Method for the determination of 4-Demethoxydaunorubicin, its quinone and hydroquinone metabolites in human plasma and urine by high-performance liquid chromatography

Abstract

4-Demethoxydaunorubicin (4-DMDNR) is a new orally active analogue of daunorubicin (DNR). We have developed a high-performance liquid chromatography (HPLC) method capable of separating and identifying 4-DMDNR, five possible fluorescent quinone metabolites and three possible non-fluorescent hydroquinone metabolites. Methods are described for high-yield synthesis of reference metabolites. The limit of detection of the fluorescence assay was less than 1 ng/ml after extraction of 1 ml plasma or urine with chloroform/propan-2-ol (2:1), with coefficients of variation in k' (HPLC column capacity factors) of less than 3% throughout the day. Efficiency of the extraction method described exceeded 80% in control experiments. Blood and urine samples were analysed from four cancer patients who had received 50 mg/m² orally as three divided doses every 8 h. A typical urinary profile of the drug and its metabolites was: parent drug, 13%; 4-demethoxydaunorubicinol (4-DMDNOL), 80%; 4-DMDNR 7-hydroxyaglycone, 4% and 4-DMDNOL 7-hydroxyaglycone, 3%. 4-DMDNOL was the major metabolite detected in plasma. A further metabolite identified as the 7-deoxyaglycone of 4-DMDNOL was detected in plasma of two patients at concentrations equal to or greater than the parent drug. In the other two patients no trace of the metabolite was detected.