FULLY AUTOMATED-DETERMINATION OF A NEW ANTHRACYCLINE N-L-LEUCYLDOXORUBICIN AND 6 METABOLITES IN PLASMA BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY WITH ONLINE SAMPLE HANDLING

N-l-Leucyldoxorubicin (Leu-Dox) was developed as a prodrug of doxorubicin (Dox) in order to diminish the cardiotoxic side-effect associated with repeated anthracycline treatment. To study the pharmacokinetics of Leu-Dox, Dox and other metabolites a sensitive and selective assay was needed. Leu-Dox and six of its known metabolites were extracted from plasma using an in-line reversed-phase precolumn (40-50-mu-m C-8 particles). The trapped analytes were subsequently flushed to the analytical column (3-mu-m C18) using 0.5 ml of phosphate buffer (pH 3.5)-acetonitrile (2:1, v/v), which also served as the isocratic mobile phase. Within 12 min, a clean baseline-resolved chromatogram is obtained by fluorescence detection. Recoveries were almost quantitative and highly reproducible, with standard deviations less-than-or-equal-to 5.4% and less-than-or-equal-to 2.7% at spiked concentrations of 10 and 100 nM. Using 300-mu-l of plasma, detection limits ranged from 0.3 to 0.8 nM at a signal-to-noise ratio of 3. The calibration curves were linear from 1 to 300 nM (r2 greater-than-or-equal-to 0.999) for each of the seven compounds. The between-day accuracy was in the range 91-99% and 99-105% at 10 and 100 nM, respectively, with standard deviations of 1-4%. Application of the assay to the analysis of plasma from patients after administration of Leu-Dox proved success