氟乙酰胺代谢产物检测新方法及家兔体内分布研究
发布时间：2018-11-04 16:23
【摘要】： 目的 1.通过氟乙酰胺体内代谢产物氟离子电化学芯片检测方法的研究,建立一种新的快速的检测手段。 2.开发便携、简便的氟乙酰胺代谢产物氟离子的芯片检测技术并完成便携式氟离子快速检测仪。 3.建立氟乙酸和氟柠檬酸的LC-MS/MS定性、定量方法。 4.建立家兔口服氟乙酰胺中毒动物模型。 5.研究氟乙酰胺的代谢规律及日常案件中常用检材的前处理方法及分布,为氟乙酰胺中毒案件的检材采取、检测、结果分析、死因判断及法医学鉴定提供科学依据。 6.研究氟乙酸和氟柠檬酸在中毒家兔体内分布情况 方法 1.动物模型 将12只家兔随机分为2组,每组6只。第一组为空白对照组,常规饲料喂养,第二组为染毒组,经口灌服致死剂量(10.0mg/kg体重)的氟乙酰胺 2.检材采取及处理 中毒组在经口服药物后自然死亡,立即解剖取血、尿、心、肝、肾、脑等常用检材,空白组耳缘静脉空气栓塞致死,取同样检材,作为对照。 3.F-前处理方法的研究：分析了不同沉淀蛋白、不同pH值条件对氟离子提取回收率的影响,优化了氟离子提取方法。 4.氟乙酸和氟柠檬酸LC-MS/MS检测方法条件优化的研究：采用保留时间(Rt)和多反应监测(MRM)方式来对常用检材中氟乙酸和氟柠檬酸进行定性定量分析。 5.氟乙酸和氟柠檬酸的提取方法 样品制备：生物组织检材样本(如肝、肾等)用匀浆机打碎混匀。取1.0ml血液或1.0g组织检材样品,加入3.0ml水／丙酮(1：4,v／v),超声8分钟,在4000r/min的离心机上离心5分钟除去蛋白质,收集提取液,重复上述提取过程3次,合并3次提取液后,用NaOH调节pH8.0左右,40℃条件下用氮气吹至2.0ml左右水溶液,用3.0ml己烷提取杂质,离心分离后,弃去己烷,用1 mo l/ml的盐酸调节pH小于2.0。用4.0ml乙酸乙酯剧烈震荡5分钟,离心分离,重复提取4次,合并上清液,加入200μL 10%的二乙醇胺丙酮液碱化,在50℃条件下氮气吹干,甲醇定容,过膜待检。 6.LC-MS／MS方法检测：采用保留时间和选择2-3对离子定性,以1对响应值相对较高的特征离子的峰面积与其溶液浓度做标准曲线,用外标法进行定量。 7.统计学方法：实验数据结果以均数±标准差(X±S)表示,用SPSS11.5统计软件对各检材中氟乙酸和氟柠檬酸的含量进行方差分析。 结果 1.氟离子选择性芯片在100～10-6mol／L范围内呈线性响应,线性相关系数为0.9994。其平均斜率为58.14,接近理论斜率59.35；氟离子选择性芯片的检测下限为5×10-7mol／L;平均回收率为76.62%(RSD%=1.21%)。 2.在10-3～10-6mol／L NaF标准溶液中,氟离子选择性芯片响应时间2min,在10-6～5×10-7mol／LNaF标准溶液中,氟离子选择性芯片响应时间5 min。 3.用同一芯片在室温下重复连续测定10-3和10-5mol／L标准溶液5次,RSD%分别小于0.43和0.90；用同一芯片在室温下保存5天,每天测定10-3和10-5 mol／L标准溶液1次,RSD%分别小于0.54和1.23。 4.芯片pH适用范围pH在5.0～6.0是氟离子敏感膜使用的最佳pH范围。 5.KF-.OH-大约为10,也就是说芯片对F-的响应比对OH-的响应灵敏10倍；KF-,Cl-大约为1000,KF-,Br-和KF-，I-大约为1300。 6.给药组家兔在给药后1～1.5小时出现强直性痉挛,阵发性抽搐症状,2小时内死亡。染毒组家兔血中氟离子含量较对照组高5-6倍；肝、肾、脑组织中氟离子含量较空白组增高2-4倍,各指标增高显著(P0.01)。 7.LC-MS／MS方法：生物样本中氟乙酰胺的主要代谢产物氟乙酸和氟柠檬酸通过多离子监测(MRM)方式可以得到较好的分离,氟乙酸和氟柠檬酸分别在5.0μg／ml～200.0μg／ml、50.0μg／ml～200.0μg／ml范围呈现良好的线性关系,相关系数R2分别为0.9995、0.9902、最低检测限(LOD)分别为5.0ng、50.0ng。 8.从家兔的血液,心,肝和肾中可以检出氟柠檬酸和氟乙酸,胃内容中可以检出氟乙酸,心脏中检出的氟柠檬酸和氟乙酸含量较低,其他个检材与心脏比较呈显著性差异(P0.01),氟柠檬酸在家兔体内的含量分布为：血肝肾心胃内容(浓度=0)；氟乙酸的浓度大小依次是：胃内容肾肝血心。 结论 1.本课题研究建立的氟乙酰胺代谢产物氟离子检测芯片结合氟离子选择电极对氟离子的高选择性、灵敏性和电位分析技术的简单,便捷等特点,本技术将大大缩短对氟乙酰胺检测的时间,降低检测成本,使检测设备便于携带,实现对氟乙酰胺的现场测定,为取缔剧毒鼠药的销售使用,预防鼠药群体染毒事件的发生提供依据。将采样至实验室的检测路线改为实验室移至现场的服务路线,将为尽快检测出染毒物质、挽救人民生命,为抢救病人赢得时间、节省大量人力物力提供简便、实用的方法。该方法特别适合在基层推广,使大量刑事染毒案件的取证检验工作可以在基层完成检测。 2.氟离子检测芯片与其他常见的F-测定方法如离子色谱法、分光光度法等比较,此检测芯片价格低,操作简便,抗干扰能力强,能够快速检测样品中F-含量,可以广泛应用于环境样品、体液、食品中F-含量的检测,是初筛实际样品中F-含量的首选方法。氟离子芯片直接给出样品氟离子浓度,响应速度快,较易实现连续测定与自动监控,有利于发展成为日常检测氟含量的一种方法。 3.本文建立了生物检材中氟乙酸和氟柠檬酸HPLC-MS/MS检测方法,回收率高,灵敏度高,重现性好,方法准确、可靠,操作简便,样品不需要衍生化处理,色谱柱寿命长,适用于生物检材中氟乙酰胺代谢产物的快速检测。 4.氟乙酰胺中毒的代谢产物氟乙酸和氟柠檬酸广泛存在家兔体内,血液中氟柠檬酸含量最高,氟乙酸胃内容中含量最高。
[Abstract]:Purpose 1. To establish a new rapid method for the detection of fluoride ion electrochemical wafer by means of fluoride ion in vivo Detection means. 2. Develop a portable, simple and convenient chip detection technology for fluoride ion metabolism products and complete the portable Rapid detector for fluoride ions. L. for the establishment of fluoroacetic acid and fluorocitric acid C-MS/ MS qualitative and quantitative methods. 4. Establish animal model of oral fluorosis in rabbits. 5. To study the metabolic rule of fluoride poisoning and the pretreatment methods and distribution of common test materials in daily cases, and take the test materials for fluorosis cases. To provide scientific basis for detection, result analysis, cause of death and forensic identification According to. 6 Fluoroacetic acid and citric acid were studied in rabbits. Methods 1. 12 rabbits were randomly divided into 2 groups, 6 in each group. The first group was blank. Control group, regular feed The second group was the fluoride group with lethal dose (10. 0mg/ kg body weight). 