Development and Validation of an HPLC–DAD Method for Analysis of the Six Major Isoflavones in Extracts from Soybean Processing

In this study, a new HPLC method was developed and validated for the determination of daidzin, genistin, glycitin, and their respective aglycones daidzein, genistein and glycitein in extracts from different stages of soy processing. The chromatographic separation was achieved using a Bonus RP 100 × 3.0 mm (3.5-μm particle size) column maintained at 35 °C. The mobile phase consisted of a gradient of water and acetonitrile both containing 0.1% formic acid. Analyses were conducted at a flow rate of 1.0 mL min −1 and the detector was set at 254 nm. Validation parameters indicated that the method has high selectivity and is linear over a range of 1.5–150 µg mL −1 for all compounds. The range of recovery was 95–102% with a RSD% <5% for intra-day and inter-day precision. The robustness study indicated that the flow rate was the only critical factor. The new HPLC–DAD method was successfully applied to real samples and excellent separation was achieved without the need of any sample pretreatment.