Development And Validation of a Rapid RP-HPLC Method For Simultaneous Quantification of Five Phytoestrogens in Pueraria Tuberosa Root Extract

Abstract

A rapid and validated RP-HPLC method was developed for the simultaneous quantification of five key phytoestrogens puerarin, genistein, daidzein, biochanin-A, and quercetin in Pueraria tuberosa root extract. Chromatographic separation was achieved within 12 minutes using a C18 column with gradient elution of 0.1% orthophosphoric acid in water and acetonitrile at a flow rate of 2.0 mL/min. Detection was performed at 205 nm with an injection volume of 10 µL. The method was validated according to regulatory guidelines, evaluating system suitability, precision, accuracy, specificity, linearity, limit of detection (LOD), and limit of quantification (LOQ). Excellent linearity (r² > 0.999) and high precision (RSD < 2%) were observed for all analytes. The novelty of this study lies in the development of a rapid and validated method for simultaneous estimation of five phytoestrogens within a short analytical run time. The method is simple, sensitive, and reliable, making it suitable for quality control, standardization, and routine analysis of P. tuberosa and its herbal formulations.