*2.3. Determination of the Physicochemical Quality Parameters*

Free fatty acid, peroxide value and spectroscopic indices (K<sup>232</sup> and K268) were carried out, following the analytical methods described in the Regulation EEC/2568/91 of the European Commission and later amendments [18]. Free fatty acid was expressed as the percentage of oleic acid and peroxide value was given as milliequivalents of active oxygen per kilogram of oil (meq O<sup>2</sup> kg−<sup>1</sup> ). K<sup>232</sup> and K<sup>268</sup> extinction coefficients were calculated from absorption at 232 and 268 nm respectively. These absorptions are expressed as specific extinctions E (the extinction of 1% *w*/*v* solution of the oil in isooctane, in a 10 mm cell) conventionally indicated by K "extinction coefficient".

#### *2.4. Determination of Sterols and Triterpene Dialcohols*

The individual sterols, total sterols, and triterpene dialcohols were determined according to the method adopted by EEC/2568/91 regulation, Annexes V with later amendments [18]. The oil sample, with added 5a-cholestan-3β-ol, as an internal standard, was saponified with potassium hydroxide in ethanolic solution and the unsaponifiable matter was extracted with diethyl ether. The sterol and triterpene dialcohol fractions were separated from the unsaponifiable matter by thin-layer chromatography on a basic silica gel plate. The fractions recovered from the silica gel were transformed into trimethylsilyl ethers (TMSE) by the addition of pyridine-hexamethyldisilizane-tri-methylchlorosilane (9:3:1, *v*/*v*/*v*). Sterols (%) and triterpene dialcohol contents were determined with a Shimadzu (GC-2010) gas chromatograph equipped with a flame ionization detector (FID), a DB-5 (30 m × 0.32 mm × 0.25 µm) capillary column and an autosampler injector. The operating conditions were as follows: injection temperature 280 ◦C, column temperature 265 ◦C, detector temperature 310 ◦C, splitting ratio (1:50), flow rate 1.4 mL/min, and injection volume of 1 µL of TMSE solution. Individual sterols were identified based on their relative retention times with respect to the internal standard, 5a-cholestan-3β-ol, according to the standardized reference method [18]. The sterols and triterpene dialcohols eluted in the following order: cholesterol, 24-methylen-cholesterol, campesterol, campestanol, stigmasterol, ∆7-campesterol, ∆5,23-stigmastadienol, clerosterol, β-sitosterol, sitostanol, ∆5-avenasterol, ∆5,24-stigmastadienol, ∆7-stigmastenol, ∆7-avenasterol, erythrodiol, and uvaol (calculated as total erythrodiol). Sterol and triterpene diol concentrations were calculated as mg/kg of oil with respect to the internal standard. Results were expressed as proportions (%) of total sterols. The sum of ∆5,23-stigmastadienol, clerosterol, β-sitosterol, sitostanol, ∆5-avenasterol, and ∆5,24-stigmastadienol represents apparent b-sitosterol. Mean values of duplicate experiments in each sample were used for further statistical analysis.

#### *2.5. Determination of Fatty Acid Composition*

The fatty acid composition was determined according to the official method of the Regulation EEC/2568/91, Annex IV with amendments [18]. The fatty acid methyl esters (FAME) were obtained by cold alkaline transesterification with methanolic potassium hydroxide solution and extracted with *n*-heptane. FAME were analyzed on a model GC-2010 Shimadzu chromatograph, equipped with a BPX-70, (60 m × 0.25 mm × 0.25 µm), capillary column and a flame ionization detector (FID). The carrier gas was helium, with a flow of 1.5 mL/min. The temperatures of the injector and detector were set at 250 and 260 ◦C respectively and the oven temperature was increased gradually from 165 to 225 ◦C in 35 min. The injection volume was 1 µL. Quantification was achieved using a FAME standard mixture. The results were expressed as a percentage of individual fatty acids.
