A simple and rapid ultra-high performance liquid chromatographic (UHPLC) method coupled with an ultraviolet detector (UV) has been developed and validated for the separation and determination of 14 major flavonoids ((±)-catechin, (−)-epicatechin, glycitin, (−)-epicatechin gallate, rutin, quercitrin, hesperidine, neohesperidine, daidzein, glycitein, quercetin, genistein, hesperetin, and biochanin A) in herbal dietary supplements. The flavonoids have been separated on a Chromolith Fast Gradient Monolithic RP-18e column utilizing a mobile phase composed of 0.05% trifluoroacetic acid in water and acetonitrile in gradient elution mode. Under these conditions, flavonoids were separated in a 5 min run. The selectivity of the developed UHPLC–UV method was confirmed by comparison with ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) analysis.
The validation parameters such as linearity, sensitivity, precision, and accuracy were found to be highly satisfactory. The optimized method was applied to determination of flavonoids in different dietary supplements. Additionally, the developed HPLC–UV method combined with 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) assay was used in the evaluation of antioxidant activity of the selected flavonoids.