ID-UPLC-MS/MS检测大鼠尿液DNA氧化标志物8-oxodGuo方法的建立  

Determination of DNA oxidation marker 8-oxodGuo in rat urine by ID-UPLC-MS/MS

作　　者：黄斌 赵素玉[1] 林董莉 吴琼碧 吴仙军[1] 张伟[1] 叶玲燕[1] 梅益斌[1] HUANG Bin;ZHAO Suyu;LIN Dongli;WU Qiongbi;WU Xianjun;ZHANG Wei;YE Lingyan;MEI Yibin(Department of Cardiology,Lishui People′s Hospital,Lishui,Zhejiang 323000,China)

机构地区：[1]浙江省丽水市人民医院心血管内科,323000

出　　处：《重庆医学》2022年第8期1392-1396,1401,共6页Chongqing medicine

基　　金：浙江省医学会临床科研资金项目(2019ZYC-A69)。

摘　　要：目的建立一种快速、高效的同位素稀释超高效液相-串联质谱法(ID-UPLC-MS/MS),用于检测大鼠尿液中DNA氧化产物8-羟基脱氧鸟苷(8-oxodGuo)。方法大鼠尿液经10 mmol/L的醋酸铵处理,加入同位素内标[^(15)N_(5)]8-oxodGuo,置于37℃生化培养箱孵育,离心后以5 mmol/L的醋酸铵(含0.1%甲酸)和甲醇为流动相,经色谱柱SB-Aq(3.0 mm×100.0 mm,1.8μm)分离,在MRM模式下监测m/z 284/168(8-oxodGuo)及m/z 289/173([^(15)N_(5)]8-oxodGuo),进行内标法定量。结果本检测方法在0.4~100 ng/mL线性范围内线性关系良好,相关系数为0.9996,定量下限为0.2 pg,低、中、高3个浓度(0.4、10和100 ng/mL)的回收率分别为(110.30±7.83)%,(104.37±2.22)%,(99.26±1.18)%,相对标准偏差为1.1%~7.1%。结论该检测方法简便、灵敏、快速、高效,可较好地应用于评价机体氧化应激所导致的DNA氧化损伤。Objective To develop a rapid and efficient isotope dilution ultra-high performance liquid-phase tandem mass spectrometry(ID-UPLC-MS/MS)method for the determination of 8-hydroxydeoxyguanosine(8-oxodGuo),a DNA oxidation product in rat urine.Methods The isotope internal standard[^(15)N_(5)]8-oxodGuo was added to the rat urine after being treated with 5 mmol/L ammonium acetate.After incubated in a biochemical incubator at 37℃and centrifuged,the analytes were separated on an SB-Aq column(3.0 mm×100.0 mm,1.8μm)with 5 mmol/L ammonium acetate(containing 0.1%formic acid)and methanol as mobile phases.The m/z 284/168 for 8-oxodguo and m/z 289/173 for[^(15)N_(5)]8-oxodGuo were monitored in MRM mode for quantification by the internal standard method.Results The detection method had a good linear relationship in the range of 0.4-100 ng/mL with the correlation coefficient was 0.9996.The lower limit of quantification was 0.2 pg,and the recovery rate of the low,medium and high concentrations(0.4,10 and 100 ng/mL)were(110.30±7.83)%,(104.37±2.22)%and(99.26±1.18)%,respectively.The relative standard deviation was 1.1%-7.1%.Conclusion The assay is simple,sensitive,rapid and efficient,and can be better applied to evaluate the oxidative DNA damage caused by oxidative stress in the body.

关 键 词：ID-UPLC-MS/MS 大鼠 尿液 8-oxodGuo 

分 类 号：R446.12[医药卫生—诊断学]