Analyzing Resveratrol in Minnesota Wines Matt Perry Chem 4101 12/4/9

What is Resveratrol? Resveratrol (3,4,5-N-trihydroxystilbene) is an antioxidant that is found in the skin of grapes. It is found in both white and red grapes but at much higher concentrations in red grapes. Resveratrol has garnered much attention for its possible implications in lowering the risk of coronary heart disease (CHD) as well as its possible anti-carcinogen properties. CHD is often linked to higher cholesterol diets, however, in France high cholesterol diets have not been accompanied by high levels of heart disease. This “French Paradox” is thought to be explained by the high levels of wine, and thus resveratrol, that are imbibed.

Problem Since U of MN hybrids are relatively new, enological practices are still being tested for the most effective winemaking techniques. A reliable method for determining resveratrol levels associated with different winemaking techniques is needed. Hypothesis: Due to the presence of V. riparia in the U of MN grape cultivars, these cultivars will have higher levels of Resveratrol than V. vinifera cultivars.

Why is this important? If U of MN grape cultivars have higher levels of Resveratrol, this could be used in marketing to boost the popularity & demand on a regional, national and possibly even international level.

Alternative Methods Method Advantages Disadvantages GC-MS HPLC Can resolve both isomers 10-8 M LOD Extraction/ derivatization procedure too involved and time consuming HPLC Good sample recovery Direct injection with fluorescence or electrochemical detection Non-destructive method Multi-step extractions Long elution times Column maintnence / short lifetime A variety of detection techniques were used including UV, MS, Fluorescence, Coulometric detection and Electrochemical detection

Capillary Electrophoresis High separation efficiency Very little sample preperation Fast elution times Relatively low cost per sample Ability to detect multiple flavonoid compounds Low sample volumes Enhanced resolution with MEKC

Instrumentation Beckman P/ACE MDQ 32 Karat 8.0 software Cost = $40,000 Parameters Fused silica capillary, 30 cm x 50 micrometer ID LOD - 0.1 micromolar Linear range 0.2 - 100 micromolar pH = 9.2 Detection - UV, 314 nm (additional filter required) Temperature - 22 C Voltage - 25 kV Resolution = 1.8

Experimental Design Select 3 different brands of wine in same vintage made from Marquette and Frontenac grapes. Select three different brands of wine in same vintage made from V. vinifera, preferably from the same area. Concentrate sample via SPE in triplicate using Ethanol/HCl/Ethyl Acetate and reconstitute with buffer solution (75 mM SDS, 30 mM boric acid, 30 mM dibasic sodium phosphate, and 15% acetonitrile, pH 9.2) prior to analysis. Run buffer solution and samples through a 0.5 micrometer filter Rinse capillary with buffer solution prior to sample injection Introduce sample under low pressure, compare elution times to that of an external standard. Co-injection of standard can also be used to verify cis/trans Resveratrol peaks as well as comparison with UV spectrums of standards.

Data Analysis Prepare standard solutions of resveratrol ranging from 0.1 micromolar to 100 micromolar. Analyze with CE and plot the known concentrations versus the peak areas or the peak heights. Determine a regression equation for each of these relationships and use the equation that is better correlated. Compare the unknown control sample peak area to standardized curve to quantify resveratrol levels. Because of the tendency for trans-resveratrol to interconvert to cis-resveratrol in short time periods, an external standard curve should be made daily.

Sample Results

Conclusions New grape hybrids developed by the U of MN could have higher levels of resveratrol than those of pure V. vinifera lineage, this could be a boost to marketing. While GC/MS and HPLC are both capable of analyzing resveratrol levels in wine, CE can also complete the task with lower costs and in less time.

References 1.) Creasy, L.; Gu, X.; Kester, A and Michael Zeece. Capillary Electrophoretic Determination of Resveratrol in Wines J. Agric. Food Chem. 1999, 47, 3223−3227 2.) Chu, Q.; Liu, F.; Peng, Y. and Jiannong Ye. Determination of Phenolic Constituents of Biological Interest in Red Wine by Capillary Electrophoresis with Electrochemical Detection. J. Agric. Food Chem. 2004, 52, 153−156. 3.) Chu, Q; Gu, X; O’Dwyer, M. and Michael Zeece. Analysis of resveratrol in wine by capillary electrophoresis. Journal of Chromatography A, 881 ( 2000) 471-481 4.) Chu, Q; O’Dwyer, M. and Michael Zeece. Direct Analysis of Resveratrol in Wine by Micellar Electrokinetic Capillary Electrophoresis. J. Agric. Food Chem. 1998, 46, 509−513 5.) Kolouchová-Hanzlíková, I.; Melzoch, K.; Filip, V. and Jan Midrkal. Rapid method for resveratrol determination by HPLC with electrochemical and UV detections in wines. Food Chemistry Volume 87, Issue 1, August 2004, Pages 151-158 6.) George J. Soleas et al. Comparative Evaluation of Four Methods for Assay of cis- and trans-Resveratrol. Am. J. Enol. Vitic. 48:2:169-176 (1997)