A sensitive high performance liquid chromatography assay for the quantification of doxorubicin associated with DNA in
Andrew T Lucas1, Sara K O'Neal1, Charlene M Santos2
1Division of Pharmacotherapy and Experimental Therapeutics, University of North Carolina at Chapel Hill (UNC) Eshelman School of Pharmacy, CB# 7569, Chapel Hill, NC 27599-7569, United States.
Abstract:
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 the intracellular active form of doxorubicin. Thus, the development of a sample processing method and a high-performance liquid chromatography (HPLC) methodology was performed in order to quantify doxorubicin that is associated with DNA in tumors and tissues, which provided 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 drug associated with DNA; a Shimadzu HPLC with fluorescence detection equipped with a Phenomenex Luna C18 (2μm, 2.0×100mm) analytical column and a gradient mobile phase of 0.1% formic acid in water or acetonitrile for separation and quantification. The assay has a lower limit of detection (LLOQ) of 10ng/mL and is shown to be linear up to 3000ng/mL. The intra- and inter-day precision of the assay expressed as a coefficient of variation (CV%) ranged from 4.01 to 8.81%. Furthermore, the suitability of this assay for measuring doxorubicin associated with DNA in vivo was demonstrated by using it to quantify the doxorubicin concentration within tumor samples from SKOV3 and HEC1A mice obtained 72h after administration of PEGylated liposomal doxorubicin (Doxil(®); PLD) at 6mg/kg IV x 1. This HPLC assay allows for sensitive intracellular quantification of doxorubicin and will be an important tool for future studies evaluating intracellular pharmacokinetics of doxorubicin and various nanoparticle formulations of doxorubicin.
Insights
A new high-performance liquid chromatography (HPLC) method accurately measures intracellular doxorubicin DNA-binding, crucial for cancer therapy assessment. This assay quantifies doxorubicin in tumors, improving understanding of drug efficacy and delivery systems.
Area of Science:
- Pharmacology
- Analytical Chemistry
- Cancer Research
Background:
- Doxorubicin is a vital chemotherapy drug, but its intracellular DNA-binding concentration is hard to measure accurately.
- Accurate intracellular doxorubicin quantification is essential for evaluating drug efficacy and delivery systems.
Purpose of the Study:
- Develop and validate a high-performance liquid chromatography (HPLC) assay to quantify intracellular doxorubicin bound to DNA in tumors and tissues.
- Establish a reliable method for measuring the intracellular cytotoxic effects of doxorubicin from various formulations.
Main Methods:
- Developed a sample processing method coupled with HPLC-fluorescence detection.
- Utilized liquid-liquid phase extraction and a Phenomenex Luna C18 column for drug isolation and separation.
- Validated the assay using daunorubicin as an internal standard, demonstrating linearity and precision.
Main Results:
- The HPLC assay achieved a lower limit of quantification (LLOQ) of 10 ng/mL and linearity up to 3000 ng/mL.
- Demonstrated high precision with intra- and inter-day coefficients of variation ranging from 4.01% to 8.81%.
- Successfully quantified doxorubicin in mouse tumor samples after administration of PEGylated liposomal doxorubicin (Doxil®).
Conclusions:
- The developed HPLC assay provides sensitive and accurate intracellular quantification of DNA-bound doxorubicin.
- This method is suitable for in vivo studies and will aid in evaluating the pharmacokinetics of doxorubicin and its nanoparticle formulations.
