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 develop a validation method for free aglycones analysis especially daidzein and genistein in soybean (Glycine max (L.) Merr.) using 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 flow rate at 1.0 mL/min and the injection volume was 10µL. 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, selectivity, 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 r² values ranged from 0,9989 - 0,9991 and all the parameters 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 value were 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 was developed and validated.