Column-switching high-performance liquid chromatographic assay for determination of apigenin and acacetin in human urine with ultraviolet absorbance detection

Abstract

A high-performance liquid chromatographic (HPLC) method is described for the determination of apigenin and the 4′-methylated derivative acacetin in human urine using column-switching and ultraviolet (UV) absorbance detection. Urine samples were enzymatically hydrolysed and solid-phase extracted prior to injection onto the HPLC system. Prior to elution of apigenin and the internal standard, 5,7,8-trihydroxyflavone, from the first column used for sample clean-up, the six-port valve was switched to the second column for analysis with UV detection. Detection of apigenin was precise and reproducible, with a limit of quantification of 10 ng ml −1 urine. Detection and quantification of acacetin was linear down to 70 ng ml −1 urine. The method has been successfully applied to determine the level of apigenin in 100 human urine samples from an intervention study with parsley.