title: A sensitive high performance liquid chromatography assay for the quantification of doxorubicin associated with DNA in tumor and tissues.
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.
authors.affiliation: Division 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. Electronic address: andrew_lucas@unc.edu.
authors.forename: Andrew T
authors.initials: AT
authors.lastname: Lucas
authors.affiliation: Division 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. Electronic address: sarakoneal@gmail.com.
authors.forename: Sara K
authors.initials: SK
authors.lastname: O'Neal
authors.affiliation: UNC Lineberger Comprehensive Cancer Center, School of Medicine, University of North Carolina at Chapel Hill, CB# 7295, Chapel Hill, NC 27599-7295, United States; LCCC Animal Studies Core Facility, University of North Carolina at Chapel Hill, 1002 Mary Ellen Jones Building, Chapel Hill, NC 27599-7295, United States. Electronic address: smross@email.unc.edu.
authors.forename: Charlene M
authors.initials: CM
authors.lastname: Santos
authors.affiliation: UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, CB#7355, Chapel Hill, NC 27599-7355, United States. Electronic address: tfwhite86@gmail.com.
authors.forename: Taylor F
authors.initials: TF
authors.lastname: White
authors.affiliation: Division 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; UNC Lineberger Comprehensive Cancer Center, School of Medicine, University of North Carolina at Chapel Hill, CB# 7295, Chapel Hill, NC 27599-7295, United States; UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, CB#7355, Chapel Hill, NC 27599-7355, United States; UNC Center for Pharmacogenomics and Individualized Therapy, 120 Mason Farm Road, CB# 7361, Chapel Hill, NC 27599, United States; Carolina Institute For NanoMedicine, University of North Carolina at Chapel Hill, 120 Mason Farm Road, 1079 Genetic Medicine Building, Chapel Hill, NC 27599-7264, United States. Electronic address: zamboni@email.unc.edu.
authors.forename: William C
authors.initials: WC
authors.lastname: Zamboni
mesh_terms: D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000032:analysis* / Q000493:pharmacokinetics; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004247:DNA / Q000737:chemistry*; D018736:DNA Adducts / Q000032:analysis* / Q000493:pharmacokinetics; D004317:Doxorubicin / Q000032:analysis* / Q000493:pharmacokinetics; D004355:Drug Stability; D005260:Female; D006801:Humans; D057230:Limit of Detection; D008099:Liver / Q000378:metabolism*; D016513:Mice, SCID; D010051:Ovarian Neoplasms / Q000378:metabolism*; D015203:Reproducibility of Results; D014018:Tissue Distribution
chemical_list: D000903:Antibiotics, Antineoplastic; D018736:DNA Adducts; C031902:doxorubicin-DNA; D004317:Doxorubicin; D004247:DNA; C427203:calf thymus DNA
keywords: Doxorubicin; HPLC; Intracellular doxorubicin; Intracellular pharmacokinetics; PEGylated liposomal doxorubicin