Uplc c18 analytical column

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.

UHPLC–QTOF-MS analysis was performed on an Agilent 6540 ultra-high definition accurate mass quadrupole time-of-flight spec-trometer with UHPLC (UHPLC–QTOF-MS, Agilent Technologies, U.S.A.). A UPLC C₁₈ analytical column (2.1 mm × 100 mm, I.D. 1.7 μm, ACQUITY UPLC®BEH, Waters, U.S.A.) was used for separation, coupled with a C₁₈ pre-column (2.1 mm × 5 mm, I.D. 1.7 μm, Van-GuardTM BEH, Waters, U.S.A.) at room temperature of 20 °C. The mobile phase was a mixture of water (A) and acetonitrile (B), both containing 0.1% formic acid, with an optimized linear gradient elution as follows: 0–5 min, 10–35% B; 5–25 min, 35–55% B; 25–28 min, 55–85% B; 28–30 min, 85–100% B. The injection volume was 4 μL. The flow rate was set at 0.35 mL/min. The mass spectra were acquired in negative mode by scanning from 100 to 1700 in mass to charge ratio (m/z). The MS analysis was performed under the following operation parameters: dry gas temperature 300 °C, dry gas (N₂) flow rate 5 L/min, nebulizer pressure 30 psi, Vcap 3000, nozzle voltage 500 V, and fragmentor voltage 200 V.