Simultaneous Determination of Nonanthocyanin Phenolic Compounds in Red Wines by HPLC-DAD/ESI-MS

Transcription

1 Determination of Nonanthocyanin Phenolic Compounds 139 Simultaneous Determination of Nonanthocyanin Phenolic Compounds in Red Wines by HPLC-DAD/ESI-MS María Monagas, Rafael Suárez, Carmen Gómez-Cordovés, and Begoña Bartolomé* Abstract: The nonanthocyanin phenolic compounds of wines from Vitis vinifera L. cv. Tempranillo, Graciano, Cabernet Sauvignon, and Merlot (vintage 00, Navarra, Spain), vinified under the same conditions, were extracted with ethyl acetate and diethyl ether and analyzed by high-performance liquid chromatography diode array detection/electrospray ionization mass spectrometry (HPLC DAD/ESI MS) (negative mode). A total of 47 phenolic compounds were identified in the different wines, including nonflavonoids (hydroxybenzoic and hydroxycinnamic acids and their derivatives, stilbenes, and phenolic alcohols and other related compounds) and flavonoids (flavanols, flavonols, and dihydroflavonols). Novel phenolic acid derivatives, such as the methyl and ethyl esters of gallic acid and some hexose esters of vanillic and p-coumaric acids, were also detected. The concentration of nonflavonoid compounds was higher for Cabernet Sauvignon (62.23 mg/l) and Graciano (57.82 mg/l) wines than for Merlot (47.52 mg/l) and Tempranillo (43.70 mg/l). The concentration of flavonoid compounds was highest for Graciano wine ( mg/l), followed by Cabernet Sauvignon ( mg/l), Merlot (77.54 mg/l), and Tempranillo (50.56 mg/l) wines. Differences between wines were found in the quantified proportion of hydroxybenzoic acids (16.3 to 29.5%), stilbenes (0.3 to 2.9%), phenolic alcohols and other related compounds (9.4 to 17.0%), flavanols (42.9 to 56.1%), and flavonols (10.1 to 15.3%). ESI MS was confirmed as a valuable tool for obtaining potentially important information on specific phenolic compounds in wine. Key words: HPLC DAD/ESI MS, nonanthocyanin phenolic compounds, red wine Phenolic compounds constitute one of the most important quality parameters of wines since they contribute to organoleptic characteristics such as color, astringency, and bitterness. Because of their antioxidant and anti-inflammatory properties, phenolic compounds are associated with several beneficial physiologic effects derived from moderate wine consumption, a phenomenon known as the French paradox (Renaud and De Lorgeril 1992). Similarly, anticarcinogenic activity of wine phenolics has also been demonstrated (Clifford et al. 1996). The phenolic composition of wines is determined by grape variety and by factors that affect berry development, such as soil, geographical location, and weather. Winemaking techniques also play a very important role in the extraction of polyphenols from the grape: the time of maceration and fermentation in contact with the grape skins and seeds, Instituto de Fermentaciones Industriales, Consejo Superior de Investigaciones Científicas, Juan de la Cierva 3, Madrid, Spain. *Corresponding author [Tel: (34) ; Fax: (34) ; bartolome@ifi.csic.es] Acknowledgments: The authors are grateful to Julián Suberviola (EVENA, Navarra, Spain) for providing the wine samples. The authors also thank the Agencia Española de Cooperación International for a MUTIS predoctoral scholarship to M.M. and the Spanish Comisión Interministerial de Ciencia y Tecnología (Project AGL C02-02) for funding. Manuscript submitted September 04; revised October 04, January 05 Copyright 05 by the American Society for Enology and Viticulture. All rights reserved. pressing, maturation, fining, and aging conditions all affect the phenolic composition of red wines. Wine phenolics belong to two main groups of compounds, nonflavonoid (hydroxybenzoic and hydroxycinnamic acids and their derivatives, stilbenes