Ionization of the molecules was acquired in negative ESI mode with capillary voltage at 4000 V, using nitrogen as drying (T = 350 °C; flow rate = 9 L/min) and nebulizing gas (40 psi). The mass acquisition in MS and MS/MS spectra ranged between m/z 50 and 1200. All data were acquired and processed using Mass Hunter Workstation software (version B.01.04; Agilent Technologies). Typically, two runs were performed during the HPLC-ESI-MS analysis of each sample. First, an MS full-scan acquisition was performed to obtain preliminary information on the predominant m/z ratios observed during the elution. Subsequently, MS/MS spectra were acquired: quadrupole 1 filtered the calculated m/z of each compound of interest, while quadrupole 3 scanned for ions produced by nitrogen collision of these ionized compounds in the chosen range at a scan time of 500 ms/cycle.
Tentative compound identification was achieved by combining different information: UV absorption, retention times (RT), and mass spectra (MS and MS/MS) which were compared with those from pure standards, when available, and/or interpreted with the help of structural models already hypothesized in the literature [11 (link),36 (link),37 (link)]. Then, the main revealed compounds were quantified by multiple reaction monitoring (MRM) as 3-hydroxytyrosol (R2 = 0.99923; LOD = 0.0033 µg/g; LOQ = 0.0113 µg/g) and tyrosol (R2 = 0.99904; LOD = 0.0041 µg/g; LOQ = 0.0125 µg/g) equivalents in the case of aromatic alcohols and secoiridoids, apigenin (R2 = 0.99937; LOD = 0.0028 µg/g; LOQ = 0.0108 µg/g) equivalents in the case of flavonoids, and pinoresinol (R2 = 0.99889; LOD = 0.0054 µg/g; LOQ = 0.0152 µg/g) equivalents in the case of lignans. The optimized parameters (fragmentor voltage and collision energy) for each selected compound together with the mass transitions adopted for MRM were acquired through Mass Hunter Optimizer software (version B.03.01; Agilent Technologies) (