乙醇生物标志性代谢物的检测及动力学研究

发布时间：2018-06-06 07:53

本文选题：乙基葡萄糖醛酸苷 + 代谢动力学　；参考：《复旦大学》2010年硕士论文

【摘要】：我国酒文化源远流长,因饮酒引发的事故和案件已经成为严重的社会问题。涉及酒精的技术鉴定,如酒后驾驶或醉酒驾驶的认定、酒后肇事逃逸的认定、尸体中酒精来源的判定等,是司法鉴定工作中经常面临的问题。乙醇进入人体后,摄入量的95%-98%经肝代谢,先被氧化为乙醛,再进一步被氧化为乙酸；其余的部分(2%-5%)主要以原体形态由尿液、呼气和汗液中排出体外；仅有微量乙醒(0.1%)与尿嘧啶核苷-5’-二磷-葡萄糖醛酸结合,生成乙基葡萄糖醛酸苷(Ethyl Glucuronide,Et(?)G)从样中排出体外；同时有微量乙醇在肝脏中代谢成乙基硫酸酯(Ethyl Sulphate, EtS),由尿液中排出体外。由于乙基葡萄糖醛酸苷和乙基硫酸酯在体内可停留时间比其原体乙醇长,近年来越来越为人们所重视,被认为是乙醇的标志性化合物。在酒后驾车事故,特别是肇事逃逸案件中,当血液中测不出乙醇时,只要检出一定量的乙基葡萄糖醛酸苷和乙基硫酸酯,也可认为是酒后驾车或生前摄入乙醇。有关乙基葡萄糖醛酸苷的检测及体内代谢动力学国外已有一些报道,国内尚未有相关研究报道。本学位论文以乙基葡萄糖醛酸苷为主要研究对象,致力于建立血液和尿液中乙基葡萄糖醛酸苷的定性、定量分析方法,并对方法的检出限、回收率、线性范围等作出评价；利用健康自愿受试者控制剂量饮酒后,定时测定其血液和尿液中乙基葡萄糖醛酸苷浓度,考察中国人体内乙基葡萄糖醛酸苷的代谢动力学；确定中国人酒后乙基葡萄糖醛酸苷的检出时限；初步实现了利用血液中EtG和乙醇浓度关系,推定饮酒时间的数据建模。全部内容按照工作展开的时间顺序分为六章。第一章介绍了国内外EtG分析研究进展和应用,阐述了本研究的研究背景、意义和主要工作。第二章建立和评价了气相色谱-串联质谱联用仪(GC-MS/MS)测定血液中EtG的方法。该方法血液中EtG的检出限(LOD)为0.05μg/mL,线性范围为0.1μg/mL-10μg/mL (R2=0.9999),准确度为90%-110%,日内精密度和日间精密度均小于15%,并成功应用于实际案例的检测。第三章建立和评价了液相色谱-线性离子阱-轨道离子阱(LC-LTQ-Orbitrap)测定血液、尿液中EtG的方法。该方法血液、尿液中EtG的检测限均为0.02μ/mL,线性范围为0.05-5.00μg/mL (R20.998),两基质中EtG检测方法的日内准确度与日间准确度均为85%-110%,日内精密度和日间精密度均小于10%。第四章建立和评价了液相色谱-串联质谱联用仪(LC-MS/MS)测定血液、尿液中EtG的方法。该方法血液和尿液中EtG的检测限均为0.05μg/mL,线性范围为0.1μg/mL-5μg/mL (R20.999),两基质中EtG检测方法的日内准确度均为95%-105%,日内精密度与日间精密度均小于15%。第五章开展了剂量控制的健康志愿者饮酒实验,采集血液与尿液样本；利用建立的LC-MS/MS方法检测样本,考察EtG的代谢动力学,探讨了血液和尿液中EtG,血液和尿液中的乙醇的相互关系。通过统计软件Stata 7.0计算,EtG在血液浓度峰值为4-6h,检测时限为8-21h,比血液中乙醇检测时限长8-10h。乙醇在EtG在尿液浓度峰值为4-6h,检测时限为20-85h,比尿液中乙醇检测时限长12-70h,并以此初步建立了由EtG和乙醇浓度推算饮酒时间的数据模型。第六章建立了血液、尿液中EtS的LC-LTQ-Orbitrap分析方法。线性范围0.1μg/mL-5μg/mL (R20.995)。本研究在国内首次建立了GC-MS/MS、LC-LTQ-Orbitrap和LC-MS/MS等多个EtG检测方法并进行了方法学验证,其中LC-LTQ-Orbitrap方法为国际上首次报道；首次考察中国人酒后乙基葡萄糖醛酸苷的代谢动力学,探讨了血液和尿液中EtG与乙醇的相互关系,提出了EtG在血液、尿液中的检出时限。研究成果为酒后驾驶逃逸者的认定、尸体检材中乙醇来源的判断提供了科学依据。
[Abstract]:The wine culture in China has a long history, and the accidents and cases caused by drinking have become serious social problems. The technical identification of alcohol, such as the identification of drunk driving or drunken driving, the identification of the escape and escape after alcohol, the determination of the source of alcohol in the corpse, is a common problem in the judicial appraisal work.
After the ethanol enters the body, the intake of 95%-98% is oxidized to acetaldehyde first and then oxidized to acetic acid; the rest (2%-5%) is mainly discharged from urine, exhaled and perspiration in the form of mycoplasma, and only a trace of awoke (0.1%) is combined with uridine -5 'two phosphorus glucuronic acid to produce ethyl glucuronic acid. Glycosides (Ethyl Glucuronide, Et (?) G) are excreted from the sample in vitro; meanwhile, there are trace amounts of ethanol in the liver that metabolize Cheng Yiji sulfate (Ethyl Sulphate, EtS) and excreted in the urine. As ethyl glucuronide and ethyl sulfate are longer in vivo than their ethanol, more and more attention has been paid to them in recent years. It is a symbol of ethanol. In the case of drunk driving, especially in the accident and escaping case, when a certain amount of ethyl glucuronide and ethyl sulfate are detected in the blood, a certain amount of ethyl glucuronide and ethyl sulfate can be considered to be drunk driving or intake of ethanol in life. There have been some reports outside of China, and no relevant research reports have been reported in China.
