*2.4. Antioxidant Activity*

Antioxidant activity was evaluated using three di fferent assays: the 2,2-diphenyl-1-picrylhydrazyl (DPPH) test, as reported by Goristen et al., [18]; the Oxygen Radical Absorbance Capacity (ORAC) test, as reported by Wang et al., [19]; and superoxide anion scavenging activity analysis, as described by Dasgupta et al. [20]. All the essays were performed in triplicate and the antioxidant activity were expressed as μmol of Trolox equivalent mg<sup>−</sup><sup>1</sup> of dry weight (DW).

#### *2.5. HPLC ESI*/*MS-TOF Analysis of Olive Extracts*

The phenolic compounds characterization was performed using Agilent 1200 High Pressure Liquid Chromatography (HPLC) System (Agilent Technologies, Palo Alto, CA, USA) equipped with a standard autosampler, as reported by Nicolì et al. [21]. The HPLC system was coupled to an Agilent diode-array detector (detection wavelength 280 nm) and an Agilent 6320 TOF mass spectrometer equipped with a dual ESI interface (Agilent Technologies) operating in negative ion mode. Detection was carried out within a mass range of 50–1700 *<sup>m</sup>*/*<sup>z</sup>*. Accurate mass measurements of each peak from the total ion chromatograms (TICs) were obtained by means of an ISO Pump (Agilent G1310B) using a dual nebulizer ESI source that introduces a low flow (20 <sup>μ</sup>L·min−1) of a calibration solution which contains the internal reference masses at *m*/*z* 112.9856, 301.9981, 601.9790, and 1033.9881, in negative ion mode. The identification of anthocyanins was carried out with the same method, but with positive ionization, using the internal reference masses at *m*/*z* 121.050873, 149.02332, 322.048121, and 922.009798.

The quantification of anthocyanins was achieved using calibration curves of authentic chemical standards cyanidin 3 glucoside and cyanidin 3 rutinoside [22], using an HPLC Agilent 1100 coupled with an Agilent DAD sensor (detection wavelength 280 nm and 520 nm). Separation was carried out at 30 ◦C with a gradient elution program at a flow rate of 0.8 mL/min using a Phenomenex Gemini C18 250 × 4.6 mm, 5-μm separation column. The mobile phases consisted of water plus 7.0% formic acid (A) and water:formic acid:acetonitrile 43:7:50 (B). The following multistep linear gradient was applied: 0 min, 6% B; 15 min, 30% B; 25 min, 50% B; 30 min, 60% B; The injection volume in the HPLC system was 5 μL.
