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Validation of the method for the determination of some wine volatile compounds

Validation of the method for the determination of some wine volatile compounds Wine aroma is influenced by a number of volatile compounds. This article describes the validation of the method for 26 volatile compounds found in wine. Volatile compounds were determined with discontinuous liquid-liquid extraction and GC-MS detection. It was determined, that the method is linear with square correlation coefficient ranging from 0.961 to 0.999. Limits of quantitative determination range from 0.52 g/L to 14.8 g/L. Recoveries range from 71.1% to 105.7% except for two compounds with lower recoveries. Measurement uncertainty ranges from 5.0% to 28.9%. According to the validation, the method is suitable for the determination of at least 24 volatile compounds common to wine. A practical method application was presented on Zelen wine variety from two different production procedures. Key words: wine, aroma, volatile compounds, GC-MS IZVLECEK VALIDACIJA METODE ZA DOLOCANJE NEKATERIH HLAPNIH SPOJIN V VINU Na aromo vina vplivajo stevilne hlapne spojine. Ta clanek opisuje validacijo metode za 26 hlapnih spojin, ki jih najdemo v vinu. Hlapne spojine so bile dolocene z diskontinuirano ekstrakcijo tekoce-tekoce in GC-MS detekcijo. Dolocili smo, da je metoda linearna, z razponom kvadrata korelacijskega koeficienta od 0,961 do 0,999. Meje kvantitativne dolocitve imajo razpon od 0,52 g/L do 14,8 g/L. Izkoristki imajo razpon od 71,1% do 105,7%, razen za dve spojini, katerih izkoristek je nizji. Merilna negotovost ima razpon od 5,0% do 28,9%. Z ozirom na validacijo lahko potrdimo primernost metode za dolocanje vsaj 24 hlapnih spojin znacilnih za vino. Prakticni prikaz uporabe metode smo predstavili na vinih sorte Zelen iz dveh razlicnih postopkov pridelave. Kljucne besede: vino, aroma, hlapne spojine, GC-MS Prispevek je del doktorske disertacije z naslovom "Vpliv maceracije na aromaticne znacilnosti primorskih belih vin", mentorica prof. dr. Tatjana Kosmerl 1 INTRODUCTION Wine aroma, a very important sensory parameter is produced by a complex balance of several volatiles. More than 800 volatile compounds such as alcohols, esters, phenols, monoterpenes, norisoprenoides, lactones, aldehydes and ketones have been identified in wine (Selli et al., 2004; Tamborra et al., 2004). The wine aroma is complex due to a large number of compounds present and their different chemical nature with a wide range of polarity, volatility, solubility and pH values. Therefore the sample preparation and particularly the extraction and concentration of volatile compound are an important factor in their determination (Cabredo Pinillos et al., 2004). Appropriate extraction of wine volatile compounds must be performed before their detection. Exceptionally so called major wine volatile compounds present in mg/l, like acetaldehyde, ethyl acetate, methanol and higher alcohols are detected directly without previous extraction where samples are only diluted and deacidificated prior to analysis (Peinado et al., 2004; Luki et al., 2008). Agricultural Institute of Slovenia, Hacquetova ulica 17, SI-1000 Ljubljana, Slovenia, Ph.D., e-mail: dejan.bavcar@kis.si, helena.basa@kis.si str. 285 - 293 Extraction of minor volatile compounds, present in wine in g/L, is done today mostly in three different ways. The first is discontinuous or continuous liquid-liquid extraction (LLE) of wine with organic solvent. Both discontinuous and continuous liquid-liquid extractions are suitable to measure volatiles, but to perform a second one special apparatus must be provided and main disadvantages, like time consuming process and large volumes of solvents, are not avoided (Cabredo Pinillos et al., 2004). As solvents mainly dichloromethane (Selli et al., 2003) or mixture of pentane: dichloromethane = 60:40 (Pérez-Coello et al., 2003; Izquierdo et al., 2008) are used. The second approach is solid phase extraction (SPE) using Sep Pack C18 cartridges (Tamborra et al., 2004) or LiChrolut EN resins (Loscos et al., 2010; Sáenz-Navajas et al., 2010). The third approach is Solid Phase Micro Extraction (SPME) with different fibers used: carbowaxdivinylbenzene (Lambropoulos and Roussis et al., 2007; Antalick et al., 2010), polydimethylsiloxane (Nasi et al., 2008; Antalick et al., 2010), polydimethylsiloxane/ divinylbenzene (Nasi et al., 2008; Antalick et al., 2010), carboxen/polydimethylsiloxane (Nasi et al., 2008; Antalick et al., 2010) or divinylbenzene/carboxen/ polydimethylsiloxane (Nasi et al., 2008; Antalick et al., 2010). Detection of volatile compounds is performed by gas chromatograph (GC) coupled with Flame Ionisation Detector (FID) (Pérez-Coello et al., 2003; Selli et al., 2003; Selli et al., 2004; Tamborra et al., 2004; Selli et al., 2006; Loscos et al., 2010) or mass spectrometer (MS) (Pérez-Coello et al., 2003; Selli et al., 2003; Selli et al., 2004; Tamborra et al., 2004; Selli et al., 2006; Lambropoulos and Roussis et al., 2007; Izquierdo Cañas et al., 2008; Nasi et al., 2008; RodriguezBencomo et al., 2008; Loscos et al., 2010; Antalick et al., 2010; Sáenz-Navajas et al., 2010). Quantification can be done with both detectors, while unequivocal identification only by MS. On Agricultural institute of Slovenia we decided to introduce discontinuous liquid-liquid extraction method with dichloromethane, chosen as the most effective organic solvent for this type of extraction (Cabredo Pinillos et al., 2004). The extraction was performed with intention to determine 26 minor volatile compounds with possible sensorial effect in wines (Schneider et al., 1998; Selli et al., 2003; Selli et al., 2006; Luki et al., 2008). Liquid-liquid extraction is actually the oldest but still the reference technique for the extraction of volatile compounds in wine (Ortega et al., 2002). 