2. Take and deal with poisoning group to take and deal with poisoning group for oral administration Natural death after the drug, immediately anatomize the common test materials such as blood, urine, heart, liver, kidney, brain, etc., the air embolism on the ear edge of the blank group will be fatal, and the same test material shall be taken as the control The effects of different precipitation proteins, different pH conditions on the extraction recovery rate of fluoride ion were analyzed, and the extraction method of fluoride ion was optimized.-MS/ MS detection method conditions optimized Study: The retention time (MRM) and multi-reaction monitoring (MRM) were used. Qualitative and quantitative analysis of fluoroacetic acid and fluorocitric acid were carried out in the test material. 5. Extraction of fluoride and citric acid was carried out. Samples of biological tissue test (e.g. liver, kidney, etc.) were crushed and mixed with a homogenizer. 1. 0ml of blood or 1. 0g of tissue samples were taken, and 3 were added. 0ml water/ acetone (1: 4, v/ v), ultrasonic for 8 min, centrifuging at 4000r/ min for 5 minutes to remove protein, collecting extractive solution, repeating the above extraction process for 3 times, mixing the extractive solutions, and adjusting with NaOH. At about pH 8.0, nitrogen was purged with nitrogen at about 40.degree. C.for 2. 0ml of a left and right aqueous solution, impurities were extracted with 3. 0ml of hexane, after centrifugation, the hexane was discarded, and the pH was adjusted to less than 2.0 with 1 mo l/ ml hydrochloric acid. shake vigorously for 5 minutes with ethyl acetate, centrifugal separation, repeat extraction for 4 times, combine supernatant, add 200 & mu; L of 10% ethanol amine acetone solution to alkalize, blow dry with nitrogen at 50 & deg; C, fix with methanol, and over-film. Detection: 6. LC-MS/ MS method detection: using retention time and selection of 2-3 pairs of ions, the peak area of the characteristic ion with relatively high response value and its solution concentration mark The quasi-curve is quantitated by external standard method. The content of fluoroacetic acid and fluorine citric acid in each test material was analyzed by SPSS 11.5. Results 1. The fluoride ion selective chip was in the range of 100 ~ 10-6mol/ L. In linear response, the linear correlation coefficient was 0. 9994. The average slope was 58. 14, close to the theoretical slope of 59. 35; the lower limit of detection of fluoride ion selective chip was 5-10-7mol/ L; the average recovery was 76. 62% (RSD).% = 1. 21%). 2. In 10-3 ~ 10-6mol/ L NaF standard solution, fluoride ion selective chip response time is 2min. In 10-6 ~ 5B10-7mol/ LNaF standard solution, the response time of fluoride ion selective chip is 5 min. 3. Use the same chip in the same chip. Continuous determination of 10-3 and 10-5mol/ L standard solution at room temperature for 5 times with RSD% less respectively At 0. 43 and 0. 90, the same chip was used for 5 days at room temperature, 10-3 and 10-5 mol/ L standard solutions were measured once a day with RSD% less than 0. 54 and 1. 23. 4. Chip p The pH range of H is 5.0 ~ 6.0, which is the optimum pH range of fluoride ion sensitive membrane.-. OH-about 10, that is, the response of the wafer to F-is 10 times sensitive to the response to OH-; KF-, Cl-is about 1000, KF-, Br-and K F-, I-about 1300. 