4.3. Determination of Timosaponin BII by LC-MS/MS

The quantitative detection of timosaponin BII and possible metabolites was performed using LCMS-8060 equipped with an ESI ion source. An XBridge^® BEH C8 column (75 × 3.0 mm, 2.5 μm, Waters, Wexford, Ireland) was applied for separation of the analytes. The flow rate was 0.4 mL/min, and the temperature of column oven was 40 °C. The injection volume was 10 μL. The mobile phase used was aqueous ammonia: water (0.075:100, v/v) as mobile phase A and methanol as mobile phase B. Gradient elution condition (B%) was as follows: 0.01 min, 30%→3.00 min, 30%→6.00 min, 50%→8.00 min, 95%→10.00 min, 95%→12.00 min, 30%→17.00 min, stop. The MRM mode was used for detection by the mass spectrometer, with mass transitions for timosaponin BII (negative MRM) of 919.40→757.40 (Q1 Pre Bias: 28.0 V, CE: 45.0 V, Q3 Pre Bias: 28.0 V, Dwell Time: 50 msec), timosaponin AIII (positive MRM) of 741.40→253.35 (Q1 Pre Bias: −20.0 V, CE: −32.0 V, Q3 Pre Bias: −11.0 V, Dwell Time: 50 msec), timosaponin AI (positive MRM) of 579.15→255.25 (Q1 Pre Bias: −24.0 V, CE: −27.0 V, Q3 Pre Bias: −17.0 V, Dwell Time: 50 msec), sarsasapogenin (positive MRM) of 417.10→255.15 (Q1 Pre Bias: −20.0 V, CE: −25.0 V, Q3 Pre Bias: −20.0 V, Dwell Time: 50 msec), and the internal standard (IS, glipizide) of 446.10→285.60 (positive MRM, Q1 Pre Bias: −16.0 V, CE: −25.0 V, Q3 Pre Bias: −19.0 V, Dwell Time: 50 msec) and 444.25→319.10 (negative MRM, Q1 Pre Bias: 18.0 V, CE: 18.0 V, Q3 Pre Bias: 24.0 V, Dwell Time: 50 msec), respectively. The mass spectrometer parameters were set as follows: nebulizer gas, 3.0 L/min; heating gas, 10 L/min; interface temperature, 300 °C; DL temperature, 250 °C; heat block temperature, 400 °C; drying gas, 10 L/min; interface voltage, −4.5 kV; and CID gas pressure, 270 kPa.

All the samples were maintained at 4 °C before injection. Sample preparation will be mentioned in Section 4.5 and Section 4.6.