2.7. Quantification of isoflavones by high-performance liquid chromatography (HPLC)

The extracted soybean sample solution was filtered using a syringe filter (0.22 μm cut-off; PALL Corporation, USA) before high-performance liquid chromatography (HPLC) analysis. Standard daidzin, genistin, glycitin, daidzein, glycitein, and genistein were dissolved in 80 % methanol. The concentration of prepared authentic standards solutions ranged from 0.01 to 1 μg/mL for isoflavone analysis. For each standard, the mobile phase and its changing ratio are shown in Table 1. The extracted sample was dissolved in 1 mL of 10 % methanol and was ultra-sonicated for 10 min. From the methanol diluted sample, 10 μL of filtrates was directly injected into a high-performance liquid chromatography (HPLC) system. Each sample was analyzed in triplicate. The samples were analyzed by a C18 column (Shiseido Cap cell pack C18 MGⅡ, 3.0 mm × 250 mm, 5 μm). Phase separation was performed at 40 °C, the flow rate was maintained at 1 mL/min and the injection volume was 10 μL. The UV detection wavelength was measured at 260 nm wavelength using HPLC (High-Performance Liquid Chromatography, Shimadzu, Japan).

HPLC analysis conditions for soybean seed isoflavones.