6. After administration, rabbits showed ankylosing spasm, paroxysmal convulsion and died in 2 hours after administration. The content of fluoride ion in rabbit blood was 5-6 times higher than that in the control group, and the fluoride ion in liver, kidney and brain tissue was higher than that in the control group. The content was increased by 2-4 times higher than that in the blank group, and the indexes increased significantly (P 0.01). 7. LC-MS/ MS method: The main metabolites of fluorobenzene in biological samples were fluoroacetic acid and fluorine citric acid through multi-ion monitoring (MRM). A good linear relationship was obtained in the range of 5. 0 ug/ ml ~ 200. 0 ug/ ml, 50. 0 ug/ ml ~ 200. 0 ug/ ml, respectively. The correlation coefficient was R2. 0. 9995, 0. 9902, minimum detection limit (LOD) of 5. 0ng, 50. 0ng. 8. from Rabbits fluorine and acetic acid can be detected in the blood, heart, liver and kidney, fluoride and acetic acid can be detected in the stomach contents, the content of fluorine and acetic acid detected in the heart is lower, and the other test materials are significantly different from the heart (P0.01). The contents of the contents of the rabbits were: the contents of liver and kidney and stomach contents (concentration = 0); the concentration of fluacetic acid was: stomach contents kidney and liver blood heart. Conclusion 1. This lesson The fluorine ion detection chip and the fluorine ion selective electrode have the characteristics of high selectivity, sensitivity, potential analysis technology and the like of the fluorine ion detection chip and the fluorine ion selective electrode, and the technology greatly shortens the detection of the fluorine ion. The time and the detection cost are reduced, the detection equipment is convenient to carry, The sales and use of the rat drug can provide a basis for preventing the occurrence of the poisoning event of the rat drug group. The detection route sampled to the laboratory is changed to a service route which is moved to the site by a laboratory, the toxicant substance is detected as soon as possible, the life of the people is saved, the time for rescuing the patient is saved, and a large amount of people are saved. The method is particularly suitable for popularization at the grass-roots level, the forensic examination of an amount of criminal contamination case can complete the detection at the base layer. 2. the fluorine ion detection chip and other common F-measuring methods such as ion chromatography, The detection chip has the advantages of low price, simple and convenient operation and strong anti-interference capability, can quickly detect the F-content in the sample, and can be widely applied to detection of F-content in environmental samples, body fluids and foods, The preferred method for the quantity is that the fluorine ion chip directly gives the sample fluorine ion concentration. high response speed, easy realization of continuous measurement and automatic monitoring, and is beneficial to development as a method for daily detection of fluorine content.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：D919
[Abstract]:Purpose 1. To establish a new rapid method for the detection of fluoride ion electrochemical wafer by means of fluoride ion in vivo Detection means. 2. Develop a portable, simple and convenient chip detection technology for fluoride ion metabolism products and complete the portable Rapid detector for fluoride ions. L. for the establishment of fluoroacetic acid and fluorocitric acid C-MS/ MS qualitative and quantitative methods. 4. Establish animal model of oral fluorosis in rabbits. 