Simultaneous determination of 8-hydroxy-2'-deoxyguanosine and 5-methyl-2'-deoxycytidine in DNA sample by high performance liquid chromatography/positive electrospray ionization tandem mass spectrometry.

8-Hydroxy-2'-deoxyguanosine (8-OHdG) and 5-methyl-2'-deoxycytidine (5-mdC) are utilized as useful biomarkers not only for early diagnosis but also for the detection and assessment of high-risk individuals. In the present study, a sensitive and specific method was developed for simultaneous determination of 8-OHdG and 5-mdC in DNA by high performance liquid chromatography/positive electrospray ionization tandem mass spectrometry. The limits of quantification for 8-OHdG and 5-mdC were 80 and 40pg/ml, respectively. The calibration curves of 8-OHdG and 5-mdC were linear over the concentration range of 0.02-100ng/ml and the correlation coefficients were higher than 0.9990. The intra-day and inter-day relative standard derivative values were in the range of 0.70-7.47% for 8-OHdG and 1.07-7.06% for 5-mdC, respectively. The recoveries were 93.4-108.5% for 8-OHdG and 87.4-104.9% for 5-mdC, respectively. This method was validated by determination of the background levels of 8-OHdG and 5-mdC in calf thymus DNA, and satisfactory results were obtained.