## 2 Results and Discussion
### Classification of Cretan VOO Samples
As a final step, the unpaired Student's t-test was performed to establish and confirm the significance of important metabolites. All statistically significant metabolites and their variations in each group are presented as box plots in Figure 6.  
Figure 5: Orthogonal Projections to Latent Structures Discriminant Analysis (OPLS-DA) on EVOO samples volatile profile, using the ‘geographical origin’ as class membership criterion, (**a**) Lasithi vs. Rethymno, (**b**) Chania vs. Lasithi. Figure 6: Box plots of identified biomarkers in VOOs samples. Boxes are drawn from the 25 th to 75 th percentiles in the concentration distribution. (**a**) Lasithi vs. Rethymno, (**b**) Chania vs. Lasithi. (e + 07 = \(\times\) 10\({}^{7}\)). The compounds found significant (\(p<0.05\)) for geographical differentiation consisted of four terpenic hydrocarbons (6-methyl-5-hepten-2-one, copaene, (\(E\))-4,8-dimethyl-1,3,7-nonatriene, and (\(Z\))-beta-ocimene), two esters ((3\(Z\))-hex-3-en-1-yl acetate and hexyl acetate), two aldehydes ((\(E\))-2-pentenal and pentanal), three hydrocarbons (dodecene, (\(E\))-2-dodecene, undecene, and 1-ethyl-2-methylbenzene), one alcohol ((\(Z\))-11-hexadecen-1-ol), and one ether (1-methoxy-2-propanol), as shown in Figure 6. Eight of them, 6-methyl-5-hepten-2-one, copaene, (\(E\))-4,8-dimethyl-1,3,7-nonatriene, (\(Z\))-beta-ocimene, (3\(Z\))-hex-3-en-1-yl acetate, hexyl acetate, (\(E\))-2-pentenal, and dodecene, have previously been reported as markers for the classification of Greek VOO according to geographical origin [42; 43]. More specifically, efforts have been put on studies that focus on the differentiation of olive oils produced in different islands of north-western Greece [43] (Zakynthos, Kefalonia, Lefkada, Kerkyra, Preveza, and Messologi) and different regions [42] (Messinia and Lakonia in the Peloponnese, Irakleio on the island of Crete, and Etoloakarnania in central Greece). In this study, when samples from Lasithi and Rethymnon were compared, seven volatiles were found to be responsible for the differentiation between the groups. Three of them ((3\(Z\))-hex-3-en-1-yl acetate, hexyl acetate and (\(E\))-2-pentenal) are produced through the LOX-pathway, contributing to olive oil aroma. The volatiles 1-methoxy-2-propanol and 1-dodecene have also been identified as potential markers in Croatian monovarietal/PDO EVOO [44], while pentanal has shown high discrimination ability for the classification of north Morocco oils [45]. A ripening indicator with fruity-like odor, 6-Methyl-5-hepten-2-one, has been detected as a marker for the characterisation of Turkish olive oils according to origin (Mediterranean, Aegean, South-eastern Anatolia, Marmara, and Black Sea) [46]. Overall, (3\(Z\))-hex-3-en-1-yl acetate, and hexyl acetate were found upregulated in samples from Lasithi while all the others were increased in samples from Rethymnon. Pentanal was found as statistically significant in both origin studies of VOOs. Samples from Lasithi showed lower levels of pentanal compared to Rethymnon and Chania. In total, eight compounds were found to differ between VOOs from Chania and Lasithi. Three terpenic hydrocarbons, copaene, with a sweet-fruity description, (\(E\))-4,8-dimethyl-1,3,7-nonatriene, and (\(Z\))-beta-ocimene, with an herb aroma description, were among the most important discriminant volatiles. This is in accordance with several studies where terpene hydrocarbons were reported as suitable markers of the geographic origin and genotype of EVOOs [29; 47; 48]. (\(E\))-2-Dodecene has been reported as a discriminant variable in a study investigating VOOs of Chemlali and Neb Jmel cultivars growing in central and south Tunisia [49]. In this study, 1-ethyl-2-methylbenzene, 1-undecene, and (\(Z\))-11-hexadecen-1-ol are reported as discriminant volatiles for geographical origin classification for the first time. In general, pentanal and 1-undecene, were found in increased amounts in oils from Chania, while the other statistically significant compounds were found in decreased levels in oils from Lasithi.