LC-MS analyses were performed on a LC-MS 2020 system
(Shimadzu, Tokyo, Japan) associated to an electrospray ionization
source (ESI). Separation was carried on a 150 × 4.6 mm
i.d. C18 reverse phase Gemini column (Phenomenex, Torrance,
CA, USA) with particle size of 3 μm and pore size of 130 Å.
The column oven was fixed at 30 °C. The mobile phase
consisted of (A) 0.5%formic acid in water and (B) acetonitrile.
The injection volume was 20 μL and the flow rate was 0.6 mL/
min. The 40 min gradient used was as follows: 0–5 min, 90:10
(A:B); 5–7min from 90:10 to 86:14 (linear gradient); 7–17 min
held at 86:14; 17–19 min from 86:14 to 75:25 (linear gradient);
19–24 min held at 75:25; 24–25 min from 75:25 to 10:90
(linear gradient); 25–31 min held at 10:90; 31–32 min from
10:90 to 90:10, 32–40 min held at 90:10 (re-equilibration step).
The electrospray ionization source was operated in negative
mode. The nebulization gas flow was set to 1.5 L/min, drying
gas flow at 20 L/min, the heat block temperature was fixed at
350 °C, and the desolvation line (DL) temperature at 250 °C.
The probe voltage was set to - 4500 V.