Resveratrol in Wine - ACS Symposium Series (ACS Publications)

Chapter 5

Resveratrol in Wine

Downloaded by NORTH CAROLINA STATE UNIV on December 18, 2012 | http://pubs.acs.org Publication Date: March 1, 1997 | doi: 10.1021/bk-1997-0661.ch005

Kenneth D. McMurtrey Department of Chemistry and Biochemistry, University of Southern Mississippi, Hattiesburg, MS 39406-5043

ABSTRACT Levels of trans-resveratrol in commercial red and white Vitis vinifera wines from several regions in Europe, South America, California, and Australia, which were purchased as individual bottles from vendors in this region, were determined using HPLC with electrochemical detection. In addition some white wines prepared in Mississippi from muscadine grapes, Vitis rotundafolia, were also analyzed. HPLC conditions suitable for simultaneous analysis of both trans-resveratrol and free quercetin were developed. Concentration of free quercetin is also reported for many of the wines analyzed. Unlike white V. vinifera wines, white V. rotundafolia wines contain appreciable levels of resveratrol. Measured values ranged from about 0.3 to 1.2 mg/L (ppm), which is similar to resveratrol levels in some red California wines. We are not aware of other analyses of muscadine wines for resveratrol. Highest resveratrol levels were found in a California Barbera (9.2 ppm), California Pinot noir (8.7 ppm), and a French Bordeaux (Merlot/Cabernet Sauvignon blend, 8.3 ppm). Bottles of the same type of wine from different vintners contained widely varying amounts of resveratrol. We also found that levels of resveratrol in multiple bottles of the same wine also varied considerably. Fourteen bottles of a California non vintage blended red wine contained resveratrol levels from 2.74 to 5.77 ppm while eight bottles of a California 1993 Cabernet Sauvignon had levels from 0.46 to 0.74 ppm, nearly the same degree of variation.

INTRODUCTION It is now reasonably well accepted that red wine has potentially positive effects on human cardiovascular health. Resveratrol (trans-3 4', 5-trihydroxystilbene, produced by certain plants as an antifungal agent) has been suggested as one of the components which may provide some of these health benefits. In addition, wine components such as the flavanol, quercetin have also been nominated as potentially positive dietary components. Others papers in this volume will provide details on potential health benefits of wine and wine components. The primary focus of this paper will be to discuss application of HPLC with electrochemical detection to measurement of resveratrol levels in wines at the point of consumption. Levels of free quercetin have also been measured for some wines. While quercetin analysis is still preliminary, available data will be presented. 9

© 1997 American Chemical Society

In Wine; Watkins, T.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.


