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

Da Pieve, Chiara; Sahgal, Natasha; Moore, Sharon A. and Velasco-Garcia, Maria N. (2013). 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. Rapid Communications in Mass Spectrometry, 27(21) pp. 2493–2503.

DOI: https://doi.org/10.1002/rcm.6709

Abstract

RATIONALE

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-carboxymethyl-guanine (O⁶CMG) and O⁶-carboxymethyl-2’-deoxiguanosine (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 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.

METHODS

An LC-MS/MS method for quantitation of O⁶CMG and the corresponding nucleoside O⁶CMdG was developed. 24 hours urine samples, collected from healthy volunteers (N=12) 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 the quantitation of O⁶-methyl guanine (O⁶MeG) and O⁶-methyl-2’-deoxyguanosine (O⁶MedG), which are possible hydrolysis products forming during the samples pre-treatment, was also developed.

RESULTS

The developed LC-MS/MS method allowed the simultaneous measurement of O⁶CMG and O⁶CMdG. The limit of detection was 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 were still found to be below the LOD.

CONCLUSIONS

Two novel, reproducible, and accurate LC-MS/MS methods were developed for the determination of O⁶CMG and O⁶CMdG, and O⁶MeG and O⁶MedG in urine. 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.

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