Ltq velos pro linear ion trap mass spectrometer

Vasicine, vasicinone, isoorientin, isovitexin, and apigenin were quantified using a Thermo Vanquish Flex UPLC System interfaced with an LTQ Velos Pro Linear Ion Trap Mass Spectrometer (ThermoFisher, San Jose, CA, USA). Chromatographic separation of the analytes was achieved using a BEH C₁₈ column (150 × 3 mm i.d.; 1.7 μm particle size; Waters, Milford, MA) at 50 °C with gradient elution. The mobile phases were (A) 0.1% formic acid in H₂O and (B) methanol and a flow rate of 300 mL/minute. The percentage of B varied as follows: 2% B at 0 min, 2%→95% (0 to 7.5 min), hold at 95% (7.5 to 8.5 min), 95%→2% (8.5 to 8.6 min), and hold at 2% from 8.6 to 10 min. A convex gradient (setting = 6 during the 0–7.5 min ramp) was used. The mass spectrometer was operated in positive electrospray ionization, selective reaction-monitoring mode. The MS/MS parameters were optimized by infusion of vasicine into the MS/MS system and the following precursor→product transitions were used: m/z 189.1→m/z 171.1 for vasicine, m/z 203.2→m/z 185.1 for vasicinone, m/z 449.1→m/z 431.1 for isoorientin, m/z 433.2→m/z 415.1 for isovitexin, and m/z 271.1→m/z 271.1 + 243.1 for apigenin. Data acquisition and processing were performed using Xcalibur software, and calibration curves were prepared in water using analyte peak area versus concentration.