and phenolic alcohols) and flavonoid (anthocyanins, flavanols, flavonols and dihydroflavonols). Past research has focused on identification of grape oligomeric and polymeric flavanols and wine anthocyanins by mass spectrometry (MS). Analysis of red wine nonanthocyanin phenolic compounds has usually been performed by HPLC with diodearray detection (DAD) (Mayén et al. 1995, Ritchey and Waterhouse 1999, Burns et al. 00, Peña-Neira et al. 00, Ibern-Gómez et al. 02), but more recently the application of HPLC-MS for these analyses has been demonstrated (Pérez-Magariño et al. 1999, Vanhoenacker et al. 01, De Pascual-Teresa et al. 00, Monagas et al. 03). Hydroxycinnamic acids, flavanols, and flavonols act as copigments of anthocyanins (Mistry et al. 1991, Eiro and Heinonen 02). Moreover, adducts of anthocyanins with flavanols and hydroxycinnamic acids have been identified recently in red wines and are implicated in color stabilization during aging (Remy et al. 00, Schwarz et al. 03). Grapes and grape products are primary sources of dietary stilbenes, which are largely known to have biological activity (Mattivi et al. 1995, Varache-Lembège et al. 00). New stilbene derivatives, including tetrahydroxystilbenes and dimeric stilbenes, have been recently isolated from wines (Baderschneider and Winterhalter 139

2 140 Monagas et al. 00, Vitrac et al. 01). Like stilbenes, astilbin (dihydroquercetin-3-o-rhamnoside), a dihydroflavonol found in wines (Baderschneider and Winterhalter 01), could be used by the vine as a phytoalexin to act against Botrytis cinerea development (Landrault et al. 02); several physiological effects of astilbin have also been demonstrated (Closa et al. 1997). Identification of phenolic compounds in wine by HPLC-DAD can be limited by coelution of two or more compounds, resulting in impure ultraviolet (UV) spectra, and by similarities in the UV spectra of compounds belonging to the same group and having close retention times (that is, flavanols and flavonols), which complicates assignment of chromatographic peaks. Screening red wine by modern mass-spectrometry techniques to confirm the structure of main nonanthocyanin phenolics and/or to detect novel compounds is therefore of great value in evaluating the peculiar characteristics of different grape varieties, optimizing enological processes, obtaining wines with original and improved characteristics, and achieving a better understanding of wine physiological properties. In the present work, HPLC DAD/electrospray ionization (ESI) MS was used to determine the different groups of nonanthocyanin phenolic compounds in wines from Vitis vinifera L. cv. Tempranillo, Graciano, Cabernet Sauvignon, and Merlot vinified under the same conditions. Tempranillo is widely cultivated in Spain, whereas Graciano is limited but highly appreciated for coupages, or wine blending. French Cabernet Sauvignon and Merlot are widely cultivated varieties. Materials and Methods Materials. Methyl gallate, ethyl gallate, quercetin-3-ogalactoside, quercetin-3-o-glucoside, kaempferol-3-o-glucoside, and (-)-epicatechin-3-o-gallate were purchased from Extrasynthèse (Genay, France). (+)-Catechin, (-)- epicatechin, myricetin, quercetin, kaempferol, tryptophol, trans-resveratrol, and gallic, trans-caffeic, trans-pcoumaric, trans-ferulic, vanillic, protocatechuic, and ellagic acids were purchased from Sigma (St. Louis, MO). Syringic acid and tyrosol were purchased from Aldrich (Deisenhofen, Germany). Cis-resveratrol was obtained from trans-resveratrol after exposure to UV light (340 nm) for one hour. Procyanidin dimers B1, B2, B3, and B4, trimer C1 [(-)-epicatechin-(4β 8)-(-)-epicatechin-(4β 8)-(-)- epicatechin], and trimer T2 [(-)-epicatechin-(4β 8)-(-)-epicatechin-(4β 8)-(+)-catechin] were isolated from a grape seed