## 3 Results and Discussion  
### Optimization of the Extraction Conditions  
Some factors such as the addition of EDTA (1%), NaCl (10%), and agitation (1100 rpm) were set according to previous studies. The addition of EDTA during the extraction of volatile sulphur compounds has demonstrated positive effects on their stability against oxidative phenomena [26], whereas the use of agitation and a certain amount of NaCl to enhance the SBSE efficiency [12,15] are clearly established. Firstly, a full factorial design (24) was employed for screening the variables that may influence the SBSE procedure for the determination of volatile sulphur compounds in wine distillates. The variables considered were the extraction time, sample volume, extraction temperature, and alcoholic grade. The range studied for all of them is presented in Table 3. All of these parameters have shown that they must be carefully studied according to the matrix and the physical and chemical properties of analytes when applying the SBSE methodology. In the case of beverages with a high amount of ethanol, the study of the dilution of the sample previous to the SBSE analysis in order to reduce its alcohol content is crucial. [PERSON] and [PERSON] [27] have shown that there is a marked decrease in the detectability of some volatile sulphur compounds by \begin{table}
\begin{tabular}{l c c c c c} \hline \hline
**Factor** & **Low (–)** & **High (+)** & **Center** & **Axial (– \(\alpha\))** & **Axial (+ \(\alpha\))** \\ \hline Temperature (\({}^{\circ}\)C) & 25 & 35 & 30 & & \\ Extraction time (min) & 30 & 100 & 65 & & \\ Sample volume (mL) & 10 & 25 & 17.5 & & \\ Alcoholic content (\%, \(\
u/v\)) & 5 & 25 & 15 & & \\ \hline
**Second Experimental Design, rotatable CCD** & & & & & \\ \hline Extraction time (min) & 15 & 45 & 30 & 8.8 & 51.2 \\ Alcoholic content (\%, \(\
u/v\)) & 2.5 & 10 & 6.25 & 0.95 & 11.6 \\ \hline \hline \end{tabular}
\end{table} 
Table 3: Factor levels. Optimization of SBSE conditions. SPME when ethanol is present in the sample. For the extraction temperature factor, its high and low values were fixed, taking into account that high temperatures may cause thermal degradation problems and that low temperatures may give low chromatographic signals in the case of volatile compounds with low boiling temperatures. The total chromatographic area and number of volatile sulphur compounds detected were considered as the experimental responses. The data were evaluated by an analysis of variance at the 5% significance level and the results are shown in Figure 1 (\(p<0.05\) indicated by blue line). As can be seen, for the total chromatographic area, the extraction time factor and the extraction time and alcoholic grade double interaction were significant at \(p<0.05\), whereas the alcoholic grade factor was the only significant variable for the number of compounds detected. In both cases, their effects were negative, that is, better chromatographic responses were obtained at lower values of these factors. Regarding the significant double interaction for the total chromatographic area, at lower extraction times, the chromatographic response  
Figure 1: 2\({}^{4}\) factorial design. Pareto diagram for total chromatographic area (**A**) and number of compounds detected (**B**). increased as the alcohol content increased, while only a slight decrease was observed at high extraction times. This seems to indicate that when low extraction times are used, the dilution effect by the addition of water to the samples is more significant than that of a higher SBSE efficiency at a low ethanol content. However, if high extraction times are used, both effects (dilution versus lower extraction competence) are compensated for. Taking into account these first results, the extraction temperature variable was fixed at 35 \({}^{\circ}\)C, whereas the sample volume was set at 10 mL. A second experimental design considering only the extraction time and alcoholic grade was carried out.