3-octanol and 4-nonanol were used as internal standards because of their high recovery (Cabredo Pinillos et al., 2004; Selli et al., 2006). By this procedure we achieved concentration factor 100. To enable qualitative and quantitative evaluation at the same time, MS was used for detection. After introduction, method was validated. Method was finally applied to real wine samples (variety Zelen) deriving from an experiment, where two different winemaking procedures were confronted. 2 MATERIALS AND METHODS 2.1 Materials Chemicals: Dichloromethane (Sigma-Aldrich) and ethanol absolute (Merck) with HPLC grade were used like solvents in our experiment, together with ultrapure water from the Milli-Q system. Similarly only the volatile compounds (Merck, SigmaAldrich, Fluka, SAFC) with the highest available purity on market (minimum of 95 %) were used with the exception of 4vinylphenol (SAFC) only sold like 10 % solution. Preparation of solutions: Stock solutions in pure dichloromethane of individual volatiles were prepared in 50 ml volumetric flasks with concentrations ranging from 1.8 ­ 2.5 g/L. From 26 stock solutions one mix solution of all 26 volatiles was prepared in 200 mL volumetric flask. All other solutions used to determine linearity, limits of detection and limits of quantification were prepared from this mix solution with proper dilutions. Internal standards 3-octanol and 4-nonanol for those dichloromethane solutions were prepared in 100 mL volumetric flask with dissolving them in quantity of 1.1 ­ 1.2 g/L in dichloromethane. They were added using 0.05 mL Hamilton syringe to 10 mL of dichloromethane solutions and mixed before determination. Preparation of model wine solution: First a mix stock solution of all volatiles in 100 % pure ethanol was prepared, with individual volatiles concentrations in range of 0.8 ­ 1.2 g/L. Stock solution was adequately diluted to model solution (mix) using 12 %vol ethanol in water to concentrations similar to ones determined in wines in average, to 0.04 ­ 0.07 mg/L, in 3000 ml volumetric flask. The pH was then adjusted to pH 3.2 with tartaric acid addition. Model wine solution was finally dispensed in twenty 125 mL flasks and they were stored in dark at 7 ºC before extraction. Internal standards 3-octanol and 4-nonanol used in our model wine solution were prepared in 100 mL volumetric flask with dissolving them in quantity of 0.04 ­ 0.06 g in ethanol absolute. They were added using 0.05 mL Hamilton syringe to model wine solution only during extraction process as described below. 286 Validation of the method for the determination of some wine volatile compounds 2.2 Procedure Liquid-liquid extraction of volatile compounds: 100 mL of model wine solution was transferred into 250 ml Erlenmeyer flask and cooled to 0 ºC in an ice bath under nitrogen. 29 g of 3-octanol and 23 g of 4-nonanol were added as internal standards using 0.05 mL Hamilton syringe from corresponding ethanol solutions. Dichloromethane (40 mL) was added and the mixture was stirred at 350 min-1 for 20 minutes (Moio et al., 1995). Then the mixture was centrifuged at 5°C (RFC = 8500, 10 minutes) and organic phase was 2.3 Determination Chromatographic conditions of GC (HP 6890)-MS (HP 5973) system: Liner Injector temperature Injection type Precolumn Column Temperature gradient Ion source temperature Auxiliary temperature Detector temperature Carrier gas Injection volume Detection Agilent 5062-3587 200 ºC Pulsed Splitless 2 m * 0.25 mm Varian, CP-WAX 57CB, 50 m x 0.25 mm ID 40 ºC; 12 min 5 ºC/min; from 40 ºC to 200 ºC 200 ºC; 20 min 230 ºC 200 ºC 150 ºC Helium 6.0; constant flow 1.0 ml/min 1 l Selective Ion Monitoring (T, Q1, Q2, Q3): 1,6-Heptadien-4-ol (71,43) 1-Hexanol (56, 43, 55, 69) 2-Phenylethyl acetate (104, 43, 91) 3-Octanol (59, 83, 101) 4-Ethylguaiacol (2-Methoxy-4-ethylphenol )(137, 152) 4-Nonanol (55, 73, 83, 101) 4-Vinylguaiacol (2-Methoxy-4-vinylphenol) (150, 135, 107, 77) 4-Vinylphenol (120, 91) Benzaldehyde (77, 105, 106) Benzyl alcohol (79, 108, 107) cis-3-hexen-1-ol (67, 41, 82) Diethyl succinate (101, 129) Ethyl butyrate (Ethyl butanoate)(71, 43, 88) Ethyl cinnamate (131, 103, 176) Ethyl decanoate (Ethyl caprate) (88, 101, 155) Ethyl dodecanoate (Ethyl laurate) (88, 101) Ethyl hexadecanoate (Ethyl palamitate) (88, 101) Ethyl hexanoate (88, 99) Ethyl lactate (45, 75) Ethyl octanoate (Ethyl caprylate) (88, 101, 57) Geraniol (69, 93, 123) Hexyl acetate (43, 56) Isoamyl acetate (70, 43, 55) Nerol (69, 84, 93) n-Hexaldehyde (Capronaldehyde) (56, 44, 57) trans-2-hexen-1-ol (55, 69, 83) -Ionone (177, 43) -Butyrolactone (42, 56, 86) recovered. The aqueous phase was re-extracted twice in the same way described above. Finally organic phases were combined and dried over sodium sulphate. They were concentrated to a final volume of 1 mL with Vigreaux distillation column and nitrogen gas flow prior to GC-MS analysis (Schneider et al., 1998). The same procedure was used for the extraction of wine samples (Moio et al., 1995; Schneider et al., 1998; Selli et al., 2006). 