
427 (152)
427 (152)
301 (152)
313 (140)
301 (126)
(E)C-(4,8)-(E)C
579
291
453 (126)
427 (152)
427 (152)
301 (152)
313 (140)
301 (126)
(E)C-(4,8)-(E)C
579
289
453 (126)
427 (152)
427 (152)
301 (152)
313 (140)
301 (126)
[M + H]+
Q
M
CD
↑/QMCD↓
Q
M
FG
↑/QMFG↓
Q
M
CD
↓/RDAI/HRFF
HRFC/QMFG↑
HRFC/RDAF
HRFC/H2O/BFFF
(E)C-(E)GC-(E)C
883
—/595
593/—
595 (288)/443(152)/317 (126)
757 (126)/467 (290)
757 (126)/589 (168)
757 (126)/601 (156)
(E)GC-(E)GC-(E)C
899
—/595
609/291
595 (304)/443(152)/317 (126)
773 (126)/483 (290)
773 (126)/605 (168)
773 (126)/617 (156)
(E)C-(E)C-(E)C
867
—/579
577/291
579 (288)/427(152)/301 (126)
741 (126)/451 (290)
741 (126)/589 (152)
741 (126)/601 (140)
(E)C-(E)C-(E)C
867
—/579
577/—
579 (288)/427(152)/301 (126)
741 (126)/451 (290)
741 (126)/589 (152)
741 (126)/601 (140)
(E)C-(E)C-(E)C
867
—/579
577/—
579 (288)/427(152)/301 (126)
741 (126)/451 (290)
741 (126)/589 (152)
741 (126)/601 (140)
[M + H]+
Diagnostic ions
(E)C-(E)C-(E)C-(E)C
1155
867, 865, 579
(E)C-(E)C-(E)GC-(E)C
1171
883, 881, 595
(E)C-(E)GC-(E)GC-(E)C
1187
899, 897, 595
(E)GC-(E)GC-(E)GC-(E)C
1203
913, 899, 595
(E)C-(E)C-(E)C-(E)C-(E)C
1443
1155, 1153, 867, 579
(E)C-(E)GC-(E)C-(E)C-(E)C
1459
1171, 1169, 867, 579
(E)C-(E)GC-(E)GC-(E)C-(E)C
1475
1187, 1185, 883, 579
(E)C-(E)C-(E)C-(E)C-(E)C-(E)C
1731
1441, 1143, 1155, 867, 579
RDA: retro-Diels-Alder fission; HRF: heterocyclic ring fission; BFF: benzofuran forming fission; QM: quinone methide fission;QMCD↓: the ion derived from the QM fission of ring-C/ring-D linkage bond by the loss of upper unit; QMCD↑: the ion derived from the QM fission of ring-C/ring-D linkage bond by the loss of lower unit; QMFG↓: the ion derived from the QM fission of ring-F/ring-G linkage bond by the loss of upper unit; QMFG↑: the ion derived from the QM fission of ring-F/ring-G linkage bond by the loss of lower unit [41].Figure 7
Fragmentation patterns ofm/z 867 trimeric procyanidins studied in ESI-MS positive-ion mode [36].Figure 8
Fragmentation pathways of monomer catechin in positive-ion mode: retro-Diels-Alder fission (RDA), heterocyclic ring fission (HRF), benzofuran forming fission (BFF), and loss of water molecule [41].Figure 9
Positive-ion mode fragmentation pathways of B-type trimerm/z  883: retro-Diels-Alder fission (RDA), heterocyclic ring fission (HRF), benzofuran forming fission (BFF), quinone methide fission (QM), and loss of water molecule. QMCD↓: the ion derived from the QM fission of ring-C/ring-D linkage bond by the loss of upper unit and QMFG↑: the ion derived from the QM fission of ring-F/ring-G linkage bond by the loss of lower unit [41].A recent study reports the identification of fourteen flavan-3-ol monoglycosides inMerlotgrape 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.Table 4
Nonanthocyanin phenolic compounds identified by LC/ESI-MS in wines fromVitis vinifera varieties Tempranillo, Graciano, 				Cabernet Sauvignon, and Merlot. Principal mass fragments and maximum UV-Vis absorption wavelengths are reported. (s): shoulder  [54].
RT (min)
Compound
(m/z)
λ
max (nm)
[M − H]−
Fragments
7.6
Gallic acid
169
125
272
14.0
Protocatechuic acid
153
109
294, 260
15.7
Dihydroxyphenylethanol
153
280
16.7
trans-caffeoyltartaric acid
311
179
330, 298(s)
19.2
2,3-Dihydroxy-1-(4-hydroxy-3-methoxyphenyl)-propan-1-one
211
310, 280
19.5
Methyl gallate
183
169, 125
272
20.5
Tyrosol
137
275
22.3
(epi)gallocatechin-(epi)catechin dimer
593
425
276
22.7
Procyanidin B3
577
425, 289
280
23.0
Procyanidin B1
577
425, 289
280
24.2
trans-coumaroyl-tartaric acid
295
163
313
27.3
(+)-catechin
289
279
28.0
Procyanidin T2
865
713, 577, 289
280
28.3
trans-feruryltartaric acid
325
193
329, 301(s)
28.7
Hexose ester of vanillic acid
329
167
nd
29.0
Procyanidin T3
865
713, 577, 289
280
29.7
Vanillic acid
167
289, 262
30.5
Procyanidin B4
577
425, 289
283
31.3
trans-caffeic acid
179
135
323
33.0
Hexose ester oftrans-p-coumaric acid (1)
325
163, 145
311
33.6
Procyanidin B2
577
425, 289
280
34.6
Syringic acid
197
277
37.0
Hexose ester oftrans-p-coumaric acid (2)
325
163, 145
312
38.7
(−)-epicatechin
289
279
40.2
Trimeric procyanidin
865
713, 577, 289
280
41.3
Ethyl gallate
197
169, 125
273
42.2
Procyanidin C1
865
713, 577, 289
282
43.2
trans-p-coumaric acid
163
119
309
43.5
Trimeric procyanidin
865
713, 577, 289
280
43.8
Procyanidin dimer gallate
729
577
278
44.0
Procyanidin B5
577
425, 289
280
48.5
Myricetin-3-O-glucuronide
493
317
349, 300(s), 261
50.1
Myricetin-3-O-glucoside
479
317
349, 300(s), 261
51.3
Epicatechin-3-O-gallate
441
289, 169
277
53.2
trans-resveratrol-3-O-glucoside
389
227
306(s), 319
55.2
Ellagic acid
301
368
57.2
Quercetin-3-O-galactoside
463
301
354, 300(s), 256
57.7
Quercetin-3-O-glucuronide
477
301
354, 300(s), 256
58.2
Astilbin
449
303
288
58.7
Quercetin-3-O-glucoside
463
301
354, 300(s), 256
60.6
Tryptophol
160
279
62.4
Kaempferol-3-O-glucoside
447
285
346, 300(s), 265
67.6
Myricetin
317
371, 300(s), 254
68.9
cis-resveratrol-3-O-glucoside
389
227
285
71.9
trans-resveratrol
227
306, 319(s)
92.7
Quercetin
301
369, 300(s), 255
95.7
cis-resveratrol
227
284Two 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 Figure10 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 MSn negative-ion mode analysis and reported in Table 5, [57].Table 5
Polyphenolic oligomers characterized by LC/ESI-MSn in wine (fractions 8 and 9 in Figure 10) and seeds extracts.
Phenolic compounds
(m/z)
[M − H]−
[M + H]+
[M]+
[
M
-
2H
]
2
-
MS2
MS3
MS4
PC2 A
577
425, 407, 451, 289, 559
407
PC2 B
577
439, 425, 393, 269, 533, 559
421, 377, 247
PC1PD1
593
441, 289
315, 289, 153
PC2-ethyl
605
315, 289, 453
229, 271, 205, 247, 191, 253, 297
PC2-G A
729
407, 559, 577, 451, 425, 441, 603, 289
285, 283, 297, 389, 256, 281, 243
PC2-G B
729
407, 559,577, 451, 425, 441, 603, 289
407, 451, 289, 559, 425
PC3 A
865
847,577, 289
451, 289
PC3 B
865
695, 577, 575 407, 543, 739, 713, 449, 587, 451, 407
543, 525, 677, 243, 405, 433, 451, 525, 363, 289, 501, 407, 557, 568, 391, 651
PC2-2G (A-type)
879
709, 727, 547, 439
557, 541, 571, 539
PC2-2G (B-type)
881
729, 559, 407, 711, 577
451, 407, 577, 603, 441, 559, 289
PC2PD1
883
847, 731,579
427, 289
PC3-ethyl A
893
603, 577, 451, 433, 407, 425, 315, 741
451, 433, 289, 287, 266
PC3-ethyl B
893
603,577, 451, 433, 407, 425, 315, 741
407, 451, 425, 541
PC3-G A
1017
729, 865, 847, 677, 891, 695, 577, 575, 557, 727, 739, 559, 771, 407
577, 559, 407, 425, 451, 603, 289, 467, 541
PC3-G B
1017
729,865, 847, 677, 891, 695, 577, 575, 557, 727, 739, 559, 771, 407
695, 407, 577, 575, 727, 425, 525, 739
PC3-G C
1017
729, 865,847, 677, 891, 695, 577, 575, 557, 727, 739, 559, 771, 407
677, 695, 525, 603, 451, 405
PC4 A
1153
863,577
451, 289
PC4 B
1153
865, 983, 575, 695, 739, 1027, 863, 577, 1001, 449
425, 803, 449, 451, 533, 559
PC4 C
1153
865,983, 575, 695, 739, 1027, 863, 577, 1001, 449
739, 577, 449, 610, 675, 447, 425, 289
PC4 D
1153
865, 983,575, 695, 739, 1027, 863, 577, 1001, 449
423, 449, 413, 405, 287, 289
PC6-4G or PC6PD1-2G
1168
1017, 729
575
PC7PD1-6G or PC7PD2-4G or PC7PD3-2G
1616
1084, 1641
PC4
1153
1155
863,577
451, 289
PC3-2G A
1169
1017, 881, 729, 847, 999, 891, 577
729, 847, 321, 621, 495, 603, 451, 289, 577
PC3-2G B
1169
1017,881, 729, 847, 999, 891, 577
729, 407, 559, 711, 577, 289
PC5
1443
1425, 1291, 1155,865, 577
847, 577
PC2-Mv-3-glu
1069
781
619
601, 331
Mv-3-glc-PC3
1357
1067,779
617
437
PCn: n units of (epi)catechin; nG: number of galloyl units. Bold/underlined ions were subjected to next fragmentation stage [43, 57].Figure 10
Fractionation of polyphenols in red wine (PA, proanthocyanidins) [43].


