摘要
目的建立一种可快速和高效检测大鼠尿液中RNA氧化产物8-羟基鸟苷(8-oxo Guo)的同位素稀释超高效液相-串联质谱法(ID-UPLC-MS/MS)。方法大鼠尿液经10 mmol的乙酸铵处理,加入同位素内标[^(15)N_(2)^(13)C_(1)]8-oxo Guo,置于37℃生化培养箱孵育10 min,4℃12000×g离心15 min,以含0.1%甲酸的5 mmol乙酸铵和甲醇为流动相,经色谱柱SB-Aq(3.0 mm×100 mm,1.8μm)分离,在多反应监测(MRM)模式下监测m/z 300/168(8-oxo Guo)及m/z 303/171([^(15)N_(2)^(13)C_(1)]8-oxo Guo),内标法定量。结果本检测方法在0.4 ng/ml~100 ng/ml线性关系良好,相关系数为0.9998,最小检测质量为0.1 pg,低、中、高3个浓度(0.4 ng/ml、10 ng/ml、100 ng/ml)的回收率分别为(106.54±11.71)%、(107.95±3.47)%、(109.70±4.18)%,相对标准偏差(n=6)为3.21%~10.99%。结论本研究建立的检测方法简便、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RNA氧化损伤。
Objective A fast and efficient isotope dilution ultra-performance liquid chromatography-triple quadrupole mass spectrometry(ID-UPLC-MS/MS)was established to detect RNA oxidation marker 8-oxo Guo in rats urine.Methods The isotope internal standard[^(15)N_(2)^(13)C_(1)]8-oxo Guo was added to the rat urine after being treated with 10 mmol ammonium acetate.Then,the mixture was incubated in a biochemical incubator at 37℃for 10 min.After centrifugation at 12000×g for 15 min at 4℃,the analytes were separated on an SB-Aq column(3.0 mm×100 mm,1.8μm)with 5 mmol ammonium acetate(containing 0.1%formic acid)and methanol as mobile phases.The m/z 300/168 for 8-oxo Guo and m/z 303/171 for[^(15)N_(2)^(13)C_(1)]8-oxo Guo were monitored in MRM mode for quantification by the internal standard method.Results The detection method,with the correlation coefficient of 0.9998,has a good linear relationship in the linear range of 0.4 ng/ml-100 ng/ml.The minimum inspection quality is 0.1 pg,and the recoveries of the low,medium and high concentrations(0.4 ng/ml,10 ng/ml,100 ng/ml)are(106.54±11.71)%,(107.95±3.47)%,(109.70±4.18)%,respectively.The relative standard deviation(n=6)was 3.21%-10.99%.Conclusion The study has established a simple,sensitive,rapid and efficient method,and it is applied to evaluate RNA oxidative damage caused by oxidative stress in the body.
作者
赵素玉
李付远
叶玲燕
吴仙军
喻晖
梅益斌
ZHAO Su-yu;LI Fu-yuan;YE Ling-yan;WU Xian-jun;YU Hui;MEI Yi-bin(Department of Cardiology,Lishui People's Hospital,Lishui,Zhejiang 323000,China)
出处
《中国卫生检验杂志》
CAS
2022年第3期341-344,349,共5页
Chinese Journal of Health Laboratory Technology
基金
浙江省医学会临床科研资金项目(2019ZYC-A69)。
关键词
同位素稀释超高效液相-串联质谱法
大鼠
尿液
8-羟基鸟苷
Isotope dilution ultra-performance liquid chromatography-tandem mass spectrometry
Rat
Urine
8-oxoGuo
