Detection of 8-hydroxydeoxyguanosine (8-OHdG) as a biomarker of oxidative damage in peripheral leukocyte DNA by UHPLC–MS/MS
2017
Wu, Danni | Liu, Baodong | Yin, Junfa | Xu, Tian | Zhao, Shuli | Xu, Qun | Chen, Xi | Wang, Hailin
8-Hydroxydeoxyguanosine (8-OHdG) is a widely-used biomarker of oxidative DNA damages. 8-OHdG in peripheral blood leukocyte is associated with mutation and cancer risk. The level of 8-OHdG in peripheral blood leukocytes can indicate a long-term response to oxidative stress rather than that in urine. Accurate identification and quantification of leukocyte 8-OHdG are essential for understanding its mechanism of formation, repair, and biological consequences. In this study, a fast and accurate ultra-performance liquid chromatography-tandem mass spectrometry (UHPLC–MS/MS) method was developed and validated to detect 8-OHdG in human peripheral leukocyte. DNA in blood samples were extracted and digested, then subjected onto UHPLC–MS/MS using an isocratic elution on a Zorbax Eclipse Plus C18 column (2.1×100mm, 1.8μm). Multiple reaction monitoring (MRM) mode was adopted by using [¹⁵N5]-8-OHdG as an internal standard. The assay was linear over the concentration range of 1.0–100nM with R²=0.999. The accuracy for spiked samples was 90.9%–94.8%, and the intra-day precision was within 3.7%. The limit of detection (LOD) is 0.30nM and limit of quantification (LOQ) is 1.0nM with 5μl of sample injection. By the analysis of human leukocyte 8-OHdG (n=121) using the developed UHPLC–MS/MS method, it demonstrates that the level of leukocyte 8-OHdG of the cancer patients group (n=46) is significantly higher than that of the health control (n=75).
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