Agilent 6540 accurate mass q tof lc ms system

UPLC-ESI-QTOF-MS analysis was performed on an Agilent 6540 accurate mass Q-TOF LC/MS system (Agilent Technologies, Santa Clara, CA, USA). Chromatography was performed on a UPLC C₁₈ analytical column (2.1 ×  5 mm, I.D. 1.7 μm, ACQUITY UPLC^® BEH, Waters, Milford, MA, USA). The mobile phase used for the elution of the column consisted of 0.1% (v/v) formic acid in water (solvent A) and 0.1% (v/v) formic acid in acetonitrile (solvent B). A gradient elution was performed as follows: 0–15 min, 10–45% B; 15–23 min, 45–70% B; 23–25 min, 70–100% B. The flow rate was set at 0.4 mL/min with an injection volume of 2 μL, whilst the column temperature was maintained at 40 °C.
Detection by ESI-QTOF-MS/MS was performed in positive ion mode. The source parameters were set as follows: gas temperature, 300 °C; gas flow, 8.0 L/min; nebulizer pressure, 45 psi; sheath gas temperature, 350 °C; sheath gas flow, 8.0 L/min. The scan source parameters were set as follows: VCap, 3500; nozzle voltage, 500 V; fragmentation voltage, 120 V. Reference masses were used at m/z 121.0508 (purine) and 922.0097 (hexakisphosphazine). Automatic MS/MS was performed using a fixed collision energy of 15 eV.

The dried aqueous fractions were resuspended in 250 μL of 50:50 of methanol:water containing 0.2% acetic acid and sonicated for 10 s, whereas the organic fractionsweresuspended in 30:70 of mobile phase A and B (Table 2—organic), followed by centrifugation at 3000 rpm for 10 min. Five microliters of the resuspended fractionswas injected into an Agilent 1260 Infinity LC System, interfaced to an Agilent 6540 accurate mass Q-TOF LC/MS system (Santa clara, CA, USA). The reference solution was prepared using API-TOF Reference Mass Solution Kit (p/n: G1969-85001 (Santa clara, California, USA).Ten microliters of HP921 and 5 μL of purine were dissolved in one liter of methanol:acetonitrile:water (750:200:50) containing 0.1% acetic acid and were sprayed using an isocratic pump at a flow rate of 0.4 mL/min. The chromatographic parameters are shown in Table 2.