Comparison of extraction methods for volatile compounds of Muscat grape juice

Talanta 79 (2009) 871–876

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Comparison of extraction methods for volatile compounds of Muscat grape juice ˜ a , M.C. Díaz-Maroto a,b,∗ , M.A. González-Vinas ˜ a , M.S. Pérez-Coello a E. Sánchez-Palomo a , M.E. Alanón a b

Área de Tecnología de los Alimentos, Facultad de Ciencias Químicas, Campus Universitario, s/n, Universidad de Castilla-La Mancha, 13071 Ciudad Real, Spain IRICA (Instituto Regional de Investigación Científica Aplicada) Universidad de Castilla-La Mancha, Campus Universitario, s/n, 13071 Ciudad Real, Spain

a r t i c l e

i n f o

Article history: Received 4 February 2009 Received in revised form 4 May 2009 Accepted 13 May 2009 Available online 22 May 2009 Keywords: Gas chromatography–mass spectrometry Extraction techniques Volatile compounds Muscat grape juice

a b s t r a c t Typical flavour of Muscat d’Alexandrie wines is mainly due to volatile compounds coming from grapes of this variety. Therefore, the choice of grapes is crucial to obtain musts with a great aromatic potential, which will contribute to the final aroma of wines derived from those musts. In this study, three sampling techniques, liquid–liquid extraction (LLE), solid phase extraction (SPE) and simultaneous distillationextraction (SDE), were compared for the analysis of volatile compounds in Muscat grape juice. Results showed that although the three techniques can be recommended for the quantitative analysis of volatile compounds from musts, LLE and SPE are better sample preparation techniques than SDE, mainly for determination of polar compounds such as acids or alcohols. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Aroma is one of the major factors that determine the nature and quality of wines. The volatile compounds derived from grapes are the main responsible for the varietal aroma of wines and they are influenced by numerous factors such as the grape variety, culture practices, soil type, climate, etc. [1,2]. These varietal aromas are very important in the enjoyment and appreciation of wine and hence in its economic valuation. Therefore, the choice of grapes is crucial to obtain musts with a great aromatic potential which will have a strong influence on the quality of wines. However, the volatile compounds responsible for the varietal aroma are present in only trace amounts, which means that to carry on their identification and quantification, an effective method of enrichment is required prior to their analysis by gas chromatography–mass spectrometry (GC–MS). Different extraction methods have been established for this purpose. Classical liquid–liquid extraction based on organic solvent extraction is certainly one of the most frequently reported methods in the literature for the isolation of volatile compounds in musts [3–7]. An advantage of this method is that all volatile compounds (low, medium and high volatility) can be analysed in one extraction step. Nevertheless, because LLE requires large amounts of high purity solvents, this technique is relatively tedious and time-consuming. Furthermore, extracts have to be concen-

∗ Corresponding author at: IRICA (Instituto Regional de Investigación Científica Aplicada) Universidad de Castilla-La Mancha, Campus Universitario, s/n, 13071 Ciudad Real, Spain. Tel.: +34 926 295300x3423; fax: +34 926 295318. E-mail address: [email protected] (M.C. Díaz-Maroto). 0039-9140/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.talanta.2009.05.019

trated. This step can bring about the loss of certain compounds as well as artefact formation and eventual interferences resulting from solvent impurities in chromatographic analysis [3–5]. However, in the last few years, numerous efforts have been made to diminished these disadvantages of LLE method, in particular with the introduction of liquid–liquid microextraction (LLEM) technique which has already been applied to volatiles analysis in grapes [8,9]. Other extraction technique which has been successfully applied to must aroma components is solid-phase extraction (SPE) [10–14]. This technique based on adsorbent materials where analytes are bound to active sites on a surface, allows the determination of a wide range of volatile compounds, requires smaller quantities of solvents and shorter time of analyses but is relatively tedious. SPE has been successfully used to study the evolution of aromatic compounds of grapes during ripening [15] and to determine the potential aroma in several varieties of Spanish grapes [16]. The simultaneous steam distillation-solvent extraction technique (SDE), proposed by Godefroot et al. [17], has been applied to grape juice in order to extract the volatile aroma components [6,18,19]. Several authors have found some limitations of the SDE technique like the low recovery of the most volatile compounds and the loss and thermal degradation of certain compounds [19–21]. Nevertheless, SDE technique achieves generally higher recoveries of volatile compounds than other isolation techniques such as headspace [22] or stir bar sorptive extraction (SBSE) [19]. Headspace techniques have also been used with the purpose of carrying out the isolation of volatile compounds from musts by means of purge and trap [6,23] or by means of SPME [24,25]. These techniques are less sensitive for the isolation of low volatile

