UPLC-HRMS法定量分析小鼠组织中的6-氧-甲基鸟嘌呤  

Quantitative analysis of 6-O-methylguanine in mouse tissues by UPLC-HRMS

作　　者：李琛[1] 王荣浩[1] 毛健[1] 霍现宽[1] 王丁众[1] 张建勋[1] 

机构地区：[1]中国烟草总公司郑州烟草研究院

出　　处：《烟草科技》2017年第5期53-59,共7页Tobacco Science & Technology

基　　金：国家自然科学基金面上项目"改性介孔材料固相萃取/UPLC-MSn方法研究烟草特有亚硝胺在动物体内代谢"(21175153);国家自然科学基金青年基金项目"烟碱在大鼠脑内代谢的活体动态分析及其神经递质化学效应的实时评价"(21307163)

摘　　要：为评价4-(N-甲基-N-亚硝氨基)-1-(3-吡啶基)-1-丁酮(NNK)的器官致癌性,建立了一种定量分析C57BL/6小鼠组织中DNA甲基化加合物6-氧-甲基鸟嘌呤(6-O-MeG)的超高效液相色谱-高分辨质谱(UPLC-HRMS)方法,并分别对NNK染毒后生理盐水对照组、吡唑处理组和5-甲氧基补骨脂素(5-MOP)处理组小鼠肝脏和肺中6-O-MeG的含量进行了测定。用试剂盒提取组织样品DNA,提取物在80℃下酸解后加入内标6-氧-甲基-d3-鸟嘌呤([CD_3]6-O-MeG),经过HLB固相萃取柱净化后进行UPLC-HRMS分析。结果表明:(1)6-O-MeG在0.05~10.00 ng/mL内线性良好(R^2=0.999 8),检出限0.011ng/mL,回收率92.1%~102.2%,日内和日间精密度分别为1.10%~4.34%和1.89%~6.58%。(2)给药4 h后,吡唑处理组小鼠肝脏和肺中6-O-MeG的含量分别为对照组的2.09和1.87倍,5-MOP处理组则分别为对照组的0.32和0.67倍,表明小鼠体内的CYP2A5在NNK的代谢活化过程中起到重要作用。该方法样品前处理简单、选择性好、灵敏度高,适用于小鼠组织中6-O-MeG的测定。In order to evaluate the carcinogenicity of 4-（N-methyl-N-nitrosamino）-1-（3-pyridyl）-1-butanone （NNK）, an ultra-high performance liquid chromatography-high resolution mass spectrometry （UPLC-HRMS） method was established to accurately determine the levels of 6-O-methylguanine （6-O-MeG）in mouse liver and lung tissues. The contents of 6-O-MeG in the liver and lung of normal saline-treated group, pyrazole-treated group and 5-methoxypsoralen （5-MOP）-treated group were measured separately after NNK administration. Tissue samples were extracted by DNA extraction kits, then the extraction was acidified at 80℃, followed by adding[CD3] 6-O-MeG as the internal standard. After going through HLB solid phase column and redissolution by acetonitrile, the extraction was analyzed by UPLC-HRMS. The results showed that：1）6-O-MeG displayed good linearity in the range of 0.05-10.00 ng/mL with R2 of 0.999 8, the limit of detection （LOD）of 0.011 ng/mL and the recoveries from 92.1% to 102.2%, the RSDs of intra-and inter-day were 1.10%-4.34% and 1.89%-6.58%, respectively. 2） The concentration of 6-O-MeG in the liver and lung of mice treated with pyrazole was 2.09 and 1.87 times as high as those treated with saline four hours after NNK administration; the 5-MOP-treated group was 0.32 and 0.67 times as high as the control group. The results indicated that cytochrome P450 2A5 （CYP2A5） enzyme played an important role in the activation of NNK metabolism in mice. This method features a simple sample preparation, good selectivity, high sensitivity, and it is suitable for determining 6-O-MeG in mouse tissue.

关 键 词：NNK 6-氧-甲基鸟嘌呤(6-O-MeG) 吡唑 5-甲氧基补骨脂素(5-MOP) C57BL/6小鼠 超高效液相色谱-高分辨质谱(UPLC-HRMS) 

分 类 号：TS416[农业科学—烟草工业]