Acquity 1.7 μm c18 column

Liquid chromatographic separation was achieved on a 100 mm × 2.1 mm Acquity 1.7-μm C18 column (Waters Corp, Milford, MA) by using an HPLC system (1200 rapid resolution system; Agilent Technologies, Santa Clara, CA). The column was maintained at 40 °C with a flow rate of 0.25 ml/min. Samples were eluted from the LC column with linear gradients of solvent A (2 mM ammonium formate in water) and solvent B (100% ACN): 0–1 min: 0% B; 1–9.6 min: 0–98% B; 9.6–15 min: 98% B; and 15–18 min 0% B.
Mass spectrometry was performed on an Agilent Q-TOFMS (6510 Q-TOF MS; Agilent Technologies, Santa Clara, CA) operated in electrospray positive-ion (ESI+) and electrospray negative-ion (ESI−) modes. The scan range was from 50 to 1000 m/z. The capillary and skimmer voltages were set at 4000 and 65 V, respectively; the liquid nebulizer was set to 30 psig, and the nitrogen drying gas was set at a flow rate of 10 l/min; and the drying gas temperature was maintained at 350 °C. To maintain a constant mass accuracy, purine ([M+H]+ = 121.050873), hexakis (1H,1H,3H-tetrafluoropropoxy) phosphazine ([M+H]+ = 922.009798), or purine ([ M – H ] − = 119.036320) and formate adduct ([M–H] − = 966.000725) were used as internal reference ions. Data were collected in the profile mode by using data acquisition software (Agilent MassHunter Workstation).