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Table 1 Calibration curves and performance characteristics obtained by LLE (n = 5). Compounds (E)-2-Hexenal 1-Hexanol (E)-3-Hexen-1-ol (Z)-3-Hexen-1-ol (E)-2-Hexen-1-ol (Z)-Linalool oxide furan (E)-Linalool oxide furan Benzaldehyde Linalool ␣-Terpineol ␤-Citronellol Nerol Geraniol Benzyl alcohol Phenylethyl alcohol

Linearity range (mg/L) 0.01–0.40 0.20–6.00 0.01–0.33 0.01–0.33 0.10–3.03 0.01–0.35 0.01–0.35 0.01–0.45 0.05–1.56 0.01–0.33 0.02–0.46 0.02–0.66 0.02–0.57 0.05–1.43 0.06–1.69

Slope

Intercept

LOL (%)

r2

Detection limit (␮g/L)

Quantification limit (␮g/L)

RSD (%)

0.43 0.68 0.78 0.75 0.57 0.64 0.42 0.82 0.89 1.05 1.11 0.88 0.72 1.28 1.11

0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 -0.02 0.00

97.89 98.43 97.83 98.74 97.52 98.95 98.72 98.27 98.35 98.97 98.35 99.29 99.35 98.30 98.33

0.998 0.998 0.999 0.998 0.997 0.999 0.998 0.997 0.997 0.999 0.995 0.999 0.999 0.997 0.997

0.02 0.01 0.01 0.01 0.01 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01

0.09 0.06 0.05 0.07 0.06 0.10 0.05 0.04 0.04 0.04 0.04 0.04 0.05 0.03 0.04

2.0 5.2 0.5 1.6 3.9 8.2 7.9 2.6 5.8 4.7 3.2 6.2 4.1 6.4 2.7

compounds and the results obtained are difficult to relate to the real concentration of compounds in the matrix. Stir bar sorptive extraction (SBSE) is one of the latest techniques developed. It has been applied to volatile constituents of musts by several authors [19,26–28]. Although it has been demonstrated that relative recoveries and reproducibility are generally lower for the SBSE technique in the analysis of volatile grape juice, it seems to be a more sensitive technique than SDE [19]. In order to select the best extraction technique for studying the volatile composition of musts, three different classical extraction techniques, LLE, SPE and SDE were evaluated for qualitative and quantitative determination of volatile components of a Muscat grape juice. 2. Experimental 2.1. Reagents and standards The chemical standards were supplied by Aldrich (Gillingham, U.K.), Fluka (Buchs, Switzerland) and Sigma (St. Louis, MO). LiChrolut EN resins, prepacked in 500 mg cartridges (6 mL total volume) or in bulk, were obtained from Merck (Darmstadt, Germany). Dichloromethane and methanol, GC-quality, was obtained from Scharlau (Barcelona, Spain); absolute ethanol glucose and fructose were from Panreac, (Barcelona, Spain). Pure water was obtained from a Milli-Q purification system (Millipore, U.S.A.). 2.2. Must samples Commercial Muscat d’Alexandrie grape samples were purchased from a local store. Grapes were gently pressed manually

(under nitrogen atmosphere), with a rapid skin separation and with the addition of SO2 (80 mg/L). 2.3. Isolation and concentration techniques 2.3.1. Liquid–liquid extraction 200 mL of sample was extracted continuously with 100 mL of n-pentane-dichloromethane (60/40, v/v) in downwards displacement extractors for 12 h using 4-nonanol as internal standard. The extract was then concentrated in a water bath using a 50 cm Vigreux column to a volume of 1 mL and then under stream of nitrogenous to a volume of 200 ␮L. 2.3.2. Solid phase extraction Volatile compounds were extracted using the method developed by Günata et al. [11]. One hundred millilitres (100 mL) were fractionated on preconditioned polypropylene-divinylbenzene cartridges (Lichrolut EN (40–120 ␮m), Merck, 0.5 g of phase) using 4-nonanol as internal standard with subsequent elution with 10 mL of dichloromethane. Extracts were concentrated under nitrogenous stream to a volume of 200 ␮L. 2.3.3. Simultaneous distillation-extraction Enrichment of the sample was achieved using a micro scale simultaneous distillation extraction apparatus (Chrompack Middelburg, The Netherlands) in the high-density solvent configuration. 70 mL volume of sample was used for analysis, using 4-nonanol as internal standard and 2 mL of dichloromethane as extractive solvent. The sample temperature was maintained at 110 ◦ C with a silicon bath at pressure atmospheric and the temperature of cold finger was −3 ◦ C. The extraction was considered to be

