High-Performance Liquid Chromatography (HPLC) Method Validation for Simultaneous Quantitation of Five Phytoestrogenic Flavonoids

Abstract

Abstract A Reversed Phase High Performance Liquid Chromatography (RP-HPLC) method for simultaneous quantitation of four isoflavone standards daidzein, genistein, formononetin and biochanin A, and one flavone standard quercetin was developed and validated through the evaluation of linearity, accuracy, precision, specificity, limit of detection and limit of quantitation in accordance with the ICH guidelines. The analysis was performed in a C18 column (150 x 4.6mm, 5µm) with an optimised gradient elution using acetonitrile-water (0.1% trifluoroacetic acid) at a flow rate of 1.0mL min-1 and sample injection volume of 10uL.The retention times of the standards in the order daidzein, quercetin, genistein, formononetin and biochanin A were 4.42, 5.24, 7.85, 10.06 and 13.55 minutes, respectively with tailing factors ranging from 1.09 – 1.12 and minimum resolution value of 3.74. Detection limits range from 0.339 to 0.964 ug/mL and quantitation limits range from 1.027 to 2.922 ug/mL with good linearity (R2 ≥ 0.9967) with a linear range of 1.25 - 20ug/mL for all standards. The method was also found to be accurate and precise based on percentage recovery ranging from 96.96% to 106.87% (intraday, n=3) and relative standard deviation of %RSD≤1.45% (intra-day, n=3) and %RSD≤2.35% (inter-day, n=5). The specificity of the method was evaluated based on the positivity of the minimum peak purity index during the quantitation of the target compounds from the spiked hydrolysed and unhydrolyzed extract of Cajanus cajan ICPL 7035.