Loop-column extraction and liquid chromatographic analysis of doxorubicin and three metabolites in plasma

A sensitive and specific high performance liquid chromatographic method for the separation and quantitation of doxorubicin and three metabolites is described. The new method involves isocratic reversed-phase separation of these compounds, with quantitation by either electrochemical or fluorescence methods. A novel loop-column method was used to extract the compounds of interest from plasma, eliminating the need for organic extraction of biological samples before injection into the chromatographic system. The limit of sensitivity with the fluorescence method was about 2.3 ng on column. Sensitivity with electrochemical detection was better, although there was more interference by early eluting plasma components. The between-run CV (%) for replicate analysis of doxorubicin in identical plasma samples was 2.0 at 112.5 ng/ml, 5.4 at 40.0 ng/ml, and 9.9 at 14.0 ng/ml. Precision for each of the three metabolites was comparable. Accuracy for doxorubicin and each metabolite ranged from 93 to 107% at concentrations of 14.0, 40.0, 65.0, 112.5, and 140.0 ng/ml. The new loop-column extraction method, with injection of plasma samples directly onto the chromatograph, is simpler and more efficient than previous methods requiring organic extraction, making it more feasible for routine processing of clinical samples.