extract and their structure elucidated as previously described (Monagas et al. 03). Winemaking. Wines from grapes of Vitis vinifera L. cv. Tempranillo, Graciano, Cabernet Sauvignon, and Merlot (vintage 00) of the same age and cultivated in the same vineyard (Navarra, Spain) were vinified at the Viticulture and Enology Station of Navarra (EVENA), Olite, Spain, under identical conditions. A lot of 2 kg of grapes of each variety was destemmed, crushed, and collected into 0-L stainless-steel wine vats. Semi-industrial scaled fermentations were performed with a yeast inoculum of 25 g/hl (80% EVENA Saccharomyces cerevisiae strain Na33; % Lallemand Saccharomyces bayanus strain EC118) at a temperature up to 27 C. The cap was punched down twice a day until it remained submerged during a 14-day maceration at the same temperature. At the end of the alcoholic fermentation (sugar level <2 g/l), the wines were racked and the grape pomace was pressed using a vertical pneumatic press at 2 bars, recovering ~50 L of wine, which was combined with nonpressed wines. After one month of stabilization at -2 C, the wines were racked, filtrated with SEITZ K250 filters (2.5 to 3.0 mm) (Sert Schenk Filter System GmB, Bad Krevznach, Germany), and bottled after correcting the free SO 2 level to 30 mg/l. Two wine samples from each variety were analyzed after 1.5 months of bottling and storage at 13ºC and 80 to 85% relative humidity. Extraction of phenolic compounds. A volume of 50 ml of wine was concentrated to 15 ml under vacuum at 30 C and extracted three times with diethyl ether (15 ml) and three times with ethyl acetate (15 ml), as described by Fernández de Simón et al. (1990). The organic phases were combined and dried with anhydrous Na 2 SO 4 for 30 min. The extract was then taken to dryness under vacuum, dissolved in 2 ml of methanol/water (1:1), then filtrated (0.45 mm) and injected into the HPLC column. Recovery of phenolic compounds in diethyl ether and ethyl acetate varies with chemical structure (mean recovery value = 87%; mean SD value = 6%) (Fernández de Simón et al. 1990). HPLC DAD/ESI MS. A Hewlett-Packard series 1100 (Palo Alto, CA) chromatography system equipped with a diode array detector (DAD) and a quadruple mass spectrometer (Hewlett-Packard series 1100 MSD) with an electrospray interface was used. Separation of 5 to 10 ml of wine extract was performed on a reversed-phase Waters Nova-Pak C 18 (300 mm x 3.9 mm, 4 mm) (Millipore, Milford, MA) column at room temperature. A gradient consisting of solvent A (water/acetic acid, 98:2, v/v) and solvent B (water/acetonitrile/acetic acid, 78::2, v/v/v) was applied at a flow rate of 0.7 ml/min as follows: 0 to 80% B linear from 0 to 55 min, 80 to 90% B linear, from 55 to 57 min, 90% B isocratic from 57 to 70 min, 90 to 95% B linear from 70 to 80 min, 95 to 100% B from 80 to 90 min, followed by washing with methanol and reequilibration of the column from 90 to 1 min. The electrospray ionization (ESI) parameters were drying gas (N 2 ) flow and temperature, 10 L/min and 350ºC, respectively; nebulizer pressure, 55 psi; and capillary voltage, 4000 V. The ESI was operated in negative mode scanning from m/z 100 to 3000 using the following fragmentation program: from m/z 0 to 0 (100 V) and from m/z 0 to 3000 (0 V). Diode array detection was performed from 2 to 380 nm. Quantification was carried out by external standard calibration curves. Hydroxybenzoic acids, stilbenes, phenolic alcohols and other related compounds, flavanols, and flavonols were quantified at 280 nm, caffeic acid and its derivatives at 340 nm, and p- coumaric acid and its derivatives at 310 nm. Caffeic and p-