Table 2 Calibration curves and performance characteristic obtained by SPE (n = 5). Compounds (E)-2-Hexenal 1-Hexanol (E)-3-Hexen-1-ol (Z)-3-Hexen-1-ol (E)-2-Hexen-1-ol (Z)-Linalool oxide furan (E)-Linalool oxide furan Benzaldehyde Linalool ␣-Terpineol ␤-Citronellol Nerol Geraniol Benzyl alcohol Phenylethyl alcohol

Linearity range (mg/L) 0.01–0.40 0.20–6.00 0.01–0.33 0.01–0.33 0.10–3.03 0.01–0.35 0.01–0.35 0.01–0.45 0.05–1.56 0.01–0.33 0.02–0.46 0.02–0.66 0.02–0.57 0.05–1.43 0.06–1.69

Slope

Intercept

LOL (%)

r2

Detection limit (␮g/L)

Quantification limit (␮g/L)

RSD (%)

0.41 0.55 0.52 0.62 0.59 0.50 0.50 0.89 0.98 1.14 1.27 1.03 0.98 1.09 1.30

0.01 0.11 0.01 0.00 −0.09 0.00 0.00 0.00 0.00 0.00 −0.04 0.00 −0.02 0.00 −0.06

98.44 98.90 97.91 98.37 98.98 97.78 98.03 97.99 98.18 97.74 98.88 97.71 97.97 98.19 99.14

0.996 0.998 0.992 0.997 0.998 0.993 0.996 0.996 0.996 0.995 0.995 0.981 0.993 0.997 0.985

0.04 0.04 0.04 0.03 0.03 0.04 0.04 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02

0.07 0.05 0.06 0.05 0.05 0.06 0.06 0.03 0.03 0.03 0.02 0.03 0.03 0.03 0.03

5.6 6.4 6.2 6.1 7.2 3.5 7.0 6.9 9.5 3.7 2.5 5.9 8.6 7.4 2.0

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Table 3 Calibration curves and performance characteristic obtained by SDE (n = 5). Compounds

Linearity range (mg/L)

(E)-2-Hexenal 1-Hexanol (E)-3-Hexen-1-ol (Z)-3-Hexen-1-ol (E)-2-Hexen-1-ol (Z)-Linalool oxide furan (E)-Linalool oxide furan Benzaldehyde Linalool ␣-Terpineol ␤-Citronellol Nerol Geraniol Benzyl alcohol Phenylethyl alcohol

0.01–0.40 0.20–6.00 0.01–0.33 0.01–0.33 0.10–3.03 0.01–0.35 0.01–0.35 0.01–0.45 0.05–1.56 0.01–0.33 0.02–0.46 0.02–0.66 0.02–0.57 0.05–1.43 0.06–1.69

Slope

Intercept

LOL (%)

r2

Detection limit (␮g/L)

Quantification limit (␮g/L)

RSD (%)

0.50 0.54 0.81 0.51 0.48 0.44 0.42 0.77 0.86 1.18 1.10 0.65 0.59 0.20 0.40

0.01 0.00 0.00 0.00 0.00 0.00 0.01 0.02 0.00 0.00 0.02 0.02 0.00 −0.02 0.00

99.17 99.03 99.33 99.03 99.35 99.28 99.52 98.34 98.91 98.57 98.16 99.45 99.16 99.44 96.17

0.999 0.999 0.999 0.999 0.999 0.999 0.999 0.997 0.999 0.998 0.995 0.999 0.997 0.998 0.998

