UHPLC Analysis of Bioactive Compounds

The UHPLC analysis was performed using an Dionex ultra high performance liquid chromatograph (Dionex UltiMate 3000, Thermo, MA, USA) equipped with a Dionex UltiMate 3000 pump, a Dionex UltiMate 3000 autosampler and a Dionex UltiMate 3000 diode array detector. The chromatographic separation was achieved on an ACQUITY UPLC column (HSS T3, 1.8 μm, 2.1 mm× 100 mm, Waters, Ireland) [49 (link)] at 30 °C. The mobile phase was composed of water containing 0.1% TFA (A) [50 (link)] and methanol (B) at a flow rate of 0.3 mL/min. The elution program was conducted as follows: 0–15 min at 0–6% B; 15–20 min at 6–11% B; 20–30 min at 11–11% B; 30–45 min at 11–20% B; 45–55 min at 20–30% B; 55–60 min at 30–90% B. The injection volume was 2 μL. DAD wavelength was set as 220 nm. Method validation was accorded to the Chinese Pharmacopeia guidelines, which were included linearity, limits of detection (LOD), limits of quantification (LOQ), repeatability, precision, stability and recovery.