3.4.2. HPLC-MS Analysis Condition

Chromatographic analysis was accomplished by means of a Shimadzu HPLC system (Kyoto, Japan) equipped with a CBM-20A controller, two LC-20AD dual-plunger parallel-flow pumps, a DGU20A5R degasser, a CTO-20AC column oven, a SIL-30AC autosampler, an SPD-M20A photodiode array detector and an LCMS-2020 single quadrupole mass spectrometer, with the employment of ESI source operated in negative and positive ionization modes.

Chromatographic separations were carried out on Ascentis Express RP C18 columns (150 × 4.6 mm; 2.7 μm) (Merck Life Science, Merck KGaA, Darmstadt, Germany). The employed mobile phase was composed of two solvents, water (solvent A) and ACN (solvent B), both acidified with 0.10% of formic acid *v*/*v*. The flow rate was set to 0.8 mL/min, and was split into 0.2 mL/min prior to ESI-MS detection, under gradient elution 0–15 min, 0–15% B; 30 min, 20% B; 60 min, 50% B; 70 min, 100% B; 79 min, 100% B. The injection volume was 5 μL. Diode array detection (DAD) was applied in the range of 200–400 nm and monitored at a wavelength of 330 nm (sampling frequency: 40.0 Hz, time constant: 0.08 s). MS conditions were as follows: scan range and scan speed were set to a mass-to-charge ratio (*m*/*z*) of 100–1000 and 2500 amu/s, respectively; event time was 0.3 s; nebulizing gas (N2) flow rate was 1.5 L/min; drying gas (N2) flow rate was 15 L/min; interface temperature was 350 ◦C; heat block temperature was 300 ◦C; desolvation line temperature was 300 ◦C; desolvation line voltage was 1 V; interface voltage was −4.5 kV.