## 3. LC/MS of Grape Anthocyanins

Liquid chromatography mass spectrometry (LC/MS) coupled with multiple mass spectrometry (MS/MS andMSn) 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.Figure 11
Profile of anthocyanins inClintonskin extract recorded by direct-injection positive-ion ESI/MS.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 Figure11 are reported in Table 6. A list of other monomer anthocyanins identified in extracts of other grape varieties is reported in Table 7, [66].Table 6
Fragments from M+ ions of anthocyanins in Clinton grape skin extract produced by direct-ESI and MSn [66].
Anthocyanin
m/z
M+
[M − C6H10O5]+
Malvidin-3-O-monoglucoside
493
331
Petunidin-3-O-monoglucoside
479
317
Delphinidin-3-O-monoglucoside
465
303
Peonidin-3-O-monoglucoside
463
301
Cyanidin-3-O-monoglucoside
449
287
M+
[M − C8H12O6]+
Malvidin-3-O-(6-O-acetyl)monoglucoside
535
331
Petunidin-3-O-(6-O-acetyl)monoglucoside
521
317
Delphinidin-3-O-(6-O-acetyl)monoglucoside
507
303
Peonidin-3-O-(6-O-acetyl)monoglucoside
505
301
Cyanidin-3-O-6-O-(acetyl)monoglucoside
491
287
M+
[M − C15H16O7]+
Malvidin-3-O-(6-O-p-coumaroyl)monoglucoside
639
331
Petunidin-3-O-(6-O-p-coumaroyl)monoglucoside
625
317
Delphinidin-3-O-(6-O-p-coumaroyl)monoglucoside
611
303
Peonidin-3-O-(6-O-p-coumaroyl)monoglucoside
609
301
Cyanidin-3-O-(6-O-p-coumaroyl)monoglucoside
595
287
M+
[M − C6H10O5]+
[M − 2(C6H10O5)]+
Malvidin-3,5-O-diglucoside
655
493
331
Petunidin-3,5-O-diglucoside
641
479
317
Delphinidin-3,5-O-diglucoside
627
465
303
Peonidin-3,5-O-diglucoside
625
463
301
Cyanidin-3,5-O-diglucoside
611
449
287
M+
[M − C6H10O5]+
[M − C15H16O7]+
[M − C15H16O7–C6H10O5]+
Malvidin-3-(6-O-p-coumaroyl),5-O-diglucoside
801
639
493
331
Petunidin-3-(6-O-p-coumaroyl),5-O-diglucoside
787
625
479
317
Delphinidin-3-(6-O-p-coumaroyl),5-O-diglucoside
773
611
465
303
Cyanidin-3-(6-O-p-coumaroyl),5-O-diglucoside
757
595
449
287
M+
[M − C15H16O8]+
Malvidin-3-O-(6-O-caffeoyl)monoglucoside
655
331Table 7
Monomer anthocyanins identified by LC/ESI-MS/MS analysis in skin extract and juice of different grape cultivars [24, 69].
Anthocyanin
m/z [M]+
Cultivar
Cy-3-O-pentoside
419
Casavecchia
Pg-3-O-glucoside
433
Concord, Salvador, Rubired
Cy-3-O-(6-O-acetyl)pentoside
461
Casavecchia
Cy-3-O-(6-O-p-coumaryl)pentoside
565
Casavecchia
Dp-3-O-glucoside-pyuvic acid
533
Isabelle
Dp-3-O-(6-O-p-coumaryl)glucoside-pyruvic acid
679
Isabelle
Pn-3-O-glucoside-acetaldehyde
487
Isabelle, Pallagrello
Mv-3-O-glucoside-acetaldehyde
517
Isabelle
Pt-3-O-(6-O-p-caffeoyl)-5-O-diglucoside
803
Isabelle, Casavecchia
Dp-3-O-(6-O-acetyl)-5-O-diglucoside
669
Isabelle
Dp-3-O-(6-O-feruloyl)-5-O-diglucoside
803
Isabelle, Casavecchia
Pn-3-O-(6-O-p-coumaryl)-5-O-diglucoside
771
Concord, Salvador, Isabelle, Casavecchia
Pg: pelargonidin; Dp: delphinidin; Cy: cyanidin; Pt: petunidin; Pn: peonidin; Mv: malvidin.In general,MSn 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 MSn 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 m/z 493) and of Mv-3,5-O-diglucoside for diglucosides (M+ at m/z 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 inClinton 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 μm) and analytical column Zorbax 300 SB-C18 (75 μm × 43 mm, 5 μm). 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 μL 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 m/z 611, 625, and 655 identified in the analysis of Clinton grape skins extract are showed in Figure 12.LC-Chip/ESI-QTOF-MS analysis ofClintongrape skins extract: (a) total ion chromatogram (TIC); (b) extracted ion chromatogram (EIC) of the signal at m/z 611; (c) EIC of the signal at m/z 625; (d) EIC of the signal at m/z 655 [49].
(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 andp-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.Table 8
Anthocyanins identified inClinton 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 m/z 611 and m/z 493.
Direct-ESI/IT-MS
LC-Chip/ESI-Q-TOF-MS
Compound
MW
M+ height %
RT (min)
M+ area %
Relative to Mv monoglucoside
Relative to Mv monoglucoside
Cyanidin-3-O-monoglucoside
449
9
3.794
11
Peonidin-3-O-monoglucoside
463
33
3.869
52
Delphinidin-3-O-monoglucoside
465
35
3.744
38
Petudinin-3-O-monoglucoside
479
44
3.794
39
Cyanidin-3-O-(6-O-acetyl)monoglucoside
491
2
4.095
tr
Malvidin-3-O-monoglucoside
493
100
3.869
100
Peonidin-3-O-(6-O-acetyl)monoglucoside
505
10
4.070
6
Delphinidin-3-O-(6-O-acetyl)monoglucoside
507
6
3.944
2
Petudinin-3-O-(6-O-acetyl)monoglucoside
521
23
3.995
5
Malvidin-3-O-(6-O-acetyl)monoglucoside
535
36
4.045
30
Cyanidin-3-(6-O-p-coumaryl)monoglucoside
595
3
4.220
5
Peonidin-3-(6-O-p-coumaryl)monoglucoside
609
6
4.295
18
Cyanidin-3,5-O-diglucoside
611
19 (m/z 611)
3.644
3
Delphinidin-3-(6-O-p-coumaryl)monoglucoside
611
4.170
15
Peonidin-3,5-O-diglucoside
625
14 (m/z 625)
3.719
3
Petudinin-3-(6-O-p-coumaryl)monoglucoside
625
4.220
3
Delphinidin-3,5-O-diglucoside
627
35
3.594
2
Malvidin-3-(6-O-p-coumaryl)monoglucoside
639
39
4.270
48
Petudinin-3,5-O-diglucoside
641
16
3.669
14
Malvidin-3,5-O-diglucoside
655
24 (m/z 655)
3.719
26
Malvidin-3-(6-O-caffeoyl)monoglucoside
655
4.170
1
Cyanidin-3-(6-O-p-coumaryl),5-O-diglucoside
757
2
3.995
1
Delphinidin-3-(6-O-p-coumaryl),5-O-diglucoside
773
2
3.944
2
Petudinin-3-(6-O-p-coumaryl),5-O-diglucoside
787
2
3.995
3
Malvidin-3-(6-O-p-coumaryl),5-O-diglucoside
801
2
4.045
3
tr: trace [49].To distinguish between isobaric pairs of anthocyanins, also accurate mass measurements were performed. Figure13 shows the Q-TOF signals recorded after LC-Chip separation of isobaric pairs at nominal mass m/z 611 (Cy-diglucoside and Dp-p-coumaroylmonoglucoside), m/z 625 (Pn-diglucoside and Pt-p-coumaroylmonoglucoside), and m/z 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 m/z 611 and m/z 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).M+ signals recorded by LC-Chip-QTOF of isobaric pairs of anthocyanins at m/z 611 (Cy-diglucoside (a) and Dp-p-coumaroylmonoglucoside (b)), at m/z 625 (Pn-diglucoside (c) and Pt-p-coumaroylmonoglucoside (d)), and at m/z 655 (Mv-diglucoside (e) and Mv-caffeoylmonoglucoside (f)) [49].