3 Determination of Nonanthocyanin Phenolic Compounds 141 coumaric acid derivatives, flavonol glycosides, and stilbene glucosides were quantified by the calibration curve of their respective free forms. Monomeric and dimeric flavan-3-ols were quantified using the (-)-epicatechin calibration curve. The remaining phenolic compounds were quantified by calibration curves produced with the same compound. Results and Discussion Identification of phenolic compounds. An HPLC-DAD chromatogram of the diethyl ether/ethyl acetate extract from the Tempranillo wine is shown in Figure 1 (peaks cited correspond to those in Table 1). Nonanthocyanin phenolic compounds were identified by peak retention time, UV spectra, and ESI-MS spectrometric data (Table 1). Nonflavonoid phenolic compounds (hydroxybenzoic and hydroxycinnamic acids and their derivatives, stilbenes, and phenolic alcohols and other related compounds) and flavonoid phenolic compounds (flavanols, flavonols, and dihydroflavonols) were identified in the wines from the four different grape varieties. Hydroxybenzoic acids and their derivatives. Gallic (1), protocatechuic (3), vanillic (), and syringic (25) acids were the main hydroxybenzoic acids identified in the different wines. For gallic and protocatechuic acids, in addition to the molecular ion, a fragment ion [M-H-44] - corresponding to the loss of the -CO 2 group (carboxylic function) was detected (Vanhoenacker et al. 01) (Table 1). Other authors (Pérez-Magariño et al. 1999) have also observed differences in the fragmentation pattern of hydroxybenzoic acids according to their chemical structure. Peaks 7 and 30 with [M-H] - at m/z 183 and 197 were identified as methyl gallate and ethyl gallate, respectively (Figure 2A). The fragment ions at m/z 169 and 125, corresponding to gallic acid after the loss of the methyl or ethyl unit and to the subsequent loss of the -CO 2 group, respectively, were detected in very low concentrations. Peak 18 presented molecular and fragment ions (m/z 329 and 167), which coincided with the mass of an hexose derivative of vanillic acid, being either the hexose ester or the glycoside. However, the peak intensity for this compound in the HPLC-DAD was too weak to allow further characterization on the basis of UV spectral characteristics. Both ethyl gallate and hexose (glucose) esters of vanillic acid have been previously identified in Riesling wine (Baderschneider and Winterhalter 01). Methyl gallate has been previously reported in red grapes (Fernández de Simón et al. 1992). Despite presenting a lactone-type structure, ellagic acid (peak 40) is included in this subgroup of compounds, as it is formed by oxidative coupling of two vicinal galloylated ester groups followed by acid hydrolysis (Haslam 1998). Hydroxycinnamic acids and their derivatives. trans- Caftaric (5), coutaric (14), and fertaric acids (17) as well as trans-caffeic (22) and p-coumaric (32) acids have been identified. For the tartaric esters, a fragment ion [M-H- 132] - corresponding to the free acid after the cleavage of the ester bond was observed (Table 1). The fragment ion corresponding to the loss of the -CO 2 group was mainly detected for the free acids, as observed in the case of hydroxybenzoic acids and their derivatives. Peaks 23 and 27 presented similar cinnamic-type UV spectra, molecular ion ([M-H] - = m/z 325), and fragments [m/z 163 ([M-H-glucose] - ); m/z 145 ([M-H-glucose-H 2 O] - )] (Figure 2B), coinciding with the mass of an hexose moiety linked to trans-p-coumaric acid (Baderschneider and Winterhalter 01, Biau et al. 1996). However, because O- glycosides present a maximum absorption wavelength AU (280 nm) x , Time (min) Figure 1 HPLC-DAD chromatogram at 280 nm of nonanthocyanin phenolic compounds of diethyl ether/ethyl acetate extract from Tempranillo wine.

