Observation and prevention of an artefactual formation of oxidized DNA bases and nucleosides in the GC-EIMS method

Gas chromatography coupled to electron impact mass spectrometry (GC-EIMS) analysis following hydrolysis of DNA is a widely used assay for the detection of oxidized nucleobases and nucleosides. However, evidence was recently provided for an oxidation of guanine residues of hydrolysed DNA during the silylation prior to GC - EIMS analysis. This reaction accounts for the overestimation of the yield of 8-oxo-7,8-dihydroguanine by GC-EIMS. In the present work, we showed that adenine, cytosine, thymine and thymidine also give rise to oxidized derivatives during the derivatization. This was inferred from the measurement of the amount of 5-formyluracil, 5-hydroxymethyluracil, 5-hydroxycytosine (5-OHCyt), 8-oxo-7,8-dihydroadenine (8-OxoAde) and 5-hydroxymethyl-2'deoxyuridine (5-HMdUrd) in a series of experiments based on the use of purified bases and nucleosides. Isotopically labelled oxidized bases and 5-HMdUrd were used as internal standards to control the quantitative aspect of the silylation reaction. Support for an artefactual oxidation of hydrolysed DNA was provided by the comparison of the amount of 8-OxoAde and 5-OHCyt detected within native and gamma-irradiated DNA by HPLC-EC and GC-EIMS. To prevent the artefactual formation of oxidized bases during the silylation, an approach based on an HPLC prepurification was developed to remove the precursors of the oxidized bases measured in the DNA sample. The HPLC/GC - EIMS assay was successfully applied to the quantitation of 8-OxoAde and 5-OHCyt in calf thymus DNA. In addition, the detection of the dose-dependent formation of 5-HMdUrd in isolated DNA exposed to ionizing radiation was achieved using the same approach.