2.4 Aromatic compounds determination in wines from two winemaking procedures - preparation of wine samples Healthy grapes of Zelen variety (40 kg) were manually harvested in 2008 at the ripeness stage corresponding to wines containing approximately 12 % vol ethanol. Grapes were divided in two equal parts. First half of grapes (Zc = control without skin contact) was immediately destemmed, crushed and pressed up to 150 kPa using a small water press (Lancman VS-A 55, Slovenia). The juice was sulphited with 30 mg/L of sulphur dioxide, left to settle at 6ºC for 12 hours, racked and divided in three glass laboratory fermentor vessels with 1.6 L juice each. The vessels were heated to 17ºC, inoculated with 0.2 g/L of dried Saccharomyces cerevisiae (CM, Lallemand), supplemented with 0.2 g/L complex yeast nutrient (Fermaid E, Lallemand) and fermented at 17ºC. After alcoholic fermentations (residual sugars <2.5 g/L) and when most of the lees had settled, the wines were racked, 50 mg/L of sulphur dioxide was added and the wines were stored at 10ºC. The second half of grapes (Zp = freezing of the pomace) was destemmed and crushed. The pomace was equally divided in three plastic vessels, frozen overnight at ­20ºC, defrozen at 20ºC and pressed up to 150 kPa. The juice from the individual plastic vessels was sulphited with 30 mg/L of sulphur dioxide, left to settle at 6ºC for 12 hours, racked and poured in 3 glass laboratory fermentor vessels with 1.6 L juice each. The remaining procedure to obtain wines was the same as described previously. In this way, two different types of Zelen wines (Zc, Zp) in three repetitions were obtained. 3 RESULTS AND DISCUSSION 3.1 Linearity, limits quantification of detection, limits of quantification (LOQ) were calculated from calibration curve and are presented in Table 1. the Linearity was verified by using the solutions of volatile compounds in dichloromethane (five repetitions for one concentration level, three to eight concentration levels for the calibration curve). Linearity and range were determined by linear regression, using the F test. Linear model is fit and remains linear over the range presented in Table 1. Limits of detection (LD) and limits of Linearity was verified for wider range also and is presented in Table 2. Concentration factor for wine samples was due to extraction 100, so realistic linearity range, LDs and LOQs are 100-times lower. Table 1: Linearity, limits of detection, limits of quantification linearity (mg/L) 0.12 - 3.35 0.19 - 3.35 0.0168 - 3.35 0.0058 - 3.35 0.05 - 3.35 0.05 - 3.35 0.0111-1.67 0.0116 - 1.67 1.67 - 10.13 0.022 - 3.35 0.0167 - 1.67 0.05 - 3.35 0.05 - 1.67 0.01 - 3.35 0.0139 - 1.67 0.0092 - 3.35 0.0099 - 1.67 0.1 - 9.51 0.011 - 3.35 0.0092 - 1.67 0.05 - 3.35 0.0058 - 3.35 0.06 - 3.35 0.05 - 3.35 0.009 - 3.35 0.024 - 3.35 R2 0.999 0.999 0.999 0.996 0.998 0.998 0.993 0.994 0.996 0.996 0.999 0.998 0.995 0.996 0.995 0.996 0.991 0.992 0.996 0.990 0.998 0.996 0.983 0.982 0.995 0.997 LD (mg/L) 0.062 0.067 0.030 0.051 0.062 0.061 0.041 0.038 0.352 0.066 0.016 0.056 0.056 0.065 0.034 0.053 0.044 0.444 0.065 0.048 0.059 0.056 0.175 0.179 0.074 0.055 LOQ (mg/L) 0.206 0.224 0.099 0.171 0.208 0.203 0.136 0.126 1.174 0.218 0.052 0.186 0.186 0.216 0.115 0.176 0.148 1.480 0.216 0.159 0.196 0.187 0.582 0.597 0.248 0.183 288 Table 2: Linearity, wider range linearity (mg/L) 0.12 - 11.92 0.19 - 19.4 0.0168 - 33.53 0.0058 - 11.5 0.05 - 9.36 0.05 - 9.08 0.0111-22.18 0.0116 - 23.14 1.67 - 10.13 0.022 - 21.62 0.0167 - 33.45 0.05 - 10.72 0.05 - 9.55 0.01 - 10.08 0.0139 - 27.7 0.0092 - 18.45 0.0099 - 10.79 0.1 - 9.51 0.011 - 10.8 0.0092 - 18.31 0.05 - 10.79 0.0058 - 11.57 0.06 - 11.31 0.05 - 3.35 0.009 - 9.32 0.024 - 24.06 R2 0.961 0.988 0.995 0.987 0.978 0.977 0.994 0.994 0.996 0.993 0.999 0.983 0.977 0.978 0.973 0.989 0.986 0.992 0.985 0.989 0.983 0.988 0.998 0.982 0.980 0.994 3.2 Trueness Trueness was verified by checking the recoveries. Two parallel extracts of model wine solution were prepared each day for ten days and injected once respectively. The average of recoveries was calculated. The results are given in Table 3. 3.3 Precision For the determination of precision (ISO 5725), i.e. repeatability and reproducibility, extracts of model wine solution was analysed (the same as for recovery evaluation). Within the period of 10 days two parallel extracts were prepared each day. Each was injected once. Then standard deviation of repeatability of the level and standard deviation of reproducibility of the level were both calculated. The results are given in Table 4. Table 3: Recoveries for model wine solution conc. in model wine solution (mg/L) 0.0435 0.0596 0.0614 0.2930 0.0543 0.2300 0.0692 0.0414 0.0713 0.0672 0.0566 0.0498 0.0599 0.0697 0.0500 0.0624 0.0524 0.0588 0.0709 0.0573 0.0495 0.0593 0.0604 0.0540 0.0433 0.0548 0.0564 0.0609 recovery (%) 84.4 98.3 91.9 85.9 92.3 87.1 95.0 98.2 95.0 94.2 84.0 91.3 77.5 95.1 81.9 67.8 27.9 76.7 79.4 71.1 105.7 80.6 78.0 96.5 82.0 102.4 89.2 