4.4. LC-MS Analysis

A collected leaf sample (1.0 g) was freeze-dried in absolute methanol (analytical grade, 20 mL) for extraction of tryptamine, loganin, tabersonine, serpentine, vindoline, catharanthine, vincamine, vinblastine and vincristine. Low-frequency ultrasonication (250 W, 40 kHz) for 40 min was used to extract the alkaloids. The methanol extract was centrifuged at 8000 rpm for 10 min, concentrated to 2 mL, and the amount of alkaloids were determined by HPLC-MS (Ultra-performance LC, Waters, Tokyo, Japan; MS, AB SCIEX, USA) using an ACQUITY UPLC BEH C18 Column (1.7 µm, 2.1 mm × 50 mm) in an oven at 30 °C, with CH₃CN/H₂O and 0.05 moL L⁻¹ ammonium acetate standard solvent system, respectively. The retention times of the alkaloids were 3.49 min (serpentine), 2.91 min (tabersonine), 3.37 min (vindoline), 3.32 min (vinblastine), 2.81 min (catharanthine), 0.76 min (loganin), 1.99 min (vincamine) and 2.49 min (vincristine). The sample injection volume was 10 μL at a flow rate of 1 mL/min. The TIAs content was quantified by tagged of the level from 10⁻⁷ to 10⁻² mg L⁻¹ based on peak area, respectively.