An improved HPLC method for therapeutic drug monitoring of daunorubicin, idarubicin, doxorubicin, epirubicin, and their 13-dihydro metabolites in human plasma

A single high-performance liquid chromatography (HPLC) method, suitable for the analysis of daunorubicin, idarubicin, doxorubicin, epirubicin, and the ir 13-dihydro metabolites is validated in the present study. Preparation of plasma samples was performed by a first extraction of analytes with a chlo roform/1-heptanol mixture (9:1) and reextraction with ortophosphoric acid 0 .1 M. The chromatographic analysis was carried out by reversed-phase isocra tic elution of anthracyclines with a Supelcosil LC-CN 5 mm column (25 cm x 4.6 mm internal diameter; Supelco) and detection was accomplished by spectr ofluorimetry at excitation and emission wavelengths of 480 and 560 nm, resp ectively. All anthracyclines eluted within 15 minutes of injection and the method appeared to be specific, because the chromatographic assay did not s how interferences at the retention time of analytes. The linearity, evaluat ed over a concentration range of 0.4-10,000 ng/mL, gave regression coeffici ents better than 0.999, with recoveries of doxorubicin-doxorubicinol and ep irubicin-epirubicinol of 67%-109% and 61%-109% respectively, and 93%-109% f or the other compounds. The limits of detection and quantification were 0.4 ng/mL in a 50-mL sample (40 pg/injection) for all anthracyclines tested. T he method proved to be precise and accurate, as the within-day and between- day coefficients of variation were less than 10% and the accuracy of the as say was in the range of 91%-107%. Overall results indicate that it is feasi ble to analyze all the anthracyclines used in clinical practice and their m ajor metabolites with a single optimized method, thereby simplifying their monitoring in chemotherapeutic regimens of cancer patients.