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研究报告

Liquid chromatography-tandem mass spectrometry method for the simultaneous detection of two DNA adducts induced by formaldehyde exposure

摘要：针对两种甲醛-DNA加合物，N⁶-羟甲基腺嘌呤（N⁶-HOMe-dA）和N²-羟甲基鸟嘌呤（N²-HOMe-dG），本实验室建立了基于液相色谱串联质谱的分析方法，以便从DNA分子水平上探讨甲醛暴露对DNA的损伤作用机理，其中[¹⁵N₅]N⁶-Me-dA和[¹³C₁₀¹⁵N₅]N²-Me-dG为内标；液相分离所用色谱柱为Thermo Polar Advantage II C18；方法的精密度较好（RSD<5%）、灵敏度较高（检出限分别为0.014和0.0064 ng·mL^-1）、回收率良好（94.9%～111%）.后用小牛胸腺DNA进行体外染毒实验，设置甲醛染毒的浓度梯度（0.001，0.01，0.05，0.1，0.5和1 mmol·L^-1）和染毒时间组（0、2、4、8、12、16、24 h），利用建立的LC-MS/MS方法检测了DNA中N⁶-HOMe-dA和 N²-HOMe-dG的含量.结果表明，两种甲醛-DNA加合物N⁶-HOMe-dA和 N²-HOMe-dG的量与甲醛暴露存在剂量-反应和时间-反应关系.

Abstract：To discuss DNA damage by formaldehyde exposure, a sensitive and selective liquid chromatography-tandem spectrometry (LC-MS/MS) method was established, which could simultaneously detect two formaldehyde-DNA adducts (N⁶-HOMe-dA and N²-HOMe-dG). In this method, [¹⁵N₅]N⁶-Me-dA and [¹³C₁₀¹⁵N₅]N²-Me-dG were used as internal standards and Thermo Polar Advantage II C18 column was used as optimized separation column. This method is in satisfied precision (RSD<5%), sensitivity (the detection limits were 0.014 and 0.064 ng·mL^-1 respectively) and recovery (94.9%~111%). Then, it was applied to two formaldehyde-DNA adducts detection in calf thymus DNA exposed to formaldehyde in vitro. The exposure experiment were prepared with various formaldehyde dose (0.001, 0.01, 0.05, 0.1, 0.5 and 1 mmol·L^-1) and different exposure time (0, 2, 4, 8, 12, 16 and 24 h). The results suggest the dose-response and time-response relationship between formaldehyde exposure and the two DNA adducts are present.