88.1 RSD (%) 3.1 4.9 2.5 3.1 4.2 2.8 8.9 2.6 3.0 4.6 3.0 2.7 3.8 2.9 15.2 8.9 9.5 3.4 3.5 7.4 3.0 4.0 3.8 2.4 3.3 8.2 2.6 3.1 1,6-Heptadien-4-ol 1-Hexanol 2-Phenylethyl acetate 3-Octanol 4-Ethylguaiacol 4-Nonanol 4-Vinylguaiacol 4-Vinylphenol Benzaldehyde Benzylalcohol cis-3-Hexen-1-ol Diethyl succinate Ethyl butyrate Ethyl cinnamate Ethyl decanoate Ethyl dodecanoate Ethyl hexadecanoate Ethyl hexanoate Ethyl lactate Ethyl octanoate Geraniol Hexyl acetate Isoamyl acetate Nerol n-Hexaldehyde trans-2-Hexen-1-ol -Ionone -Butyrolactone Table 4: Standard deviation of repeatability and reproducibility of the method, in mg/L conc. in model wine solution (mg/L) 0.0435 0.0596 0.0614 0.0543 0.0692 0.0414 0.0713 0.0672 0.0566 0.0498 0.0599 0.0697 0.0500 0.0624 0.0524 0.0588 0.0709 0.0573 0.0495 0.0593 0.0604 0.0540 0.0433 0.0548 0.0564 0.0609 means of the levels (mg/L) 0.0367 0.0583 0.0563 0.0501 0.0659 0.0406 0.0676 0.0632 0.0475 0.0455 0.0463 0.0662 0.0404 0.0419 0.0147 0.0449 0.0562 0.0405 0.0524 0.0476 0.0470 0.0521 0.0354 0.0559 0.0503 0.0536 standard deviation of repeatability (sr) 0.0007 0.0011 0.0012 0.0020 0.0055 0.0009 0.0013 0.0014 0.0010 0.0012 0.0010 0.0014 0.0010 0.0008 0.0012 0.0010 0.0015 0.0009 0.0014 0.0011 0.0012 0.0012 0.0007 0.0044 0.0010 0.0012 standard deviation of reproducibility (sR) 0.0011 0.0029 0.0014 0.0024 0.0056 0.0010 0.0021 0.0028 0.0014 0.0012 0.0018 0.0019 0.0064 0.0039 0.0014 0.0016 0.0020 0.0031 0.0015 0.0019 0.0017 0.0012 0.0012 0.0050 0.0013 0.0016 290 3.4 Uncertainty of repeatability and uncertainty of reproducibility Uncertainty of repeatability and uncertainty of reproducibility were calculated by multiplying standard deviation of repeatability and standard deviation of reproducibility by Student's t factor for 9 degrees of freedom and 95% confidence level (t95;9 = 2.262). Ur = t95; 9 x sr ; UR = t95; 9 x sR The results are presented in Table 5. Table 5: Uncertainty of repeatability and reproducibility of the method, in mg/L conc. in model wine solution (mg/L) 0.0435 0.0596 0.0614 0.0543 0.0692 0.0414 0.0713 0.0672 0.0566 0.0498 0.0599 0.0697 0.0500 0.0624 0.0524 0.0588 0.0709 0.0573 0.0495 0.0593 0.0604 0.0540 0.0433 0.0548 0.0564 0.0609 uncertainty of repeatability (Ur) 0.0016 0.0025 0.0026 0.0046 0.0124 0.0021 0.0029 0.0031 0.0022 0.0026 0.0022 0.0031 0.0023 0.0017 0.0027 0.0023 0.0034 0.0021 0.0031 0.0025 0.0028 0.0027 0.0016 0.0100 0.0022 0.0028 uncertainty of reproducibility (UR) 0.0026 0.0066 0.0031 0.0055 0.0127 0.0023 0.0047 0.0064 0.0032 0.0027 0.0040 0.0042 0.0144 0.0088 0.0031 0.0036 0.0044 0.0071 0.0034 0.0044 0.0040 0.0028 0.0026 0.0113 0.0029 0.0036 3.5 Aromatic compounds determination in wines from two winemaking procedures To determine volatile compound in real wine samples, the method proposed in this article was applied and results are presented in Table 6. Results are in correlation with previously observed aromatics content in wines and differences due to two winemaking procedures are comparable to other skin contact procedures (Moio et al., 1995; Ortega et al., 2002; Selli et al., 2003; Selli et al., 2006; Rodriguez-Bencomo et al., 2008). Table 6 : Concentrations of individual aromatic compounds in the Zelen wines produced by two different procedures (Zc - control without skin contact, Zp - freezing of pomace), in g/L. Zc wine 1,6-Heptadien-4-ol 1-Hexanol 2-Phenylethyl acetate 4-Ethylguaiacol 4-Vinylguaiacol 4-Vinylphenol Benzaldehyde Benzylalcohol cis-3-Hexen-1-ol Diethyl succinate Ethyl butyrate Ethyl cinnamate Ethyl decanoate Ethyl dodecanoate* Ethyl hexadecanoate* Ethyl hexanoate Ethyl lactate Ethyl octanoate Geraniol Hexyl acetate Isoamyl acetate Nerol n-Hexaldehyde trans-2-Hexen-1-ol -Ionone -Butyrolactone 17 ± 1 1349 ± 67b 466 ± 49b 516 ± 8a 117 ± 5a 2 ± 0a 20 ± 3a 21 ± 1b 112 ± 16a 366 ± 10b 593 ± 6b 37 ± 2a 6 ± 1a 570 ± 16b 4276 ± 475a 1239 ± 56b 275 ± 40b 3260 ± 423b 4 ± 1b 2501 ± 60a Zp wine 12 ± 1a 1118 ± 18a 253 ± 12a 908 ± 53b 354 ± 23b 9 ± 2b 90 ± 9b 18 ± 0a 129 ± 8a 217 ± 11a 443 ± 9a 38 ± 4a 8 ± 1a 409 ± 0a 6350 ± 328b 933 ± 25a 46 ± 5a 1291 ± 97a -a 2569 ± 62a Values are the mean value ± error at 95 % confidence level (n = 3). Significant differences between procedures are indicated a, b at p 0.05. - = not detected * = volatile compounds with low recoveries 4 CONCLUSIONS According to the validation, the method is suitable for the determination of at least 24 volatile compounds in wine (the ones with recoveries >70%). The system is linear with R2 higher than 0.96. Limits of detection range from 0.16 g/L for ethyl butyrate to 4.44 g/L for ethyl octanoate. Limits of quantitative determination range from 0.52 g/L for ethyl butyrate to 14.8 g/L for ethyl octanoate. Recoveries range from 71.1% (ethyl octanoate) to 105.7% (geraniol), except for ethyl dodecanoate (67.8%) and ethyl hexadecanoate (27.9%). Uncertainty of reproducibility ranges from 5.0% for 2phenylethyl acetate to 28.9% for ethyl decanoate. A practical application was checked and presented for Zelen wines from two different winemaking procedures. 5 ACKNOWLEGEMENTS The authors thank those who contributed to the work: Mr. Tomaz Sket and co-workers at the Central Laboratories of the Agricultural Institute of Slovenia. 292 6 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Acta Agriculturae Slovenica de Gruyter

