Chromatographic determination of 8-oxo-7,8-dihydro-2 '-deoxyguanosine in cellular DNA: A validation study

Although a series of biomarkers are widely used for the estimation of oxida tive damage to biomolecules, validations of the analytical methods have sel dom been presented. Formal validation, that is the study of the analytical performances of a method, is however recognized as the best safeguard again st the generation and publication of data with low reliability. Classical v alidation parameters were investigated for the determination of an oxidativ e stress biomarker, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) in cellu lar DNA, by high-performance liquid chromatography coupled to amperometric detection (HPLC-EC); this modified base is increasingly considered as a mar ker of oxidative damage to DNA, but many questions are still raised on the analytical methods in use. Upon a rigorous statistical evaluation of the qu ality criteria currently required for assays in biological media, including selectivity, Linearity, accuracy, repeatability, sensitivity, limits of de tection and quantification, ruggedness and storage at different stop points in the procedure, the HPLC-EC assay method is found mostly reliable. The present validation attempt demonstrates that (i) the HPLC-EC assay of 8 -oxo-dG provides consistent data allowing to reliably detect an increase of this biomarker in cellular DNA; (ii) a harsh oxidative stress does not hin der the enzymatic digestion of DNA by nuclease pi; and (iii) the analytical results must be expressed relative to the internal standard dG which signi ficantly improves both repeatability and sensitivity. Whereas the described assay minimizes the artifactual production of the analyte from processing and storage, this cannot be totally ruled out; the true 8-oxo-dG base level s still lack a definitive assay method, which remains a considerable analyt ical challenge and the object of controversy.