Development and validation for free aglycones daidzein and genistein in soybeans (Glycine max (l.) Merr.) using RP HPLC method

Abstract

Objective: The aim of this research was to develop a validation method for free aglycones analysis especially daidzein and genistein in soybean (Glycine max (L.) Merr.) using High Performance Liquid Chromatography (HPLC). Methods: In this study, reversed-phase HPLC (RP-HPLC) equipped with an endcapped Sun Fire TM C-18 column (150 mm x 4.6 mm, 5 μm) was used for the separation. The binary mobile phase consisted of methanol and 0.1% acetid acid (53:47). An isocratic program was used with a flow rate at 1.0 ml/min and the injection volume was 10 µl. The analytes were detected by using Photo-diode array (PDA) at 254 nm and the samples in various soybean varieties from Balitkabi Malang, Indonesia. Results: The developed method showed that the parameters of system suitability and selectivity meet the requirements of method validation. The accuracy values of recoveries for daidzein 83.0%-100.95% and genistein 80.07%-108.79%, and the precision was calculated%RSD ≤ 2% respectively, linearity of the method was the r2values ranged from 0,9989-0,9991 and all the parameters meet the acceptable criteria of validated method. The Limit of detection (LoD) and Limit of quantitation (LoQ) indicated a good sensitive method, with LoDs values obtained, were 0.05192 μg/ml and 0,0600 μg/ml for daidzein and genistein, respectively. Meanwhile, the LoQs values was 0.1731 μg/ml and 0.2000 μg/ml for daidzein and genistein, respectively. Conclusion: A simple, accurate, precise and being specific HPLC method coupled to PDA for the analysis of daidzein and genistein in various soybeans varieties were developed and validated.