(a)
(b)
(c)
(d)
(e)
(f)Figure14 shows the MS/MS mass spectra of the isobaric anthocyanin pairs Cy-diglucoside and Dp-p-coumaroylmonoglucoside (M+ at m/z 611), Pn-diglucoside and Pt-p-coumaroylmonoglucoside (M+ at m/z 625), and Mv-diglucoside and Mv-caffeoylmonoglucoside (M+ at m/z 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 m/z 449, 463, and 493 for Cy-diglucoside, Pn-diglucoside, and Mv-diglucoside, respectively, and the aglycone ions at m/z 287, 301, and m/z 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 m/z 303, 317, and m/z 331, respectively.MS/MS spectra of isobaric anthocyanin pairs recorded by LC-Chip/Q-TOF: Cy-diglucoside (a) and Dp-p-coumaroylmonoglucoside (b) with M+ at m/z 611, Pn-diglucoside (c) and Pt-p-coumaroylmonoglucoside (d) with M+ at m/z  625, and Mv-diglucoside (e) and Mv-caffeoylmonoglucoside (f) with M+ at m/z 655 [49].
(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 Figure15.Figure 15
Calibration curve calculated by spiking theClintongrape skins extract with Mv-3,5-O-diglucoside standard at three different concentration. Mean data of the M+ signal area at m/z 655 extrapolated from EIC of two replicated analyses and variability bars are shown [49].Quantitative percentage data of LC-Chip/ESI-QTOF-MS and ESI/IT-MS analysis ofClinton extract are reported in Table 8. The two methods showed good agreement, except for Pt-3-O-(6-O-acetyl)monoglucoside (m/z 521) and Dp-3,5-O-diglucoside (m/z 627) for which the M+ signal area resulted overestimated in the ESI/IT-MS analysis. CID of the species at m/z 627 in the ESI/MS spectrum, other than the ions at m/z 465 and 303 corresponding to Dp-3,5-O-diglucoside, produced the ion at m/z 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 m/z 521, other than the aglycone base peak signal at m/z 317, showed two intense signals at m/z 522 and 488 with relative abundance of about 40% each. MS3 of the ion at m/z 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 m/z 449 and m/z 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 Table9 were identified [56, 59]. Figure 16 shows the fragmentation scheme proposed for two anthocyanin dimers.Table 9
Oligomeric anthocyanins identified inShiraz grape skins.
m/z
Assignment
617F
MvCy
631F
MvPn
633F
MvDp
647F
MvPt
661F
MvMv
779F
MvCy + G
793F
MvPn + G
795F
MvDp + G
809F
MvPt + G
823F
MvMv + G
941M
MvCy + 2G
955M
MvPn + 2G
957M
MvDp + 2G
971M
MvPt + 2G
985M
MvMv + 2G
1087M
MvCy + G·pCG
1101M
MvPn + G·pCG
1103M
MvDp + G·pCG
1117M
MvPt + G·pCG
1131M
MvMv + G·pCG
1271F
MvMvCy + 2G
1285F
MvMvPn + 2G
1287F
MvMvDp + 2G
1301F
MvMvPt + 2G
1315F
MvMvMv + 2G
1417F
MvMvCy + G·pCG
1431F
MvMvPn + G·pCG
1433F
MvMvDp + G·pCG
1433M
MvMvCy + 3G
1447F
MvMvPt + G·pCG
1447M
MvMvPn + 3G
1449M
MvMvDp + 3G
1461F
MvMvMv + G·pCG
1463M
MvMvPt + 3G
1477M
MvMvMv + 3G
1579M
MvMvCy + 2G·pCG
1593M
MvMvPn + 2G·pCG
1595M
MvMvDp + 2G·pCG
1609M
MvMvPt + 2G·pCG
1623M
MvMvMv + 2G·pCG
F: fragment ion; M: molecular ion. Dp: delphinidin; Cy: cyanidin; Pt: petunidin; Pn: peonidin; Mv: malvidin; G: glucose, pCG:p-coumaroylglucoside [56].Figure 16
Fragmentation scheme proposed for two anthocyanin dimers. HCR: heterocyclic ring fission and RDA: retro-Diels-Alder fission [56].In a recent work anthocyanins of 21 hybrid red grape varieties produced by crossing of differentVitis vinifera,riparia, labrusca,Lincecumii, andrupestris 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 m/z 331 (corresponding to Mv), the signals of precursors at m/z 493 (the monoglucoside derivative), m/z 535 (acetyl monoglucoside), m/z 639 (p-coumaroylmonoglucoside), m/z 655 (diglucoside), and m/z 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].Figure17 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.Table 10
Precursors of the aglycone fragment ions identified by UPLC/MS precursor-ion analysis in the study of 21 hybrid grape varieties.
Varieties
Precursor ions ofm/z 331 (Mv)
Precursor ions ofm/z 317 (Pt)
Precursor ions ofm/z 303 (Dp)
Precursor ions ofm/z 301 (Pn)
Precursor ions ofm/z 287 (Cy)
M+
Rt
Compound
M+
Rt
Compound
M+
Rt
Compound
M+
Rt
Compound
M+
Rt
Compound
Bacò 1
655
1.65
Digluc.
479
1.82
Monogluc.
465
1.57
Monogluc.
463
2.04
Monogluc.
449
1.79
Monogluc.
493
2.06
Monogluc.
625
2.79
(p-coum.)-Monogluc.
801
2.60
(p-coum.)-digluc.
639
2.96
(p-coum.)-monogluc.
Bacò 30-12
655
1.65
Digluc.
641
1.41
Digluc.
465
1.57
Monogluc.
625
1.63
Digluc.
611
1.43
Digluc.
493
2.06
Monogluc.
479
1.82
Monogluc.
773
2.24
(p-coum.)-digluc.
463
2.04
Monogluc.
449
1.79
Monogluc.
801
2.63
(p-coum.)-digluc.
625
2.79
(p-coum.)-monogluc.
611
2.62
(p-coum.)-monogluc.
609
2.58
(p-coum.)-monogluc.
595
2.74
(p-coum.)-monogluc.
639
2.96
(p-coum.)-monogluc.
Bertille Seivè 1808
493
2.06
Monogluc.
479
1.82
Monogluc.
465
1.57
Monogluc.
449
1.79
Monogluc.
611
2.62
(p-coum.)-monogluc.
Bertille Seivè 4825
655
1.65
Digluc.
479
1.82
Monogluc.
465
1.57
Monogluc.
625
1.63
Digluc.
611
1.43
Digluc.
493
2.06
Monogluc.
625
2.79
(p-coum.)-monogluc.
611
2.62
(p-coum.)-monogluc.
463
2.04
Monogluc.
449
1.79
Monogluc.
609
2.58
(p-coum.)-monogluc.
595
2.74
(p-coum.)-monogluc.
Burdin 4077
493
2.06
Monogluc.
479
1.82
Monogluc.
465
1.57
Monogluc.
463
2.04
Monogluc.
449
1.79
Monogluc.
639
2.96
(p-coum.)-monogluc.
611
2.62
(p-coum.)-monogluc.
Clinton
655
1.65
Digluc.
479
1.82
Monogluc.
465
1.57
Monogluc.
625
1.63
Digluc.
449
1.79
Monogluc.
493
2.06
Monogluc.
625
2.79
(p-coum.)-monogluc.
611
2.62
(p-coum.)-monogluc.
463
2.04
Monogluc.
595
2.74
(p-coum.)-monogluc.
639
2.96
(p-coum.)-monogluc.
Couderc 25
655
1.61
Digluc.
479
1.82
Monogluc.
465
1.57
Monogluc.
625
1.63
Digluc.
611
1.43
Digluc.
493
2.06
Monogluc.
625
2.79
(p-coum.)-monogluc.
611
2.62
(p-coum.)-monogluc.
463
2.04
Monogluc.
449
1.79
Monogluc.
639
2.96
(p-coum.)-monogluc.
595
2.74
(p-coum.)-monogluc.
Galibert 238-35
493
2.06
Monogluc.
479
1.82
Monogluc.
465
1.57
Monogluc.
463
2.04
Monogluc.
449
1.79
Monogluc.
535
2.56
(Acetyl)-monogluc.
521
2.35
(Acetyl)-monogluc.
611
2.62
(p-coum.)-monogluc.
639
2.96
(p-coum.)-monogluc.
625
2.79
(p-coum.)-monogluc.
Seibel 10878
493
2.06
Monogluc.
479
1.82
Monogluc.
465
1.57
Monogluc.
463
2.04
Monogluc.
449
1.79
Monogluc.
639
2.96
(p-coum.)-monogluc.
521
2.36
(Acetyl)-monogluc.
625
2.79
(p-coum.)-monogluc.
Seibel 8357
493
2.06
Monogluc.
479
1.82
Monogluc.
465
1.57
Monogluc.
625
1.63
Digluc.
449
1.79
Monogluc.
801
2.60
(p-coum.)-digluc.
521
2.30
(Acetyl)-monogluc.
611
2.62
(p-coum.)-monogluc.
463
2.04
Monogluc.
639
2.96
(p-coum.)-monogluc.
787
2.45
(p-coum.)-digluc.
505
2.54
(Acetyl)-monogluc.
625
2.79
(p-coum.)-monogluc.
Seibel 8745
655
1.65
Digluc.
479
1.82
Monogluc.
465
1.57
Monogluc.
625
1.63
Digluc.
449
1.79
Monogluc.
493
2.06
Monogluc.
625
2.79
(p-coum.)-monogluc.
611
2.62
(p-coum.)-monogluc.
639
2.96
(p-coum.)-monogluc.
Seyve Villard 12-347
655
1.65
Digluc.
479
1.82
Monogluc.
465
1.57
Monogluc.
625
1.63
Digluc.
449
1.79
Monogluc.
493
2.06
Monogluc.
611
2.62
(p-coum.)-monogluc.
639
2.96
(p-coum.)-monogluc.
Seyve Villard 12-390
493
2.06
Monogluc.
