# Profiling Croatian Whisky Using GC/MS-MS and UHPLC-QTOF
## 3 Results and Discussion
### Aromatic Profile of Croatian Whisky Determined by SPME-GC-MS
#### 3.3.7 Fatty Acids and Related Compounds
Clusters observed in the plot may indicate distinct whisky types, such as single malt versus blended whiskies, or differences arising from production techniques, maturation periods, or geographical origins. The presence of outliers suggests certain samples have unique chemical signatures that could correspond to experimental variations or deliberate differences in their formulations. The dendrogram (Figure 4), constructed from a similarity matrix, groups whiskies based on their compositional similarities. Close clustering between certain samples implies a shared production method, source, or aging process, whereas samples located in distant branches highlight their distinctiveness. Such clustering is particularly useful for identifying counterfeit whiskies or assessing the consistency of products from the same distillery. As seen clearly in the figure, Croatian whisky (whisky 1 and 2) forms a distinct group, setting it apart from the other analyzed whiskies. Furthermore, in the Supplementary Materials (Figure S3), the data analysis methods clearly illustrate the distinction between Croatian whisky and Scotch whisky based on untargeted QToF analysis of non-volatile compounds. Principal Component Analysis (PCA) provides an unsupervised overview, revealing clear clustering patterns where Croatian and Scotch whisky occupy distinct regions of the score plot. The Partial Least Squares Discriminant Analysis (PLS-DA) refines these distinctions by maximizing variance between the groups, confirming the separation initially observed in PCA. Sparse PLS-DA (sPLS-DA) further identifies key non-volatile compounds contributing most significantly to the differentiation, narrowing down the specific markers of each whisky type. A Scores Heat Map visually emphasizes these findings, with rows corresponding to samples and columns to key compounds. The clustering within the heatmap highlights unique compound profiles for Croatian and Scotch whiskies, underscoring their biochemical differences. Lastly, the dendrogram derived from hierarchical clustering corroborates these results, organizing the samples into two distinct branches that align with their respective geographic and production origins. Together, these analytical approaches robustly demonstrate that Croatian whisky is compositionally distinct from Scotch whisky, providing a comprehensive molecular-level insight into their unique chemical signatures and regional characteristics. Key differences between Croatian and Scotch whisky were observed, particularly in the presence and concentrations of certain esters, phenolic compounds, and aging markers. Croatian whisky exhibited a higher prevalence of D-limonene and linalool, contributing to citrus and floral notes, whereas Scotch whisky samples demonstrated higher concentrations of traditional oak-derived compounds such as vanillin and whisky lactones. Additionally, Croatian whisky displayed unique signatures of indigenous grain metabolites, further reinforcing the significance of regional influences on whisky chemistry. By limiting the scope of this study to Croatian whisky, a comprehensive and in-depth analysis was achievable, providing foundational data for further research and industry development. Future studies may expand the comparative aspect by including additional whisky-producing regions, exploring how geographical and production variations influence whisky's chemical and sensory profiles.