5. McMURTREY

Resveratrol in Wine

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Analysis of wines for resveratrol began with an account published in 1992 by Siemann and Creasy, the seminal paper of this area. These researchers used an analytical technique which involved a complicated multistep process requiring extraction with ethyl acetate, freezing overnight to remove water, rotary evaporation, normal phase preparative HPLC, irradiation with U V light to convert trans resveratrol to the ds-isomer and finally analysis by reversed-phase HPLC with U V detection. Jeandet, et al. (1993) analyzed wines using a process involving multistep extraction followed by derivitization to give trimethylsilyl ethers followed by analysis on gas chromatography. Lamuela-Raventos and Waterhouse (1993) shortened the extraction procedure required somewhat and analyzed the resulting extracts using HPLC with diode-array U V detection. Both of the above reports found relatively low levels of resveratrol. Analysis of resveratrol was simplified further by Mattivi (1993), who used disposable extraction cartridges follow by analysis of extracts using HPLC with U V detection. This report found somewhat higher levels of resveratrol than had previously been reported. Pezet, etal. (1994) reported analysis of Jraws-resveratrol and related pterostilbenes in grape berries and wines using HPLC with fluorescence detection. Because of the greater sensitivity of fluorescence to /ra/w-resveratrol, wines were analyzed by direct injection without the sample treatments which had previously been used. These authors found levels of resveratrol consistent with those reported by Mattivi and higher than those given in earlier reports. Pezet, etal., reported that trans-resveratrol was unstable to conditions used during rotary evaporation in earlier accounts. We employed direct injection of 10 μ\, samples of wine on liquid chromatographs with electrochemical detectors operated in an oxidative mode (McMurtrey, et al., 1994). We examined eleven wines and found low levels of transresveratrol (< 0.02 mg/L) in four white wines and levels of from 1 to 5 mg/L in the seven red wines studied. These levels were consistent with those reported by Mattivi and Pezet, et al. Goldberg et al. (1994) has reported details of a method for determining transresveratrol using octadecylsilyl extraction cartridges eluted with ethyl acetate followed by analysis using combined gas chromatography/mass spectrometry. These authors report finding resveratrol levels in red wines from 0.1 to 12 mg/L. They indicate that resveratrol concentrations follow geographical distribution with relatively low levels in wines from California, Australia, and Italy, and relatively high levels in wines from Oregon, Canada, and France. Lamuela-Raventos and co-workers (1995) have extended analysis of wines to include determination of both cis- and /ra/w-resveratrol and their glycoside conjugates, the piceids, in wine using direct injection (after filtration) and diode array U V detection. In this account these researchers found combined cis-, and transresveratrol and the cis-, and trans-piceid levels of up to 13.8 mg/L in a Spanish Merlot with high levels also in Pinot noir wines from that country. The cisresveratrol and piceid and trans-piceid compounds could apparently be converted in the body to trans-resveratrol by combined isomerization and hydrolysis reactions. Further details of the analytical procedures used by the research groups of Drs. Lamuela-Raventos and Goldberg, as well as results from application of these procedures, are to be found in papers in this collection. R E S U L T S A N D DISCUSSION We were initially interested in the analysis of wine for the trans -isomer of resveratrol and have not performed analyses specifically for the cis analog.



WINE: NUTRITIONAL AND THERAPEUTIC BENEFITS

CH OH 2

HO

OH

Ε Figure 1. Substances readily detected using HPLC with electrochemical detection in the oxidative mode. A . aniline derviatives, B. tryptamine, tryptophan and other indoles; phenols such as C. trans-resveratrol, D. quercetin; and enols such as E. ascorbic acid.



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Resveratrol levels quoted below are for trans-resveratrol only, and unless specified the use of the term resveratrol is meant to indicate the trans -isomer only. Analytical Conditions: Analysis of substances such as resveratrol by chromatography or other analytical techniques is dependent on a balance of selectivity and sensitivity. In this regard, for a successful analytical method based on HPLC the column must be capable of separating the components of interest from interfering substances while the detector must be capable of detecting and quantitating the analyte at the levels available. The chromatography column can affect sensitivity primarily through peak sharpness, while the detector may also contribute to selectivity if it is insensitive to materials in the matrix. We chose an electrochemical detection as the method for monitoring resveratrol in wines primarily because of its excellent sensitivity to phenolic substances. When used under the conditions which were employed (glassy carbon working electrode, silver/silver chloride reference electrode, stainless steel auxiliary electrode, oxidative mode), the detector responds very well to two groups of organic compounds: aromatic amines and phenols, Figure 1. Aromatic amines including substituted analines and biologically important substances such as tryptamine and tryptophane are readily detected as are phenols including resveratrol, quercetin, and any of the other many phenolic substances contained in wine. Some enolic materials such as ascorbic acid are also detected. Thus, the electrochemical detector would also respond well to cw-resveratrol, the piceid derivatives of the two isomers of resveratrol, and to various sugar conjugates of flavonoids such as quercetin, assuming that at least one phenolic hydroxyl group remains unconjugated. Sensitivity levels obtained with the electrochemical detector are more than sufficient for quantitation of resveratrol or quercetin at the parts-per-million or partsper-billion levels. We have not tested the ultimate limit of sensitivity but they appear to be at least in the mid ppt (ng/L) range for resveratrol, well below the levels currently of interest. Initial conditions for resveratrol analysis are those used in Figure 2. A mobile phase of 25% acetonitrile afforded separation of resveratrol from other components with a retention time of about 16 minutes. Although resveratrol was easily quantitated, quercetin eluted partially overlapping an interfering peak, ca. 25 and 26 min, respectively. We have modified the original conditions of analysis with the goal of being able to quantitate both trans-resveratrol and free quercetin during the same chromatographic analysis. The mobile phase was changed from acetonitrile-ammonium phosphate buffer to one composed of methanol and formic acid. To keep the analysis time within the same interval (approximately 30 min, or less) the percent of organic modifier was increased to 45% and the counterion used was formic acid, which also decreased the pH of the mobile phase from a value of about 4.5 to 2.5. A n example of analysis of a red wine with these conditions is given if Figure 3. In this case while separation of quercetin in near optimum, the resveratrol peak elutes early in the chromatogram and may provide less than ideal conditions for quantitation. We next employed a mixture of acetonitrile and methanol as the organic modifier of the mobile phase. In addition, it appeared that formic acid may lead to early failure of the reference electrodes. Ammonium phosphate (0.05 M , pH adjusted to 3.0) was used instead. We desired a blend of methanol and acetonitrile which would provide good conditions for analysis of both £ra/w-resveratrol and quercetin and have an analysis time of approximately 25 minutes. A simple graph was constructed, see Figure 4. Boundary mobile phases of 25% CH3CN with 0.05 M ammonium phosphate, pH 3.0, and 47% methanol with the same buffer were used as graph end points. These two mobile phases provided equal retention time for quercetin although resveratrol is eluted more rapidly in the methanolic phase. This graph allow rapid selection of percent acetonitrile and methanol which should give approximately equal total analysis time for quercetin. Thus, a mixture of 20%


