RAPID AND SIMPLE METHOD TO DETERMINE TAMOXIFEN AND ITS MAJOR METABOLITES IN HUMAN LIVER-MICROSOMES BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY WITH FLUORESCENCE DETECTION

Sensitive and reproducible analyses were developed for assaying tamoxi fen, desmethyltamoxifen, didesmethyltamoxifen and hydroxytamoxifen in human liver microsomes by high performance liquid chromatography (H.P. L.C.) following protein precipitation. Detection was by fluorimetry of phenanthrene derivatives formed by on-stream U.V. irradiation with a photochemical reactor enhanced detection (P.H.RE.D.) for post-column i rradiation of the H.P.L.C, stream. The method is highly sensitive (1 n g/ml). A C8, 5 mu m column (4.6 x 150 mm) was used with a mobile phase of acetonitrile / 100 mM ammonium acetate (1:1) pH 5. Precipitation o f the microsomal protein was carried out by adding methanol and concen trated chloric acid to the reaction mixture and extraction efficiencie s were approximately 100 % for tamoxifen and its metabolites. The assa y has been optimised for the identification and quantitation of the 4- hydroxylated metabolite, as well as the two metabolites formed by N-ox idative demethylation of the side chain. The latter catalytic activity involves cytochrome P450 3A enzymes.