Determination of Volatile Compounds in Romanian White Wines by Headspace Solid-phase Micro-extraction and Gas Chromatography Mass Spectrometry
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1 Determination of Volatile Compounds in Romanian White Wines by Headspace Solid-phase Micro-extraction and Gas Chromatography Mass Spectrometry ANAMARIA HANGANU 1*, MARIA-CRISTINA TODASCA 2, FULVIA-ANCUTA MANOLACHE 3, SORIN ROSCA 2 1 Romanian Academy, Organic Chemistry Center Costin D. Nenitzescu 202B Splaiul Independentei, , Bucharest, Romania 2 University Politehnica of Bucharest, Faculty of Applied Chemistry and Materials Science, Costin D. Nenitzescu Organic Chemistry Department, 1-7 Polizu Str., , Bucharest, Romania 3 National Institute of Research&Development for Food Bioresources IBA, 6 Dinu Vintila, Bucharest, Romania A headspace solid-phase micro-extraction (HS-SPME) and gas chromatography (GC) coupled with mass spectrometry (MS) method was developed for identification and quantitative measurement of sixteen volatile compounds in thirty-seven Romanian white wines. The HS-SPME step was carried out using a divinylbenzene/ Carboxen/ polydimethylsiloxane (DVB/CAR/PDMS) 100 µm fiber. One fiber was used for all wine samples, control and blank experiments. Esters and alcohols have been the volatiles present in wines in the highest concentration. In HS-SPME step was used an internal standard 2-octanol. Based on the concentration of the internal standard added in every wine sample was determined the relative concentration of volatile compounds identified through GC-MS spectrometry. The data obtained by using HS-SPME-GC-MS method were processed using the chemometrical method Principal Component Analysis (PCA). From the diagram obtained trough HS-SPME-GC-MS coupled with PCA method it was observed that the wines samples have a tendency to group according to their variety; in this way were authenticated Romanian white wines in terms of variety. To validate the authentication method were analyzed five samples of commercial white wines. Keywords: wine, HS-SPME-GC-MS, PCA, grape variety, authentication, volatile compounds The volatile composition of wines is complex because several organic structures contribute to flavour. The volatile components present in wines comprise several classes of organic compounds: esters, alcohols, organic acids, ketones, aldehydes and monoterpene alcohols [1]. Concentration of the volatile components in wine is different depending on the grape variety, cultural practices and climatic or biological factors [2]. The amounts of volatile compounds in wine range from nanograms to micrograms; these components have distinct physicochemical properties regarding, for example, polarity, volatility and odor impact as result of the functional groups (alcohol, aldehyde, acid, etc.) present in the molecule [3]. Volatile compounds in wine are commonly analyzed by gas-chromatography coupled to at least one detector, such as flame ionization detector (FID) or mass spectrometer (MS). The mass spectrometer detector has the advantage that it provides fragmentation data that is useful for the identifications of molecule. Most studies on wine aroma utilizing gas-chromatography deal with improved extraction methods including liquid-liquid extraction, headspace extraction, purge and trap extraction, solid phase extraction (SPE) and solid phase micro-extraction (SPME) in order to capture volatile compounds from wine samples [4-6]. The SPME method is sufficiently sensitive to absorb small amounts of volatile compounds and the wide range of fiber types allows a large diversity of compounds analyzed [7-9]. Extraction and desorption parameters, such as time and temperature, are commonly investigated to optimize SPME method. Addition of salt to wine samples can increase the concentration of same compound in the headspace and hence their extraction [10-11]. SPME technique can also have disadvantages. The degradation of the fiber can often impart lower response as well as * ana.hanganu@cco.ro tel : poor repeatability and reproducibility. Changing fibers during an extended study introduces complications if the fiber does not have identical quantitative responses to analytes under examination. To compensate for these phenomena, internal standards can be added to the samples in such a way as to detect fiber degradation and to provide a reliable comparison of extraction