Oxidative attack to DNA is of particular interest since DNA modifications can lead to heritable mutations. The most studied product of DNA oxidation is 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG). While 8-oxodG determination in blood and tissue cells is prone to artifacts, its measurement in urine employing liquid chromatography tandem mass spectrometry (LC-MS/MS) has gained more and more interest for increased reliability. LC-MS/MS can be affected by matrix effects and this is particularly true when ion trap is used as MS analyzer, due to ion accumulation in the trap and related space charge effect. In the present work, we have developed a LC-MS/MS method where the combination of cation exchange and reverse phase solid phases resulted in LC separation optimization. This together with the employment of an isotopically labeled internal standard, allowed the usage of ion trap LC-MS/MS, typically not employed for quantitative measurement in biological samples, for the measurement of 8-oxodG in urine samples from control populations. Four different urine matrices were employed for method validation. Limit of quantitation was set at least at 0.5 ng/ml. While analyzing urine samples from healthy volunteers, 8-oxodG levels reported as ng/ml were statistically different comparing males with females (p<0.05, Mann Whitney test); while comparing results normalized for creatinine no statistical significant difference was found. Mean urinary 8-oxodG level found in healthy volunteers was 1.16±0.46 nmol/mmol creatinine. The present method by enhancing at best the chromatographic performances allows the usage of ion trap LC-MS/MS for the measurement of 8-oxodG in urine samples from control populations.
Keywords: 15[N5]2-dG, 15[N5]2′-deoxyguanosine; 15[N5]8-oxodG, 8-oxo-7, 8-dihydro-15[N5]2′-deoxyguanosine; 8-oxo-7,8-dihydro-2′-deoxyguanosine; 8-oxodG, 8-oxo-7, 8-dihydro-2′-deoxyguanosine; EIC, Extracted Ion Chromatogram; ESI, electrospray ionization; IQC, internal quality control; IS, internal standard; Ion trap; LC-MS/MS; LC-MS/MS, liquid chromatography tandem mass spectrometry; LOQ, limit of quantitation; MRM, multiple reaction monitoring; MTH1, Nudix hydrolase mut T homologue 1; NER, nucleotide excision repair system; NIR, nucleotide incision repair system; Oxidative stress; ROS, reactive oxygen species; Reactive oxygen species; SACI, surface-activated ionization; TIC, Total Ion Chromatogram; Urine.