2.7. HPLC-DAD-ESI-MS/MS Analyses for Phenolic Characterization

Polyphenol analysis was performed according to method described by Bosiljkov et al. [74] and modified with the following elution gradient: solvent B: 0–20 min, 14–23%; 20–40 min, 23–35%; 40–50 min, 40–60 min, 60%; 60–65 min 95%. Method in brief description analysis was carried out using LC-ESI-MS/MS an Agilent 1260 series LC and Agilent LC-QQQ-MS G6460A mass spectrometer (Agilent Technologies, Palo Alto, CA, USA) equipped with an electrospray ionization (ESI) interface. The LC system includes a G1322A on-line degasser, a G1312B Bin Pump, a G1367E autosampler, a G1330B thermostatic column control, and a G4218B DAD, all of which were controlled by the Agilent MassHunter B 6.0 software. The HPLC separation was performed on a Poroshell 120 EC-C18 column (120 × 2.1 mm i.d. 2.7 μm particle size, Agilent Technologies, Palo Alto, CA, USA) at 30 °C. The mobile phase consisted of 1% formic acid in water (solvent A) and methanol (solvent B) by applying the following gradient: 0−20 min: 2−23% B, 20−40 min: 23−35% B, 40−46 min: 35−38% B, 46−60 min: 60% B, 60−65 min: 95% B. The flow rate was 0.2 mL min^-1. The injection volume was 1.0 μL with the UV detector set to an absorbance wavelength of 280 nm for phenolic acid, 520 nm for anthocyanins and 360 nm for flavonol glycosides. The mass spectrometer was equipped with electrospray ion source (ESI), parameters were as follows: nebulizer 35 psi; dry gas (N2) flow, 6 L min⁻¹; and dry gas temp. 300 °C; capillary voltage, 4 kV where the ion trap mass spectrometer was operated in negative/positive ion mode with a scanning range from m/z 100 to m/z 1000. Individual phenolic compounds were identified by comparing their retention times MS/MS and UV/Vis spectra with those of authentic standards [89,90,91]. Quantification of phenolic compounds were calculated from the peak areas of the samples and corresponding standards. For the compounds lacking standards, the quantification was achieved using similar compounds. Trans-resveratrol glucoside was quantified in equivalents of trans-resveratrol, malvidin-3-(6-O-p-coumaroyl) glucoside and malvidin-3-(6-O-acetyl) glucoside were quantified in equivalents of malvidin 3-O glucoside.