Determination of Isoflavones in Human Urine by High Performance Liquid Chromatograph

A reversed-phase high performance liquid chromatography (RP-HPLC) method was established for the determination of isoflavones (daidzein, glycitein, equol, and genistein) in human urine. After the addition of flavone as internal standard and the enzymatic hydrolysis in urine sample, the isoflavones were extracted with ethyl acetate (1:1) in medium of pH 7.0 and then separated by gradient elution with a ternary eluant of 0.02% trifluoroacetic acid-methanol-acetonitrile. The detection limits were 12.9 nM, 13.9 nM, 71.6 nM, and 11.8 nM for daidzein, glycitein, equol, and genistein, respectively. The recovery of the isoflavones was more than 89%. The proposed method is easier to carry out, accurate, reproducible, and suitable for the analysis of a large number of samples.