QUANTITATION OF CELL-ASSOCIATED DOXORUBICIN BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY AFTER ENZYMATIC DESEQUESTRATION

A method for measuring cellular concentrations of the anthracycline do xorubicin was developed. The assay involves cell lysis and protein deg radation by detergent and proteinase K treatment followed by DNA hydro lysis using DNase I. Prior to high-performance liquid chromatography, samples are deproteinized by the addition of ZnSO4 and methanol. The a ssay is linear with respect to both the cellular drug content and the number of cells assayed over the ranges tested, and drug recovery is c lose to 100%. The method has a limit of detection of 50 fmol injected doxorubicin. Within run and between-day coefficients of variation have consistently been found to be in the 5% and 10% range, respectively, in different cell lines exposed to doxorubicin in vitro. The method ha s been evaluated in analyses of doxorubicin levels in mononuclear bloo d cells of patients. The assay offers several advantages over commonly used organic extraction techniques and may improve cellular drug moni toring during anthracycline therapy in patients.