Validation of the method for the determination of some wine volatile compounds

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de Gruyter
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Abstract

Wine aroma is influenced by a number of volatile compounds. This article describes the validation of the method for 26 volatile compounds found in wine. Volatile compounds were determined with discontinuous liquid-liquid extraction and GC-MS detection. It was determined, that the method is linear with square correlation coefficient ranging from 0.961 to 0.999. Limits of quantitative determination range from 0.52 g/L to 14.8 g/L. Recoveries range from 71.1% to 105.7% except for two compounds with lower recoveries. Measurement uncertainty ranges from 5.0% to 28.9%. According to the validation, the method is suitable for the determination of at least 24 volatile compounds common to wine. A practical method application was presented on Zelen wine variety from two different production procedures. Key words: wine, aroma, volatile compounds, GC-MS IZVLECEK VALIDACIJA METODE ZA DOLOCANJE NEKATERIH HLAPNIH SPOJIN V VINU Na aromo vina vplivajo stevilne hlapne spojine. Ta clanek opisuje validacijo metode za 26 hlapnih spojin, ki jih najdemo v vinu. Hlapne spojine so bile dolocene z diskontinuirano ekstrakcijo tekoce-tekoce in GC-MS detekcijo. Dolocili smo, da je metoda linearna, z razponom kvadrata korelacijskega koeficienta od 0,961 do 0,999. Meje kvantitativne dolocitve imajo razpon od 0,52 g/L do 14,8 g/L. Izkoristki imajo razpon od 71,1% do 105,7%, razen za dve spojini, katerih izkoristek je nizji. Merilna negotovost ima razpon od 5,0% do 28,9%. Z ozirom na validacijo lahko potrdimo primernost metode za dolocanje vsaj 24 hlapnih spojin znacilnih za vino. Prakticni prikaz uporabe metode smo predstavili na vinih sorte Zelen iz dveh razlicnih postopkov pridelave. Kljucne besede: vino, aroma, hlapne spojine, GC-MS Prispevek je del doktorske disertacije z naslovom "Vpliv maceracije na aromaticne znacilnosti primorskih belih vin", mentorica prof. dr. Tatjana Kosmerl 1 INTRODUCTION Wine aroma, a very important sensory parameter is produced by a complex balance of several volatiles. More than 800 volatile compounds such as alcohols, esters, phenols, monoterpenes, norisoprenoides, lactones, aldehydes and ketones have been identified in wine (Selli et al., 2004; Tamborra et al., 2004). The wine aroma is complex due to a large number of compounds present and their different chemical nature with a wide range of polarity, volatility, solubility and pH values. Therefore the sample preparation and particularly the extraction and concentration of volatile compound are an important factor in their determination (Cabredo Pinillos et al., 2004). Appropriate extraction of wine volatile compounds must be performed before their detection. Exceptionally so called major wine volatile compounds present in mg/l, like acetaldehyde, ethyl acetate, methanol and higher alcohols are detected directly without previous extraction where samples are only diluted and deacidificated prior to analysis (Peinado et al., 2004; Luki et al., 2008). Agricultural Institute of Slovenia, Hacquetova ulica 17, SI-1000 Ljubljana, Slovenia, Ph.D., e-mail: dejan.bavcar@kis.si, helena.basa@kis.si str. 285 - 293 Extraction of minor volatile compounds, present in wine in g/L, is done today mostly in three different ways. The first is discontinuous or continuous liquid-liquid extraction (LLE) of wine with organic solvent. Both discontinuous and continuous liquid-liquid extractions are suitable to measure volatiles, but to perform a second one special apparatus must be provided and main disadvantages, like time consuming process and large volumes of solvents, are not avoided (Cabredo Pinillos et al., 2004). As solvents mainly dichloromethane (Selli et al., 2003) or mixture of pentane: dichloromethane = 60:40 (Pérez-Coello et al., 2003; Izquierdo et al., 2008) are used. The second approach is solid phase extraction (SPE) using Sep Pack C18 cartridges (Tamborra et al., 2004) or LiChrolut EN resins (Loscos et al., 2010; Sáenz-Navajas et al., 2010). The third approach is Solid Phase Micro Extraction (SPME) with different fibers used: carbowaxdivinylbenzene (Lambropoulos and Roussis et al., 2007; Antalick et al., 2010), polydimethylsiloxane (Nasi et al., 2008; Antalick et al., 2010), polydimethylsiloxane/ divinylbenzene (Nasi et al., 2008; Antalick et al., 2010), carboxen/polydimethylsiloxane (Nasi et al., 2008; Antalick et al., 2010) or divinylbenzene/carboxen/ polydimethylsiloxane (Nasi et al., 2008; Antalick et al., 2010). Detection of volatile compounds is performed by gas chromatograph (GC) coupled with Flame Ionisation Detector (FID) (Pérez-Coello et al., 2003; Selli et al., 2003; Selli et al., 2004; Tamborra et al., 2004; Selli et al., 2006; Loscos et al., 2010) or mass spectrometer (MS) (Pérez-Coello et al., 2003; Selli et al., 2003; Selli et al., 2004; Tamborra et al., 2004; Selli et al., 2006; Lambropoulos and Roussis et al., 2007; Izquierdo Cañas et al., 2008; Nasi et al., 2008; RodriguezBencomo et al., 2008; Loscos et al., 2010; Antalick et al., 2010; Sáenz-Navajas et al., 2010). Quantification can be done with both detectors, while unequivocal identification only by MS. On Agricultural institute of Slovenia we decided to introduce discontinuous liquid-liquid extraction method with dichloromethane, chosen as the most effective organic solvent for this type of extraction (Cabredo Pinillos et al., 2004). The extraction was performed with intention to determine 26 minor volatile compounds with possible sensorial effect in wines (Schneider et al., 1998; Selli et al., 2003; Selli et al., 2006; Luki et al., 2008). Liquid-liquid extraction is actually the oldest but still the reference technique for the extraction of volatile compounds in wine (Ortega et al., 2002). 