efficiency from one fiber to the next [12]. In previous studies of Romanian wines, our research team, used for identification and quantitative determination of wine composition spectroscopic methods ( 1 H-NMR and FT-IR) and Principal Component Analysis for wine classification in terms of variety, geographical origin, crop year and technological practices [13-19]. In the current study we developed another method for wine analysis and classification. The objective of this study was to use HS-SPME-GC-MS for the analysis of the Romanian white wines aroma compounds. An elastic DVB/CAR/PDMS 100 μm fiber was used for the headspace extraction of analytes from wines samples. GC-MS analysis was performed for identification and quantitative determination of volatile compounds present in white wines. The volatile compounds in various samples of Muscat Ottonel, Riesling, Sauvignon Blanc, Feteascã Albã, Pinot Gris, Chardonny and Columna wines were compared by utilizing PCA as a classification method. It was observed that the white wines were grouped according to their variety. To validate the authentication method were analyzed five samples of commercial white wines from Jidvei. Experimental part Wine sample analyzed in this study were from different variety, geographical area and crop year (table 1). The first 32 wines samples are authentic samples provided by REV. CHIM. (Bucharest) 63 No
2 Table 1 WINE SAMPLES ANALYZED IN THIS STUDY Urlati Murfatlar, Odobesti, Valea Cãlugãreascã, Medgidia, Tohani, Urlati and Panciu vineyards. The last five samples are commercial wines from Jidvei vineyard. HS-SPME-GC-MS analysis of white wines An elastic DVB/CAR/PDMS 100 μm fiber was used in the headspace SPME mode to isolate compounds from wines samples. Sample preparation: 10 ml of wine sample with additional 3g of NaCl and 50 μl 2-octanol (internal standard) 1mg/mL solution were introduced in a 25 ml vial. The sample was incubated 30 min at 40 o C with an agitation speed of 200 rmp; the fiber was exposed to the headspace above the mixture for 30 min (at 40 o C and agitation speed of 200 rmp). Following the sample incubation and extraction, analytes were thermally desorbed into the GC (Agilent 7890A) injector. Split-less mode was carried out and the flow-rate of the carrier gas (helium) was maintained at 1mL/min. A linear GC oven temperature program was used, starting at 40 o C (2 min) and then increased at 10 o C/min to 250 o C and maintained at the 250 o C for another 10 min. The total GC run time was approximately 30 min. The volatile compounds separated on GC column were identified using the mass spectrometer (Agilent 5975C). One fiber was used for all wine samples, control and blank experiments. Blank experiments consisting of the: (a) blank of the fiber, (b) blank of the empty vial, (c) blank of the vial including 3g NaCl, (d) blank of the vial including 10 ml of deionized water and (e) blank of the vial including 3g NaCl and 10 ml of deionized water were carried out after every 15 samples [20]. Statistical analysis The statistical analyses (Principal Component Analysis) used to investigate the compositional differences between white wines samples were carried out using the XLSTAT program (from Addinsoft). Results and discussions Based on gas-chromatograms and mass spectra were identified and quantitative determined sixteen volatile compounds present in white wines. To determine the relative concentration of those compounds was used an internal standard 2-cotanol (it was added in known concentration in each sample analyzed). The volatile compounds extracted from wines were separated by gas-chromatography and analyzed by mass spectrometry. Figure1 presents the volatile compounds extracted from Chardonnay wine, produced at Murfatlar in For each pick of the gas-chromatogram was recorded a mass spectrum which allowed structure identification for volatile compounds. The volatile compounds identified in Chardonnay wine are listed in table 2. REV. CHIM. (Bucharest) 63 No
