High-performance liquid chromatography (HPLC) of flavonoids

High-performance liquid chromatography was conducted according to a modified method of Dai & Row (2019), using a Shimadzu LC-20A HPLC with a UV/VIS detector set at a wavelength of 285 nm. The column used was an Agilent C18 column (250 mm ×4.6 mm, i.d. 5 µm) coupled to an Agilent C18 guard column (4.6 mm ×12.5 mm, i.d. 5 µm). The mobile phase was 0.2% acetic acid (in HPLC grade water) and acetonitrile at a ratio of 65:35 (v/v). The injection volume was 20 µL, and flow rate was 1.0 mL/min. At least three separate injections were prepared for each sample. The standard flavonoid compounds used were quercetin, kaempferol, and quercetin-3-β-D-glucoside. Serial dilutions ranged from 10 to 200 µg/mL for each standard.