This thesis focuses on the determination of ethyl glucuronide in blood and urine, quantitative analysis, detection limit, recovery, linear range, etc. The metabolic kinetics of ethyl glucuronide in Chinese human body was investigated by ethyl glucuronide concentration, and the detection time limit of ethyl glucuronide in Chinese people was determined. The data modeling of using the relationship between the concentration of EtG and ethanol in the blood and determining the time of alcohol consumption was preliminarily realized. The whole content was divided according to the time sequence of work. It is the six chapter.
The first chapter introduces the progress and application of EtG analysis at home and abroad, and expounds the background, significance and main work of this research.
In the second chapter, the method for the determination of EtG in blood by gas chromatography tandem mass spectrometry (GC-MS/MS) is established and evaluated. The detection limit (LOD) of EtG in the blood is 0.05 mu g/mL, the linear range is 0.1 mu g/mL-10 mu g/mL (R2=0.9999), the accuracy is 90%-110%, the intraday precision and the day precision are less than 15%, and are successfully applied to the actual cases. Testing.
In the third chapter, the method for the determination of EtG in blood and urine by liquid chromatography linear ion trap orbital ion trap (LC-LTQ-Orbitrap) is established and evaluated. The detection limit of EtG in blood and urine is 0.02 /mL, and the linear range is 0.05-5.00 mu g/mL (R20.998). The daily accuracy and day accuracy of the two matrix EtG test methods are all 85%-110%, The intraday precision and the day precision are less than 10%.
In the fourth chapter, the method for the determination of EtG in blood and urine by liquid chromatography tandem mass spectrometry (LC-MS/MS) is established and evaluated. The detection limit of EtG in blood and urine is 0.05 g/mL, and the linear range is 0.1 mu g/mL-5 g/mL (R20.999). The intra day accuracy of EtG detection method in the two matrix is all 95%-105%, intraday precision and day precision The degree is less than 15%.
In the fifth chapter, a dose controlled healthy volunteers drinking experiment was carried out to collect blood and urine samples. The metabolic kinetics of EtG was examined by the established LC-MS/MS method. The relationship between the blood and urine in EtG, blood and urine was investigated. The peak of the blood concentration of EtG was calculated by the statistical software Stata 7. The peak of the blood concentration was 4-6 H, the detection time limit is 8-21h, the peak of 8-10h. ethanol at the urine concentration of EtG at EtG is 4-6h, the time limit is 20-85h, and the time limit of the urine ethanol detection is 12-70h, and the data model of calculating the drinking time by the concentration of EtG and ethanol is preliminarily established.
In the sixth chapter, LC-LTQ-Orbitrap analysis of EtS in blood and urine was established. The linear range was 0.1 g/mL-5 g/mL-5 (R20.995).
In this study, multiple EtG detection methods, such as GC-MS/MS, LC-LTQ-Orbitrap and LC-MS/MS, were established and verified for the first time in China. The LC-LTQ-Orbitrap method was first reported in the world. The metabolic kinetics of Chinese alcohol ethyl glucuronide was first investigated, and the relationship between EtG and ethanol in blood and urine was discussed. The time limit for the detection of EtG in the blood and urine was put forward. The research results provided a scientific basis for the identification of the drunk driving runaway and the determination of the sources of ethanol in the corpse inspection.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：D919.2

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