2.6. Analysis of Phenolic Compounds

One extract with the best qualities (antioxidant and antiproliferative activities) was selected and analyzed by HPLC–ESI-MS, in order to identify some phenolic compounds. The analyses were performed using an Agilent Technologies 1260 chromatograph (Palo Alto, CA, USA) equipped with a binary pump (G1312B), a degasser, an autosampler, and a thermostatted column compartment. The extract was dissolved in methanol at 20 mg/mL and the injected sample was of 1 μL. Chromatographic separation was performed on a Luna Phenyl-hexyl column (Phenomenex 150 mm × 4.6 mm × 5 μm) kept at 30 °C. The mobile phase was composed of 0.1% formic acid (A) and acetonitrile (B) at a flow rate of 0.5 mL/min. The gradient program was as follows: 0% B (0 min), 15% B (20 min), 50% B (28 min), 70% B (30 min), 90% B (50 min), and 0% B (55 min); finally, the initial conditions were maintained for 10 min. The chromatographic system was coupled to a quadrupole time-of-flight mass spectrometer (MS) detector (Agilent Technologies 6520 Q-TOF, Palo Alto, CA, USA), equipped with an electrospray ionization (ESI) source operating in the negative ionization mode. The MS analysis settings were as follows: ESI capillary voltage, 4.0 kV; nitrogen as a drying and nebulizing gas at flow rate 10 L/min, temperature 350 °C, and pressure 40 psi; collimator voltage 175 V; and octopole voltage 750 V. The spectra were acquired over a mass to charge ratio (m/z) ranging from 50 to 1100. All results were analyzed using the MassHunter Workstation Agilent Technologies software. The phenolic compounds were identified using their mass spectra information, the molecular formula, and the bibliographic data.