2.8. Characterization of Phenolic Compounds by LC-ESI-QTOF-MS/MS Analysis

Extensive characterization of free and bound phenolic compounds from the extracts were carried out using liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry (LC-ESI-QTOF-MS/MS) analysis utilizing an Agilent 1200 series HPLC system equipped with an Agilent 6520 Accurate-Mass Q-TOF LC-MS (Agilent Technologies) with an electrospray ionization (ESI) source according to the method described by Duan, Subbiah, Xie, Agar, Barrow, Dunshea, and Suleria [17]. HPLC buffers (Mobile phase A: 100% MilliQ water with 0.1% formic acid, Mobile phase B: acetonitrile/MilliQ water/formic Acid (95:5:0.1)) were firstly deaerated by sonication in an Ultrasonic water bath (Power sonic 505, Gyeonggi-do, Republic of Korea) at 25 °C for 10 min. The separation process was conducted using a Synergi Hydro-Reverse Phase 80 Å, LC column 250 × 4.6 mm, 4 µm (Phenomenex, Torrance, CA, 202 USA) with column temperature set at 25 °C. The sample injection volume was 6 µL. The mobile phase was applied at a flow rate of 0.8 mL/min with gradient generation as follow: 10–25% B (0–25 min), 25–35% B (25–35 min), 35–40% B (35–45 min), 40–55% B (45–75 min), 55–80% B (75–79 min), 80–90% B (79–82 min), 90–100% B (82–84 min), 100–10% B (84–87 min), and isocratic 10% B (87–90 min). Nitrogen gas nebulization was fixed at 45 psi at 5 L/min and 300 °C while the sheath gas was set at 11 L/min and 250 °C. The voltages for capillary and nozzle were fixed at 3.5 kV and 500 V, respectively. Mass scan within the range of 50–1300 m/z was utilized. MS/MS analyses were performed in automation with collision energy of 10, 15, and 30 eV for fragmentation purposes. Finally, peak identification was carried out in both positive and negative mode based on comparing fragmentation pattern with database. Instrument control data acquisition and processing were conducted using MassHunter Workstation software (Qualitative Analysis, version B.03.01) (Agilent Technologies).