## 3 Results and Discussion
### Classification of Greek Distillates According to Geographical Region  
Tispuo samples contain hundreds of volatile and semi-volatile metabolites, which are indicators of geographic origin, and distillation techniques [18]. For example, [PERSON] et al. [25] found a lower content of volatile fermentation compounds (both qualitatively and quantitatively) in Italian spirits (Grappa) compared to Spanish Orujos, with 2-phenyl ethanol, hexenols, aldehydes, and most ethyl esters and acetates being responsible for the specific characteristics of these distillates. On the other hand, [PERSON] et al. [21], who studied the effect of the distillation technique on the aromatic profile of tispuoro, found that the fractional column distillation gave the richest volatile profile, followed by home distillation and copper alembic distillation, mainly due to the high values of ethyl octanoate, ethyl decanoate, and DL-limonene, which were much higher when fractional column distillation was applied. A PCA scores plot, presenting the differentiation of all analyzed samples in this study according to their geographical origin, is illustrated in Figure 5, and it is shown that distillates of Limnos were clearly differentiated from other samples, while samples from Crete produced from native grape cultivars were not discriminated from those produced from Soultania cultivar. The three groups of Macedonian samples, Thessaloniki, Pella,and the rest of Macedonia, presented a small separation, as they were all produced from Muscat pomaces and with the addition of arise, so OPLS-DA models were used for further investigation of the observed differences between production areas. The OPLS-DA models were used to further study the differences among samples, and the statistical significance of each model was confirmed by permutations, Hotelling lines, and CV-ANOVA analysis (\(p<0.05\)). Six statistically significant OPLS-DA models between the production areas of the analyzed distillates are presented in Figures S3 and S4. The statistically significant metabolites of these models were determined by assessing the VIP and p(corr) vectors and the two-tailed \(t\)-test, with thresholds of VIP \(>0.5\), p(corr) \(>0.5\), and \(p<0.05\). Tables S3-S8 show the values of these vectors for each metabolite. The OPLS-DA models between Macedonia-Thessaloniki and Macedonia-Pella samples were not statistically significant (\(p>0.05\)), as well as in the case of Crete and Cretaceous-Soutlanina samples. This observation could lead to the conclusion that distillates produced from either the same or partially the same grape cultivar, or by a similar production process but in a common region, are difficult to distinguish; however, several metabolites were characteristic for some of these products, as mentioned in Section 3.1. The responsible biomarkers for the differentiation of Crete and Thessaloniki samples were found to be six ethyl esters and one acetate ester, three alcohols, three flavored volatiles, four terpen, one acid, and two aldehydes. In the case of Crete and Limnos spirits, four ethyl esters, two flavored compounds, two aldehydes, two acids, five terpen, and two alcohols were the statistically significant metabolites, while four ethyl esters, four flavored compounds, four terpen, three aldehydes, two alcohols, and two acids were responsible for the separation between Thessaloniki and Limnos distillates. Moreover, five esters, five flavored compounds, three carbonyl compounds, two alcohols, three terpenoids, and two acids were found to be statistically significant for the model between Macedonia and Limnos distillates, while nine esters, three alcohols, three carbonyl compounds, two acids, three terpenoids, and one flavored compound were responsible for the differentiation between Crete and Thessaloniki samples. The statistically significant metabolites between Crete and Macedonia grape marc spirits were seven esters, three alcohols, three flavored compounds, one acid, four terpenoids, and two carbonyl compounds.