An approach based on ultra-high pressure liquid chromatography–tandem mass spectrometry to quantify O6-methyl and O6-carboxymethylguanine DNA adducts in intestinal cell lines

► Optimisation of the extraction of O6-carboxymethylguanine and O6-methylguanine. ► Development and proper validation of an UHPLC–MS/MS detection method. ► Working with pre-extracted DNA instead of cultured cell lines is more efficient. ► Applicability of the detection method for in vitro and in vivo experiments. O6-methylguanine (O6-MeG) and O6-carboxymethylguanine (O6-CMG) are characteristic promutagenic and toxic DNA adducts formed by nitrosated glycine derivates and N-nitrosopeptides. Since endogenous nitrosation has been hypothesised as a plausible origin for the association between red and processed meat intake and colorectal cancer, a highly sensitive, fast and specific quantitative assay is needed to correlate the dose of individual DNA adducts with the effects of food consumption and individual digestive and metabolic processes. An ultra-high pressure liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) assay for quantitation of O6-MeG and O6-CMG, using the deuterated analogues as internal standards (ISTD), was developed. Samples of calf thymus DNA containing O6-MeG and O6-CMG were purified by acid hydrolysis and solid phase extraction prior to quantification by UHPLC–MS/MS in the selected reaction monitoring mode. The method was successfully validated in terms of repeatability (RSD