2.4. LC-ESI-MS/MS Analysis of Phenolic Acids

Phenolic acid content was determined according to a modified method described by Oniszczuk et al. [21]. Experiments were carried out using an Agilent 1200 Series HPLC system (Agilent Technologies, Santa Clara, CA, USA) connected to a 3200 QTRAP Mass spectrometer (AB Sciex, Redwood City, CA, USA) equipped with electrospray ionization source (ESI). Both were controlled with Analyst 1.5 software (AB Sciex, USA). This was also used for data interpretation. Separations were carried out on a Zorbax SB-C18 column (2.1 × 100 mm, 1.8-µm particle size; Agilent Technologies, Santa Clara, CA, USA) at 20 °C. The gradient method was used with mobile phases: Water with 0.1% HCOOH (A) and acetonitrile with 0.1% HCOOH (B). Herein, the injection volume was 3 µL, the flow rate was 250 µL/min and the gradient were as follows: 0–2 min: 25% B, 3–6 min: 35% B, 8–10 min: 55% B, 12–16 min: 75% B, 19–25 min: 25% B. ESI operated in the negative-ion mode at the following conditions: Capillary temperature 400 °C, curtain gas at 30 psi, nebulizer gas at 50 psi, negative ionization mode source voltage −4500 V. Triplicate injections were made for each standard solution and sample. The analytes were identified by comparing retention time and m/z values obtained by MS and MS2 with the mass spectra from corresponding standards tested under the same conditions. The identified phenolic acids were quantified based on their peak areas and by comparison with a calibration curve obtained via the corresponding standards.