Liquid chromatography was performed on a Waters Acquity UPLC system (Waters Corp., Milford, MA, USA). Separation was achieved with a Waters ethylene-bridged hybrid (BEH) C18 column (50 mm × 2.1 mm, 1.7 μm) held at 30°C. Gradient elution with a mobile phase composed of water and acetonitrile with 0.1% formic acid was performed at a flow rate of 0.3 mL/min (Table 1 ).
Mass spectrometric detection was performed on a Micromass Quattro Micro API mass spectrometer (Waters Corp.) with an electrospray ionization interface and triple quadrupole mass analyzer. The electrospray ionization source was set in the positive mode. The following parameters were used: capillary voltage, 3.2 kV; cone voltage, 30 V; source temperature, 120°C; and desolvation temperature, 300°C. Nitrogen was used for desolvation and as the cone gas at flow rates of 600 L/h and 50 L/h, respectively. The full scan mode was used in a mass range of 100–1,000 amu. For MS/MS, argon was used as the collision gas, and the collision energy was set according to the metabolite composition. NaCsI was used for mass correction before the experiment was commenced. Data were collected in centroid mode.
Mass spectrometric detection was performed on a Micromass Quattro Micro API mass spectrometer (Waters Corp.) with an electrospray ionization interface and triple quadrupole mass analyzer. The electrospray ionization source was set in the positive mode. The following parameters were used: capillary voltage, 3.2 kV; cone voltage, 30 V; source temperature, 120°C; and desolvation temperature, 300°C. Nitrogen was used for desolvation and as the cone gas at flow rates of 600 L/h and 50 L/h, respectively. The full scan mode was used in a mass range of 100–1,000 amu. For MS/MS, argon was used as the collision gas, and the collision energy was set according to the metabolite composition. NaCsI was used for mass correction before the experiment was commenced. Data were collected in centroid mode.