## 3 Results and Discussion  
### Identification of Volatile Metabolites in Analyzed Grape Marc Spirits  
Higher alcohols, acids, ethyl and acetate esters, carbonyl compounds, and terpen were the major compound groups identified in analyzed distillates. A chromatogram of a QC grape marc spirit sample is illustrated in Figure 1. More than 90 metabolites were detected, identified, and semi-quantified with the proposed method, and their names, chemical formulae, molecular weights, and relative concentrations are presented in Table S2. The volatile compound groups that are very important for the aromatic profile of Greek grape marc spirits are terpenes and terpenoids. Although higher concentrations of terpenes are expected in the initial fractions of distillation due to their good solubility in ethanol and poor solubility in water, their behavior during distillation is characterized by an increase in their concentration in the final fractions at higher temperatures because their relatively high boiling points are crucial in determining the distillation behavior [20]. The presence of monoterenes in the aromatic profile of distillates is reflected by the grape variety from which the beverage is produced. Distillates produced by Muscat and Muscat of Alexandria grapes, such as Macedonia and Limnos spirits, have been found as richer in terpenes than spirits produced in other regions in Greece using different grape cultivars. Linalool, _alpha_-terpineol, nerol oxide, and geraniol were the major terpenoids identified in the analyzed samples. The concentration of the first two was 5-to-10-fold higher than the other terpenoids. These differences in concentrations have also been observed by several researchers. [PERSON] et al. [27] noticed that these terpenoids were also dominant in Muscat Ottonel distillates. [PERSON] et al. [39] detected them in Moscato and Prosecco grappas, while [PERSON] et al. [20] found that, in some cases under acidic conditions, nerol and geraniol were converted to linalool and _alpha_-terpineol. Linalool and nerol oxide were the major forms of terpenoids in Limnos and Thessaloniki samples, while the more stable forms of terpenoids, _alpha_-terpineol, and geraniol were found in higher levels in Thessaly and Thessaloniki samples, while terpinen-4-ol was the most abundant terpenoid in Thessaly and Macedonia samples, as illustrated in Figure 2. The concentration of botrienol was found to be 10-fold  
Figure 1: Representative HS-SPME-GC-MS chromatogram of volatile compounds identified in a QC grape marc spirit sample. higher in Muscat of Alexandria grape marc samples from Limnos than any other analyzed distillate. \(o\)-Cymene [34; 40; 41], _alpha_-pinene [42; 43; 44], _beta_-myrcene [21], D-limonene [21; 45], ylapgene [18; 29], _alpha_-cedrene [46] and _alpha_-terpinene [14; 29] were detected mostly in Muscat, Muscat of Alexandria and Merlot wines, grape musts, and spirits, as they are characteristic metabolites of these cultivar grapes; however, in this study, their concentration was found to be over 50-fold higher in some Muscat grape marc spirit samples originated from Macedonia. Caryophyllene has been previously found in Cabernet Sauvignon berries [47], while [PERSON] et al. [35] found terpenols in orujo samples and [PERSON] et al. [14] and [PERSON] et al. [19] detected terpenols, such as _alpha_-terpinene, \(p\)-cymene, _alpha_-cubebene, and _alpha_-calacorene in several European spirit samples. The distillation of pressed fermented saccharated raw materials flavored with star aniseed (_Illicium verum_), fennel (_Foeniculum vulgar_), green aniseed (_Pimpinella anisum_), or other plants yields aniseed spirits. Anise (_Pimpinella anisum L._; Umbelliferae family) has been cultivated throughout Europe and has been used as a popular aromatic herb and spice since antiquity. Anise is a plant native to the Middle East that has been used since ancient Egypt, and its major volatile compound is anethol, which is responsible for aniseed's fragrant characteristics [29; 48; 49].