液相色谱-串联质谱法检测甲醛暴露产生的两种DNA加合物

针对两种甲醛-DNA加合物,N~6-羟甲基腺嘌呤(N~6-HOMe-d A)和N2-羟甲基鸟嘌呤(N_2-HOMe-d G),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[~(15)N_5]N~6-Me-d A和[~(13)C_(10)~(15)N_5]N_2-Me-d G为内标;液相分离所用色谱柱为Thermo Polar Advantage II C_(18);方法的精密度较好(RSD5%)、灵敏度较高(检出限分别为0.014和0.0064 ng·m L~(-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N~6-HOMe-d A和N_2-HOMe-d G的含量.结果表明,两种甲醛-DNA加合物N~6-HOMe-d A和N2-HOMe-d G的量与甲醛暴露存在剂量-反应和时间-反应关系.

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

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.