2.6. Analysis of HT and Its Metabolites
All samples were run in duplicate. Measurement was conducted using UHPLC-DAD-MS/MS, with an Acquity UPLC I-Class system coupled to a XEVO-TQS micro mass spectrometer (Waters, Milford, MA, USA). The instrument consisted of a sample manager cooled at 10 °C, a binary pump, a column oven, and a diode array detector (DAD) measuring at 280 nm for confirmation of the peaks. The column oven temperature was set at 40 °C. Eluent B was water with 0.1% formic acid, eluent A was acetonitrile with 0.1% formic acid, and the flow was 0.4 mL/min on an Acquity BEH C18 RP column (50 mm × 2.1 mm, 1.7 µm particle size) combined with an Acquity BEH C18 precolumn (2.1 mm × 5 mm, 1.7 µm), both from Waters (Milford, MA, USA). The gradient started with 1% A for 3 min and rose linearly to 20% A within 1 min, then to 80% A within 1.3 min, then to 100% A in 0.3 min and holding for 1.5 min as a washing step; then back to 1% A within 0.2 min and equilibrating for 1 min. The injection volume was 5 µL.The peaks were identified using MS/MS in negative mode. The source voltage was kept at 3.4kV, and the cone voltage was 77 V. The source temperature was set at 150 °C and the desolvation temperature at 400 °C with a desolvation gas flow of 800 L/h and a cone gas flow of 50 L/h. Data were acquired and processed using MassLynx 4.1 (Waters, Milford, MA, USA).