| 同位素稀释-亲水作用色谱串联质谱快速测定尿液中8-羟基脱氧鸟苷和可替宁的研究 |
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| 引用本文： | 杨明岐,袁悦,任建伟,朱婧,王焱,王文佳,邹晓莉. 同位素稀释-亲水作用色谱串联质谱快速测定尿液中8-羟基脱氧鸟苷和可替宁的研究[J]. 四川大学学报(医学版), 2020, 51(1): 74-80. DOI: 10.12182/20200160102 |
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| 作者姓名： | 杨明岐 袁悦 任建伟 朱婧 王焱 王文佳 邹晓莉 |
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| 作者单位： | 四川大学华西公共卫生学院/四川大学华西第四医院 卫生检验与检疫系 （成都 610041） |
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| 基金项目： | 四川省科技厅重点项目;四川省卫生和计划生育委员会科研课题 |
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| 摘 要： | 目的 建立尿液中8-羟基脱氧鸟苷和可替宁的同位素稀释-亲水作用色谱串联质谱的快速测定方法。 方法 离心后的尿样上清液加入15N5-8-羟基脱氧鸟苷、D3-可替宁两种内标，以3 mmol/L甲酸铵水溶液稀释5倍，经0.22 μm水系滤器过滤后，引入液相色谱-串联质谱法（ultra-performance liquid chromatography-tandem mass spectrometry，UPLC-MS/MS）分析。以ACQUITY UPLC? BEH HILIC色谱柱（50 mm×3.0 mm, 1.7 μm）分离，3 mmol/L甲酸铵水溶液与乙腈为流动相(10∶90) 等度洗脱，柱温40 ℃，流速0.3 mL/min；质谱以电喷雾电离源（electrospray ionization, ESI）+扫描多反应监测模式检测，内标法定量。 结果 8-羟基脱氧鸟苷和可替宁在各自线性范围内线性良好，方法检出限分别为0.064 μg/L和0.035 μg/L；定量限分别为0.21 μg/L和0.12 μg/L；加标回收率分别为92.6%～102.0%和102.0%～106.0%。采用所建立的方法测定实际尿样，并通过40例样本统计分析发现，主、被动吸烟者的烟草烟气暴露量、烟草特有亚硝胺摄入量存在差异（P<0.05），NNAL和可替宁含量在主动吸烟者尿样中含量更高；主、被动吸烟者的烟草烟气暴露与烟草特有亚硝胺暴露呈正相关（其相关系数r分别为0.487和0.786，P<0.05）。 结论 本研究成功建立同时测定尿液中8-羟基脱氧鸟苷和可替宁含量的同位素稀释-亲水作用色谱串联质谱法，该方法前处理简单、快速，通过同位素内标校正，定量灵敏准确，能够满足批量分析的需求。
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| 关 键 词： | 亲水作用色谱串联质谱 同位素稀释 尿液 8-羟基脱氧鸟苷 可替宁 |
| 收稿时间： | 2019-06-04 |
Determination of 8-oxo-2'-deoxyguanosine and Cotinine in Urine by Hydrophilic Chromatography-tandem Mass Spectrometry with Isotope Dilution |
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| YANG Ming-qi,YUAN Yue,REN Jian-wei,ZHU Jing,WANG Yan,WANG Wen-jia,ZOU Xiao-li. Determination of 8-oxo-2'-deoxyguanosine and Cotinine in Urine by Hydrophilic Chromatography-tandem Mass Spectrometry with Isotope Dilution[J]. Journal of Sichuan University. Medical science edition, 2020, 51(1): 74-80. DOI: 10.12182/20200160102 |
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| Authors: | YANG Ming-qi YUAN Yue REN Jian-wei ZHU Jing WANG Yan WANG Wen-jia ZOU Xiao-li |
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| Affiliation: | Department of Laboratory Technology and Science of Public Health, West China School of Public Health and West China Fourth Hospital, Sichuan University, Chengdu 610041, China |
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| Abstract: | Objective To develop an assay for determination of 8-oxo-2'-deoxyguanosine and cotinine in human urine by hydrophilic chromatography tandem mass spectrometry (HILIC-MS/MS) with isotope dilution. Methods The urine supernatant was 1∶5 diluted with 3 mmol/L ammonium formate aqueous solution containing 15N5-8-OHdG and D3-cotinine as internal standard. After being filtered through a 0.22 μm water filter, the sample solution was injected into ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) for analysis. Separation was performed on ACQUITY UPLC? BEH HILIC column (50 mm×3.0 mm, 1.7 μm) with isocratic elution (A∶B=10∶90) at 40 ℃. The mobile phase was composed with acetonitrile (B) and 3 mmol/L ammonium formate water soulution (A). The flow rate was 0.3 mL/min. Positive ion scan-multiple reaction monitoring (MRM) mode were used for monitoring and internal standard curves were applied for quantification. Results Good linearity was obtained under the optimal conditions. Detection limits for 8-OHdG and cotinine were 0.064 μg/L and 0.035 μg/L respectively, the quantitation limits were 0.21 μg/L and 0.12 μg/L respectively, and the recoveries of the spiked urine samples were 92.6%-102% and 102%-106% respectively. Statistical analysis of 40 urine sample determination results obtained by using the above assay showed that there were significant differences in tobacco smoke exposure and tobacco-specific nitrosamine intake between active and passive smoker (P<0.05). The concentration of NNAL and cotinine were higher in urine samples of active smoker. Tobacco smoke exposure was positively correlated with tobacco specific nitrosamine intake in both active and passive smokers (the correlation coefficients were 0.487 and 0.786 respectively, P<0.05). Conclusion We successfully established a simple and fast assay for simultaneously detecting 8-oxo-2'-deoxyguanosine and cotinine in human urine. It was sensitive and accurate for quntification via the calibration by the isotope internal standards, and can meet the needs of batch analysis. |
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