4 142 Monagas et al. Table 1 Nonanthocyanin phenolic compounds identified by HPLC DAD/ESI MS in wines from Vitis vinifera L. cv. Tempranillo, Graciano, Cabernet Sauvignon, and Merlot. Peak Rt a λ max [M-H] - Fragments Wine b,c no. (min) (nm) (m/z) (m/z) Compound TM GR CS MR Gallic acid * * * * Unknown * * * * , Protocatechuic acid * * * * Dihydroxyphenylethanol * * * , 298(s) d trans-caftaric acid * * * * , ,3-Dihydroxy-1-(4-hydroxy-3- * * * methoxyphenyl)-propan-1-one , 125 Methyl gallate * * * * Tyrosol * * * * Unknown * * Dimer (epi)gallocatechin-(epi)catechin * , 289 Procyanidin B3 * * * * , 289 Procyanidin B1 * * * * Unknown * * * * trans-cutaric acid * * * * (+)-Catechin * * * * , 577, 289 Procyanidin T2 * * * * , 301(s) trans-fertaric acid * nd e Hexose ester of vanillic acid * * * * , 577, 289 Procyanidin T3 * * * , Vanillic acid * * * * , 289 Procyanidin B4 * * * * trans-caffeic acid * * * * , 145 Hexose ester of trans-p-coumaric acid (1) * * * * , 289 Procyanidin B2 * * * * Syringic acid * * * * Unknown * * * , 145 Hexose ester of trans-p-coumaric acid (2) * * * * (-)-Epicatechin * * * * , 577, 289 Trimeric procyanidin * * * , 125 Ethyl gallate * * * * , 577, 289 Procyanidin C1 * * * * trans-p-coumaric acid * * * * , 577, 289 Trimeric procyanidin * Procyanidin dimer gallate * , 289 Procyanidin B5 * * * * , 300(s), Myricetin-3-O-glucuronide * , 300(s), Myricetin-3-O-glucoside * * * * , 169 Epicatechin-3-O-gallate * (s), trans-resveratrol-3-o-glucoside * * * * Ellagic acid * * * * , 300(s), Quercetin-3-O-galactoside * * * * , 300(s), Quercetin-3-O-glucuronide * * * * Astilbin * * * *

5 Determination of Nonanthocyanin Phenolic Compounds 143 Table 1 continued Peak Rt λ max [M-H] - Fragments Wine no. (min) (nm) (m/z) (m/z) Compound TM GR CS MR , 300(s), Quercetin-3-O-glucoside * * * * , 302(h), Unknown * * * * Tryptophol * * * nd 697 Unknown * Unknown * * * * , 300(s), Kaempferol-3-O-glucoside * * * * Unknown * * * * , 300(s), Myricetin * * * * cis-resveratrol-3-o-glucoside * * * * , 319(s) 227 trans-resveratrol * * * * Unknown * * * * , 300(s), Quercetin * * * * cis-resveratrol * * * * a Rt: retention time referenced to Tempranillo. b TM: Tempranillo; GR: Graciano; CS: Cabernet Sauvignon; MR: Merlot. c *: detected; : not detected. d (s): shoulder. e nd: not determined. hypsochromically shifted with respect to that of the free acid (Baderschneider and Winterhalter 01, Määttä et al. 03), which was not observed for peaks 23 and 27, we tentatively propose the ester-type structure. The origin of the intermediate fragment at m/z 265 (Figure 2B), corresponding to the loss of 60 amu (-C 2 H 4 O 2 ), could not be elucidated, although it has also been detected in berries for two compounds exhibiting the same UV and mass spectra characteristics as peaks 23 and 27 (Määttä et al. 03). After considering similar descriptions in the literature (Macheix et al. 1990), we believe these compounds are either forms composed of two different hexoses or isomers composed of the same hexose esterified through different -OH positions of the molecule. Hexose (glucose) esters of trans-p-coumaric acid have been identified previously in Riesling wines (Baderschneider and Winterhalter 01). Another compound with phenylpropanoid structure corresponded to peak 6, which presented a UV spectra and molecular ion equivalent to 2,3-dihydroxy-1-(4-hydroxy- 3-methoxyphenyl)-propan-1-one (Table 1), previously identified in both brandies and wines (Gómez-Cordovés et al. 1997, Peña-Neira et al. 00, Baderschneider and Winterhalter 01). Glutathionyl adducts derived from hydroxycinnamic acids, which were previously reported in wines (Ibern-Gómez et al. 02), were not detected under our conditions. Stilbenes. cis- and trans-resveratrol (peaks 56 and 53) and their respective glucosides (peaks 52 and 39) were identified. In the case of the latter forms, a fragment ion [M-H-162] - corresponding to resveratrol after the loss