2.3. Polyphenol analysis and free radical scavenging capacity

To isolate the phenolic fraction of the three EVOOs, 1.5 g of sample was mixed with 1.5 ml of hexane and charged onto cartridges SPE C₁₈. Polyphenols were eluted through 3 ml of methanol 100% and recovered; this step was repeated other two times. The three residues were collected, grouped, dried, and re‐suspended with 1 ml of methanol. The samples were filtered (mesh = 0.20 μm). The method of Singleton and Rossi (Singleton & Rossi, ¹⁹⁶⁵) was used to evaluate the content of total polyphenols present in the three EVOO samples. Quantification was determined by using gallic acid as standard and reading the absorbance at 760 nm through a Cary UV/Vis spectrophotometer (Varian). Results were expressed as μg gallic acid equivalent (GAE)/g of EVOO ± standard deviation (SD).

The free radical scavenging activity was determined using the stable radical 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH assay) (Brand‐Williams, Cuvelier, & Berset, ¹⁹⁹⁵). The analysis was performed in microplates by adding 15 μl of extract to 300 μl of a methanol DPPH solution (0.153 M). Next, the absorbance at λ = 517 nm was spectrophotometrically measured (Cary 50 MPR, Varian). The absorbance of DPPH without antioxidant (control sample) was used for baseline measurements. The scavenging activity was expressed as effectiveness (%) of the sample to inhibit DPPH radical activity during a 60‐min incubation.

Polyphenol profile was determined through UPLC (ultra high‐performance liquid chromatography) by using an ACQUITY Ultra Performance system linked to a PDA 2996 photodiode array detector (Waters), setting the UV detection wavelength at 280 nm, following the method of Fratianni and coworkers (Fratianni et al., ²⁰¹⁶). Quantification of known components was performed by comparing the peak areas on the chromatograms of samples with those obtained from standard solutions.