Method for the Detection of Nucleosides, Bases, and Hydroxylated Adducts Using Gradient HPLC with Coulometric Array and Ultraviolet Detection

Abstract

Free radicals are compounds with an unpaired electron capable of independent existence. These highly reactive species have been implicated in many disease states and can react with cell membranes, lipids, proteins, and DNA. When an oxygen radical reacts with DNA, base damage, cross-linking (e.g., DNA-DNA or DNA-protein), or DNA backbone damage (e.g., single- or double-strand breaks) can occur and often result in cell death. The field of oxidative metabolism as it relates to DNA damage has grown tremendously, with more DNA adducts being identified as biomarkers. These biomarkers are indicative of DNA damage. Measurement of these biomarkers has proved to be a challenge because of their relatively low occurrence (1 per 10(5)-10(6) bases). Methodologies for the measurement of DNA damage include thin-layer chromatography, enzyme-linked immunosorbent assay, gas chromatography-mass spectrometry, DNA sequencing, high-performance liquid chromatography (HPLC)-ultraviolet, and HPLC-ECD. HPLC-ECD (electrochemical detection) is a powerful technique that is both sensitive and selective. However, HPLC-ECD is generally not amenable to gradient analyses, so its utility is restricted. In addition, many of the bases and nucleosides are not electrochemically active. Gradient HPLC separation coupled to both a coulometric electrochemical array detector and an ultraviolet detector overcomes these limitations. Presented here is a gradient HPLC method that measures a wide variety of nucleosides, bases, and hydroxylated adducts using the inherent stability, sensitivity, and wide dynamic range of a coulometric electrochemical array detector and the universal detection qualities of an ultraviolet detector. Linear ranges, limits of detection, and detailed methods development are presented.