5. To study the metabolic rule of fluoride poisoning and the pretreatment methods and distribution of common test materials in daily cases, and take the test materials for fluorosis cases. To provide scientific basis for detection, result analysis, cause of death and forensic identification According to. 6 Fluoroacetic acid and citric acid were studied in rabbits. Methods 1. 12 rabbits were randomly divided into 2 groups, 6 in each group. The first group was blank. Control group, regular feed The second group was the fluoride group with lethal dose (10. 0mg/ kg body weight). 2. Take and deal with poisoning group to take and deal with poisoning group for oral administration Natural death after the drug, immediately anatomize the common test materials such as blood, urine, heart, liver, kidney, brain, etc., the air embolism on the ear edge of the blank group will be fatal, and the same test material shall be taken as the control The effects of different precipitation proteins, different pH conditions on the extraction recovery rate of fluoride ion were analyzed, and the extraction method of fluoride ion was optimized.-MS/ MS detection method conditions optimized Study: The retention time (MRM) and multi-reaction monitoring (MRM) were used. Qualitative and quantitative analysis of fluoroacetic acid and fluorocitric acid were carried out in the test material. 5. Extraction of fluoride and citric acid was carried out. Samples of biological tissue test (e.g. liver, kidney, etc.) were crushed and mixed with a homogenizer. 1. 0ml of blood or 1. 0g of tissue samples were taken, and 3 were added. 0ml water/ acetone (1: 4, v/ v), ultrasonic for 8 min, centrifuging at 4000r/ min for 5 minutes to remove protein, collecting extractive solution, repeating the above extraction process for 3 times, mixing the extractive solutions, and adjusting with NaOH. At about pH 8.0, nitrogen was purged with nitrogen at about 40.degree. C.for 2. 0ml of a left and right aqueous solution, impurities were extracted with 3. 0ml of hexane, after centrifugation, the hexane was discarded, and the pH was adjusted to less than 2.0 with 1 mo l/ ml hydrochloric acid. shake vigorously for 5 minutes with ethyl acetate, centrifugal separation, repeat extraction for 4 times, combine supernatant, add 200 & mu; L of 10% ethanol amine acetone solution to alkalize, blow dry with nitrogen at 50 & deg; C, fix with methanol, and over-film. Detection: 6. LC-MS/ MS method detection: using retention time and selection of 2-3 pairs of ions, the peak area of the characteristic ion with relatively high response value and its solution concentration mark The quasi-curve is quantitated by external standard method. The content of fluoroacetic acid and fluorine citric acid in each test material was analyzed by SPSS 11.5. Results 1. The fluoride ion selective chip was in the range of 100 ~ 10-6mol/ L. In linear response, the linear correlation coefficient was 0. 9994. The average slope was 58. 14, close to the theoretical slope of 59. 35; the lower limit of detection of fluoride ion selective chip was 5-10-7mol/ L; the average recovery was 76. 62% (RSD).% = 1. 21%). 2. In 10-3 ~ 10-6mol/ L NaF standard solution, fluoride ion selective chip response time is 2min. In 10-6 ~ 5B10-7mol/ LNaF standard solution, the response time of fluoride ion selective chip is 5 min. 3. Use the same chip in the same chip. Continuous determination of 10-3 and 10-5mol/ L standard solution at room temperature for 5 times with RSD% less respectively At 0. 43 and 0. 90, the same chip was used for 5 days at room temperature, 10-3 and 10-5 mol/ L standard solutions were measured once a day with RSD% less than 0. 