*3.1. Triacylglycerol Fraction*

The results of this research confirm that EVOOs show a high percentage (from 76.2% of Leccino to 78.0% of Dolce Agogia) of oleic acid, a medium content of palmitic (from 12.3% of Dolce Agogia to 13.2% of Leccino) and stearic (about 2%) acids, a good percentage of linoleic acid (from 6.0% of Dolce Agogia to 7.1% of Frantoio), and a low content of α-linolenic (about 0.7%) acid. Table 1 shows the data of the acidic compositions of the total TAG percent composition of monovarietal EVOO samples.


**Table 1.** Total fatty acid (FA) percent composition of triacylglycerol (TAG) fraction of monovarietal extra virgin olive oil (EVOO) samples (% mol, mean values ± SD, *n* = 3).

The linearity parameters, evaluated in the range 12.5–200 μg/mL of the considered FAME, have been calculated together with their limits of detection (LOD) and limits of quantification (LOQ), according to the statistical method, reported in Federal Register [28]. The coefficients of correlations for the considered FAME were always greater than 0.9969. The LOD values change from 10 ng/mL of α-linolenic acid to 20 ng/mL of palmitic acid. The LOQ values change from 30 ng/mL of α-linolenic acid to 66 ng/mL of palmitic acid.

Similar FA composition was obtained from other analyzed EVOO samples [20]. In a previous work, the leaves of the same olive tree varieties (Dolce Agogia, Frantoio, Leccino, and Moraiolo) have been studied to evaluate the seasonal variations of antioxidant compounds, and significant differences in hydroxytyrosol and oleuropein contents among the cultivars have been observed [29].

Some slight differences (*p* > 0.05) of total FA percent compositions were observed. Considering, for example, the Dolce Agogia and Leccino varieties, a higher percent content of oleic acid and a lower percent content of linoleic acid, the first in respect to the second cultivar, were found. Recently, the effects of different cultivar (Arbequina, Leccino, Maurino and Moraiolo) on the qualitative and quantitative profile of EVOOs have been studied by determining the profiles of acylglycerides, sterols, phenolics, hydrocarbons, pigments, and volatile components [8]. Regarding FA composition (palmitic, oleic, linoleic acids), as a function of cultivar and harvest date (2015), significantly different data were obtained in respect to those reported in Table 1.

It is known that the structure of glycerol backbone of TAG fraction influences the physicochemical, physiological, and nutritional properties of lipids. A deeper knowledge of these aspects can be useful to determine the geographical origin, species, varieties of a fat, as well as to detect food fraud, and to predict nutritional value. The dietary fats are absorbed mainly as *sn*-2-MAG and also as free FA, produced by lipase hydrolysis. They are re-esterified as TAG and incorporated into chylomicrons. In this way, the absorbed TAG molecules maintain the FA in the *sn*-2 position as in the dietary TAG, whereas the FA in the *sn*-1 and *sn*-3 positions are randomized and partly substituted by endogenous FA. Consequentially, the positions esterified by FA in the glycerol backbone become important for physiological/nutritional reasons [30]. In addition, another interesting application of enzymatic-instrumental methods is the monitoring of the synthesis of structured lipids to obtain better healthy fats [31–33]. For example the production of TAG with interesting FA esterified in *sn*-2 position, with nutritional advantages due to their real bioavailability, or medium chain triglycerides useful for enteral nutrition.

Table 2 shows the data of the acidic percent compositions of the *sn*-1, *sn*-2, and *sn*-3 positions of the TAG. With regard to the intrapositional acidic compositions, some significant differences among the cultivars were observed (*p* < 0.05). For example, considering the acidic composition of the *sn*-1 position, a lesser incorporation of palmitic acid in the *sn*-1 position (15.4 vs. 18.6/17.5/17.6) in Dolce Agogia variety, was observed compared to Frantoio, Leccino, and Moraiolo. A lower content of α-linolenic acid in the *sn*-1 position was found in Dolce Agogia variety compared to Leccino and Moraiolo (*p* < 0.05). A minor incorporation of linoleic acid in the *sn*-2 position of TAG of Dolce Agogia variety was observed compared to Frantoio and Leccino (8.9 vs. 10.5/10.8). Moreover, lower percent content of palmitic acid in *sn*-2 position has been highlighted in Dolce Agogia variety, compared to Frantoio (*p* < 0.05).

**Table 2.** Intrapositional FA percent composition of TAG fraction of monovarietal EVOO samples (% mol, mean values ± SD, *n* = 3).



The data of the intrapositional compositions of the TAG could also be used to obtain all the TAG molecular species, including the isomeric and enantiomeric species in order to evaluate the differences in the content of individual species among the different cultivars. It is, however, important to emphasize that the procedure for TAG stereospecific analysis has the disadvantage of being laborious, time-consuming, and difficult to automate.

In this study, the little differences highlighted from stereospecific analysis data were better revealed applying a chemometric procedure as LDA, reported below.