479
1.82
Monogluc.
465
1.57
Monogluc.
449
1.79
Monogluc.
535
2.56
(Acetyl)-monogluc.
521
2.35
(Acetyl)-monogluc.
611
2.62
(p-coum.)-monogluc.
595
2.74
(p-coum.)-monogluc.
639
2.96
(p-coum.)-monogluc.
625
2.79
(p-coum.)-monogluc.
Seyve Villard 23-369
655
1.65
Digluc.
479
1.82
Monogluc.
465
2.62
Monogluc.
493
2.06
Monogluc.
Seyve Villard 23-399
655
1.65
Digluc.
641
1.41
Digluc.
465
1.57
Monogluc.
611
1.40
Digluc.
493
2.06
Monogluc.
479
1.82
Monogluc.
611
2.62
(p-coum.)-monogluc.
449
1.79
Monogluc.
Seyve Villard 23-512
655
1.65
Digluc.
625
2.79
(p-coum.)-monogluc.
595
2.74
(p-coum.)-monogluc.
493
2.06
Monogluc.
801
2.60
(p-coum.)-digluc.
Seyve Villard 29-522
655
1.65
Digluc.
479
1.82
Monogluc.
465
1.57
Monogluc.
493
2.06
Monogluc.
625
2.79
(p-coum.)-monogluc.
611
2.62
(p-coum.)-monogluc.
801
2.60
(p-coum.)-digluc.
639
2.96
(p-coum.)-monogluc.
Terzi 100-31
655
1.65
Digluc.
479
1.82
Monogluc.
465
1.57
Monogluc.
625
1.63
Digluc.
449
1.79
Monogluc.
493
2.06
Monogluc.
463
2.04
Monogluc.
639
2.96
(p-coum.)-monogluc.
Terzi 108-6
493
2.06
Monogluc.
479
1.82
Monogluc.
465
1.57
Monogluc.
625
1.63
Digluc.
449
1.79
Monogluc.
639
2.96
(p-coum.)-monogluc.
611
2.62
(p-coum.)-monogluc.
463
2.04
Monogluc.
Terzi 97-46
655
1.65
Digluc.
479
1.82
Monogluc.
465
1.57
Monogluc.
463
2.04
Monogluc.
449
1.79
Monogluc.
493
2.06
Monogluc.
787
2.45
(p-coum.)-digluc.
611
2.62
(p-coum.)-monogluc.
535
2.60
(Acetyl)-monogluc.
625
2.79
(p-coum.)-monogluc.
639
2.96
(p-coum.)-monogluc.
Unknown
493
2.06
Monogluc.
479
1.82
Monogluc.
465
1.57
Monogluc.
463
2.04
Monogluc.
449
1.79
Monogluc.
639
2.96
(p-coum.)-monogluc.
625
2.79
(p-coum.)-monogluc.
611
2.62
(p-coum.)-monogluc.
609
2.95
(p-coum.)-monogluc.
595
2.74
(p-coum.)-monogluc.
Rt: column retention time (min) [58].UPLC/MS total ion chromatogram (TIC) (a) and precursor-ion chromatograms from anthocyanin analysis ofBacò 30-12 grape skins extract. (b) Precursor-ion chromatogram of fragment at m/z 331 (corresponding to Mv); (c) precursor-ion chromatogram of fragment at m/z 317 (Pt); (d) precursor-ion chromatogram of fragment at m/z 303 (Dp); (e) precursor-ion chromatogram of fragment at m/z 301 (Pn); (f) precursor-ion chromatogram of fragment at m/z 287 (Cy) [58].
(a)
(b)
(c)
(d)
(e)
(f)The precursors identified by precursor-ion analysis of monoglucoside anthocyanins are reported in Table11. 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 m/z 697, Dp-3,5-diglucoside at m/z 627, Pn-3-O-(6-O-p-coumaroyl)diglucoside at m/z 771, and Cy-3-O-(6-O-p-coumaroyl)diglucoside at m/z 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.Table 11
Precursors of the monoglucoside anthocyanin fragment ions identified by UPLC/MS precursor-ion analysis in 21 hybrid grape varieties.
Varieties
Precursor ions ofm/z 493 (Mv)
Precursor ions ofm/z 479 (Pt)
Precursor ions ofm/z 465 (Dp)
Precursor ions ofm/z 463 (Pn)
Precursor ions ofm/z 449 (Cy)
M+
Rt
Compound
M+
Rt
Compound
M+
Rt
Compound
M+
Rt
Compound
M+
Rt
Compound
Bacò 1
655
1.65
Digluc.
641
1.40
Digluc.
773
2.32
(p-coum.)-digluc.
625
1.65
Digluc.
611
1.44
Digluc.
801
2.61
(p-coum.)-digluc.
787
2.45
(p-coum.)-digluc.
770
2.65
(p-coum.)-digluc.
Bacò 30-12
655
1.65
Digluc.
641
1.40
Digluc.
773
2.32
(p-coum.)-digluc.
625
1.65
Digluc.
611
1.44
Digluc.
801
2.61
(p-coum.)-digluc.
Bertille Seivè 1808
655
1.65
Digluc.
641
1.40
Digluc.
627
1.27
Digluc.
625
1.65
Digluc.
611
1.44
Digluc.
Bertille Seivè 4825
655
1.65
Digluc.
641
1.40
Digluc.
625
1.65
Digluc.
611
1.44
Digluc.
801
2.61
(p-coum.)-digluc.
770
2.65
(p-coum.)-digluc.
757
2.41
(p-coum.)-digluc.
Burdin 4077
655
1.65
Digluc.
641
1.47
Digluc.
801
2.61
(p-coum.)-digluc.
787
2.45
(p-coum.)-digluc.
Clinton
655
1.65
Digluc.
641
1.47
Digluc.
627
1.27
Digluc.
625
1.65
Digluc.
611
1.44
Digluc.
801
2.61
(p-coum.)-digluc.
787
2.45
(p-coum.)-digluc.
Couderc 25
655
1.65
Digluc.
641
1.40
Digluc.
627
1.27
Digluc.
625
1.65
Digluc.
611
1.44
Digluc.
801
2.60
(p-coum.)-digluc.
Galibert 238-35
655
1.65
Digluc.
801
2.61
(p-coum.)-digluc.
Seibel 10878
Seibel 8357
655
1.65
Digluc.
641
1.47
Digluc.
773
2.32
(p-coum.)-digluc.
625
1.65
Digluc.
611
1.44
Digluc.
801
2.61
(p-coum.)-digluc.
787
2.45
(p-coum.)-digluc.
770
2.65
(p-coum.)-digluc.
757
2.45
(p-coum.)-digluc.
Seibel 8745
655
1.65
Digluc.
641
1.40
Digluc.
627
1.27
Digluc.
625
1.65
Digluc.
611
1.44
Digluc.
801
2.61
(p-coum.)-digluc.
773
2.32
(p-coum.)-digluc.
770
2.65
(p-coum.)-digluc.
Seyve Villard 12.347
655
1.65
Digluc.
641
1.47
Digluc.
627
1.27
Digluc.
625
1.65
Digluc.
611
1.44
Digluc.
801
2.61
(p-coum.)-digluc.
787
2.45
(p-coum.)-digluc.
Seyve Villard 12.390
655
1.65
Digluc.
641
1.40
Digluc.
625
1.65
Digluc.
611
1.44
Digluc.
801
2.61
(p-coum.)-digluc.
770
2.65
(p-coum.)-digluc.
Seyve Villard 23.369
655
1.65
Digluc.
641
1.40
Digluc.
625
1.65
Digluc.
611
1.44
Digluc.
Seyve Villard 23.399
655
1.65
Digluc.
641
1.47
Digluc.
627
1.27
Digluc.
625
1.65
Digluc.
611
1.44
Digluc.
801
2.61
(p-coum.)-digluc.
787
2.45
(p-coum.)-digluc.
Seyve Villard 23.512
655
1.65
Digluc.
641
1.47
Digluc.
625
1.65
Digluc.
611
1.44
Digluc.
697
2.06
(Acetyl)-digluc.
787
2.45
(p-coum.)-digluc.
770
2.65
(p-coum.)-digluc.
801
2.61
(p-coum.)-digluc.
Seyve Villard 29.522
655
1.65
Digluc.
641
1.47
Digluc.
627
1.27
Digluc.
625
1.65
Digluc.
611
1.44
Digluc.
801
2.61
(p-coum.)-digluc.
Terzi 100.31
655
1.65
Digluc.
641
1.40
Digluc.
625
1.65
Digluc.
611
1.44
Digluc.
801
2.61
(p-coum.)-digluc.
770
2.65
(p-coum.)-digluc.
Terzi 108.6
655
1.65
Digluc.
641
1.40
Digluc.
627
1.27
Digluc.
625
1.65
Digluc.
611
1.44
Digluc.
801
2.61
(p-coum.)-digluc.
787
2.45
(p-coum.)-digluc.
773
2.32
(p-coum.)-digluc.
757
2.41
(p-coum.)-digluc.
Terzi 97.46
655
1.65
Digluc.
641
1.47
Digluc.
627
1.27
Digluc.
625
1.66
Digluc.
611
1.44
Digluc.
801
2.61
(p-coum.)-digluc.
787
2.45
(p-coum.)-digluc.
773
2.32
(p-coum.)-digluc.
Unknown
655
1.65
Digluc.
641
1.47
Digluc.
625
1.65
Digluc.
611
1.44
Digluc.
801
2.61
(p-coum.)-digluc.
787
2.45
(p-coum.)-digluc.
770
2.65
(p-coum.)-digluc.
Rt: column retention time (min) [58].