of the glucose moiety was observed in addition to the molecular ion (Table 1). Phenolic alcohols and other related compounds. Peak 8 corresponded to tyrosol (p-hydroxyphenylethanol), an alcohol formed from tyrosine (3-(4-hydroxyphenyl)-alanine) during yeast fermentation. Peak 4 presented a molecular ion ([M-H] - = m/z 153) 16 amu higher than tyrosol ([M-H] - = m/z 137), which is equivalent to an additional hydroxyl group (-OH) and therefore consistent with the structure of a dihydroxyphenylethanol (Table 1). Tryptophol ([M-H] - = m/z 160), a nonphenolic alcohol synthesized from tryptophan (2-amino-3-(3-indolyl)-propionic acid), corresponded to peak 46. Flavonols. The aglycones myricetin (51) and quercetin (55) were identified in the different wines. Kaempferol ([M- H] - = m/z 285) and isorhamnetin ([M-H] - = m/z 315) were detected at very high retention times (<100 min) and were not evaluated quantitatively. The derivatives myricetin-3- O-glucuronide (36), myricetin-3-o-glucoside (37), quercetin-3-o-galactoside (41), quercetin-3-o-glucuronide (42), quercetin-3-o-glucoside (44), and kaempferol-3-o-glucoside (49) were also identified. In addition to the molecular ion, the fragment ions [M-H-162] - (for glucose/galactose derivatives) and [M-H-176] - (for glucuronide derivatives) were detected (Table 1). The nature of the hexose was determined by comparison with the retention time of glycosylated commercial standards from the same aglycone under identical chromatographic conditions. Dihydroflavonols. Peak 43 corresponded to astilbin (dihydroquercetin-3-o-rhamnoside), a compound recently identified both in white and red wines (Baderschneider and Winterhalter 01, Landrault et al. 02). Flavanols. Nongalloylated and galloylated monomeric ([M-H] - = m/z 289 and 441, respectively) and

6 144 Monagas et al. dimeric ([M-H] - = m/z 577 and 729, respectively) flavan-3- ols and trimeric procyanidins ([M-H] - = m/z 865) were identified in the different wine extracts. (+)-Catechin (15), (-)-epicatechin (28), and epicatechin-3-o-gallate (38) were detected as monomeric flavan-3-ols. The procyanidins identified included the dimers B3 (11), B1 (12), B4 (21), B2 (24), and B5 (35); the trimers T2 (16), T3 (19), and C1 (31); one dimer gallate (peak 34); and two trimers (peaks 29 and 33) of unknown structure (Table 1). Identification was also performed in comparison with the retention time of commercial standards and of grape seed procyanidins previously characterized (Monagas et al. 03). The fragmentation pathway for oligomeric flavanols under ESI-MS in negative mode consisted of ions derived from: (1) the interflavanic bond cleavage ([M (base) -H] -, [M (middle-base) -H] -, [M (top) -3H] -, [M (top-middle) -3H] - ); (2) the Retro-Diels-Alder (RDA) fission on the C ring followed by the loss of a water molecule ([M-H-152] -, [M-H-152-H 2 O] - ); and (3) the elimination of a phloroglucinol molecule ([M-H-126] - ), as reported by other authors (Friedrich et al. 00, Monagas et al. 03). Only the most abundant ions for each type of procyanidin are reported in Table 1. In the case of epicatechin-3-o-gallate (38), fragment ions resulting from the cleavage of the ester bond were detected at m/z 289 for the (-)-epicatechin unit and at m/z 169 for the gallic acid moiety (Table 1). The unknown dimer gallate (34) presented the ions corresponding to the loss of a gallic acid moiety (m/z 577) (Table 1), although OH O OCH 2 -CH 3 Absorbance A Methyl gallate Ethyl gallate Relative abundance (%) HO OH Ethyl gallate 1000 m/z OH O OCH nm Relative abundance (%) HO OH Methyl gallate 1000 m/z B Peak 23 Absorbance Peak 27 Relative abundance (%) HO O C OGlc nm m/z Figure 2 Comparison of ultraviolet and ESI-MS spectra: (A) methyl and ethyl gallates; (B) hexose esters of trans-p-coumaric acid.