54 and 1. 23. 4. Chip p The pH range of H is 5.0 ~ 6.0, which is the optimum pH range of fluoride ion sensitive membrane.-. OH-about 10, that is, the response of the wafer to F-is 10 times sensitive to the response to OH-; KF-, Cl-is about 1000, KF-, Br-and K F-, I-about 1300. 6. After administration, rabbits showed ankylosing spasm, paroxysmal convulsion and died in 2 hours after administration. The content of fluoride ion in rabbit blood was 5-6 times higher than that in the control group, and the fluoride ion in liver, kidney and brain tissue was higher than that in the control group. The content was increased by 2-4 times higher than that in the blank group, and the indexes increased significantly (P 0.01). 7. LC-MS/ MS method: The main metabolites of fluorobenzene in biological samples were fluoroacetic acid and fluorine citric acid through multi-ion monitoring (MRM). A good linear relationship was obtained in the range of 5. 0 ug/ ml ~ 200. 0 ug/ ml, 50. 0 ug/ ml ~ 200. 0 ug/ ml, respectively. The correlation coefficient was R2. 0. 9995, 0. 9902, minimum detection limit (LOD) of 5. 0ng, 50. 0ng. 8. from Rabbits fluorine and acetic acid can be detected in the blood, heart, liver and kidney, fluoride and acetic acid can be detected in the stomach contents, the content of fluorine and acetic acid detected in the heart is lower, and the other test materials are significantly different from the heart (P0.01). The contents of the contents of the rabbits were: the contents of liver and kidney and stomach contents (concentration = 0); the concentration of fluacetic acid was: stomach contents kidney and liver blood heart. Conclusion 1. This lesson The fluorine ion detection chip and the fluorine ion selective electrode have the characteristics of high selectivity, sensitivity, potential analysis technology and the like of the fluorine ion detection chip and the fluorine ion selective electrode, and the technology greatly shortens the detection of the fluorine ion. The time and the detection cost are reduced, the detection equipment is convenient to carry, The sales and use of the rat drug can provide a basis for preventing the occurrence of the poisoning event of the rat drug group. The detection route sampled to the laboratory is changed to a service route which is moved to the site by a laboratory, the toxicant substance is detected as soon as possible, the life of the people is saved, the time for rescuing the patient is saved, and a large amount of people are saved. The method is particularly suitable for popularization at the grass-roots level, the forensic examination of an amount of criminal contamination case can complete the detection at the base layer. 2. the fluorine ion detection chip and other common F-measuring methods such as ion chromatography, The detection chip has the advantages of low price, simple and convenient operation and strong anti-interference capability, can quickly detect the F-content in the sample, and can be widely applied to detection of F-content in environmental samples, body fluids and foods, The preferred method for the quantity is that the fluorine ion chip directly gives the sample fluorine ion concentration. high response speed, easy realization of continuous measurement and automatic monitoring, and is beneficial to development as a method for daily detection of fluorine content.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：D919
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