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Proceeding of the 4 rd International Symposium“NEW RESEARCH IN BIOTECHNOLOGY” USAMV Bucharest, Romania, 2011Sample handling - In this study 24 wine samples (10 white and 14 red) fromMuntenia and Oltenia region were analyzed. The analyzed red wine varieties were FeteascaNeagra, Cabernet Sauvignon, Merlot, Pinot Noire and those of white wines varieties wereFeteasca Regala, Tamaioasa Romaneasca, Sauvignon Blanc, Feteasca Alba, Riesling Italianand Muscat Ottonel. All samples were filtered through 0.45 µm filters prior to analysis andkept protected from light at 4 °CHPLC analysisThe chromatographic analysis was carried out with a Thermo Finnigan SurveyorPlus chromatograph equipped with a photodiode array detector at 310 nm, Surveyorautosampler, Surveyor LC Pump (Quaternary gradient). Data analysis was done using theChrome Quest Chromatography Workstation.The analytical column aquasil C18, 250×4.6mm with the sorbent particle size of 5 µm. The mobile phase mixed ofwater:acetonitrile:acetic acid = 70:29.9:0.1 has flown through the system at a rate of 1ml/min. Samples of 20 µl volume were injected into the analytical column. All analyseswere carried out at ambient temperature. Quantification was based on the peak area, usingthe external standard method.3. RESULTS AND DISCUSSIONSValidation studiesCalibration curve was performed by analysis in duplicate of eight standards oftrans-resveratrol covering the range 0.25-10 mg/L. The constructed calibration curveshoved excellent linearity (correlation coefficient: 0.9991) (fig. 2).500000040000003000000Area2000000100000017800 1 2 3 4 5 6 7 8 9 10 11concentration (mg/L)Figure 2. Calibration curve for trans-resveratrolThe linearity of the detector response for the prepared standards was assessed bymeans of a linear regression analysis regarding the amounts of each standard introduced inthe loop of the chromatographic system and the area of the corresponding peak on thechromatogram.Detection and quantification limits were calculated by measuring the response ofanalytical background by running six blanks (12% alcoholic solution), using the maximumsensitivity allowed by the system and calculating the standard deviation of this response
Proceeding of the 4 rd International Symposium“NEW RESEARCH IN BIOTECHNOLOGY” USAMV Bucharest, Romania, 2011(STD). The detection limit, DL was estimated by multiplying the STD by a factor of 3, andthe quantification limit, QL was defined as 10 times the STD. The values obtained were0.008 mg/L for DL and 0.026 mg/L for QL.The precision or the degree of reproducibility is expressed as the coefficient ofvariation (CV). Precision was determined by performing six replicate analyses of the samesample on six different days. The value obtained for precision was 9.8 % for direct injectionof the sample in chromatographic system.The uncertainty was estimate according EURACHEM/CITAC. This estimationaccounts for the most significant sources of uncertainty that are thought to interfere in thefinal result. In this work, the contributions to uncertainty were ascribed to: the uncertaintyin the preparation of standards, the uncertainty of the calibration curve and the uncertaintyin the precision of the method. The global uncertainty was estimated at 23.5 % forconcentration above 0.25 mg/L.Quantification of trans-resveratrol in Romanian wineThe determination of the trans-resveratrol alone proved to be accurate using asimple procedure, without sample pre-treatment other than filtration, allowing for a lessthan 15 minute analysis run time.However, the chromatograms of the standards are free from interferences, with thepeak of trans-resveratrol clearly separated (Fig. 3a). Regarding the wine samples it isnoticeable that in the red wine (Fig. 3b) the interferences are more significant than in whitewines (Fig. 3c), in spite of the lower amounts found in the latter samples.In the work of Ratola et all [9], Portuguese red wine from different regions hadtrans-resveratrol content from 0.55 to 1.24 mg/L and white wines had values ranging fromnot detected to 0.19 mg/L. Other studies report trans-resveratrol content ranging from 0.508to 2.534 mg/L for greek wines [10] and values ranged from 0.25 to 8.87 mg/L for Italianwines [11].The amount of trans-resveratrol in the different analyzed wines variedconsiderably depending on the grape variety, environmental factors in the vineyard andwine-processing techniques. In our study, the occurrence of trans-resveratrol was confirmedin all of the analyzed 14 red wine samples, with concentration ranging between 0.025 and6.598 mg/L (Table 1). For the white wine, 5 of the 10 tested revealed the presence of thecompound at concentration between 0.132- 0.159 mg/L (Table 2).It is known that trans-resveratrol is produced by grape berries in response to fungalinfection and UV irradiation [12]. Thus, it is possible that sun exposure of grapes grown atMuntenia region may be a factor in increased resveratrol level compared with the grapesfrom Oltenia region. In addition, the red grape varieties Feteasca Neagra may be geneticallyricher in this compound than other varieties.Graphical representation of mean concentration of trans-resveratrol found in thisresearch, depending on the red wine variety show that the content of trans-resveratroldecreased in order Feteasca Neagra, Merlot, Pinot Noire, Cabernet Sauvignon (Fig. 4).Concerning white wine, higher concentration of trans-resveratrol was found in MuscatOttonel variety.179
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