Collections > UNC Chapel Hill Undergraduate Honors Theses Collection > A Sensitive High Performance Liquid Chromatography (HPLC) Assay for the Quantification of Doxorubicin Bound to DNA
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Doxorubicin, a widely used anticancer agent, exhibits antitumor activity against a wide variety of malignancies. The drug exerts its cytotoxic effects by binding to and intercalating within the DNA of tumor and tissue cells. However, current assays are unable to accurately determine the concentration of intracellular active form of doxorubicin. Thus, we have developed a high performance liquid chromatography (HPLC) methodology in order to quantify the concentrations of doxorubicin that are bound to DNA in tumors and tissues as an intracellular cytotoxic measure of doxorubicin exposure after administration of small molecule and nanoparticle formulations of doxorubicin. The assay uses daunorubicin as an internal standard; liquid-liquid phase extraction to isolate bound drug; a Shimadzu HPLC with fluorescence detection equipped with a Phenomenex Luna C18 (2 um, 2.0 x 100 mm) analytical column; and a gradient mobile phase of 0.1% formic acid in water and acetonitrile. The assay has a lower limit of quantification (LLOQ) of 10 ng/mL and is shown to be linear up to 3,000 ng/mL. We demonstrated the suitability of this assay for doxorubicin bound to DNA in vivo by using it to quantify the doxorubicin concentration within tumor samples from SKOV3 and HEC1A mice obtained 72 hours after administering PEGylated liposomal doxorubicin (Doxil®; PLD) IV at 6 mg/kg. This HPLC assay allows for a sensitive and simple intracellular quantification of doxorubicin as compared to other methods and will be an important tool for future studies evaluating intracellular pharmacokinetics of doxorubicin and various nanoparticle carriers.