## 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. Table12 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].Table 12
Anthocyanin derivatives identified by positive-ion LC/ESI-MS analysis inGraciano, Tempranillo, Cabernet Sauvignon, and Primitivo wines [36, 82].
Compound
m/z
[M]+
Main MS2 fragments
Main MS3 fragments
(epi)catechin-peonidin-3-O-glucoside
751
589
571, 437, 463
(epi)catechin-malvidin-3-O-glucoside
781
619
601, 493, 467
Delphinidin-3-O-glucoside
465
303
/
Di(epi)catechinMalvidin-3-O-glucoside
1069
907, 781, 619
/
Cyanidin-3-O-glucoside
449
287
/
(epi)catechin-malvidin-3-O-glucoside
781
619
601, 493, 467
Di(epi)catechin-malvidin-3-O-glucoside
1069
907, 781, 619
/
Petunidin-3-O-glucoside
479
317
/
Peonidin-3-O-glucoside
463
301
/
Malvidin-3-O-glucoside
493
331
/
Malvidin-3-O-glucoside pyruvate
561
399
/
Malvidin-3-O-glucoside acetaldehyde
517
355
/
Malvidin-3-O-glucoside-8-ethyl-(epi)catechin
809
647, 519, 357
/
Malvidin-3-O-glucoside-8-ethyl-(epi)catechin
809
647, 519, 357
/
Malvidin-3-O-glucoside-4-vinyl-di(epi)catechin
1093
931, 803
641
Malvidin-3-O-glucoside-8-ethyl-(epi)catechin
809
647, 519, 357
/
Malvidin-3-O-glucoside-4-vinyl-di(epi)catechin
1093
931, 803
641
Malvidin-3-O-glucoside-8-ethyl-(epi)catechin
809
647, 519, 357
/
Malvidin-3-(6-O-p-coumaroylglucoside)-(epi)catechin
927
619
601, 493, 467
Malvidin-3-(6-O-p-coumaroylglucoside)  pyruvate
707
399
/
Peonidin-3-(6-O-acetylglucoside)
505
301
/
Malvidin-3-(6-O-acetylglucoside)
535
331
/
Delphinidin-3-(6-O-p-coumaroylglucoside)
611
303
/
Malvidin-3-(6-O-acetylglucoside)-4-vinyl-(epi)catechin
847
643
491
Malvidin-3-(6-O-caffeoylglucoside)
655
331
/
Cyanidin-3-(6-O-p-coumaroylglucoside)
595
287
/
Petunidin-3-(6-O-p-coumaroylglucoside)
625
317
/
Malvidin-3-(6-O-p-coumaroylglucoside)
639
331
/
Malvidin-3-(6-O-p-coumaroylglucoside)-4-vinyl-di(epi)catechin
1239
931, 641
641
Malvidin-3-O-glucoside-4-vinyl-(epi)catechin
805
643
491
Peonidin-3-(6-O-p-coumaroylglucoside)-8-ethyl-(epi)catechin
925
635, 617, 327
/
Malvidin-3-(6-O-p-coumaroylglucoside)-8-ethyl-(epi)catechin
955
665, 357
/
Peonidin-3-(6-O-p-coumaroylglucoside)
609
301
/
Malvidin-3-(6-O-p-coumaroylglucoside)
639
331
/
Malvidin-3-O-glucoside-4-vinylcatechol
625
463
/
Malvidin-3-O-glucoside-4-vinyl-(epi)catechin
805
643
491
Malvidin-3-(6-O-p-coumaroylglucoside)-4-vinyl-(epi)catechin
951
643
491
Malvidin-3-(6-O-p-coumaroylglucoside)-4-vinyl-(epi)catechin
951
643
491
Malvidin-3-O-glucoside-4-vinylphenol
609
447
/
Malvidin-3-O-glucoside-4-vinylguaiacol
639
477
/
Malvidin-3-(6-O-p-coumaroylglucoside)-8-ethyl-(epi)catechin
955
665, 357
/
Malvidin-3-O-glucoside-(epi)catechin
781
619
Delphinidin-3-O-glucoside
465
303
Peonidin-3-O-glucoside pyruvate
531
369
Malvidin-3-O-glucoside pyruvate
561
399
Malvidin-3-(6-O-acetylglucoside)  pyruvate
603
399
Malvidin-3-O-glucoside-8-ethyl-(epi)catechin
809
647, 519, 357
Malvidin-3-(6-O-p-coumaroylglucoside)  pyruvate
707
399
Peonidin-3-(6-O-caffeoylglucoside)
625
301
Malvidin-3-(6-O-caffeoylglucoside)
655
331
Malvidin-3-O-glucoside-4-vinyl-catechin
805
643
Malvidin-3-(6-O-p-coumaroylglucoside)-8-ethyl-(epi)catechin
955
665, 357
Malvidin-3-(6-O-acetylglucoside)-4-vinyl-catechin
847
643
Malvidin-3-O-glucoside-4-vinylcatechol
625
463
Malvidin-3-(6-O-acetylglucoside)-4-vinyl-epicatechin
847
643
Malvidin-3-O-glucoside-4-vinyl-epicatechin
805
643
Malvidin-3-O-glucoside-4-vinylphenol
609
447
Malvidin-3-O-glucoside-4-vinylguaiacol
639
477
Malvidin-3-(6-O-acetylglucoside)-4-vinylphenol
651
447
Malvidin-3-(6-O-p-coumaroylglucoside)-4-vinylphenol
755
447Several 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 m/z 781) and for Mv-3-glu-(epi)catechin with A-type linkage (M+ at m/z 783).Figure 18
MS/MS fragmentation scheme proposed for (epi)catechin-Mv-3-glu B-type linkage (M+ at m/z 781) and for Mv-3-glu-(epi)catechin A-type linkage (M+ at m/z 783) [67, 68]. RDA: retro-Diels-Alder fission.A list of anthocyanin derivatives identified in wines at different aging stages is reported in Table13. 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.Table 13
Anthocyanin derivatives identified in 4–23 months aged wines.
RT (min)
Compound
[M]+ (m/z)
M
S
n fragment ions
λ
max (nm)
Aged wine (months)
(m/z)
4
8
13
16
23
21.7
Dp-3-glc
465
303
277, 342, 524
*
*
*
*
*
26.1
Cy-3-glc
449
287
279, 516
*
*
*
*
*
28.1
Pt-3-glc
479
317
277, 347, 525
*
*
*
*
*
34.1
Pn-3-glc
463
301
280, 517
*
*
*
*
*
35.5
Mv-3-glc
493
331
277, 348, 527
*
*
*
*
*
38.3
Dp-3-acetylglc
507
303
276, 346, 527
*
*
*
*
*
41.0
Cy-3-acetylglc
491
287
280, 523
*
*
*
*
*
41.6
Pt-3-acetylglc
521
317
270, 529
*
*
*
*
*
43.6
Pn-3-acetylglc
505
301
280, 522
*
*
*
*
*
44.3
Mv-3-acetylglc
535
331
278, 350, 530
*
*
*
*
*
43.1
Dp-3-p-coumglccis
611
303
280, 301, 534
*
*
*
*
*
44.3
Dp-3-p-coumglctrans
611
303
282, 313, 531
*
*
*
*
*
45.1
Cy-3-p-coumglc cis
595
287
280, 301, 533
*
*
*
*
*
46.3
Cy-3-p-coumglctrans
595
287
284, 314, 524
*
*
*
*
*
45.3
Pt-3-p-coumglccis
625
317
281, 301, 536
*
*
*
*
*
46.6
Pt-3-p-coumglctrans
625
317
282, 313, 532
*
*
*
*
*
47.5
Pn-3-p-coumglccis
609
301
283, 300, 535
*
*
*
*
*
48.6
Pn-3-p-coumglctrans
609
301
283, 313, 526
*
*
*
*
*
47.5
Mv-3-p-coumglccis
639
331
280, 301, 535
*
*
*
*
*
48.7
Mv-3-p-coumglctrans
639
331
282, 313, 532
*
*
*
*
*
41.1
Dp-3-cafglctrans
627
303
283, 331, 532
*
n.d.
n.d.
n.d.
n.d.
43.6
Pt-3-cafglctrans
641
317
283, 328, 531
*
*
n.d.
n.d.
n.d.
45.6
Pn-3-cafglctrans
625
301
283, 328, 525
*
n.d.
n.d.
n.d.
n.d.
44.8
Mv-3-cafglccis
655
331
*
*
n.d.
n.d.
n.d.
45.7
Mv-3-cafglctrans
655
331
282, 328, 534
*
*
*
*
*
16.7
Dp-3,7-diglc
627
303
279, 523
*
n.d.
n.d.
n.d.
n.d.
20.4
Pt-3,5-diglc
641
317
275, 521
*
n.d.
n.d.
n.d.
n.d.
24.6
Pt-3,7-diglc
641
317
275, 349, 522
*
*
*
*
*
28.7
Pn-3,7-diglc
625
301
*
*
n.d.
n.d.
n.d.
23.5
Mv-3,5-diglc
655
331
275, 524
*
*
n.d.
n.d.
n.d.
30.8
Mv-3,7-diglc
655
331
278, 350, 526
*
*
*
*
*
34.7
Dp-3-glc + L(+)lactic acid
537
*
*
*
*
*
37.5
Pt-3-glc + D(−)lactic acid
551
*
*
*
*
n.d.
39.2
Pt-3-glc + L(+)lactic acid
551
317
278, 526
*
*
*
*
*
40.5
Pn-3-glc + D(−)lactic acid
535
*
*
n.d.
n.d.
n.d.