3-octanol and 4-nonanol were used as internal standards because of their high recovery (Cabredo Pinillos et al., 2004; Selli et al., 2006). By this procedure we achieved concentration factor 100. To enable qualitative and quantitative evaluation at the same time, MS was used for detection. After introduction, method was validated. Method was finally applied to real wine samples (variety Zelen) deriving from an experiment, where two different winemaking procedures were confronted. 2 MATERIALS AND METHODS 2.1 Materials Chemicals: Dichloromethane (Sigma-Aldrich) and ethanol absolute (Merck) with HPLC grade were used like solvents in our experiment, together with ultrapure water from the Milli-Q system. Similarly only the volatile compounds (Merck, SigmaAldrich, Fluka, SAFC) with the highest available purity on market (minimum of 95 %) were used with the exception of 4vinylphenol (SAFC) only sold like 10 % solution. Preparation of solutions: Stock solutions in pure dichloromethane of individual volatiles were prepared in 50 ml volumetric flasks with concentrations ranging from 1.8 ­ 2.5 g/L. From 26 stock solutions one mix solution of all 26 volatiles was prepared in 200 mL volumetric flask. All other solutions used to determine linearity, limits of detection and limits of quantification were prepared from this mix solution with proper dilutions. Internal standards 3-octanol and 4-nonanol for those dichloromethane solutions were prepared in 100 mL volumetric flask with dissolving them in quantity of 1.1 ­ 1.2 g/L in dichloromethane. They were added using 0.05 mL Hamilton syringe to 10 mL of dichloromethane solutions and mixed before determination. Preparation of model wine solution: First a mix stock solution of all volatiles in 100 % pure ethanol was prepared, with individual volatiles concentrations in range of 0.8 ­ 1.2 g/L. Stock solution was adequately diluted to model solution (mix) using 12 %vol ethanol in water to concentrations similar to ones determined in wines in average, to 0.04 ­ 0.07 mg/L, in 3000 ml volumetric flask. The pH was then adjusted to pH 3.2 with tartaric acid addition. Model wine solution was finally dispensed in twenty 125 mL flasks and they were stored in dark at 7 ºC before extraction. Internal standards 3-octanol and 4-nonanol used in our model wine solution were prepared in 100 mL volumetric flask with dissolving them in quantity of 0.04 ­ 0.06 g in ethanol absolute. They were added using 0.05 mL Hamilton syringe to model wine solution only during extraction process as described below. 286 Validation of the method for the determination of some wine volatile compounds 2.2 Procedure Liquid-liquid extraction of volatile compounds: 100 mL of model wine solution was transferred into 250 ml Erlenmeyer flask and cooled to 0 ºC in an ice bath under nitrogen. 29 g of 3-octanol and 23 g of 4-nonanol were added as internal standards using 0.05 mL Hamilton syringe from corresponding ethanol solutions. Dichloromethane (40 mL) was added and the mixture was stirred at 350 min-1 for 20 minutes (Moio et al., 1995). Then the mixture was centrifuged at 5°C (RFC = 8500, 10 minutes) and organic phase was 2.3 Determination Chromatographic conditions of GC (HP 6890)-MS (HP 5973) system: Liner Injector temperature Injection type Precolumn Column Temperature gradient Ion source temperature Auxiliary temperature Detector temperature Carrier gas Injection volume Detection Agilent 5062-3587 200 ºC Pulsed Splitless 2 m * 0.25 mm Varian, CP-WAX 57CB, 50 m x 0.25 mm ID 40 ºC; 12 min 5 ºC/min; from 40 ºC to 200 ºC 200 ºC; 20 min 230 ºC 200 ºC 150 ºC Helium 6.0; constant flow 1.0 ml/min 1 l Selective Ion Monitoring (T, Q1, Q2, Q3): 1,6-Heptadien-4-ol (71,43) 1-Hexanol (56, 43, 55, 69) 2-Phenylethyl acetate (104, 43, 91) 3-Octanol (59, 83, 101) 4-Ethylguaiacol (2-Methoxy-4-ethylphenol )(137, 152) 4-Nonanol (55, 73, 83, 101) 4-Vinylguaiacol (2-Methoxy-4-vinylphenol) (150, 135, 107, 77) 4-Vinylphenol (120, 91) Benzaldehyde (77, 105, 106) Benzyl alcohol (79, 108, 107) cis-3-hexen-1-ol (67, 41, 82) Diethyl succinate (101, 129) Ethyl butyrate (Ethyl butanoate)(71, 43, 88) Ethyl cinnamate (131, 103, 176) Ethyl decanoate (Ethyl caprate) (88, 101, 155) Ethyl dodecanoate (Ethyl laurate) (88, 101) Ethyl hexadecanoate (Ethyl palamitate) (88, 101) Ethyl hexanoate (88, 99) Ethyl lactate (45, 75) Ethyl octanoate (Ethyl caprylate) (88, 101, 57) Geraniol (69, 93, 123) Hexyl acetate (43, 56) Isoamyl acetate (70, 43, 55) Nerol (69, 84, 93) n-Hexaldehyde (Capronaldehyde) (56, 44, 57) trans-2-hexen-1-ol (55, 69, 83) -Ionone (177, 43) -Butyrolactone (42, 56, 86) recovered. The aqueous phase was re-extracted twice in the same way described above. Finally organic phases were combined and dried over sodium sulphate. They were concentrated to a final volume of 1 mL with Vigreaux distillation column and nitrogen gas flow prior to GC-MS analysis (Schneider et al., 1998). The same procedure was used for the extraction of wine samples (Moio et al., 1995; Schneider et al., 1998; Selli et al., 2006). 