0.03 0.03 0.02 0.03 0.03 0.04 0.04 0.02 0.02 0.01 0.01 0.07 0.03 0.08 0.05

0.04 0.03 0.02 0.03 0.04 0.04 0.04 0.02 0.02 0.01 0.02 0.20 0.03 0.08 0.05

5.4 2.6 5.1 3.7 2.2 7.5 0.8 7.3 6.9 6.2 2.3 10.2 5.6 4.0 5.0

complete in 2 h. Extracts were then concentrated under nitrogenous stream to a volume of 200 ␮L. 2.4. Synthetic must solutions Calibration curves were obtained using LLE, SPE and SDE, and subsequent injection onto the GC-system, in duplicate, of synthetic must solutions (100 g of glucose and 100 g of fructose in 1 L of MilliQ water, pH adjusted to 3.5 with tartaric acid), containing defined amounts of the compounds of interest, at six increasing concentrations included in the ranges in which each compounds are normally found in grape musts [1,2]. The ranges of concentrations of defined amounts of compounds are shown in Tables 1–3.

Peak identifications were based on comparison of their mass fragmentation with those of pure standards and/or with those reported by commercial libraries such as Wiley A and NBS75K. The quantitative analysis of positively identified compounds was performed by total ion current using the calibration curves proposed for each method. 2.6. Statistical analysis The Student–Newman–Keuls test was applied to discriminate among the chemical data. Statistical processing was carried out by using the SPSS 14.0 for Windows statistical package. 3. Results and discussion

2.5. GC–MS analysis

3.1. Calibration curves and performance characteristics

An Agilent gas chromatograph model 6890 N coupled to a mass selective detector model 5973 inert was used. An amount of 1 ␮L of extract was injected in splitless mode on a BP-21 capillary column (50 m × 0.32 mm i.d.; 0.32 ␮m film thickness). Oven temperature program was: 70 ◦ C (5 min)–1 ◦ C/min–95 ◦ C (10 min)–2 ◦ C/min–190 ◦ C (40 min). Injector and transfer line temperatures were 250 ◦ C and 280 ◦ C, respectively. Mass detector conditions were: electronic impact (EI) mode at 70 eV; source temperature: 178 ◦ C; scanning rate: 1 scan/s; mass acquisition range: 40–450 amu. As carrier gas was used helium under a flow rate of 0.7 mL/min.

To study the linear character and the performance characteristics of the three extraction methods tested (LLE, SPE and SDE), six solutions containing defined amounts of synthetic volatile compounds normally found in grape musts (Tables 1–3) were prepared in synthetic must. The volatile compounds selected together with the ranges of linearity and performance parameters of calibration curves of each method are summarized in Tables 1–3. Each compound was added in the concentration range expected in grape musts. Linear range covered the volatile compound concentrations expected in the samples since good regression coefficients (r2 ) were

Table 4 Regression analysis of LLE compared with SPE and SDE. Compounds

(E)-2-Hexenal 1-Hexanol (E)-3-Hexen-1-ol (Z)-3-Hexen-1-ol (E)-2-Hexen-1-ol (Z)-Linalool oxide furan (E)-Linalool oxide furan Benzaldehyde Linalool ␣-Terpineol ␤-Citronellol Nerol Geraniol Benzyl alcohol Phenylethyl alcohol *

Significant differences.

LLE-SPE

LLE-SDE

r2

Slope

0.998 0.997 0.992 0.998 0.992 0.992 0.991 0.993 0.994 0.997 0.999 0.997 0.999 0.997 0.998

0.978 0.997 1.030 0.994 1.009 0.983 1.022 0.975 1.015 1.035 0.987 1.022 0.993 1.015 1.023

Confidence limits for the slope 0.946–1.011 0.938–1.017 0.962–1.097 0.961–1.026 0.944–1.075 0.922–1.048 0.951–1.093 0.902–1.048 0.956–1.074 0.998–1.073 0.962–1.012 0.983–1.061 0.971–1.015 0.976–1.055 0.991–1.051

Intercept

Confidence limits for the intercept

r2

Slope

0.013 0.142 0.004 0.0002 0.009 0.002 0.011 −0.007 −0.019 0.077 −0.003 −0.008 0.001 0.007 0.015

−0.006–0.032 0.036–0.248* −0.006–0.014 −0.004–0.004 −0.078–0.097 −0.008–0.012 −0.001–0.022 −0.022–0.008 −0.059–0.022 0.0131–0.002* −0.008–0.002 −0.020–0.003 −0.005–0.006 −0.017–0.031 −0.009–0.039

