*3.1. Characterization of Olive Oils Using International Olive Oil Trade Standards*

Olive oils were grouped as extra virgin olive oil (EVOO) (*n* = 77), virgin olive oil (VOO)/olive oil (OO) (*n* = 27), and adulterated olive oil with vegetable oils (corn, sunflower, soybean, and canola oil) (*n* = 47) according to information provided by the Aydin Commodity Exchange Laboratories (Aydin, Turkey) and California Olive Oil Council. Table 1 summarizes the information on reference analysis with regard to the levels of major fatty acids, free fatty acids (FFA), peroxide value (PV), pyropheophytins (PPP), and total polar compounds (TPC).


**Table 1.** Reference concentration levels for the compounds measured in olive oil samples.

<sup>a</sup> EVOO: Extra virgin olive oil, <sup>b</sup> VOO/OO: Blend of virgin olive oil and olive oil, <sup>c</sup> Mixture: Adulterated olive oil with vegetable oils (corn, sunflower, soybean, and canola oil).

Fatty acid (FA) composition of the EVOO group (Table 1) showed that the five major FAs (16:0, 18:0, 18:1*n*-9, 18:2*n*-6 and 18:3*n*-3) fell within specified ranges set by the United States standards for grades of olive oil [41] and International Olive Council [42]. EVOO variation in FA levels among samples can be related to differences in geographic origin, variety, stage of maturity of the fruit, latitude, climatic

conditions, storage, and extraction process of samples [43–45]. EVOO and VOO showed similar fatty acid profiles, except for a sample obtained from Peru that showed higher palmitic (18.1%) and linoleic (17.7%) but lower oleic (57.7%) compared to other VOO samples. On the contrary, adulterated olive oils with vegetable oils showed marked variation in FA composition (Table 1). For instance, olive oil adulterated with canola oil had lower palmitic acid (5.7%), while linoleic (28.5%) and linolenic (4.4%) acids were higher than pure olive oil. Adulteration of EVOO with corn oil resulted in a decrease in the levels of oleic acid (29.9%) and an increase in linoleic acid (58.6%) content.

The average FFA content of the EVOO and VOO/OO samples ranged from 0.4 ± 0.2% and 0.5 ± 0.5%, respectively. The main difference between EVOO and VOO resulted from their FFA content. According to the trade standards of the International Olive Council (IOC) (2018), the FFA content of EVOO, VOO, and OO cannot exceed 0.8%, 2.0%, and 1.0%, respectively [42]. FFA levels of adulterated EVOO samples with other vegetable oils ranged from 0.1% to 10.3% (2.1 ± 2.7%). In particular, two adulterated EVOO samples showed FFA levels of 9.0% and 10.3% that could be related to mixing olive oils with crude vegetable oil or waste cooking or frying oil. There is no FFA limit for the crude vegetable oils, van Doosselaere (2013) reported that crude palm oil FFA levels could reach levels of 20–25% because of the lipolytic enzymes of the fruit that were not handled properly [46]. The frying or cooking process increases the FFA content of vegetable oils since oils that contain high levels of polyunsaturated fatty acids are highly susceptible to hydrolysis, oxidation, and polymerization under a frying environment [47].

Peroxide value of olive oil samples were 9.8 ± 2.0, and 10.0 ± 2.5 meqO2/kg for EVOO and VOO/OO samples, respectively. According to the European Union Commission Regulations (EEC/2568/91), the PV limit for EVOO and VOO are 20 meqO2/kg, whereas the limit for OO is 15 meqO2/kg [48], and our findings were under the established limits for different grades of olive oils. Similar values for PV of EVOO and VOO, ranging from 6.2 to 11 meqO2/kg, were reported by Casal and others (2010) [49]. A high PV indicates that olives or paste were likely mishandled [50]. Adulterated olive oils with other vegetable oils showed PV ranging from 2.5 to 32.7 meqO2/kg, indicating that counterfeiters employ a wide array of oil quality, including freshly deodorized to highly oxidized vegetable oils.

Pyropheophytin (PPP) values of the samples were 11.5 ± 2.3, 13.2 ± 3.0, 19.8 ± 3.0% for EVOO, VOO/OO blends, and olive oil mixtures with vegetable oil samples, respectively. The PPPs are the breakdown products of chlorophyll in olive oil. The chlorophyll pigment initially breaks down to pheophytin (a and a'), and then into pyropheophytins, due to the decarbomethoxylation of chlorophyll and pheophytins, upon the effect of heat [51]. The elevated level of PPP indicates that the samples were oxidized and/or adulterated with cheaper refined oils and the limit of the total PPP should be lower than 15% in EVOO [52].

Average total polar compounds (TPC) of the EVOO, VOO/OO, and adulterated olive oils ranged from 5.2 ± 1.1%, 6.6 ± 1.5%, and 8.7 ± 2.4%, respectively. The TPC measures the polar fraction in oils that are composed of polymers (dimers, trimers, and highly polymerized compounds) and decomposition products (mono and diacylglycerols, FFAs, volatile compounds, cyclic, and non-cyclic monomers) [53]. The TPC limit for frying oil is 25% according to international legislation, and if an oil exceeds this limit it becomes unsuitable for human consumption [53].

Overall, the chemical quality parameters of EVOO and OO showed strong overlapping within minimum and maximum limits, making it challenging to use these parameters as reliable markers to identify potential adulteration to consumers.