48



100 nAFS

0

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20

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MINUTES Figure 2. Analysis of three wines by HPLC with electrochemical detection. Chromatographic conditions were: Nucleosil C18,5 //m, 4.6 X 150 mm, mobile phase 25 % (v/v) acetonitrile 75 % 0.05 M NH4H2PO4,1.0 mL/min. Resveratrol elutes at ca. 16 min. Sensitivity in trace A is 5 times that in trace Β and 10 times that of trace C. Quercetin elutes at about 25 min with an unresolved unknown substance (trailing edge). The electrochemical detector was operated at 0.5 V vs. the Ag/AgCl reference electrode.


McMURTREY

Resveratrol in Wine Chianti


Quercetin

Resveratrol

Γ'

0

MINUTES Figure 3. Analysis of a Chianti wine using methanolic mobile phase. Chromatography conditions similar to those employed in Figure 2 were used except that the mobile phase was 45% (v/v) methanol in 0.5% formic acid.

Figure 4. Graph for selecting acetonitrile-methanol mixtures. Methanol ( acetonitrile ( ).


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Figure 5. Analysis of Barbera wine under conditions that allow quantitation of both iraws-resveratrol and quercetin in a single chromatographic analysis. Nucleosil C18 column, 4.6 X 150 mm, 16% each C H C N and MeOH + 68% 0.05 M N H 4 H 2 P O 4 , pH 3.0, 1.0 mL/min. EC detector 0.700 V vs. Ag/AgCl, 1000 nAFS 3



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Resveratrol in Wine

acetonitrile and 9% methanol + 71% ammonium phosphate buffer (1, Figure 4), a mixture of 16% of each organic solvent + 68% ammonium phosphate (2, Figure 4), or 10% acetonitile and 28% methanol + 62% ammonium phosphate (3, Figure 4) should give approximately equivalent quercetin retention with respective decreasing resveratrol retention time. It was found that a 16% mixture of the two solvents provided a mobile phase which separated the two analytes well, see Figure 5. These conditions are now used in the analysis of wines in our laboratory. Results of Wine Analyses: A number of commercial wines were analyzed for resveratrol, with some analyses also quantitating free quercetin. As others have found, white wines from vinifera grapes contain little resveratrol, see Table 1. Even wines from grapes grown in England (with its relatively moist climate) contain little.

Table 1 Resveratrol and Free Quercetin Levels in Commercial White Vitis vinifera Wine Ouercetin

Vintage

Origin

Resveratrol

Chardonnay (Marcus James) Chardonnay (Blossom Hill)

NA NV

Brazil Calif.

Resveratrol in Wine - ACS Symposium Series (ACS Publications)

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