7 Determination of Nonanthocyanin Phenolic Compounds 145 the ions resulting from the cleavage of the interflavanic bond previous to the loss of the gallic acid moiety (m/z 439 [for epicatechin-3-o-gallate-(epi)catechin] or m/z 441 [for (epi)catechin-epicatechin-3-o-gallate]) were not detected, complicating the final assignment of the galloylated unit within the dimer. Based on its molecular ion and fragment ion resulting from the RDA fission, peak 10 (m/z 593, 425) corresponded to the dimer (epi)gallocatechin- (epi)catechin (Table 1) (De Pascual-Teresa et al. 00, Friedrich et al. 00). Contents and relative distributions of nonanthocyanin phenolic compounds. In order to apply the information provided by ESI-MS, nonanthocyanin phenolic compounds were quantified in Tempranillo, Graciano, Cabernet Sauvignon, and Merlot wines. Because of the complexity of the chromatograms (Figure 1), the different compounds quantified were selected on the basis of their concentration in wine and their chromatographic resolution. The concentration of nonanthocyanin phenolic compounds, either individual, grouped, or as a total, is presented in Table 2. Since the mean recovery value of phenolic compounds in diethyl ether and ethyl acetate is relatively high (87% as a mean; Fernández de Simón et al. 1990), the data presented refers to the wine extracts. Of the nonflavonoid phenolics, hydroxybenzoic acids and their derivatives and phenolic alcohols and related compounds were the most abundant subgroups in the different wines, accounting for 16.3 to 29.5% and 9.4 to 17.0% of total nonanthocyanin phenolics quantified, respectively (Table 2). Mayén et al. (1995) also found that hydroxybenzoic acids represented the largest proportion of nonflavonoid phenolic compounds in wines from Tempranillo and Cabernet Sauvignon grape varieties cultivated in the south of Spain. The total concentration of hydroxybenzoic acids and their derivatives was similar among varieties, although Graciano was slightly higher. However, greater differences were found in the subgroup of phenolic alcohols and other related compounds; Cabernet Sauvignon had the highest concentration, especially in tyrosol (Table 2). Gallic acid was the most abundant hydroxybenzoic acid quantified in the wines, as reported by other authors (Ritchey and Waterhouse 1999, Peña-Neira et al. 00), followed by syringic acid. For gallic acid derivatives, ethyl gallate had a higher concentration than methyl gallate. Quantitative data on these latter compounds in wines is reported here for the first time. For hydroxycinnamic acids and their derivatives, the percentage distribution was very similar among varieties, although Cabernet Sauvignon wine had the highest total concentration because of its high trans-caftaric acid content, followed by Graciano, Merlot, and Tempranillo (Table 2). The concentration of tartaric esters of hydroxycinnamic acids was generally higher than that of free acids (Ritchey and Waterhouse 1999, Burns et al. 00). The concentration of hexose esters of trans-p-coumaric acid was similar in all four grape varieties. Concentration of stilbenes was extremely low in Cabernet Sauvignon compared with the other varieties, especially Graciano, which had the highest concentration (Table 2). As expected, the trans- forms of resveratrol-3-o-glucoside and its aglucone were the most abundant ones (Mattivi et al. 1995, Burns et al. 00). Flavanols were the principal flavonoid compounds found. They comprised 42.9 to 56.1% of total nonanthocyanin phenolics in the different varieties (Table 2). Graciano and Cabernet Sauvignon had the highest total flavanol concentrations followed by Merlot and then Tempranillo. Other authors (Mayén et al. 1995, Monagas et al. 03) also found low concentrations of flavanols in Tempranillo wines when compared to other varieties, probably because of the low total flavanol content of Tempranillo seeds. Flavan-3-ol monomers were present in higher concentration than the procyanidin dimers in three wines, with (+)-catechin more abundant than (-)-epicatechin. The exception was Graciano, which exhibited similar levels of both monomers (Burns et al. 00, Monagas et al. 03). Graciano and Merlot had similar total concentrations of flavonols, which were higher than in Cabernet Sauvignon and Tempranillo, especially with respect to quercetin-3-o-glucuronide (Table 2). Graciano and Cabernet Sauvignon wines had the highest total concentration of nonflavonoid, flavonoid, and total nonanthocyanin phenolic compounds quantified, followed by Merlot and Tempranillo (Table 2). Flavonoid compounds represented the highest proportion of total nonanthocyanin phenolics in the four varieties, as reported by other authors (Mayén et al. 1995). In general, the concentration of individual compounds is in the range presented for Spanish wines from different geographical origins (Peña-Neira et al. 00, Mayén et al. 1995), although lower for some compounds when compared with wines from other countries (Ritchey and Waterhouse 1999, Burns et al. 00). Since the different grape varieties were cultivated in the same vineyard and the respective wines were vinified under identical conditions, the differences in phenolic profiles could be due to grape variety. However, more wines from the same varieties and from other production areas and vintages should be studied in order to confirm the results found in this work. Conclusion Results confirmed that ESI-MS operated in negative mode coupled to HPLC-DAD is a valuable tool for the unambiguous identification of a wide range of well-known phenolic compounds in red wines, as well as for the preliminary identification of novel compounds. The method used allowed the simultaneous determination of nonflavonoid (hydroxybenzoic and hydroxycinnamic acids and their derivatives, stilbenes, and phenolic alcohols and other related compounds) and flavonoid (flavanols, flavonols, and dihydroflavonols) phenolic compounds in a single HPLC run. Other compounds not reported before in red wines and tentatively identified by ESI-MS need to be isolated and finally characterized by NMR.