2.4 Aromatic compounds determination in wines from two winemaking procedures - preparation of wine samples Healthy grapes of Zelen variety (40 kg) were manually harvested in 2008 at the ripeness stage corresponding to wines containing approximately 12 % vol ethanol. Grapes were divided in two equal parts. First half of grapes (Zc = control without skin contact) was immediately destemmed, crushed and pressed up to 150 kPa using a small water press (Lancman VS-A 55, Slovenia). The juice was sulphited with 30 mg/L of sulphur dioxide, left to settle at 6ºC for 12 hours, racked and divided in three glass laboratory fermentor vessels with 1.6 L juice each. The vessels were heated to 17ºC, inoculated with 0.2 g/L of dried Saccharomyces cerevisiae (CM, Lallemand), supplemented with 0.2 g/L complex yeast nutrient (Fermaid E, Lallemand) and fermented at 17ºC. After alcoholic fermentations (residual sugars <2.5 g/L) and when most of the lees had settled, the wines were racked, 50 mg/L of sulphur dioxide was added and the wines were stored at 10ºC. The second half of grapes (Zp = freezing of the pomace) was destemmed and crushed. The pomace was equally divided in three plastic vessels, frozen overnight at ­20ºC, defrozen at 20ºC and pressed up to 150 kPa. The juice from the individual plastic vessels was sulphited with 30 mg/L of sulphur dioxide, left to settle at 6ºC for 12 hours, racked and poured in 3 glass laboratory fermentor vessels with 1.6 L juice each. The remaining procedure to obtain wines was the same as described previously. In this way, two different types of Zelen wines (Zc, Zp) in three repetitions were obtained. 3 RESULTS AND DISCUSSION 3.1 Linearity, limits quantification of detection, limits of quantification (LOQ) were calculated from calibration curve and are presented in Table 1. the Linearity was verified by using the solutions of volatile compounds in dichloromethane (five repetitions for one concentration level, three to eight concentration levels for the calibration curve). Linearity and range were determined by linear regression, using the F test. Linear model is fit and remains linear over the range presented in Table 1. Limits of detection (LD) and limits of Linearity was verified for wider range also and is presented in Table 2. Concentration factor for wine samples was due to extraction 100, so realistic linearity range, LDs and LOQs are 100-times lower. Table 1: Linearity, limits of detection, limits of quantification linearity (mg/L) 0.12 - 3.35 0.19 - 3.35 0.0168 - 3.35 0.0058 - 3.35 0.05 - 3.35 0.05 - 3.35 0.0111-1.67 0.0116 - 1.67 1.67 - 10.13 0.022 - 3.35 0.0167 - 1.67 0.05 - 3.35 0.05 - 1.67 0.01 - 3.35 0.0139 - 1.67 0.0092 - 3.35 0.0099 - 1.67 0.1 - 9.51 0.011 - 3.35 0.0092 - 1.67 0.05 - 3.35 0.0058 - 3.35 0.06 - 3.35 0.05 - 3.35 0.009 - 3.35 0.024 - 3.35 R2 0.999 0.999 0.999 0.996 0.998 0.998 0.993 0.994 0.996 0.996 0.999 0.998 0.995 0.996 0.995 0.996 0.991 0.992 0.996 0.990 0.998 0.996 0.983 0.982 0.995 0.997 LD (mg/L) 0.062 0.067 0.030 0.051 0.062 0.061 0.041 0.038 0.352 0.066 0.016 0.056 0.056 0.065 0.034 0.053 0.044 0.444 0.065 0.048 0.059 0.056 0.175 0.179 0.074 0.055 LOQ (mg/L) 0.206 0.224 0.099 0.171 0.208 0.203 0.136 0.126 1.174 0.218 0.052 0.186 0.186 0.216 0.115 0.176 0.148 1.480 0.216 0.159 0.196 0.187 0.582 0.597 0.248 0.183 288 Table 2: Linearity, wider range linearity (mg/L) 0.12 - 11.92 0.19 - 19.4 0.0168 - 33.53 0.0058 - 11.5 0.05 - 9.36 0.05 - 9.08 0.0111-22.18 0.0116 - 23.14 1.67 - 10.13 0.022 - 21.62 0.0167 - 33.45 0.05 - 10.72 0.05 - 9.55 0.01 - 10.08 0.0139 - 27.7 0.0092 - 18.45 0.0099 - 10.79 0.1 - 9.51 0.011 - 10.8 0.0092 - 18.31 0.05 - 10.79 0.0058 - 11.57 0.06 - 11.31 0.05 - 3.35 0.009 - 9.32 0.024 - 24.06 R2 0.961 0.988 0.995 0.987 0.978 0.977 0.994 0.994 0.996 0.993 0.999 0.983 0.977 0.978 0.973 0.989 0.986 0.992 0.985 0.989 0.983 0.988 0.998 0.982 0.980 0.994 3.2 Trueness Trueness was verified by checking the recoveries. Two parallel extracts of model wine solution were prepared each day for ten days and injected once respectively. The average of recoveries was calculated. The results are given in Table 3. 3.3 Precision For the determination of precision (ISO 5725), i.e. repeatability and reproducibility, extracts of model wine solution was analysed (the same as for recovery evaluation). Within the period of 10 days two parallel extracts were prepared each day. Each was injected once. Then standard deviation of repeatability of the level and standard deviation of reproducibility of the level were both calculated. The results are given in Table 4. Table 3: Recoveries for model wine solution conc. in model wine solution (mg/L) 0.0435 0.0596 0.0614 0.2930 0.0543 0.2300 0.0692 0.0414 0.0713 0.0672 0.0566 0.0498 0.0599 0.0697 0.0500 0.0624 0.0524 0.0588 0.0709 0.0573 0.0495 0.0593 0.0604 0.0540 0.0433 0.0548 0.0564 0.0609 recovery (%) 84.4 98.3 91.9 85.9 92.3 87.1 95.0 98.2 95.0 94.2 84.0 91.3 77.5 95.1 81.9 67.8 27.9 76.7 79.4 71.1 105.7 80.6 78.0 96.5 82.0 102.4 