0.997 0.995 0.995 0.998 0.992 0.999 0.999 0.990 0.994 0.999 0.998 0.999 0.999 0.999 0.994

0.973 0.962 1.024 0.994 1.015 0.975 1.007 0.999 0.989 1.016 0.988 1.029 1.020 0.990 0.996

Confidence limits for the slope 0.935–1.015 0.912–1.012 0.970–1.077 0.962–1.026 0.949–1.082 0.972–1.034 0.982–1.038 0.927–1.071 0.934–1.045 0.988–1.043 0.956–1.020 1.013–1.045* 0.998–1.042 0.970–1.009 0.932–1.048

Intercept

Confidence limits for the intercept

0.002 0.095 −0.005 0.0002 −0.026 −0.001 −0.001 −0.004 0.004 −0.003 0.019 0.001 0.001 −0.012 0.003

−0.005–0.009 −0.039–0.228 −0.013–0.003 −0.004–0.004 −0.115–0.060 −0.021–0.005 −0.005–0.005 −0.019–0.010 −0.034–0.043 −0.008–0.0002 0.012–0.025* −0.003–0.006 −0.005–0.006 −0.020–1.20* −0.041–0.046

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Fig. 1. Total ion chromatogram from must grapes of Muscat obtained by SPE. Time scale in minutes. For peak identification see Table 5.

obtained for all methods in all cases. These results were also corroborated by the term “limit of linearity”, the concentration at which the calibration curve departs from linearity” (LOL) which displayed in all cases values superior to 95% (Tables 1–3). This parameter is obtained by the following equation in which RSDb is the relative standard deviation of the slope expressed as a percentage. LOL (%) = 100 − RSDb Detection limit (calculated as three times the standard deviation of the noise of the baseline) and the quantification limit (calculated as ten times the standard deviation of the noise of the baseline) obtained by interpolation of calibrated curves of each compound analyzed in the three extraction techniques were in all cases inferior to the minimum concentration found in musts [1,2]. The low detection and quantification limits observed, revealed that the three techniques have a high sensitivity and they are suitable for the quantitative analysis. In general, the detection limits of volatile compounds extracted by LLE were lower than those detected in SDE and SPE techniques. Meanwhile, the quantification limits of certain compounds such as linalyl oxides and (E)-2-hexanal extracted by LLE and SPE were slightly higher than those of the rest of the compounds. The quantification limits of nerol, benzyl alcohol and phenylethyl alcohol calculated for the SDE were also slightly higher probably due to the lower volatility of these compounds [29]. To study the reproducibility of the three extraction methods, five analyses of the same synthetic must solution containing defined amounts of volatile components were carried out. Tables 1–3 show, respectively, the relative standard deviation (RSD) obtained for each compound with the three extraction techniques evaluated. The reproducibility of the three methods were satisfactory since RSDs for all compounds studied ranged from 0.5 to 10.2%. In order to compare the three extraction methods tested, a regression analysis were made between data obtained for each point of the calibrated curve with each compounds analyzed by means of LLE and SPE, as well as by means of LLE and SDE. Table 4 shows the coefficient of determination (r2 ), slopes and intercepts calculated. In no cases were ideal results obtained (intercept equal to zero and slope equal to one), which is the reason why the confidence limits for both were calculated (˛ = 0.05). In order to carry out these operations, the standard deviation of the slope and intercept were calculated determining the statistical Sy/x previously.

If the value 1 is included in the confidence interval belonging to the slope, it can be considered that slope is not significantly different from 1. Similarly, if the value 0 is included in the confidence interval belonging to the intercept, it is possible to assume that intercept is not significantly different from 0. Results obtained when comparing the SPE with the LLE showed that results calculated from both methods are not significantly different except for 1-hexanol and ␣-terpineol. These two compounds presented concentrations slightly superior when the LLE is used. Comparing LLE with the SDE, only two compounds, ␤-citronellol and nerol, displayed significant differences between results obtained by both methods. 3.2. Analysis of volatile compounds in musts of Muscat grapes Although the validation and comparison of the three extraction techniques studied were made with synthetic must solutions, natural must is a complex matrix in which other components exist and could influence on the recovery of volatile compounds responsible for the aroma. The effect of the matrix must be considered when an extraction technique is used with quantitative purposes. Therefore, the three extraction methods were applied to real samples of Muscat must. Gas chromatographic analysis of the extracts obtained by means of LLE, SPE and SDE made it possible to identify 38 volatile components of Muscat grape juice by their mass spectral data. The quantification process only was applied to the compounds included in the calibration set by means of the calibration curves obtained previously. The total ion chromatogram of the extract obtained by SPE is showed in Fig. 1. Table 5 shows the detected compounds in the three extracts. The qualitative composition of the three organic extract obtained was similar, characterizing the volatile profile of must derived from Muscat grapes, a very aromatic variety. Among the compounds identified were aldehydes and alcohols of six carbon atoms like hexanal or 1-hexanol, compounds give rise to the herbaceous aromas of the grapes and musts [30]. The majority of compounds detected are monoterpenes (linalool, terpineol, nerol, geraniol) and poly-oxygenated terpenes. This group of terpenes has a great sensorial relevance since they are responsible for the floral and fruit aromas of this variety of grape [30]. An important group of benzoid compounds was also found. Among them, the most remarkable benzoid compounds from the