3 Fig.1 Gas-chromatogram of volatile compounds extracted from Chardonnay wine Table 2 VOLATILE COMPOUNDS IDENTIFIED IN CHARDONNAY WINE The relative concentrations of volatile compounds present in white wines are listed in table 3. Esters and alcohols have been the volatiles present in wines in the highest concentration. To authenticate white wines varieties, in the PCA statistical processing, were used the relative concentrations of volatile compounds identified in the authentic wines using HS-SPME-GC-MS method. The diagram resulted from the PCA processing of data is illustrated in figure 2. From the graphical representation of PC1/PC2 scores plot it can be observed a very good separation of wines according to their variety. Wines from the same variety are grouped into distinct area very well defined. Using the HS- SPME-GC-MS method coupled with PCA chemometrical method we were able to authenticate white wines in terms of variety. To validate the authentication method we also study commercial wine from Jidvei vineyard. The relative concentrations of volatile compounds from Muscat Ottonel, Sauvignon Blanc, Riesling, Feteascã Albã and Pinot Gris wines from Jidvei were introduced in the matrix of data obtained for authentic white wines. The set of data, obtained in this way, was process also using PCA (fig. 3). From figure3 it can be seen that the commercial wines are distributed in the specific areas of their variety origin. In this way, it was validated the authentication method of white wines. REV. CHIM. (Bucharest) 63 No
4 Table 3 RELATIVE CONCENTRATION OF VOLATILE COMPOUNDS IDENTIFIED IN WHITE WINES BY HS-SPME-GC-MS METHOD REV. CHIM. (Bucharest) 63 No
5 continued table 3 Fig.2 Graphical representation of PC1/PC2 scores plot derivates from the relative concentration of volatile compounds identified in authentic white wines Conclusions The HS-SPME-GC-MS analytical method for determination of white wine aroma compounds was used to study thirty-seven wine samples for characterization and classification purposes. First the volatile components of wines were extracted using an elastic SPME fiber, then the Fig.3 Graphical representation of PC1/PC2 scores plot derivates from the relative concentration of volatile compounds identified in authentic and commercial white wines compounds were desorbed into a gas-chromatograph and identified by mass spectrometry. Sixteen volatile compounds were identified and quantitatively measured by HS-SPME-GC-MS. REV. CHIM. (Bucharest) 63 No
6 The data provided by HS-SPME-GC-MS relative concentrations of volatile compounds were process by PCA chemometrical method. The graphical representation of PC1/PC2 scores plot shows a very good separation of wines according to their variety. In this way, we were able to authenticate wines in term of variety. The validation of the authentication method was performed using commercial wine samples. References 1. HOWARD, K.L., MIKE, J.H., RIESEN, R., Am. J. Enol. Vitic., 56, 2005, p SANCHEZ-PALOMO, E., DIAZ-MAROTO, M.C., PEREZ-COELLO, M.S., Talanta, 66, 2005, p BOSCH-FUSTE, J., RIU-AUMATELL, M., GUADAYOL, J.M., CAIXACH, J., LOPEZ-TAMAMES, E., BUXADERAS, S., Food Chem., 105, 2007, p CABREDO-PINILLOS, S., CEDRON-FERNANDEZ, T., SAENZ-BARRIO, C., Anal. Lett., 37, 2004, p REBIERE, L., CLARK, A.C., SCHMIDTKE L.M., PRENZLER, P.D., SCOLLARY, G.R., Anal. Chim. Acta, 660, 2010, p MINUTI, L., PELLEGRINO, R., J. Cromatogr. A, 1185, 2008, p RODRIGUEZ-BENCOMO, J.J., SCHNEIDER, R., LEPOUTRE, J.P., RIGOU, P., J. Cromatogr. A, 1216, 2009, p CAMPILLO, N., PENALVER, R., LOPEZ-GARCIA, I., HERNANDEZ- CORDOBA, M., J. Cromatogr. A, 1216, 2009, p CARRILLO, J.D., GARRIDO-LOPEZ A., TENA, M.T., J. Cromatogr. A, 1102, 2006, p PENA, R.M., BSRCIELA, J., HERRERO, C, GARCIA-MARTIN, S., J. Sci. Food Agric., 85, 2005, p SETKOVA, L., RISTICEVIC, S., PAWLISZYN, J., J. Cromatogr. A, 1147, 2007, p KALUA, C.M., BEDGOOD Jr., D.R., PRENZLER, P., Anal. Chim. Acta, 556, 2006, p HANGANU, A., TODAªCÃ, M.C., CHIRA, N.A., ROSCA, S., Rev. Chim.(Bucharest), 62, no. 7, 2011, p TODASCA, M.C., FOTESCU, L., HÎNCU, F.A., HANGANU, A., CHIRA, N.A., ROSCA, S., Rev. Chim.(Bucharest), 61, no. 11, 2010, p TODASCÃ, M.C., FOTESCU, L., CHIRA, N.A., DELEANU, C., ROSCA, S., Rev. Chim.(Bucharest), 62, no. 2, 2011, p HANGANU, A., TODAªCÃ, M.C., CHIRA, N.A., ROªCA, S., U.P.B Sci. Bull., 73 (2), 2011, p BUZAª, M.C., CHIRA, N., DELEANU, C., ROªCA, S., Rev. Chim.(Bucharest), 54, no. 10, 2003, p TODAªCÃ, M.C., CHIRA, N., DELEANU, C., ROªCA, S., U.P.B Sci. Bull., 69 (4), 2007, p TODAªCÃ, M.C, CHIRA, N., AVRAMESCU, M., RUBELI, A., DELEANU, C., ROªCA, S., Rev. Chim.(Bucharest), 59, no. 10, 2008, p SETKOVA, L., RISTICEVIC, S., PAWLISZYN, J., J. Cromatogr. A, 1147, 2007, p.224. Manuscript received: REV. CHIM. (Bucharest) 63 No
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