3.2. HPLC–DAD Analysis of Fisetin

In vitro FST content was determined by high-performance liquid chromatography (HPLC), coupled with a diode array detector (DAD) modified from the previous study [49]. The HPLC system (Hitachi 7000, Hitachi, Tokyo, Japan) consisted of a D−7100 pump, an L−7200 autosampler, and an L−7455 DAD detector. FST was separated by a reversed-phase column (Synergi fusion RP, 250 × 4.6 mm, 4 µm; Phenomenex, Inc., Torrance, CA, USA). The determination was conducted at room temperature (27 ± 2 °C). The mobile phase consisted of acetonitrile 0.01 M NaH₂PO₄ (pH 2.5) with a ratio of 35:65 (v/v), and the flow rate was set to 1.2 mL/min. The injection volume was 20 μL and detected at 358 nm. The run time of FST analysis was 10 min. Analytical method validation for FST analysis was performed according to our previous work [50].

For the standard preparation, FST (5.0 mg) standard was dissolved in 50% (v/v) methanol as stock solution, and diluted to the concentration of 50.0, 25.0, 12.5, 5.0, 1.25, and 0.5 μg/mL with 50% (v/v) methanol. After dilution, A 25 μL luteolin standard (100.0 μg/mL) was added as internal standard for the preparation of calibration curves.