41.7
Pn-3-glc + L(+)lactic acid
535
301
281, 525
*
*
*
*
*
40.8
Mv-3-glc + D(−)lactic acid
565
331
278, 350, 530
*
*
*
*
*
42.1
Mv-3-glc + L(+)lactic acid
565
331
278, 348, 531
*
*
*
*
*
5.7
Dp-3-glc-GC
769
607, 439
531
*
*
*
*
*
7.1
Cy-3-glc-GC
753
591, 453
282, 524
*
*
*
*
*
7.2
Pt-3-glc-GC
783
621, 453
279, 532
*
*
*
*
*
10.8
Pn-3-glc-GC
767
605, 437
*
*
*
*
*
10.6
Mv-3-glc-GC
797
635, 467
281, 531
*
*
*
*
*
22.3
Mv-3-glc-EGC
797
635, 467
*
*
*
*
*
24.4
Mv-3-acetylglc-GC
839
n.d.
*
*
*
n.d.
30.9
Dp-3-p-coumglc-GC
915
607, 439
n.d.
*
*
*
n.d.
35.1
Cy-3-p-coumglc-GC
899
n.d.
*
*
n.d.
n.d.
35.4
Pt-3-p-coumglc-GC
929
n.d.
*
*
*
n.d.
38.5
Pn-3-p-coumglc-GC
913
605, 437
n.d.
*
*
*
*
38.3
Mv-3-p-coumglc-GC
943
635, 467
n.d.
*
*
*
*
41.1
Mv-3-p-coumglc-EGC
943
n.d.
*
*
*
*
10.8
Dp-3-glc-C
753
591, 439
282, 534
*
*
*
*
*
14.8
Dp-3-glc-EC
753
591, 439
*
*
*
*
*
14.9
Cy-3-glc-C
737
575, 423
286, 526
*
*
*
*
*
18,0
Cy-3-glc-EC
737
575, 423
*
*
*
*
*
16.2
Pt-3-glc-C
767
605, 453
279, 532
*
*
*
*
*
21.6
Pt-3-glc-EC
767
605, 453
*
*
*
*
*
20.3
Pn-3-glc-C
751
589, 437
283, 524
*
*
*
*
*
24.3
Pn-3-glc-EC
751
589, 437
*
*
*
*
*
21.0
Mv-3-glc-C
781
619, 467
280, 532
*
*
*
*
*
29.9
Mv-3-glc-EC
781
619, 467
279, 533
*
*
*
*
*
35.9
Mv-3-acetylglc-C
823
619, 467
n.d.
*
*
*
*
39.0
Dp-3-p-coumglc-C
899
591, 439
n.d.
*
*
*
*
40.5
Dp-3-p-coumglc-EC
899
n.d.
*
n.d.
n.d.
n.d.
39.5
Cy-3-p-coumglc-C
883
n.d.
*
*
*
n.d.
41.4
Cy-3-p-coumglc-EC
883
n.d.
*
n.d.
n.d.
n.d.
41.0
Pt-3-p-coumglc-C
913
605, 453
n.d.
*
*
*
*
42.6
Pt-3-p-coumglc-EC
913
n.d.
*
n.d.
n.d.
n.d.
41.8
Pn-3-p-coumglc-C
897
589, 437
n.d.
*
*
*
*
43.8
Pn-3-p-coumglc-EC
897
n.d.
*
n.d.
n.d.
n.d.
43.4
Mv-3-p-coumglc-C
927
619, 467
290, 538
*
*
*
*
*
46.0
Mv-3-p-coumglc-EC
927
n.d.
*
n.d.
n.d.
n.d.
35.8
Dp-3-glc-ethyl-C
781
329, 329
*
n.d.
n.d.
*
n.d.
36.7
Dp-3-glc-ethyl-EC
781
*
n.d.
n.d.
*
n.d.
39.5
Cy-3-glc-ethyl-C
765
*
n.d.
n.d.
n.d.
n.d.
39.6
Pt-3-glc-ethyl-C
795
343
*
n.d.
n.d.
*
*
42.0
Pn-3-glc-ethyl-C
779
327
*
*
n.d.
*
*
43.1
Pn-3-glc-ethyl-EC
779
*
n.d.
n.d.
*
*
41.1
Mv-3-glc-ethyl-C
809
357
*
*
*
*
*
42.2
Mv-3-glc-ethyl-C
809
357
282, 539
*
*
*
*
*
43.1
Mv-3-glc-ethyl-C
809
357
276, 537
*
*
*
*
*
45.8
Mv-3-acetylglc-ethyl-C
851
357
*
*
n.d.
n.d.
n.d.
47.4
Mv-3-p-coumglc-ethyl-C
955
357
*
*
*
*
n.d.
34.7
Dp-3-glc-ethyl-GC
797
329
*
n.d.
n.d.
n.d.
*
38.6
Cy-3-glc-ethyl-GC
781
*
n.d.
n.d.
n.d.
n.d.
38.7
Pt-3-glc-ethyl-GC
811
343
*
n.d.
n.d.
*
*
40.9
Pn-3-glc-ethyl-GC
795
*
n.d.
n.d.
n.d.
n.d.
40.6
Mv-3-glc-ethyl-GC
825
357
*
n.d.
*
*
*
41.1
Mv-3-glc-ethyl-GC
825
357
539
*
*
*
*
*
41.8
Mv-3-glc-ethyl-GC
825
357
*
n.d.
*
*
*
45.0
Mv-3-acetylglc-ethyl-GC
867
*
n.d.
n.d.
n.d.
n.d.
21.0
A-type vitisin of Dp-3-glc
533
371
297, 368, 507
n.d.
*
*
*
*
27.0
A-type vitisin of Cy-3-glc
517
n.d.
n.d.
n.d.
*
n.d.
28.7
A-type vitisin of Pt-3-glc
547
385
299, 371, 508
n.d.
*
*
*
*
35.0
A-type vitisin of Pn-3-glc
531
369
503
n.d.
n.d.
n.d.
*
*
36.0
Vitisin A
561
399
299, 372, 510
n.d.
*
*
*
*
40.7
A-type vitisin of Pt-3-p-coumglc
693
n.d.
n.d.
*
*
n.d.
43.8
A-type vitisin of Pn-3-p-coumglc
677
369
284, 508
n.d.
*
*
*
*
44.1
A-type vitisin of Mv-3-p-coumglc
707
399
271, 514
n.d.
*
*
*
*
24.4
B-type vitisin of Dp-3-glc
489
327
n.d.
*
*
*
*
33.5
B-type vitisin of Pt-3-glc
503
341
492
n.d.
*
*
*
*
38.5
B-type vitisin of Pn-3-glc
487
325
*
*
*
*
n.d.
39.5
Vitisin B
517
355
294, 358, 490
*
*
*
*
*
41.4
B-type vitisin of Pn-3-acetylglc
529
325
*
*
n.d.
n.d.
n.d.
42.4
B-type vitisin of Mv-3-acetylglc
559
355
298, 361, 494
*
*
*
*
n.d.
41.1
Acetone derivative of Pn-3-glc
501
339
475
n.d.
*
*
*
n.d.
42.1
Acetone derivative of Mv-3-glc
531
369
480
n.d.
*
*
*
*
45.5
Dp-3-glc 4-vinylphenol adduct
581
419
264, 412, 503
*
*
*
*
*
47.5
Cy-3-glc 4-vinylphenol adduct
565
n.d.
*
*
*
*
48.3
Pt-3-glc 4-vinylphenol adduct
595
433
264, 413, 502
*
*
*
*
*
50.5
Pn-3-glc 4-vinylphenol adduct
579
417
278, 406, 500
*
*
*
*
*
51.0
Mv-3-glc 4-vinylphenol adduct
609
447
263, 412, 504
*
*
*
*
*
53.2
Mv-3-acetylglc 4-vinylphenol adduct
651
447
298, 416, 505
*
*
*
*
*
49.9
Dp-3-p-coumglc 4-vinylphenol adduct
727
419
n.d.
n.d.
n.d.
*
*
52.4
Pt-3-p-coumglc 4-vinylphenol adduct
741
433
314, 504
n.d.
n.d.
n.d.
*
*
54.6
Pn-3-p-coumglc 4-vinylphenol adduct
725
417
314, 501
n.d.
n.d.
n.d.
*
*
55.2
Mv-3-p-coumglc 4-vinylphenol adduct
755
447
264, 313, 416, 505
n.d.
*
*
*
*
43.5
Dp-3-glc 4-vinylcatechol adduct
597
435
509
*
*
*
*
*
46.5
Pt-3-glc 4-vinylcatechol adduct
611
449
510
*
*
*
*
*
48.6
Pn-3-glc 4-vinylcatechol adduct
595
433
506
*
*
*
*
*
49.2
Mv-3-glc 4-vinylcatechol adduct
625
463
510
*
*
*
*
*
50.9
Mv-3-acetylglc 4-vinylcatechol adduct
667
463
513
n.d.
n.d.
*
*
*
47.6
Dp-3-p-coumglc 4-vinylcatechol adduct
743
435
*
*
*
*
*
50.5
Pt-3-p-coumglc 4-vinylcatechol adduct
757
449
n.d.
*
*
*
*
53.2
Mv-3-p-coumglc 4-vinylcatechol adduct
771
463
312, 511
*
*
*
*
*
52.0
Mv-3-glc 4-vinylguaiacol adduct
639
477
511
*
*
*
*
*
54.0
Mv-3-acetylglc 4-vinylguaiacol adduct
681
477
514
n.d.
*
*
*
*
55.7
Mv-3-p-coumglc 4-vinylguaiacol adduct
785
477
514
n.d.
