**1. Introduction**

Extra virgin olive oil (EVOO) is appreciated among consumers because of its specific flavor and nutritional properties. Due to its economic importance, EVOO is among the most common commodities subject to fraud and mislabeling. The European Union (EU) protects EVOO by the regulation mostly based on analytical and sensory controls [1] which generally succeed in detecting illegal manipulation with EVOO intrinsic properties (adulteration with cheaper refined and/or extraneous oils) and EVOO extrinsic properties (fraudulent misrepresentation of quality category). In the EU, most EVOOs of high economic value are additionally protected by Protected Denomination of Origin (PDO) [2]. Each PDO EVOO is produced according to a set of specific requirements prescribed by the holder of a designation in a specification document, governing aspects such as olive varieties used, cultivation, harvest and processing conditions, physico-chemical parameters, and sensory characteristics.

Many PDO EVOOs are produced from olives of a single variety (monovarietal EVOOs), while the blends also owe a large part of their typicity to the unique olive assortment of a particular region. Nowadays, the information on the label about the varietal origin of EVOO is becoming more and more important and attracting, especially for the market segment of informed consumers interested in healthy, quality products with remarkable diversity and clear identity. Similar as in the case of wine, besides being linked to a given geographical origin and PDO, EVOOs from particular varieties are recognized, appreciated, and demanded on the market because of their specific nutritional and sensory properties. As a consequence, they often reach higher prices, and, given the obvious financial benefits associated with them, are very likely subject to fraud by mislabeling with respect to varietal origin.

As regards EVOO varietal authentication within the process of protection by designation of origin (PDO), controls against counterfeiting include auditing of mandatory documentation and records that prove traceability in production and compliance with the requirements set up in PDO specification. The other part is the assessment of the conformity of EVOO with physico-chemical parameters and sensory characteristics laid down in the specification. The analytical parameters controlled more often (e.g., acidity, peroxide value, measurements in ultraviolet, etc.) do not specifically reflect varietal origin, and the limits that are established, although usually stricter than those prescribed by the official EU regulation [1], are regularly not designed to identify olive variety used. Similar applies for the sensory profiles commonly used to describe monovarietal/PDO EVOO [3] which are not highly discriminative.

The mentioned measures are not sufficient to control varietal origin and avoid fraud. Fraud or false labelling might also be detected or confirmed chemically by analysis of other, minor EVOO compounds. The general strategy that is followed in various research laboratories is the detection of as many as possible EVOO constituents from a larger set of samples and application of multivariate statistical analysis to the analytical data in order to build up classification/prediction models based on varietal origin [4,5]. Many EVOO compounds were found useful for this purpose, including sterols [6], tocopherols [7], fatty acids [6,8], etc. The chemical compounds whose amounts are regulated neither by the official EU regulation nor the PDO specifications, but are certainly the most involved in the typical sensory identity of PDO and monovarietal EVOOs and could serve as differentiators based on such criteria, are volatile aroma compounds and phenols [9–14]. In fact, many successful reports were published which confirmed the utility of these constituents for EVOO varietal differentiation [12,14–20]. Volatile fraction of high quality EVOO, which is responsible for its characteristic so-called *green* and *fruity* flavor, consists mainly of C5 and C6 volatiles (aldehydes, ketones, alcohols, and esters) generated enzymatically in the so-called lipoxygenase (LOX) pathway and other subsequent bioprocesses during olive processing. LOX-derived compounds are accompanied by those from other chemical classes, such as hydrocarbons, terpenes, benzenoids, etc. with mostly unknown or minor sensory relevance [12,14,21–23]. Besides being among the most important contributors to EVOO antioxidant activity, phenols, especially secoiridoids which are the most abundant, are responsible for the characteristic EVOO bitterness and pungency [12–14,24,25]. Olive oil phenols are formed mainly by cleavage of their glycosides by hydrolytic enzymes during olive fruit processing and their concentrations are further affected by oxidative degradation catalyzed by polyphenoloxidases and peroxidases [26,27]. The activity of the mentioned enzymes responsible for the formation of both volatile compounds and phenols is strongly genetically predetermined [28,29], which makes these compounds a logical choice for potential varietal markers in EVOO varietal characterization and differentiation studies.

Croatia is the latest country that joined EU in 2013, and some of the most recently registered PDOs are Croatian [30]. Despite relatively small quantities produced in relation to the leading olive oil producing countries, such as Spain, Italy, Greece etc. [31], EVOOs from Croatia are emerging rapidly on the global market and are much appreciated. For example, Croatian EVOOs are often among those awarded with the highest prizes at relevant international competitions, while Istria, one of the most important olive growing and EVOO producing regions in Croatia, has been represented in the first and leading global EVOO guide *Flos Olei* by the largest number of EVOOs among all the regions for the last

four years in a row (2015–2018). The olive plantations in Croatia have high genetic diversity, including many native varieties which concentrate close to their area of origin and show a limited geographical dispersion [32]. For this reason, Croatian EVOOs protected by various PDOs owe a significant part of their typicity to the varietal origin of the olives, which certainly becomes most pronounced in the case of monovarietal EVOO. In spite of that, and despite existing reports on the chemical and sensory characteristics of Croatian monovarietal EVOO [33–40], the potential of Croatian native olive varieties to produce diverse and specific EVOO has not been investigated enough to be adequately exploited in designing more unique and robust PDOs.

The main aim of this study was to investigate the inter-varietal diversity of typical volatile and phenolic profiles of Croatian monovarietal EVOOs by gas chromatography-ion trap mass spectrometry (GC-IT-MS) and ultra-performance liquid chromatography with diode array detection (UPLC-DAD), respectively. The approach was tested for the characterization and differentiation of EVOOs made from native varieties grown in the two most important olive growing regions in Croatia, Istria and Dalmatia, with each monovarietal EVOO represented by a heterogeneous sample group in terms of geographical microlocations, growing conditions, harvest date, olive processing technology, and EVOO finalization and storage parameters. It was expected that the results obtained would be useful for improving the understanding of the origins of the typical sensory characteristics of the investigated Croatian monovarietal EVOOs. However, the main premise was that the instrumental techniques utilized would be effective in tracing robust chemical markers among the investigated compounds despite the aforementioned sample heterogeneity, able to provide complementary information about varietal origin to that obtained by sensory analysis. Besides allowing better quality management and control in production, such findings would contribute strengthening the PDO identities and position on the market of Croatian EVOO.