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Table 5 Volatile compounds identified in Muscat grape must by LLE, SPE and SDE. Compounds

KIa

m/zb

LLE

SPE

SDE

(1) Hexanal (2) (E)-2-Hexenal (3) 1-Hexanol (4) (Z)-3-Hexen-1-ol (5) (E)-2-Hexen-1-ol (6) (Z)-linalol oxyde furan (7) 2-Furancarboxaldehyde (8) (E)-linalool oxyde furan (9) Benzaldehyde (10) Linalool (11) Diethyl malonate (12) Ho-trienol (13) ˛-terpineol (14) 2(5H)-Furanone, 3-methyl(15) p-mentha-1(7). 2-dien-8-ol (isomer I) (16) (Z)-linalol oxyde pyran (17) (E)-linalool oxyde pyran (18) ˇ-citronellol (19) p-mentha-1(7). 2-dien-8-ol (isomer II) (20) Nerol (21) ˇ-Damascenone (22) Hexanoic acid (23) Geraniol (24) Benzyl Alcohol (25) 2(5H)-Furanone, 5-methyl(26) Phenylethyl alcohol (27) 2,6-dimethyl-3,7-octadiene-2,6-diol (28) 3,7-dimethyl-1-octen-3,7-diol (29) 2,6-dimethyl-1,7-octadiene-3,6-diol (30) Nonanoic acid (31) Benzoic acid, 4-ethoxy, ethyl ester (32) (Z)-8-Hydroxylinalool (33) Geranic acid (34) Benzoic acid (35) Dodecanoic acid (36) Tetradecanoic acid (37) Methylvanillil eter (38) Hexadecanoic acid

1080 1225 1354 1371 1410 1432 1448 1459 1529 1554 1576 1623 1720 1743 1743 1768 1791 1798 1802 1812 1820 1841 1861 1864 1892 1902 1953 1967 1983 2202 2243 2310 2329 2457 2517 − − −

41/44/56 55/69/83 56/43/41/55 41/67/55/82 57/82/41 153/152/135 97/96/98 153/152 77/105/106 71/93 115/133/88 71/82/43 59/93/121/136 41/69/98 94/79/59 68/94 68/94/155 69/81/123 59/79/94 69/93 69/121/190 60/73/87 69/93/123 79/108/107 69/98/41 91/92/65 82/71/43 71/59/121 67/43/71 60/73/129 121/138/166 71/121/137 69/100/123 77/105/122 60/73/157 73/60/43 137/168/122 43/60/73

+ + + + + + − + + + + + + + + + + + + + − + + + + + + + + + + + + + + + + +

+ + + + + + − + + + + + + + + + + + + + − + + + + + + + + + + + + + + + + +

+ + + + + + + + + + + + + + + + + + + + + + + + + − − − + − + + − − − −

a b

Kovat’s index calculated on a BP-21 capillary column. Characteristic ions in the mass spectrum.