*
*
*
*
49.0
Mv-3-glc 4-vinylepicatechin adduct
805
*
*
*
*
*
Dp: delphinidin; Cy: cyanidin; Pt: petunidin; Pn: peonidin; Mv: malvidin; glc: glucose; p-coumglc: p-coumaroylglucoside; cafglc: caffeoylglucoside; acetylglc: acetylglucoside; catechin; GC: gallocatechin; EC: epicatechin; ECG: epigallocatechin; *detected; n.d.: not detected [30].Also, oligomeric pigments F-A-A+ type (F, flavanol; A, anthocyanin) were identified in wines and characterized by ESI/MSn. Table 14 reports the compounds identified in Tempranillo aged wines. Possible structures proposed for the M+ species at m/z 1273 are shown in Figure 19, [60].Table 14
Molecular and fragment ions of the flavanol-anthocyanin-anthocyanin (F-A-A+) trimers identified in Tempranillo aged wines. The fragment ions are reported in order of abundance.
m/z
Proposed identity
[M]+
Fragment ions
MS2
MS3
MS4
(E)C-DpG-MvG
1245
1083 [M+ − 162]
921 [MS2+ − 162]
795 [M+ − 450]
903 [MS2+ − 180]
921 [M+ − 324]
657 [MS2+ − 426]
903 [M+ − 342]
633 [MS2+ − 450]
837 [MS2+ − 246]
(E)C-CyG-MvG
1229
1067 [M+ − 162]
905 [MS2+ − 162]
904 [M+ − 324]
917 [MS2+ − 150]
(E)C-PtG-MvG
1259
1097 [M+ − 162]
935 [MS2+ − 162]
629 [MS3+ − 306]
971 [M+ − 288]
917 [MS2+ − 180]
935 [M+ − 324]
899 [MS2+ − 198]
809 [M+ − 450]
671 [MS2+ − 426]
1079 [M+ − 180]
747 [MS2+ − 350]
971 [MS2+ − 126]
(E)C-PnG-MvG
1243
1081 [M+ − 162]
919 [MS2+ − 162]
1063 [M+ − 180]
(E)C-MvG-MvG
1273
1111 [M+ − 162]
949 [MS2+ − 162]
949 [M+ − 324]
931 [MS2+ − 180]
661 [M+ − 612]
823 [MS2+ − 288]
931 [M+ − 342]
685 [MS2+ − 426]
823 [M+ − 450]
661 [MS2+ − 450]
737 [MS2+ − 374]
913 [MS2+ − 198]
535 [MS2+ − 576]
331 [MS2+ − 780]
(E)GC-DpG-MvG
1261
1099 [M+ − 162]
937 [MS2+ − 162]
(E)GC-PtG -MvG
1275
1113 [M+ − 162]
951 [MS2+ − 162]
647 [MS2+ − 466]
(E)GC-MvG-MvG
1289
1127 [M+ − 162]
965 [MS2+ − 162]
965 [M+ − 324]
929 [MS2+ − 198]
661 [M+ − 628]
947 [MS2+ − 180]
823 [M+ − 466]
823 [MS2+ − 304]
865 [MS2+ − 262]
661 [MS2+ − 466]
535 [MS2+ − 592]
839 [MS2+ − 288]
467 [MS2+ − 660]
331 [MS2+− 796]
MS2+: major fragment ion obtained in MS2 analysis; MS3+: major fragment ion obtained in MS3 analysis; Dp: delphinidin; Cy: cyanidin; Pt: petunidin; Pn: peonidin; Mv: malvidin; G: glucose; (E)C: (epi)catechin; (E)GC: (epi)gallocatechin [60].Possible structures proposed for the M+ species at m/z 1273: (a) (E)C-MvG-Mv+G trimer in flavene-flavylium form (B-type linkage) and (b) (E)C-MvG-Mv+G trimer in flavan-flavylium form (A-type linkage) [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 m/z 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 m/z 331, 479, 493, 535, and 609 increase (marked peaks in the spectrum in the following).Mass spectra of aCabernet Sauvignonwine recorded by application of spray capillary voltage 3 kV (a) and 0.5 kV (b). *: main anthocyanin signals [70].
(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 m/z 531, 533, and 561, resp.) and malvin-vinyl(epi)catechin (m/z 805) (in particular in aged wines), and other several anthocyanin derivatives. The list of compounds identified is reported in Table 15.Table 15
Compounds identified by DIPS and MS/MS in 12 wines at different aging stages.
Compound
m/z
Loss Da
M+
MS/MS ions
Cy-glu
449
287
−162
Pn-glu
463
301
−162
Dp-glu
465
303
−162
Pt-glu
479
317
−162
Vitisin B-Pn-glu
487
325
−162
Vitisin B-Dp-glu
489
327
−162
Mv-glu
493
331
−162
Vitisin B-Pt-glu
503
341
−162
Pn-glu-Ac
505
301
−204
Dp-glu-Ac
507
303
−204
Pt-glu-Ac
521
317
−204
Vitisin A-Pn-glu
531
369
−162
Vitisin A-Dp-glu
533
371
−162
Mv-glu-Ac
535
331
−204
Vitisin A-Pt-glu
547
385
−162
Vitisin B-Mv-glu-Ac
559
355
−204
Vitisin A
561
399
−162
4-Vinylphenol-Pn-glu
579
417
−162
4-Vinylphenol-Dp-glu
581
419
−162
Cy-glu-pcoum
595
287
−308
4-Vinylphenol-Pt-glu
595
433
−162
4-Vinylcatechol-Dp-glu
597
435, 303
−162
4-Vinylphenol-Mv-glu
609
447
−162
Pn-glu-pcoum
609
301
−308
Dp-glu-pcoum
611
303
−308
4-Vinylcatechol-Mv-glu (Pinotin A)
625
463
−162
Mv-glu-pcoum
639
331
−308
4-Vinylguaiacol-Mv-glu
639
477
−162
4-Vinylphenol-Mv-glu-Ac
651
447
−204
Mv-diglu (IS)
655
493, 331
−162
4-Vinylcatechol-Mv-glu-Ac
667
463
−204
Vitisin A-Pt-glu-pcoum
693
385
−308
Vitisin A-Mv-glu-pcoum
707
399
−308
4-Vinylphenol-Pt-glu-pcoum
741
433
−308
Cat-Pn-glu
751
589
−162
Cat-Dp-glu
753
591
−162
4-Vinylphenol-Mv-glu-pcoum
755
447
−308
4-Vinylcatechol-Pt-glu-pcoum
757
449
−308
Cat-Et-Cy-glu
765
603
−162
GCat-Pn-glu or Cat-Pt-glu
767
605
−162
4-Vinylcatechol-Mv-glu-pcoum
771
463, 331
−308
Cat-Mv-glu
781
619, 467, 331
−162
GCat-Pt-glu
783
621
−162
Cat-Dp-glu-Ac
795
591, 507
−204
Cat-Et-Pt-glu
795
633, 507, 343, 317
−162
GCat-Mv-glu
797
635, 331
−162
4-VinylCat-Mv-glu
805
643
−162
Cat-Et-Mv-glu
809
657, 647, 357, 331
−162
Cat-Pt-glu-Ac
809
605, 519, 317
−204
GCat-Et-Pt-glu
811
649, 343
−468
Cat-Mv-glu-Ac
823
619, 535, 331
−204
GCat-Et-Mv-glu
825
663, 331
−162
GCat-Mv-glu-Ac
839
635
−204
GCat-Et-Mv-glu-Ac
867
663
−204
Cat-Pn-glu-pcoum
897
589, 301
−308
GCat-Dp-glu-pcoum
915
607
−308
Cat-Mv-glu-pcoum
927
619
−308
GCat-Pt-glu-pcoum
929
621
−308
GCat-Mv-glu-pcoum
943
635
−308
Cat-Et-Mv-glu-pcoum
955
647
−308
M+: molecular ion; Cat: (epi)catechin; GCat: gallo(epi)catechin; Ac: acetyl; pcoum: p-coumaroyl; Et: ethyl; Dp: delphinidin; Cy: cyanidin; Pt: petunidin; Pn: peonidin; Mv: malvidin; glu: glucoside [70].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 Figure21. Signals of pyranoanthocyanin and anthocyanin-flavanol derivatives arise in two m/z ranges of the spectrum, the first at m/z 579–771 and the second at m/z 751–929, respectively.Figure 21
Direct-infusion pneumatic spray (DIPS with capillary voltage 0.5 kV) of aCabernet Sauvignonwine aged several years. Mv: malvidin; IS: internal standard Mv-3,5-diglucoside [70].For a semiquantitative analysis, Mv-3,5-diglucoside (compound usually not present inV. 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 ratioΣanthocyanin-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 m/z 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]. α-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 m/z 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 290+288c+152g+23 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, g number of galloyl esters, 23 Na atomic mass) [89].Table 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].
Polymer
no. galloyl ester
Calculated [M + Na]+
Observed [M + Na]+
Positive linear
Positive reflectron
0
601
600
601
Dimer
1
753
752
753
2
905
905
905
Trimer
0
889
889
889
1
1041
1041
1041
2
1193
1193
1193
3
1345
1346
1345
0
1177
1177
1177
1
1329
1329
1329
Tetramer
2
1481
1482
1482
3
1634
1634
1634
4
1786
1785
1786
Pentamer
0
1466
1465
1466
1
1618
1618
1618
2
1770
1770
1770
3
1922
1922
1922
4
2074
2074
2074
5
2226
n.d.
n.d.
0
1754
1754
1754
1
1906
1907
1906
2
2058
2059
2058
Hexamer
3
2210
2211
n.d.
4
2362
2362
2362
5
2514
2513
n.d.
6
2666
2667
n.d.
