| Doxorubicin (DOX) is a widely used anthracycline effective against a variety of human malignancies, including leukemia and breast cancer. However, cardiac and liver toxicity and drug resistance result from prolonged treatment with DOX. Studies have implicated DOX metabolites as key in the occurrence of such side effects. There is a need for sensitive analytical techniques to characterize DOX metabolism and the role these metabolites play in DOX chemotherapy. Therefore, this thesis focuses on the design of sensitive analytical techniques to characterize DOX metabolism and subcellular localization.; In this research, a capillary electrophoresis with post-column laser-induced fluorescence detection (CE-LIF) system was built and used to separate and detect DOX metabolites produced by DOX treated NS-1 mouse hybridoma cells. The high sensitivity of this CE-LIF method has allowed us to separate and detect a greater number of DOX metabolites then previously reported. CE-LIF was also used to study DOX metabolite abundance, distribution, and identity in subcellular fractions. A full characterization of methods for analysis, including cell lysis, metabolite extraction and capillary electrophoresis separation of a DOX resistant (CEM/C2) and DOX sensitive (CCL-119) human leukemia cell line was done. The methods developed were used to study the accumulation of doxorubicin in subcellular environments of the CEM/C2 and CCL-119 cell lines. Lastly, the accumulation of doxorubicin in mitochondria was determined in a dual-channel detection experiment using an organelle specific fluorescent probe to label the isolated mitochondria. One of the aims of this work was to develop CE-LIF methods that addressed a relevant scientific problem and offered immediate advantages over existing assays and techniques. This thesis represents the most comprehensive work with capillary electrophoresis and DOX metabolites produced in cellulo to date. The results reported here outline significant advances in developing clinically relevant and sensitive methods for separation, detection and quantification of DOX and DOX metabolites. |
