Analysis of 4-demethoxydaunorubicin and metabolites in plasma and urine.

A high-performance liquid chromatographic procedure, including sample pretreatment, is presented for the analysis of 4-demethoxydaunorubicin, the C13 hydroxy metabolite 4-demethoxy-daunorubicinol and four possible aglycone metabolites, 4-demethoxydaunorubicinone, 4-demethoxydaunorubicinolone, 4-demethoxy-7-deoxydaunorubicinone and 4-demethoxy-7-deoxydaunorubicinolone, in plasma and urine samples. Doxorubicin has been utilized as the internal standard of the assay. The sample pretreatment involves liquid-liquid extraction of the buffered (pH 9) biological matrix with chloroform-1-propanol (4 : 1, v/v). Separation of the compounds has been achieved by using a Lichrosorb RP-8 (5 μm) column and by isocratic elution with a mobile phase composed of acetonitrile-phosphate buffer, pH 2.4 (70 : 30, w/w). The flow rate is 1.5 ml/min. Fluorescence detection with excitation at 460 nm and monitoring at 540 nm was applied. The detection limit is 1 ng/ml (using 1.0 ml samples). The applicability of the assay has been demonstrated in a pharmacokinetic study with two rabbits. In plasma, 4-demethoxydaunorubicinol and 4-demethoxydaunorubicinolone were observed as metabolites, whereas 4-demethoxydaunorubicinol was the main metabolite present in urine. Fitting of the plasma concentration versus time curves showed a three-compartment model to give a better description of the plasm concentration-time curves than a two-compartment model.