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Diagnostic Technologies and Molecular and Cellular Profiling: Biomarker Assay Development, Validation, and Qualification

UPLC-ESI-MS/MS Quantitation of cisplatin 1,2 guanine-guanine intrastrand cross links

University of North Carolina, Chapel Hill, NC

Abstract

A6

Platinum chemotherapeutic agents have been successfully used in the treatment of various epithelial cell based cancers for over 30 years. However, much remains unknown about the mode of action of these agents. It is generally believed that about 1% of the total cellular platinum binds to DNA, causing the induction of intra- and interstrand cross links. These DNA cross links are repaired to some extent by nucleotide excision repair. However, if not repaired they may lead to the induction of apoptosis via the inhibition of DNA replication and transcription, or cause mutations leading to cancer. It remains to be seen if specific adducts are responsible for varied responses to treatment, as current methods measure total platinum. The most prevalent DNA modification is the formed between two adjacent guanines at the N7 position producing the intrastrand cross link between cisplatin and 2'-deoxyguanylyl(3',5')-2'-deoxyguanosine or CP-d(GpG). We report here a new sensitive and specific quantitation method employing electro spray ionization mass spectrometry (ESI-MS) to measure the formation of the intrastrand cross link. Analyte standards have been synthesized and characterized by UV, ESI-MS/MS and NMR. Platinum has a characteristic isotopic cluster which aids in MS identification, but complicates quantitation. The following mass to charge (m/z) ratios therefore list the most prevalent ion. ESI-MS and ESI-MS/MS analysis revealed ions at [M+H]⁺ and [M+H-phosphate -2 deoxyribose - 2 amine]⁺ (m/z 824 and 496, respectively) for CP-d(GpG). Synthesis and characterization of stable isotope internal standard CP-[¹³C₅]d(GpG) are underway. Preliminary studies have shown that anion exchange columns are an effective way of separating this adduct from unmodified nucleosides. Ultra performance liquid chromatography (UPLC) was used in combination with ESI-MS/MS to gain further sensitivity. Currently, analysis of CP-d(GpG) spiked DNA after anion exchange clean-up with selected reaction monitoring (SRM; m/z 824 496) resulted in a preliminary limit of detection of 9 adducts per 10⁸ nucleotides. The addition of an internal standard and further method optimization is anticipated to allow accurate quantitation at lower concentrations of CP-d(GpG). The goal of this project is to measure CP-d(GpG) in specimens from human patients undergoing chemotherapy as a marker for tissue specific uptake at therapeutic doses. This work is supported by in part by grants from the NIEHS (T32ES07126, R42ES011746, P30ES10126) and the NCI (RO1CA084480).