Subcellular Quantification of Doxorubicin and Its Metabolite in Cultured Human Leukemia Cells Using Liquid Chromatography-Tandem Mass Spectrometry

Doxorubicin (DOX) is widely used in the world as an anticancer agent for the treatment of leukemia and solid tumors. However, its clinical use is largely limited by the emergence of cardiotoxicity. One of the most frequently proposed mechanisms for DOX induced cardiotoxicity is the formation of metabolites. However, the enzymatic pathways involved in DOX intracellular metabolism have not been fully elucidated thus far. To provide a more detailed description of DOX metabolism, an assay using liquid chromatography-tandem mass spectrometry (LC/MS/MS) was developed in our lab to simultaneously determine DOX and its primary metabolite doxorubicinol (DOXol) in subcellular compartments. Sample cleanup and enrichment were achieved using solid phase extraction, and the validated calibration ranges for DOX and DOXol were 5.00–1000 ng/mL and 0.50–50.0 ng/mL, respectively. Good accuracy and precision were achieved. Using this assay, the accumulation of DOX and DOXol in whole cells, nuclear-enriched fraction (NEF) and organelle-enriched fraction (OEF) were compared between two human T leukemia cell lines (i.e., Jurkat and CCRF-CEM). A time-course analysis was also carried out. The resulting varieties of DOX and DOXol subcellular distributions and concentration-time profiles might be attributed to the differential expression, activities, and localization of reductive enzymes within these cell lines. More importantly, this work demonstrated that simultaneous determination of drug and its metabolite in subcellular compartments could be achieved using LC/MS/MS.