UHPLC-QTOF-MS Analysis of Compounds

Both chromatographic conditions and mass spectrometer parameters were previously described in other publication [29] (link). Briefly, analysis was performed on Agilent 1290 UHPLC system with XTerra MS C18 Column (30 mm×2.1 mm; 3.5 µm). The mobile phase A was ultrapure water with 0.05% FA and B was MeOH at a flow rate 0.4 ml min⁻¹ in gradient elution (95% A to 0% A in 15 min, back to 95% A in 1 min and kept at 95% A for 5 min). The column was maintained at 22°C and the injection volume was 5 µL.
The UHPLC system was coupled to a hybrid quadrupole time-of-flight (QTOF) mass spectrometer (Agilent 6540 Series Accurate Mass QTOF-MS) with Dual ESI interface and operated in positive ion mode. Operation conditions were as follow: sheath gas temperature 400°C at flow rate of 12 L min⁻¹, capillary voltage 4000 V, nebulizer pressure 20 psig, drying gas 10 L min⁻¹, gas temperature 325°C, skimmer voltage 45 V, octopole RF peak 750 V and fragmentor voltage 100 V. Analysis were performed using MS/MS or TargetMS/MS mode with variable collision energies (10, 20 or 30 V; however, 20 V was the optimal value for all the target compounds) and in mass range 50–1000 m/z. The instrument was operated in the 4 GHz high-resolution mode and acquisition rate was 1.5 spectra per second. Acquisition data were processed with Agilent MassHunter Workstation software.