sensorial point of view are benzaldehyde, phenylethyl alcohol and bencylalcohol [31]. The most noticeable differences found in the qualitative profile of must were the occurrence of 2-furancarboxaldehyde and ␤-damascenone which only were identified in the extracts obtained by SDE technique. The presence of 2-furancarboxaldehyde can be attributable to sugar thermal degradation produced during the sample heating, fact that is in a good agreement with the comments of others authors [19,29]. Whereas, the occurrence of ␤-damascenone can be explained by the release of its glycosides precursors by acid hydrolysis during the sample heating. Despite this fact, SDE is a very convenient isolation technique to carry out a characterization of volatile profile of musts since, with a good calibration in a good model system, the occurrence of these artefacts should not be a problem. However, these products might be co-eluted with target compounds and upset the identification and quantification process. On the other hand, high polar compounds like acids (geranoic, benzoic, dodecanoic, tetradecanoic and hexadecanoic acid) and alcohols (2,6-dimethyl-3,7-octadiene-2,6-diol, 3,7-dimethyl1-octen-3,7-diol, 2,6-dimethyl-1,7-octadiene-3,6-diol and hydroxylinalool) were not detected by SDE (Table 5). This fact can be attributed to a poor extraction due to their high affinity for the aqueous phase and consequently their low water/solvent partition coefficients or because their low volatility [29]. Nevertheless, these compounds are minor components of musts and the majority of them do not play an important sensorial role.

Table 6 shows quantitative results obtained using the calibration curves calculated previously. In general, results obtained by the three techniques were quite similar, although small differences were found in some compounds. The must contained significant quantities of C6 alcohols, mainly (E)-2-hexen-1-ol and 1-hexanol

Table 6 Volatile compounds concentration (␮g/L) and relative standard deviations in the three organic extracts of Muscat must. Compounds

LLE

SPE *

Mean (E)-2-Hexenal 1-Hexanol (E)-3-Hexen-1-ol (Z)-3-Hexen-1-ol (E)-2-Hexen-1-ol (Z)-Linalool oxide furan (E)-Linalool oxide furan Benzaldehyde Linalool ␣-Terpineol ␤-Citronellol Nerol Geraniol Benzyl alcohol Phenylethyl alcohol

a

7.73 123a n.d. 16.0a 148a 163a 64.9a 7.44a 639a 8.25a 2.04a 8.82a 91.2a 8.11a,b 6.34a

RSD (%) 1.68 0.93 – 1.54 3.76 1.97 3.21 0.02 2.24 1.64 1.68 2.56 3.94 0.57 2.52

SDE *

Mean b

10.5 123a n.d. 10.9b 118b 145b 60.3a 8.31b 693a 5.04a 3.24b 5.87b 82.6a 8.53a 6.46a

RSD (%)

Mean*

RSD (%)

2.49 2.66 – 0.30 0.34 0.12 1.00 2.44 1.93 3.62 5.76 2.02 0.51 5.57 0.31

9.60b 95.6b n.d. 11.3b 124b 148b 68.6a 8.38b 629a 7.04a 3.13b 3.17c 90.4a 7.73b 5.04b

0.72 1.34 – 1.81 1.75 1.07 0.96 2.15 0.70 4.17 0.50 5.04 2.60 2.46 2.16

n.d.: not detected. * According to the result of the Student–Newman–Keuls test, mean values in the same row with different superscript (a, b, c) are significantly different (p < 0.05).

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whose concentrations extracted were slightly higher when the LLE technique is used. The concentrations of almost monoterpenes and polyoxigenated terpenes were independent of the extraction technique used, except in the case of ␤-citronellol, nerol and (Z)-linalool oxide furan whose concentrations were slightly superior in the extracts obtained by LLE. The benzene derivates (benzaldehyde, benzyl alcohol and phenylethyl alcohol) are known to be characteristic compounds in grape must and SDE seems not to extract them efficiently. 4. Conclusions Results show that the three extraction methods studied can be used for the qualitative and quantitative determination of volatile compounds from musts. Nevertheless, when wishing to choose one of these isolation techniques, it would be convenient to consider other factors such as the amount of solvent required or the time consumption. Despite the fact that extracts obtained by means of SPE and LLE displayed a similar qualitative composition. LLE seems to extract slightly higher concentration of compounds from must in comparison with the other techniques tested. However, SPE required smaller amount of solvent and sample and shorter extractions times. On the other hand, extraction procedure by means of SDE was the fastest and it used minor amount of sample and solvent, obtaining good quantitative results for the more relevant shift aroma to compounds of aroma must. However, it should be taken into account the difficulty of SDE in extracting compounds with low volatility and the possible appearance of artefacts formed during sample heating such as furancarboxaldehyde (product of Maillard reaction) and the liberation of volatile compounds from their glycosylated forms by acid hydrolysis such as ␤-damascenone.

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