LC–MS/MS analysis was carried out using a Thermo Scientific UHPLC instrument coupled to a TSQ Quantum Vantage (Thermo Fischer Scientific, San José, CA, USA) triple-stage quadrupole mass spectrometer. Chromatographic separation was achieved using a C18 reversed-phase analytical column, Kinetex C18 (2.1 × 50 mm, 1.7 μm particle size, 100 Å, Phenomenex, Torrance, CA, USA). The elution gradient consisted of mobile phase (A) H2O (0.1% HCOOH) and (B) CH3OH. The gradient used was the following: at t = 0.0 min, 90% A and 10% B; at t = 1 min, 90% A and 10% B; at t = 6 min, 65% A and 35% B; at t = 9 min, 65% A and 35% B; at t = 11 min, 5% A and 95% B; at t = 13 min, 5% A and 95% B; at t = 14 min, 90% A and 10% B; at t = 15 min, 90% A and 10% B. The flow rate was set at 0.3 mL/min, and the sample injection volume was 10 μL. Mass spectrometric analysis was performed using a heated electrospray ionization (HESI II) source operating in negative ion mode. The following operating conditions were applied: spray voltage, 4.0 kV; vaporizer and capillary temperatures, 280 and 280 °C, respectively; sheath and auxiliary gas at 60 and 20 arbitrary units (au), respectively; S-lens rf amplitude was fixed at 165 V.
Instrument control and data processing was carried out using Xcalibur software v2.0.0 (Thermo Fischer Scientific, San José, CA, USA). The total LC–MS/MS method runtime was 16 min.
Instrument control and data processing was carried out using Xcalibur software v2.0.0 (Thermo Fischer Scientific, San José, CA, USA). The total LC–MS/MS method runtime was 16 min.