Analytical uflc system

The LC analyses were performed on a Shimadzu analytical UFLC system (Kyoto, Japan) consisting of two LC-20AD XR pumps (pump A and pump B), a CTO-20A column oven, a DGU-20A3 degasser, a SPD-20A UV detector, and an SIL-20A XR autoinjector. An ACQUITY UPLC^® BEH C18 column (1.7 um, 2.1 mm, 150 mm, Waters, USA) with a column temperature of 40 °C was used for sample separation. The UFLC mobile phases were pumped by pump A (water) and pump B (acetonitrile), respectively; the water phase contained 0.1% formic acid (pH 2.70), and the flow rate was 0.3 mL/min. The on-line chromatogram of HBX-5 was recorded by UV1 at 254 nm in the UFLC system. A gradient elution was programmed as follows: 0–20 min, 5.0–90.0% (v/v) B; 20–24 min, 90–90% B; 24–25 min, 90–5% B; 25–30 min, maintaining 5.0% B. Injection volume was 1.0 μL. The above UFLC system was connected to a hybrid IT-TOF mass spectrometer (Shimadzu LCMS-IT-TOF, Kyoto, Japan) via an ESI interface. The detector voltage was fixed at 1.67 kV and the curved desolvation line (CDL) temperature and block heater temperature were both maintained at 200 °C. The flowrate of nitrogen as nebulizer gas was 1.5 L/min. For full scan MS analyses, spectra were recorded in the range m/z 100–1000. Data were processed later by LC/MS solution software (version 3, Shimadzu, Kyoto, Japan), which included a formula predictor.