*2.2. Analytical Methods*

The oil yield (% oil dry weight) was determined in drupes after stone removing by extraction with petroleum ether in a Soxhlet apparatus (Bicasa s.r.l., Bernareggio, MI, Italy). For the olive oil extraction, about 15 kg of drupes were milled with a hammer mill. The obtained paste was mixed at a temperature below 20–25 ◦C for 30 min and pressed using a hydraulic press (pressure up to 200 bar) in a small olive oil press mill Mini 30 system (Agrimec Valpesana, Firenze, Italy). After centrifugation and filtration through paper, olive oils were then stored in dark glass bottles at room temperature and analyzed for the total free acidity value, peroxide index, and UV light absorption coefficients according to EC regulations [21,22]. Pigments were extracted from the oil samples (5 mL of oil and 5 mL of cyclohexane) following the method reported by Minguez-Mosquera et al. [23], and the total

contents of chlorophylls and carotenoids were determined spectrophotometrically (670 nm and 470 nm, respectively). Total tocopherols were evaluated according to Bakre et al. [24]. The oil samples were diluted in isopropanol (1:10) and filtered (0.45 μ pore size). An aliquot of 5 μL of samples was injected in an ultra-high performance liquid chromatography (UHPLC) system (UHPLC PLATINblue, Knauer, Germany), coupled with a fluorescence detector RF-20A/RF-20Axs model (Shimadzu Corporation, Kyoto, Japan) and analyzed (flow rate of 0.4 mL min−1) through a mobile phase of methanol/acetonitrile (50:50). The detector was set at a 290 nm excitation wavelength and 330 nm emission wavelength. The identification and quantification were performed by calibration curve, using pure α-tocopherol as the standard and concentrations ranging from 10 to 500 mg kg<sup>−</sup>1. Results were expressed as mg kg<sup>−</sup>1. Determination of total polyphenols was performed following Baiano et al. [25]. Two mL of methanol/water (70:30, *v*/*v*) and 2 mL of hexane were added to 5 g of oil samples and mixed with a vortex (10 min). The hydro-alcoholic phase containing phenols was separated from the oil phase by several centrifugations; 100 μL of phenolic extract were mixed with 100 μL of Folin–Ciocal teau reagent (2N) and, after 4 min, with 800 μL of an aqueous solution of Na2CO3 (5%). The mixture was heated in a 40 ◦C water bath for 20 min, and the total phenol content was determined calorimetrically at 750 nm. The total phenolic content was expressed as milligrams of gallic acid equivalents per kilogram of oil. The total antioxidant activity of the olive oils was detected by 2,2 -azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS)/Trolox equivalent antioxidant capacity (TEAC), according to Re et al. [26], and 2,2-diphenyl-1-picrylhydrazyl (DPPH), following the opportunely modified method of Brand-Williams et al. [27]. Fatty acid composition was determined as methyl esters (FAME) following the official method [22].
