Agilent 1260 lc series instrument

Isolation and purification were carried out by column chromatography. Agilent 1260 HPLC and thin-layer chromatography were used to monitor the separation, and thin-layer chromatography was performed on precoated silica gel 60 GF254 plates and visualized using UV illumination at 254 nm and 365 nm or by spraying with a 10% solution of sulfuric acid and 1% vanillin in ethanol. ¹H and ¹³C nuclear magnetic resonance spectra were recorded on a Bruker Avance 400 MHz spectrometer (Bruker BioSpin GmbH, Beijing, China) with tetramethylsilane as the internal standard. Chemical shifts are expressed in δ values. HPLC quantitative analysis was performed on Agilent 1260 LC Series instrument (Agilent, Santa Clara, CA, USA) equipped with a G4212B DAD using a Luna C-18 column (5 μm, 4.6 mm i.d. × 250 mm; Phenomenex, Inc., Torrance, CA, USA). Flow rate was 1.0 mL/min. The mobile phase was a mixture of 0.2% (v/v) phosphoric acid–water solution (A) and methanol (B) with a gradient elution as follows: 0–6 minutes, 0–50% B; 6–13 minutes, 50–57% B; 13–25 minutes, 57–60% B; 25–40 minutes, 60–70% B; 40–50 minutes, 70–100% B; 50–57 minutes, 100% B; 57–60 minutes, 100–0% B. The injection volume was 10 μL, and the column oven was maintained at 25°C. DAD detection wavelength was set at 295 nm for all analytes.