MEASUREMENT OF OXIDIZED BASES IN DNA - COMPARISON BETWEEN HPLC-EC ANDGC-MS ASSAYS

The detection of oxidized bases in cellular DNA is a challenging probl em due to the sensitivity required that should be high enough in order to detect one modification per 10(6) DNA bases. In this respect, gas chromatography mass spectrometry (GC-MS) has been extensively used dur ing the last ten years. However, comparison of the results obtained by this method and those inferred from high performance liquid chromatog raphy analyses coupled with an electrochemical detection (HPLC-EC) ind icated an overestimation in the level of the oxidized DNA bases in the GC-MS measurement. A comparative evaluation of the two analytical met hods allows us to demonstrate that this overestimation is due to an ar tefactual oxidation of normal DNA bases during the derivatization reac tion that is required prior to GC-MS analysis. Purification of the mod ified DNA bases prior to the derivatization reaction prevents the arte fact to occur. Under these conditions, the results obtained using the GC-MS method and the HPLC-EC assay are similar.