Heptamer
0
2042
2043
2042
1
2194
2195
2194
2
2346
2346
2346
3
2398
2499
2499
4
2651
2651
n.d.
5
2803
2800
n.d.
6
2955
2954
n.d.
7
3107
n.d.
n.d.
0
2330
2330
2330
1
2483
2483
2483
2
2635
2635
n.d.
Octamer
3
2787
2787
n.d.
4
2939
2938
n.d.
5
3091
3090
n.d.
6
3243
n.d.
n.d.
0
2619
2618
2618
1
2771
2770
n.d.
Nonamer
2
2923
2923
n.d.
3
3075
3075
n.d.
4
3227
n.d.
n.d.
Decamer
0
2907
2907
n.d.
1
3059
3060
n.d.
2
3211
3212
n.d.
3
3363
n.d.
n.d.
Undecamer
0
3195
3194
n.d.
1
3347
3349
n.d.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 andt-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 Table16 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 andV. 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.Table 17
Main PAs identified in the MALDI-TOF positive-ion spectra of seeds extracts of 35 hybrid andV. vinifera grape varieties. Signals were divided in A and B series in reference to the interflavanic bond type: series B only B-type interflavanic bonds, and also A-type bonds are included in series A. Signals of sodium and protonated ions are reported. G: galloyl ester.
Procyanidin
Series B
Series A
[M + H]+
[M + Na]+
[M + H]+
[M + Na]+
Dimer A-type
577
Dimer B-type
601
Dimer, 1G
753
Trimer
867
863, 865
Trimer
889
887
Dimer, 2G
905
903
Trimer, 1G
1041
1039
Tetramer
1177
1175
Trimer, 2G
1193
1191
Tetramer, 1G
1305
1329
1303
1327
Trimer, 3G
1345
1343
Pentamer
1465
1463
Tetramer, 2G
1481
1629, 1631
Pentamer, 1G
1617
1613, 1615
Tetramer, 3G
1633
1629, 1631
Hexamer
1753
1751
Pentamer, 2G
1769
1765, 1767
Tetramer, 4G
1785
1781, 1783
Hexamer, 1G
1905
1903
Pentamer, 3G
1921
1917, 1919
Heptamer
2041
Hexamer, 2G
2057
2053, 2055
Pentamer, 4G
2073
2069, 2071
Heptamer, 1G
2193
2189, 2191
Hexamer, 3G
2209
2205, 2207
Pentamer, 5G
2225
2221, 2223
Heptamer, 2G
2345
2341, 2343
Hexamer, 4G
2361
2357, 2359
Heptamer, 3G
2497
2495
Hexamer, 5G
2513
2511
Octamer, 2G
2633
2631
Heptamer, 4G
2649
2647
Hexamer, 6G
2665
2663
Octamer, 3G
2785
2783
Heptamer 5G
2802
Nonamer, 2G
2922
2919
Octamer, 4G
2937
2935
Heptamer, 6G
2953
2951
Nonamer, 3G
3073
3071
Octamer, 5G
3089
3087
Decamer, 2G
3209
3207
Nonamer, 4G
3225
3223
Octamer, 6G
3241
Decamer, 3G
3361
Nonamer, 5G
3377
3376
Undecamer, 2G
3496Figure 22
Signals of PAs in the MALDI-TOF spectrum of aCabernet Sauvignongrape seeds extract.


## 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 calledGrape 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% (Table18). 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.Table 18
Phenolic compounds identified by LC/QTOF mass spectrometry and “suspects screening” analysis using the libraryGrapeMetabolomics in the study of metabolomics of Raboso Piave grape extract. The compounds identified with identification score (id.) higher than 95% are reported.
Phenolic compound
Id. score
Exact mass
Formula
Vanillic acid
99.9
168.0423
C8H8O4
cis-piceid
99.9
390.1315
C20H22O8
Pentahydroxyflavone A
99.8
302.0427
C15H10O7
Myricetin-3-O-rhamnoside
99.8
464.0955
C21H20O12
(epi)catechin gallate
99.8
442.0900
C22H18O10
Hydroxycoumarin
99.8
162.0317
C9H6O3
Rhamnetin
99.8
316.0583
C16H12O7
trans-resveratrol
99.8
228.0786
C14H12O3
Myricetin
99.8
318.0376
C15H10O8
Coumarin
99.8
146.0368
C9H6O2
Resveratrol tetramer 3
99.8
906.2676
C56H42O12
Pentahydroxyflavone B
99.7
302.0427
C15H10O7
Pyrogallol
99.7
126.0317
C6H6O3
Syringetin
99.7
346.0689
C17H14O8
Taxifolin-deoxy hexoside B
99.7
450.1162
C21H22O11
Gingerol
99.7
294.1831
C17H26O4
Dimethoxyphenol
99.6
154.0630
C8H10O3
Barbaloin
99.6
418.1264
C21H22O9
trans-caffeoyl-tartaric acid
99.6
312.0481
C13H12O9
Ferulic acid
99.6
194.0579
C10H10O4
Delphinidin p-coumarylmonoglucoside
99.6
611.1401
C30H27O14
Laricitrin-3-O-glucoside
99.6
494.1060
C22H22O13
Emodin 8-glucoside
99.5
432.1057
C21H20O10
E-ε-viniferin
99.5
454.1416
C28H22O6
Isorhamnetin-3-O-glucoside
99.5
478.1111
C22H22O12
Laricitrin
99.4
332.0532
C16H12O8
Cyanidin p-coumarylmonoglucoside
99.4
595.1452
C30H27O13
Esahydroxyflavanone-C-deoxy hexoside
99.4
466.1111
C21H22O12
Pentahydroxyflavanone-C-hexoside
99.4
466.1111
C21H22O12
Kaempferol-deoxy hexoside
99.4
432.1056
C21H20O10
Z-ε-viniferin
99.4
454.1416
C28H22O6
(epi)gallocatechin-catechin
99.3
594.1373
C30H26O13
Caffeic acid
99.3
180.0423
C9H8O4
Taxifolin-deoxy hexoside A
99.3
450.1162
C21H22O11
Taxifolin-pentoside B
99.3
436.1006
C20H20O11
Guaiacol
99.2
124.0524
C7H8O2
Resveratrol dimer 4
99.2
454.1416
C28H22O6
Methylnaringenin
99.2
286.0841
C16H14O5
Petunidin-3-O-monoglucoside
99.2
479.1190
C22H23O12
Tamarixetin
99.2
316.0583
C16H12O7
Peonidin acetylmonoglucoside
99.2
505.1346
C24H25O12
Dihydroquercetin
99.2
304.0583
C15H12O7
Syringetin-3-O-glucoside
99.1
508.1217
C23H24O13
Quercetin-3-O-rhamnoside
99.1
448.1006
C21H20O11
Fisetin
99.1
286.0477
C15H10O6
Luteolin
99.1
286.0477
C15H10O6
Vanillic acid beta-D-glucopyranoside
98.9
330.0951
C14H18O9
Peonidin p-coumaroylmonoglucoside
98.9
609.1608
C31H29O13
Piceatannol
98.7
244.0736
C14H12O4
Petunidin acetylmonoglucoside
98.6
521.1295
C24H25O13
Myricetin-3-O-glucoside
98.5
480.0904
C21H20O13
p-hydroxybenzoic acid
98.4
138.0317
C7H6O3
Methylsyringate  beta-D-glucopyranoside
98.3
374.1213
C16H22O10
Quercetin-3-O-glucoside
98.0
464.0955
C21H20O12
Resveratrol dimer 3
98.0
454.1416
C28H22O6
Silybin
97.9
482.1213
C25H22O10
Methylvanillate  beta-D-glucopyranoside
97.9
344.1107
C15H20O9
Syringaldehyde 4-O-beta-D-glucopyranoside
97.9
344.1107
C15H20O9
Procyanidin (B1, B2, B3, B4, B5)
97.8
578.1424
C30H26O12
Tetrahydroxy-dimethoxyflavanone-hexoside
97.8
510.1373
C23H26O13
Malvidin-3-O-monoglucoside
97.7
493.1346
C23H25O12
Resveratrol trimer 1
97.7
680.2046
C42H32O9
Pelargonidin p-coumaroylmonoglucoside
97.6
579.1503
C30H27O12
Cyanidin-3-O-monoglucoside
97.6
449.1084
C21H21O11
Resveratrol trimer 2
97.5
680.2046
C42H32O9
Di-O-methylquercetin B
97.5
330.0740
C17H14O7
p-cumaric acid  beta-D-glucopyranoside
97.5
326.1002
C15H18O8
Delphinidin-3-O-monoglucoside
97.4
465.1033
C21H21O12
Quercetin-3-O-glucuronide
97.4
478.0747
C21H18O13
p-cumaric acid
97.2
164.0473
C9H8O3
Delphinidin acetylmonoglucoside
97.2
507.1139
C23H23O13
Procyanidin (T2, T3, T4)
97.0
866.2058
C45H38O18
Petunidin p-coumaroylmonoglucoside
96.8
625.1557
C31H29O14
Malvidin acetylmonoglucoside
96.7
535.1452
C25H27O13
Di-O-methylquercetin A
96.5
330.0740
C17H14O7
Malvidin p-coumaroylmonoglucoside
96.3
639.1714
C32H31O14
Methylgallic acid isomer 2
96.0
184.0372
C8H8O5
Resveratrol dimer 5
95.5
454.1416
C28H22O6
Coniferaldehyde 4-O-beta-D-glucopyranoside
95.3
340.1158
C16H20O8
Myricetin-3-O-glucuronide
95.0
494.0697
C21H18O14


## 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.



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*Source: 813563-2013-01-27.xml*