UPLC Analysis of IGF Compounds

The chemical composition of the IGF was analyzed using UPLC. Crude extracts were dissolved in methanol to produce a final concentration of 0.5 mg/mL. The standards (verproside, catalposide, and amphicoside) were dissolved in methanol to produce a final concentration of 0.1 mg/mL, respectively. Our UPLC-photodiode array (PDA) used a Waters Acquity System (Waters Co., Milford, MA, USA) that consisted of a PDA, binary autosampler, online degasser, and column oven. The UPLC system was fitted with an Acquity UPLC HSS T3 column (100 × 2.1 mm, 1.7 μm; Waters Co.). The column and autosampler temperatures were maintained at 40°C and 25°C, respectively. The mobile phases A and B were ultrapure water and acetonitrile, respectively. The following gradient profile was used: 10–25% B (0–0.8 min), 25–35% B (0.8–2.4 min), 35–95% B (2.4–2.8 min), 95% B (2.8–4 min), and 95–100% B (4–8 min). The flow rate was 0.5 mL/min and the injection volume was 1.0 µL. Compounds were identified by a comparison of the retention times between samples and standards. The PDA detection was conducted at 260 nm. Levels of the IGF were quantitated using Empower software version 3.0 (Waters) [10 (link), 16 (link)].