4.2. Determination of Triterpene Saponins by UPLC-PDA-MS

The leaves, flowers and seeds were lyophilized and milled into powder. The samples were extracted with 70% methanol (1:15, w/v) at 70 °C for 30 min, and the supernatant was collected after centrifugation at 12,000× g for 5 min. The saponins were determined using the ACQUITY UPLC/MS system equipped with a PDA detector (Waters, Milford, MA, USA) and an electrospray ionization (ESI) source (Waters, Milford, MA, USA) using a method developed in our lab [26]. Briefly, a 5.0 μL sample was injected and eluted on a Waters Acquity UPLC HSS T3 column (1.8 µm, 150 mm × 2.1 mm i.d., Waters, Milford, MA, USA) at a flow rate of 0.2 mL/min. The mobile phase A and B were water and acetonitrile, both containing 0.1% formic acid. The gradient elution was as follows: 0–4 min, 35–37% B; 4–32 min 37% B; 32–58 min, 37–45% B; 58–60 min, 35% B. The detection wavelength was set at 210 nm, and the column temperature was 30 °C. For mass spectrometry (MS) analysis, the conditions were: negative ion mode; scan range, m/z 100–2000; capillary voltage, 3 kV; cone voltage, 40 V; extractor voltage, 4 V; source temperature, 150 °C; desolvation temperature, 350 °C; cone gas flow, 50 L/h; desolvation gas flow, 600 L/h. Theasaponin E1 (purity 98.0%) was prepared as previously described and used as the external standard for saponin quantification [26]. The content of saponins was calculated by bringing the area of each individual peak under the PDA detector into the standard curve of theasaponin E1 (Supplemental Table S4).