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Quantitative High-Performance Liquid Chromatography−Electrospray Ionization Tandem Mass Spectrometry Analysis of Bis-N7-Guanine DNA−DNA Cross-Links in White Blood Cells of Cancer Patients Receiving Cyclophosphamide Therapy

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Departments of Medicinal Chemistry and Experimental and Clinical Pharmacology, College of Pharmacy, and the Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota 55455
* To whom correspondence should be addressed.
†Department of Medicinal Chemistry.
‡Department of Experimental and Clinical Pharmacology.
Cite this: Anal. Chem. 2010, 82, 9, 3650–3658
Publication Date (Web):April 2, 2010
Copyright © 2010 American Chemical Society

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    Cyclophosphamide (CPA) is a DNA alkylating agent widely used in cancer chemotherapy. CPA undergoes metabolic activation to phosphoramide mustard and nornitrogen mustard (NOR) which alkylate the N-7 position of guanine in DNA to produce N-[2-(N7-guaninyl) ethyl]-N-[2-hydroxyethyl]-amine (G-NOR-OH) monoadducts and N,N-bis[2-(N7-guaninyl) ethyl] amine cross-links (G-NOR-G). G-NOR-G cross-links are strongly cytotoxic and are thought to be responsible for the biological activity of CPA. In the present work, an isotope dilution high-performance liquid chromatography−electrospray ionization (positive ion) tandem mass spectrometry (HPLC−ESI+-MS/MS) methodology was developed to accurately quantify G-NOR-G adducts in human blood. In our approach, DNA extracted from white blood cells (5−20 μg) is spiked with an internal standard of [15N10]-G-NOR-G and subjected to thermal hydrolysis to release G-NOR-G adducts from the DNA backbone. Following solid phase extraction, G-NOR-G conjugates are quantified by capillary HPLC−ESI-MS/MS in the selected reaction monitoring mode. The application of the new methodology is demonstrated for DNA extracted from blood of three cancer patients receiving 50−60 mg/kg of intravenous CPA. The highest numbers of G-NOR-G adduct (up to 18 adducts per 106 normal nucleotides) were observed 4−8 h following CPA administration, followed by a gradual decrease over time, probably due to adduct hydrolysis, DNA repair, and white blood cell death. This methodology will be useful for future investigations of the interindividual differences for CPA-induced DNA−DNA cross-linking.

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    HPLC−UV traces for CPA, calibration curves, and additional method validation results. This material is available free of charge via the Internet at

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