Sensitive method for the determination of daunorubicin and all its known metabolites in plasma and heart by high-performance liquid chromatography with fluorescence detection.

The cytostatic agent daunorubicin is effective against leukaemia. An important side-effect is cardiomyopathy, common to all anthracyclines. Since anthracycline metabolites are thought to contribute to the observed cardiotoxicity, a method for the quantitative determination of all metabolites in plasma as well as in tissues is needed as a basis for the further investigation of the correlation between toxicity and the amount of each metabolite formed. Using Sep-Pak C18 cartridges we were able to extract daunorubicin and its five metabolites, including the aglycones, with recoveries in the range 50-90%. Depending on the chemical properties of each metabolite, fluorescence detection following high-performance liquid chromatographic separation permitted detection limits as low as 0.2-0.9 nM in plasma and 0.8-3.10(-11) mol/g in tissue, at a signal-to-noise ratio of 2, which compare favourably with literature data. The method showed linearity in the ranges 1-250 nM in plasma and 0.04-4.0 nmol/g in tissue (r greater than or equal to 0.998). The accuracy determined at 10 and 100 nM for plasma and at 0.1 and 1.0 nmol/g for tissue, was in the range 86-103 and 85-110% for plasma and tissue, respectively. The within-day and between-day repeatability values were acceptable (between 2 and 12%). Because of large inter-compound differences, separate calibration curves were used for each anthracycline. Application of the assay to the analysis of plasma and tissue samples of mice after intravenous injection of daunorubicin proved successful.