TY - JOUR
T1 - Quantitative high-performance liquid chromatography-electrospray ionization tandem mass spectrometry analysis of bis- n 7-guanine DNA-DNA cross-links in white blood cells of cancer patients receiving cyclophosphamide therapy
AU - Malayappan, Bhaskar
AU - Johnson, L'aurelle
AU - Nie, Bei
AU - Panchal, Dolly
AU - Matter, Brock
AU - Jacobson, Pamala
AU - Tretyakova, Natalia
PY - 2010/5/1
Y1 - 2010/5/1
N2 - 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.
AB - 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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U2 - 10.1021/ac902923s
DO - 10.1021/ac902923s
M3 - Article
C2 - 20361772
AN - SCOPUS:77951779215
SN - 0003-2700
VL - 82
SP - 3650
EP - 3658
JO - Analytical Chemistry
JF - Analytical Chemistry
IS - 9
ER -