Development of a liquid chromatography/tandem mass spectrometry method to investigate the presence of biomarkers of DNA damage in urine related to red meat consumption and risk of colorectal cancer


Correspondence to: C. Da Pieve and M. Velasco-Garcia, Department of Life, Health and Chemical Sciences, Faculty of Science, The Open University, Walton Hall, Milton Keynes MK7 6AA, UK.




The consumption of red meat is known to enhance the endogenous formation of N-nitroso compounds (NOCs), which are potent carcinogens. DNA damage related to NOCs, and hence red meat, has been detected in colorectal cells and in blood. We proposed to extend previous studies to a non-invasive approach for the detection of O6-carboxymethylguanine (O6CMG) and O6-carboxymethyl-2'-deoxyguanosine (O6CMdG) in urine in relation to red meat intake using liquid chromatography/tandem mass spectrometry (LC/MS/MS). The presence of the adduct in urine samples either as the free base or as 2'-deoxynucleoside could help in determining the repair mechanism involved when such lesions are produced. A non-invasive assessment of DNA adducts could also allow for large-scale analyses in the population and cancer prevention dietary strategies.


An LC/MS/MS method for the quantitation of O6CMG and O6CMdG was developed. Urine samples collected from healthy volunteers on red meat and vegetarian diets were analysed either by direct injection or after purification by solid-phase extraction (SPE). A separate LC/MS/MS method for O6-methylguanine (O6MeG) and O6-methyl-2'-deoxyguanosine (O6MedG), which are possible hydrolysis products forming during the sample pre-treatment, was also developed.


The developed LC/MS/MS method allowed the simultaneous measurement of O6CMG and O6CMdG. The limits of detection (LODs) were 0.38 ng/mL for O6CMG and 0.18 ng/mL for O6CMdG. The direct injection analysis of the clinical samples showed low sensitivity due to high background signal that was improved by SPE purification. However, the concentrations of the adducts in clinical samples were still found to be below the LOD.


Novel, reproducible, and accurate LC/MS/MS methods were developed for the determination of the urinary content of O6CMG and O6CMdG, and of the possible formation of O6MeG and O6MedG by decarboxylation. Clinical samples from volunteers on different diets were analysed. Further studies are required to discover a link between the presence of these biomarkers in urine and red meat consumption. Copyright © 2013 John Wiley & Sons, Ltd.