Separation of oxidatively damaged DNA nucleobases and nucleosides on packed and monolith C18 columns by HPLC-UV-EC

This study involves the incorporation of a commercially available Phenomenex Onyx C18 monolith column into the separation and detection of oxidative DNA damage. It includes thorough investigation of monolith performance and a comparison of the performance of monolith columns with a commercially available packed Restek reverse phase Ultra C18 column for the separation of DNA bases and nucleosides. The performance of the monolith was examined using efficiency, resolution, plate height, asymmetry and retention times, and each case showed improved or at least comparable results in the separation of a mix of DNA bases and nucleosides. A 90% reduction, from just under 40 min to just under 4 min, was obtained in the elution time of this separation. To the best of our knowledge, this is the first report of a fast monolith column separation successfully coupled to both a UV-vis and EC detector, which is especially useful for the analysis of oxidative DNA damage. The determination of 8-oxoG and 8-OH-dG, oxidation products of guanine and 2′-deoxyguanosine, respectively, may be compromised by their ease of oxidation and therefore the fast separation, selective and sensitive detection, with no artifactual oxidation, detailed in this report, is ideal.