8 146 Monagas et al. Table 2 Concentration (mg/l) of nonanthocyanin phenolic compounds in wine extracts from Vitis vinifera L. cv. Tempranillo, Graciano, Cabernet Sauvignon, and Merlot. Compound Tempranillo (mg/l) Graciano (mg/l) Cabernet (mg/l) Merlot (mg/l) Nonflavonoid phenolics Hydroxybenzoic acids/derivatives ± 1.30 a (29.5%) ± 0.52 (17.7%) ± 0.60 (16.3%) ± 0.17 (21.7%) Gallic acid ± ± ± ± 0.03 Protocatechuic acid 1.25 ± ± ± ± 0.01 Vanillic acid 1.24 ± ± ± ± 0.06 Syringic acid 4.26 ± ± ± ± 0.01 Methyl gallate 2.26 ± ± ± ± 0.01 Ethyl gallate 4.60 ± ± ± ± 0.05 Hydroxycinnamic acids/derivatives 2.93 ± 0.12 (3.1%) 4.54 ± 0.14 (2.6%) 6.26 ± 0.29 (3.8%) 4.81 ± 0.07 (3.8%) trans-caftaric acid 0.72 ± ± ± ± 0.01 trans-coutaric acid 0.77 ± ± ± ± 0.01 trans-caffeic acid 0.42 ± ± ± ± 0.01 trans-p-coumaric acid 0.31 ± ± ± ± 0.01 Hexose ester trans-p-coumaric acid (1) 0.37 ± ± ± ± 0.02 Hexose ester trans-p-coumaric acid (2) 0.34 ± ± ± ± 0.01 Stilbenes 1.49 ± 0.07 (1.6%) 5.18 ± 0.27 (2.9%) 0.44 ± 0.01 (0.3%) 1.84 ± 0.06 (1.5%) trans-resveratrol-3-o-glucoside 0.41 ± ± ± ± 0.03 cis-resveratrol-3-o-glucoside 0.23 ± ± ± ± 0.01 trans-resveratrol 0.73 ± ± 0.13 tr c 0.21 ± 0.01 cis-resveratrol 0.12 ± ± 0.01 tr 0.05 ± 0.01 Alcohols/related compounds ± 0.49 (12.2%) ± 0.07 (9.4%) ± 0.38 (17.0%) ± 0.07 (11.0%) Tyrosol 6.99 ± ± ± ± 0.04 Tryptophol 4.51 ± 0.03 nd 2.95 ± ± 0.03 Total nonflavonoid phenolics ± ± ± ± 0.37 Flavonoid phenolics Flavanols ± 1.15 (42.9%) ± 2.29 (56.1%) ± 2.17 (52.6%) ± 0.35 (46.7%) (+)-Catechin ± ± ± ± 0.07 (-)-Epicatechin 9.89 ± ± ± ± 0.08 Procyanidin B ± ± ± ± 0.07 Procyanidin B ± ± ± ± 0.13 Flavonols ± 0.63 (10.8%) ± 0.67 (11.3%) ± 0.73 (10.1%) ± 0.21 (15.3%) Myricetin-3-O-glucoside 2.60 ± ± ± ± 0.05 Quercetin-3-O-galactoside nd b nd 1.05 ± 0.03 tr Quercetin-3-O-glucuronide 1.30 ± ± ± ± 0.10 Kaempferol-3-O-glucoside 1.23 ± ± ± 0.06 tr Myricetin 3.15 ± ± ± ± 0.04 Quercetin 1.88 ± ± ± ± 0.02 Total flavonoid phenolics ± ± ± ± 0.56 Total nonanthocyanin phenolics ± ± ± ± 0.93 a Mean value ± standard deviation. b nd: not determined. c tr: trace.

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