4.4. Quantification of Saponarin Using High-Performance Liquid Chromatography (HPLC)

HPLC analyses were conducted with a Shiseido SI-2 series HPLC system (Shiseido, Tokyo, Japan), equipped with a Shiseido Capcell Pak C18 MG II S-5 (250 × 4.6 mm; i.d., 5 µm) column. The mobile phase consisted of water (solvent A) and acetonitrile (solvent B), both containing 1% formic acid, and was applied as follows: 0–20 min, 0–10% (B), 21–31 min, 10–40% (B), and 32–35 min, 40–60% (B), followed by re-equilibration with 10% (B) for 36–42 min, at a flow rate of 1.0 mL/min and an injection volume of 5 µL. The detection wavelength of SA was set to 254 nm. The SA content in the BSE was verified by comparing the retention times to the external standards. Stock mixed standard solutions with SA were prepared in methanol at the following concentrations: 0, 6.25, 12.5, 25, 50, and 100 µg/mL. The BSEs were immersed in 300 µL methanol, filtered, and injected into the HPLC system.