Validation of an optimized HPLC–UV method for the quantification of formononetin and biochanin A in Trifolium pratense extract

Abstract Here, two important isoflavones present in Trifolium pratense, formononetin and biochanin A, were analyzed by high performance liquid chromatography coupled with UV (HPLC–UV). These isoflavones are marker compounds with anti-inflammatory, anti-cancer, and anti-oxidant properties, and are also potent phytoestrogens that can be used in hormonal therapies. Till date, no study regarding rapid identification and quantification of T. pratense extract has been conducted. This study developed and validated an optimized method for quantifying formononetin and biochanin A using HPLC–UV. The results showed excellent linearity of the calibration curve (r 2 ≥ 0.999), and good resolutions of chromatographic peaks were obtained. Other validation parameters such as specificity, accuracy, and precision demonstrated that our method had good reliability and sensitivity. Furthermore, our method for quantifying formononetin and biochanin A in T. pratense extract is convenient.