4.2. Isoflavone Extraction and Quantification

Lyophilized whole soybean seeds were finely ground with a mortar. Each ground sample (7 mg) was immersed in 1 mL of 58% (v/v) aqueous acetonitrile. The mixture was sonicated for 30 min and centrifuged at 13,000 rpm for 5 min, after which the supernatant was retained. The pellet was resuspended in an equal volume of solvent, and both retained supernatants were combined and diluted with distilled water. The extract volume was adjusted to 4 mL for each extraction from a 7-mg freeze-dried seed sample. The diluted extracts were filtered through a 0.45-μm syringe filter (Futecs Co., Ltd., Daejeon, Korea) and used for reversed-phase high performance liquid chromatography (HPLC) analysis.

Extracts were analyzed using reversed-phase HPLC (Waters 2695 Alliance HPLC; Waters Inc., Milford, MA, USA) with an octadecylsilane column (Prontosil 120–5-C18-ace-EPS 5.0 μm (250 × 4.6 mm; Bischoff, Leonberg, Germany). The flow rate of the mobile phase was 1.0 mL/min and the sample injection volume was 5 μL. The mobile phase was a combination of (A) water with 0.1% formic acid and (B) acetonitrile with 0.1% formic acid. Gradient elution was performed by adding 15% of solvent B at the initial running time and increasing the concentration to 34% over 60 min. Peaks were monitored at 254 nm using a Waters 996 photodiode array detector (Waters Inc.). Twelve isoflavone standards were purchased from Sigma-Aldrich (St. Louis, MO, USA) and used for quantification of the isoflavones from soybean seeds in the HPLC analysis.