*2.5. Analysis of Phenols by UPLC-DAD*

Extraction of phenols from EVOO was performed according to the modified method proposed by Jerman Klen, Golc Wondra, Vrhovšek, and Mozetiˇc Vodopivec [42]. Ten grams of EVOO were dissolved in 10 mL of n-hexane, and 5 mL of methanol was added. The mixture was vortexed for 2 min, sonicated for 10 min, and then centrifuged at 5000 rpm for 5 min. The extraction was repeated 2 more times, and unified methanol extracts were defatted by 3 portions of 10 mL n-hexane. The methanol extracts (or standard solutions) were evaporated to dryness, the residue was re-dissolved in a 2 mL of a mixture of HPLC eluents (A (95:5 water—acetic acid (*v*/*v*)):B (methanol) = 90:10 (*v*/*v*)), and filtered through 0.45 µm PTFE filters.

Analysis of phenols was performed by ultra-performance liquid chromatography with diode array detection (UPLC-DAD) using an Agilent Infinity 1260 system (Agilent Technologies, Palo Alto, CA, USA) equipped with a G1311B quaternary pump, a G1329B autosampler, a G1316A column oven, and a G4212B DAD detector. A Kinetex PFP column (2.6 µm, 100 mm × 4.6 mm) with a guard was used (Phenomenex, Sydney, Australia) at 27 ◦C. Solvents were water with glacial acetic acid (95:5, *v*/*v*) (A) and methanol (B), with a flow rate of 1 mL/min. Ten microliters of the extract were injected. A 20-step gradient run used was reported previously [42]. Identification was performed by comparing retention times and UV/Vis spectra with those of pure standards when available, and with UV/Vis spectra from the literature [42]. Detection wavelengths were 280 nm (for simple phenols, vanillic acid, lignans, and secoiridoids), 320 nm (vanillin and *p*-coumaric acid), and 365 nm (flavonoids), while spectra were registered from 200 to 600 nm. Standard calibration curves were constructed for tyrosol, hydroxytyrosol, vanillic acid, vanillin, *p*-coumaric acid, luteolin, apigenin, pinoresinol, and oleuropein. For other compounds semi-quantitative analysis was carried out: secoiridoids were expressed in mg/kg as oleuropein, and acetoxypinoresinol as pinoresinol equivalents, respectively.
