Determination of doxorubicin and its metabolites in rat serum and bile by LC: application to preclinical pharmacokinetic studies.

A simple, accurate and precise high-performance liquid chromatographic method was developed and validated for the simultaneous determination of doxorubicin and its three metabolites, including doxorubicinol, doxorubicinolone and doxorubicinone, in rat serum and bile. Following a single protein precipitation step, chromatographic separation was accomplished using a C-18 column with a mobile phase consisting of 50 mM sodium phosphate buffer-acetonitrile-1-propanol (65:25:2, v/v), pH 2.0. The analytes were measured by fluorescence detection with excitation wavelength of 480 nm and emission wavelength of 560 nm. The lower limits of quantitation were 10 ng/ml for doxorubicin, and 5 ng/ml for the three metabolites. The calibration curves were linear over a concentration range of 10-2500 ng/ml for doxorubicin, and 5-1250 ng/ml for its three metabolites. The average recoveries were greater than 89% for all analytes. The within-day and between-day coefficients of variation were generally less than 13%. Doxorubicin and its metabolites were stable in the precipitated serum and bile samples at room temperature in darkness for at least 48 h. This method permitted the analysis of samples without the presence of the anticoagulant sodium citrate and thus was applied to serum and bile samples collected from rats that were administered doxorubicin intravenously in a pharmacokinetic study.