## 2 Results
### Optimization of HS-SPME-GC \(\times\) GC-TOFMS Methodology
In the grape varieties under analysis, it was possible to identify and quantify a total of fifty-two free varietal volatile compounds, with seventeen compounds identified as belonging to the monoterpene class, twenty-eight compounds assigned to the sesquiterpene family, and seven compounds attributed to the C\({}_{13}\)-norisopenroid group (Table 2). Compounds like p-cymene, d-limonene, p-cymenene, linalool, \(\alpha\)-copaene, \(\alpha\)-gurjunene, longfolene, \(\beta\)-caryophyllene, \(\alpha\)-humulene, \(\alpha\)-cadinene, \(\beta\)-cadinene, cis-calamenene, \(\alpha\)-calacorene, cadalene, \(\beta\)-cyclocitral, geranylacetone, and \(\beta\)-ionone were found in all the varieties studied. However, certain compounds are exclusive to specific varieties. For instance, \(\beta\)-myrorene, hotrienol, and \(\beta\)-citronellol are exclusively found in the TB variety, while \(\beta\)-elemene, \(\alpha\)-cedrene, and aromadendrene are unique to the Sy variety. Additionally, isocaryophyllene is solely present in the Trinc variety. Moreover, the compounds \(\gamma\)-elemene, \(\gamma\)-muurolene, and \(\alpha\)-amorphene are present in all varieties except TB, while tetrahydrolinalool is present in all varieties under study, except Trinc. As expected, an inter-annual variability is observed; therefore, there are compounds that are only present, for example, in 2021 or 2022. In the case of the Cast variety compounds, such as, \(\gamma\)-terpinene, \(\alpha\)-terpinolene, \(\gamma\)-terpineol, \(\alpha\)-terpineol, vitispirane, theaspirane B, and \(\beta\)-damascenone, they are only present in the year 2021 and were not found in 2022. However, compounds such as dihydromycenol, \(\delta\)-elemene, aristolene, \(\beta\)-copaene, \(\beta\)-guaiene, zonanere, and the unknown compound m/z 105/161/189/204 are only present in the year 2022 and not in 2021. A similar trend is observed for the other varieties under study; for example, in TB, the compounds cis-myrtanol and \(\gamma\)-selinene were only found in 2021 and not in 2022, but \(\alpha\)-ylangene and \(\beta\)-damascenone were only found in 2022 and not in 2021. In Sy grapes, compounds like \(\gamma\)-terpinene, \(\alpha\)-terpinolene, \(\gamma\)-terpineol, and cis-myrtanol were only identified in the year 2021, and nerol and \(\delta\)-selinene were identified in the year 2022 but not in 2021. As for the CS variety, compounds such as \(\gamma\)-terpinene, \(\alpha\)-terpinolene, \(\gamma\)-terpineol, nerol, cis-myrtanol, \(\gamma\)-selinene, and \(\beta\)-copaene were only found in the year 2021, while valencene was only found in the year 2021. In the case of the Trinc variety, it also presents compounds that are only present in 2022 and not in 2021, such as \(\delta\)-elemene, \(\alpha\)-cubebene, \(\alpha\)-ylangene, aristolene, and \(\delta\)-selinene. According to [PERSON] et al. 2020 [40], volatile compounds are rarely assigned as variety markers. Indeed, the concentration of volatile compounds changes from variety to variety, but other factors such as the effect of the terror, climatic conditions, and harvest dates also have to be considered due to their remarkable effect on the volatile composition of the grapes. According to [PERSON] et al. 2021 [41], the same volatile compounds were found in different varieties, revealing that the individual aromatic potential of these varieties is related to infinite combinations and modulated by environmental characteristics. Other studies also corroborate the results obtained in this work, showing differences in the volatile varietal composition of grapes between years and between varieties [1, 4, 42, 43]. Figure 3 shows the polar heatmaps with a dendrogram for the varieties under study. These data evidenced differences between varieties and, apparently, there seems to be a clear hierarchical grouping between the Portuguese varieties Trinc, Cast, and TB, and between the CS and SY varieties.