HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC ASSAY FOR GENISTEIN IN BIOLOGICAL-FLUIDS

A specific, sensitive and technically convenient HPLC method for assay ing genistein in biological fluids has been developed. The compound an d 4-hydroxybenzophenone, added as an internal standard, were efficient ly isolated from both plasma and urine by extraction with tert.-butyl methyl ether. Following evaporation of the organic solvent, the extrac t was reconstituted with methanol-0.05 M ammonium acetate buffer, pH 4 .7 (30:70, v/v), and loaded onto a 4 mu m Nova-Pak C-8 column (15 cm x 3.9 mm I.D.). Chromatography was performed at ambient temperature usi ng a mobile phase of acetonitrile-0.05 M ammonium formate buffer, pH 4 .0 (27:73, v/v), at a flow-rate of 1.0 ml/min, with UV detection at 26 0 nm. Mean values of the t(R) for the drug and internal standard, dete rmined from chromatograms of the 1 mu g/ml plasma standard during a 6 month period, were 8.27 +/- 0.55 and 11.92 +/- 0.71 min, respectively (S.D., n = 29). With a sample volume of 50 mu l, the lowest concentrat ion of genistein included in the plasma standard curve, 0.020 mu g/ml, was quantified with a 10.7% R.S.D. over a 5 month period. Plasma stan dards having concentrations of 0.050 to 1.02 mu g/ml exhibited R.S.D. values ranging from 2.3 to 6.1%. The drug was quantified in urine with similar reproducibility. The sensitivity of the assay was adequate fo r characterizing the plasma pharmacokinetics of genistein in the mouse and dog. However, a 10-fold improvement in sensitivity was afforded b y increasing the sample size to 250 mu l, without otherwise modifying the method. Thus, this procedure may prove suitable for determining pl asma and urine levels of genistein in humans consuming dietary isoflav onoids in a much more convenient manner than permitted by existing met hodology.