## 2 Results and Discussion
### Optimization of SPME-Arrow Conditions for Analysis of Bound VOCs  
Before starting the optimization experiments, the heating of samples in order to remove free volatiles was carried out. The effect of different heating times on the content of different classes of free VOCs is presented on Figure 4. As expected, the prolonged heating time decreased the content of free VOCs. Thus, the heating time of 4 h was chosen as a suitable time for removing free VOCs from the sample. Acid hydrolysis is a complex process accompanied by several steps. In the first step, the solvation of dry grape skins occurs when skins are put in contact with the citric buffer. After solvation is complete, dissolution of bound forms of VOCs occurs. To enhance the process of hydrolysis, the reaction is conducted at elevated temperature (95 \({}^{\circ}\)C) in the nitrogen atmosphere to inhibit any oxidation reaction. Before performing the SPME-Arrow extraction procedure, it is necessary to increase the ionic strength of the sample solution. Aqueous solubility of many organic compounds decreases with the addition of the salt to the sample. The distribution coefficient between coating and the sample increases with the decreasing of aqueous solubility [19]. The effect of salting out leads to the more readily passage of the analyte from liquid sample to the headspace, and hence to the coating of SPME-Arrow. The weight of NaCl (2 g) was chosen based on the previously reported study [11]. Classes of bound volatile compounds identified in the Merlot grape skins were monoterpenes (21), C\({}_{13}\)-norisoprenoids (3), alcohols (33), acids (7), carbonyl compounds (18), and others (2). In total 84 volatile compounds were identified (Supplemental Table S2). Figure 4: The effect of different heating times on different classes of free volatiles represented as absolute peak areas. The glycosylated bound monoterpenes are usually 3 to 10 times more abundant than free aglycones and the proportion of the glycosidic aroma substances changes among grape varieties [4; 36]. Norisoprenoids are carotenoid-derived aroma compounds. During development carotenoids are degraded to produce glycosidically bound norisoprenoids [37]. Although the other classes of VOCs are mostly synthesized during alcoholic fermentation, they can also be found in bound forms in grape berries [38]. All detected compounds have a greater water solubility in comparison with the compounds detected as free direct from the solid matrix. Similar to the optimization of SPME-Arrow conditions for analysis of free VOCs, the BBD experimental design was chosen. The experimental factors were again extraction temperature (40, 50, 60 \({}^{\circ}\)C), incubation time (10, 20, 30 min), and exposure time (30, 45, 60 min). Desorption time was not included, as it was shown that it did not affect the extraction efficiency, and was set to 7 min. The BBD experimental design generated 15 experiments including three central points. The experiments were again randomly performed. For the evaluation of the significance for all the studied factors, the area of the responses based on the sum of the peak areas was applied. Fitting the data with various models showed that contents of alcohols, carbonyls, acids, and monoterpenes were best represented by quadratic polynomial models. In Table 3 are presented parameters of analyses of variance (ANOVA) parameters for all studied models. The determination coefficients (R\({}^{2}\)) are in range from 0.8991 to 0.9931 while \(p\)-values are in range from \(<\)0.0001 to 0.0364, indicating that models are highly significant. Models also showed statistically insignificant lack of fit because all determined \(p\)-values are higher than 0.2500. All responses parameters of quadratic polynomial equations are depicted in Table 3.  
The results obtained showed that the most significant factors were extraction temperature and exposure time (Table 3). For both factors an increase in their values positively affected the extraction efficiency for almost all classes of VOCs.