89.2 88.1 RSD (%) 3.1 4.9 2.5 3.1 4.2 2.8 8.9 2.6 3.0 4.6 3.0 2.7 3.8 2.9 15.2 8.9 9.5 3.4 3.5 7.4 3.0 4.0 3.8 2.4 3.3 8.2 2.6 3.1 1,6-Heptadien-4-ol 1-Hexanol 2-Phenylethyl acetate 3-Octanol 4-Ethylguaiacol 4-Nonanol 4-Vinylguaiacol 4-Vinylphenol Benzaldehyde Benzylalcohol cis-3-Hexen-1-ol Diethyl succinate Ethyl butyrate Ethyl cinnamate Ethyl decanoate Ethyl dodecanoate Ethyl hexadecanoate Ethyl hexanoate Ethyl lactate Ethyl octanoate Geraniol Hexyl acetate Isoamyl acetate Nerol n-Hexaldehyde trans-2-Hexen-1-ol -Ionone -Butyrolactone Table 4: Standard deviation of repeatability and reproducibility of the method, in mg/L conc. in model wine solution (mg/L) 0.0435 0.0596 0.0614 0.0543 0.0692 0.0414 0.0713 0.0672 0.0566 0.0498 0.0599 0.0697 0.0500 0.0624 0.0524 0.0588 0.0709 0.0573 0.0495 0.0593 0.0604 0.0540 0.0433 0.0548 0.0564 0.0609 means of the levels (mg/L) 0.0367 0.0583 0.0563 0.0501 0.0659 0.0406 0.0676 0.0632 0.0475 0.0455 0.0463 0.0662 0.0404 0.0419 0.0147 0.0449 0.0562 0.0405 0.0524 0.0476 0.0470 0.0521 0.0354 0.0559 0.0503 0.0536 standard deviation of repeatability (sr) 0.0007 0.0011 0.0012 0.0020 0.0055 0.0009 0.0013 0.0014 0.0010 0.0012 0.0010 0.0014 0.0010 0.0008 0.0012 0.0010 0.0015 0.0009 0.0014 0.0011 0.0012 0.0012 0.0007 0.0044 0.0010 0.0012 standard deviation of reproducibility (sR) 0.0011 0.0029 0.0014 0.0024 0.0056 0.0010 0.0021 0.0028 0.0014 0.0012 0.0018 0.0019 0.0064 0.0039 0.0014 0.0016 0.0020 0.0031 0.0015 0.0019 0.0017 0.0012 0.0012 0.0050 0.0013 0.0016 290 3.4 Uncertainty of repeatability and uncertainty of reproducibility Uncertainty of repeatability and uncertainty of reproducibility were calculated by multiplying standard deviation of repeatability and standard deviation of reproducibility by Student's t factor for 9 degrees of freedom and 95% confidence level (t95;9 = 2.262). Ur = t95; 9 x sr ; UR = t95; 9 x sR The results are presented in Table 5. Table 5: Uncertainty of repeatability and reproducibility of the method, in mg/L conc. in model wine solution (mg/L) 0.0435 0.0596 0.0614 0.0543 0.0692 0.0414 0.0713 0.0672 0.0566 0.0498 0.0599 0.0697 0.0500 0.0624 0.0524 0.0588 0.0709 0.0573 0.0495 0.0593 0.0604 0.0540 0.0433 0.0548 0.0564 0.0609 uncertainty of repeatability (Ur) 0.0016 0.0025 0.0026 0.0046 0.0124 0.0021 0.0029 0.0031 0.0022 0.0026 0.0022 0.0031 0.0023 0.0017 0.0027 0.0023 0.0034 0.0021 0.0031 0.0025 0.0028 0.0027 0.0016 0.0100 0.0022 0.0028 uncertainty of reproducibility (UR) 0.0026 0.0066 0.0031 0.0055 0.0127 0.0023 0.0047 0.0064 0.0032 0.0027 0.0040 0.0042 0.0144 0.0088 0.0031 0.0036 0.0044 0.0071 0.0034 0.0044 0.0040 0.0028 0.0026 0.0113 0.0029 0.0036 3.5 Aromatic compounds determination in wines from two winemaking procedures To determine volatile compound in real wine samples, the method proposed in this article was applied and results are presented in Table 6. Results are in correlation with previously observed aromatics content in wines and differences due to two winemaking procedures are comparable to other skin contact procedures (Moio et al., 1995; Ortega et al., 2002; Selli et al., 2003; Selli et al., 2006; Rodriguez-Bencomo et al., 2008). Table 6 : Concentrations of individual aromatic compounds in the Zelen wines produced by two different procedures (Zc - control without skin contact, Zp - freezing of pomace), in g/L. Zc wine 1,6-Heptadien-4-ol 1-Hexanol 2-Phenylethyl acetate 4-Ethylguaiacol 4-Vinylguaiacol 4-Vinylphenol Benzaldehyde Benzylalcohol cis-3-Hexen-1-ol Diethyl succinate Ethyl butyrate Ethyl cinnamate Ethyl decanoate Ethyl dodecanoate* Ethyl hexadecanoate* Ethyl hexanoate Ethyl lactate Ethyl octanoate Geraniol Hexyl acetate Isoamyl acetate Nerol n-Hexaldehyde trans-2-Hexen-1-ol -Ionone -Butyrolactone 17 ± 1 1349 ± 67b 466 ± 49b 516 ± 8a 117 ± 5a 2 ± 0a 20 ± 3a 21 ± 1b 112 ± 16a 366 ± 10b 593 ± 6b 37 ± 2a 6 ± 1a 570 ± 16b 4276 ± 475a 1239 ± 56b 275 ± 40b 3260 ± 423b 4 ± 1b 2501 ± 60a Zp wine 12 ± 1a 1118 ± 18a 253 ± 12a 908 ± 53b 354 ± 23b 9 ± 2b 90 ± 9b 18 ± 0a 129 ± 8a 217 ± 11a 443 ± 9a 38 ± 4a 8 ± 1a 409 ± 0a 6350 ± 328b 933 ± 25a 46 ± 5a 1291 ± 97a -a 2569 ± 62a Values are the mean value ± error at 95 % confidence level (n = 3). Significant differences between procedures are indicated a, b at p 0.05. - = not detected * = volatile compounds with low recoveries 4 CONCLUSIONS According to the validation, the method is suitable for the determination of at least 24 volatile compounds in wine (the ones with recoveries >70%). The system is linear with R2 higher than 0.96. Limits of detection range from 0.16 g/L for ethyl butyrate to 4.44 g/L for ethyl octanoate. Limits of quantitative determination range from 0.52 g/L for ethyl butyrate to 14.8 g/L for ethyl octanoate. Recoveries range from 71.1% (ethyl octanoate) to 105.7% (geraniol), except for ethyl dodecanoate (67.8%) and ethyl hexadecanoate (27.9%). Uncertainty of reproducibility ranges from 5.0% for 2phenylethyl acetate to 28.9% for ethyl decanoate. A practical application was checked and presented for Zelen wines from two different winemaking procedures. 5 ACKNOWLEGEMENTS The authors thank those who contributed to the work: Mr. Tomaz Sket and co-workers at the Central Laboratories of the Agricultural Institute of Slovenia. 292 6

Journal

Acta Agriculturae Slovenicade Gruyter

Published: Sep 1, 2011

References