Polyphenol Characterization by RP-LC-DAD-ESI-MS

Polyphenol characterization of LE and RE was carried out by RP-LC-DAD-ESI-MS analysis according to Smeriglio et al. [32 (link)]. Chromatographic elution was carried out by a Luna Omega PS C18 column (150 mm × 2.1 mm, 5 µm; Phenomenex, Torrance, CA, USA) at 25 °C by using mobile phase 0.1% formic acid (Solvent A) and methanol (Solvent B) according to the following program: 0–3 min, 0% B; 3–9 min, 3% B; 9–24 min, 12% B; 24–30 min, 20% B; 30–33 min, 20% B; 33–43 min, 30% B; 43–63 min, 50% B; 63–66 min, 50% B; 66–76 min, 60% B; 76–81 min, 60% B; 81–86 min, 0% B and equilibrated 4 min. The injection volume was 5 µL. The UV–Vis spectra were recorded ranging from 190 to 600 nm, and chromatograms were acquired at different wavelengths (220, 260, 292, 330, and 370 nm) to identify all polyphenol classes. The experimental parameters of the mass spectrometer (ion trap, model 6320, Agilent Technologies, Santa Clara, CA, USA) operating in the negative (ESI−) and positive (ESI+) ionization mode were set as follows: 3.5 kV capillary voltage, 40 psi nebulizer (N₂) pressure, 350 °C drying gas temperature, 9 L/min drying gas flow and 40 V skimmer voltage. Acquisition was carried out in full-scan mode (90–1000 m/z). Data were acquired by Agilent ChemStation software version B.01.03 and Agilent trap control software version 6.2.