# Profiling Croatian Whisky Using GC/MS-MS and UHPLC-QTOF
## 1 Introduction
During fermentation, the conversion of sugar into alcohol and other compounds generates a wide spectrum of volatile compounds, including esters, fatty acids, and higher alcohols, which contribute to the complexity and depth to the final products of aroma. Esters are an important group of compounds predominantly formed by yeast during alcoholic fermentation. They play a critical role in imparting fruity (e.g., ethyl hexanoate and isoamyl acetate) and floral (e.g., 2-phenylethyl) acetate) aromatics to the final product [1; 14; 15]. Inaddition to esters, other alcohols, such as 2-methyl-1-butanol and 3-methyl-1-butanol, are generated by yeast through the decarboxylation of \(\alpha\)-keto acids, followed by the reduction of the resultant aldehydes to alcohols. Other chemical groups, including terpen, ketones, aldehydes, sulfur-containing compounds, and furans, also play key roles in forming the complex aroma profile of newly distilled spirits [16; 17; 18; 19]. Distillation is a crucial step in whisky production where ethanol and congeners are separated and incorporated into the distillate [20]. The distillation process further shapes the whisky's aroma profile by concentrating certain compounds and removing others. The volatile compounds with boiling points lower than ethanol are concentrated during distillation, while heavier, less volatile compounds remain in the stillage. This separation process leads to the refinement and concentration of specific aroma compounds, contributing to the distinctive scent and flavor of the final product [14; 15]. Additionally, the particular distillation methods used, such as pot still and column distillation, have a significant impact on the flavor and aromatic profile of the distilled spirit [21; 22]. Pot stills are commonly used in Scotch production and in some part in Irish whiskies, while column stills are more typical in American bounbon and rye whisky production. The number of distillation runs and the shape and size of the still can contribute to the final aroma and flavor profile of the whisky. Moreover, fractional separation during distillation permits the modulation of the quantity and the characteristics of volatile aromatic compounds in the distillates. Such distillation processes lead to the concentration of volatile aromatic compounds, thereby enhancing their sensory impact. Finally, the maturation process in oak casks plays a crucial role in enriching the whisky's complexity and depth of aromas. During this stage, flavors are extracted from the wood, imparting unique characteristics to the spirit [23]. Some compounds associated with longer ageing of the whisky are octadecanoic acid, esuletin, ellagic acid, flavonoids, and lyoniresinol and its derivatives: dihydroxy- or hydro-peroxy, methylated, and lyoniresinol xyloside. Understanding the interplay between these compounds and the production process is essential for characterizing the unique attributes of Croatian whisky. This comprehensive profiling helps in identifying key biomarkers for quality control and offers insights into how specific production variables impact the final sensory experience. Additionally, it provides a scientific basis for differentiating Croatian whisky in the global market, emphasizing its distinctiveness rooted in local raw materials and traditional craftsmanship. This study represents the first comprehensive analytical characterization of Croatian whisky using solid-phase microextraction gas chromatography-mass spectrometry (SPME-GC-MS) for volatile compounds and untargeted UHPLC-QTOF for non-volatile compounds. Unlike previous studies focused on whiskies from established regions, this research highlights the unique chemical composition of Croatian whisky, shaped by indigenous raw materials, local fermentation practices, and specific maturation conditions. The novelty of this study lies in its dual analytical approach, combining SPME-GC-MS and UHPLC-QTOF, to deliver an integrated profile of Croatian whisky's volatile and non-volatile constituents.