# Profiling Croatian Whisky Using GC/MS-MS and UHPLC-QTOF
## 1 Introduction  
Whisky or whiskey production relies on three main ingredients: cereal, yeast, and water. Whisky production follows a relatively homogeneous fundamental process that is in some way regulated by the EU Regulation 2019/787 [1,2]. EU Regulations define whisky or whiskey as a spirit drink produced following three main production operations:* Distilling a mash from malted cereals, with or without unmalted cereal grains that have been saccharified by malt enzymes or other natural enzymes and then fermented by the action of yeast;
* Every distillation is carried out at less than 94.8% vol., preserving flavor and aroma from the raw materials;
* Maturation of the final distillate from at least three years in wooden casts no larger than 700 L. The final distilled product, to which only water and plain caramel (for coloring) may be added, must retain the color, aroma, and flavor imparted by the production processes outlined in points (i), (ii), and (iii). Additionally, the EU regulation requires that whisky has a minimum alcohol content of 40% and prohibits the addition of any extra alcohol. Despite its relatively standardized production framework, governed by EU Regulation 2019/787, the final whisky profile is significantly influenced by numerous variables such as cereal variety, fermentation conditions, distillation techniques, maturation time, and cash type. These factors result in whisky with distinct flavors, shaped by numerous active flavor compounds known as congeners. The main congeners in whisky include alcohols, aldehydes, acids, esters, phenols, terpen, tannins, and lactones, which are produced at different stages of the production process and may be more prominent depending on the specific process involved [1]. Traditional whisky-producing regions such as Scotland, Ireland, and the United States have well-documented profiles, yet the chemical composition of whiskies from emerging regions like Croatia remains underexplored. Croatian whisky production, influenced by local ingredients, environmental conditions, and modern techniques, offers a distinctive character that warrants detailed scientific investigation. Advanced analytical techniques such as gas chromatography-mass spectrometry (GC-MS/MS) and ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-QTOF) provide robust platforms for comprehensive profiling of both volatile and non-volatile compounds. These methods enable the identification and quantification of key flavor compounds, offering insights into the biochemical pathways and production processes that define the sensory attributes of whisky. The types of grains used in the mash provide a range of aroma and flavor compounds to the new distillate, contributing to the distinct characteristics of each whisky. For example, compounds like lyoniresino and its glycosides have been identified in whisky by [PERSON] et al. [3], while syringaresinol is commonly found in grains such as barley, rye, and corn [4]. [PERSON] et al. [5] also detected syringaresinol and other lignans in enzymatically fractionated extracts of brewer's spent grain, which consists of grain and malt residues left after mashing [6; 7]. Additionally, (E)-2-nonenal, a compound formed through the oxidation of linoleic and linolenic acids during malting, can also be introduced from wood during maturation [8], and it can be found in trace levels in barley [6; 9]. (E)-2-nonenal contributes to the green notes in Scotch malt whisky [10], and it was recognized as an important compound in the aroma profile of both new make and aged Bourbon [7; 11]. Aldehydes could be derived from raw materials and be produced post-harvest throughout the malting process via enzymatic transformations of lipids in the presence of oxygen. This process occurs primarily through the lipoxygenase pathway (e.g., hexanal) [6; 12; 13]. After malting, fermentation is a crucial step in whisky production and significantly influences its aroma profile.