氟乙酰胺代謝產(chǎn)物檢測新方法及家兔體內(nèi)分布研究
發(fā)布時間：2018-11-04 16:23
【摘要】： 目的 1.通過氟乙酰胺體內(nèi)代謝產(chǎn)物氟離子電化學芯片檢測方法的研究,建立一種新的快速的檢測手段。 2.開發(fā)便攜、簡便的氟乙酰胺代謝產(chǎn)物氟離子的芯片檢測技術(shù)并完成便攜式氟離子快速檢測儀。 3.建立氟乙酸和氟檸檬酸的LC-MS/MS定性、定量方法。 4.建立家兔口服氟乙酰胺中毒動物模型。 5.研究氟乙酰胺的代謝規(guī)律及日常案件中常用檢材的前處理方法及分布,為氟乙酰胺中毒案件的檢材采取、檢測、結(jié)果分析、死因判斷及法醫(yī)學鑒定提供科學依據(jù)。 6.研究氟乙酸和氟檸檬酸在中毒家兔體內(nèi)分布情況 方法 1.動物模型 將12只家兔隨機分為2組,每組6只。第一組為空白對照組,常規(guī)飼料喂養(yǎng),第二組為染毒組,經(jīng)口灌服致死劑量(10.0mg/kg體重)的氟乙酰胺 2.檢材采取及處理 中毒組在經(jīng)口服藥物后自然死亡,立即解剖取血、尿、心、肝、腎、腦等常用檢材,空白組耳緣靜脈空氣栓塞致死,取同樣檢材,作為對照。 3.F-前處理方法的研究：分析了不同沉淀蛋白、不同pH值條件對氟離子提取回收率的影響,優(yōu)化了氟離子提取方法。 4.氟乙酸和氟檸檬酸LC-MS/MS檢測方法條件優(yōu)化的研究：采用保留時間(Rt)和多反應(yīng)監(jiān)測(MRM)方式來對常用檢材中氟乙酸和氟檸檬酸進行定性定量分析。 5.氟乙酸和氟檸檬酸的提取方法 樣品制備：生物組織檢材樣本(如肝、腎等)用勻漿機打碎混勻。取1.0ml血液或1.0g組織檢材樣品,加入3.0ml水／丙酮(1：4,v／v),超聲8分鐘,在4000r/min的離心機上離心5分鐘除去蛋白質(zhì),收集提取液,重復(fù)上述提取過程3次,合并3次提取液后,用NaOH調(diào)節(jié)pH8.0左右,40℃條件下用氮氣吹至2.0ml左右水溶液,用3.0ml己烷提取雜質(zhì),離心分離后,棄去己烷,用1 mo l/ml的鹽酸調(diào)節(jié)pH小于2.0。用4.0ml乙酸乙酯劇烈震蕩5分鐘,離心分離,重復(fù)提取4次,合并上清液,加入200μL 10%的二乙醇胺丙酮液堿化,在50℃條件下氮氣吹干,甲醇定容,過膜待檢。 6.LC-MS／MS方法檢測：采用保留時間和選擇2-3對離子定性,以1對響應(yīng)值相對較高的特征離子的峰面積與其溶液濃度做標準曲線,用外標法進行定量。 7.統(tǒng)計學方法：實驗數(shù)據(jù)結(jié)果以均數(shù)±標準差(X±S)表示,用SPSS11.5統(tǒng)計軟件對各檢材中氟乙酸和氟檸檬酸的含量進行方差分析。 結(jié)果 1.氟離子選擇性芯片在100～10-6mol／L范圍內(nèi)呈線性響應(yīng),線性相關(guān)系數(shù)為0.9994。其平均斜率為58.14,接近理論斜率59.35；氟離子選擇性芯片的檢測下限為5×10-7mol／L;平均回收率為76.62%(RSD%=1.21%)。 2.在10-3～10-6mol／L NaF標準溶液中,氟離子選擇性芯片響應(yīng)時間2min,在10-6～5×10-7mol／LNaF標準溶液中,氟離子選擇性芯片響應(yīng)時間5 min。 3.用同一芯片在室溫下重復(fù)連續(xù)測定10-3和10-5mol／L標準溶液5次,RSD%分別小于0.43和0.90；用同一芯片在室溫下保存5天,每天測定10-3和10-5 mol／L標準溶液1次,RSD%分別小于0.54和1.23。 4.芯片pH適用范圍pH在5.0～6.0是氟離子敏感膜使用的最佳pH范圍。 5.KF-.OH-大約為10,也就是說芯片對F-的響應(yīng)比對OH-的響應(yīng)靈敏10倍；KF-,Cl-大約為1000,KF-,Br-和KF-，I-大約為1300。 6.給藥組家兔在給藥后1～1.5小時出現(xiàn)強直性痙攣,陣發(fā)性抽搐癥狀,2小時內(nèi)死亡。染毒組家兔血中氟離子含量較對照組高5-6倍；肝、腎、腦組織中氟離子含量較空白組增高2-4倍,各指標增高顯著(P0.01)。 7.LC-MS／MS方法：生物樣本中氟乙酰胺的主要代謝產(chǎn)物氟乙酸和氟檸檬酸通過多離子監(jiān)測(MRM)方式可以得到較好的分離,氟乙酸和氟檸檬酸分別在5.0μg／ml～200.0μg／ml、50.0μg／ml～200.0μg／ml范圍呈現(xiàn)良好的線性關(guān)系,相關(guān)系數(shù)R2分別為0.9995、0.9902、最低檢測限(LOD)分別為5.0ng、50.0ng。 8.從家兔的血液,心,肝和腎中可以檢出氟檸檬酸和氟乙酸,胃內(nèi)容中可以檢出氟乙酸,心臟中檢出的氟檸檬酸和氟乙酸含量較低,其他個檢材與心臟比較呈顯著性差異(P0.01),氟檸檬酸在家兔體內(nèi)的含量分布為：血肝腎心胃內(nèi)容(濃度=0)；氟乙酸的濃度大小依次是：胃內(nèi)容腎肝血心。 結(jié)論 1.本課題研究建立的氟乙酰胺代謝產(chǎn)物氟離子檢測芯片結(jié)合氟離子選擇電極對氟離子的高選擇性、靈敏性和電位分析技術(shù)的簡單,便捷等特點,本技術(shù)將大大縮短對氟乙酰胺檢測的時間,降低檢測成本,使檢測設(shè)備便于攜帶,實現(xiàn)對氟乙酰胺的現(xiàn)場測定,為取締劇毒鼠藥的銷售使用,預(yù)防鼠藥群體染毒事件的發(fā)生提供依據(jù)。將采樣至實驗室的檢測路線改為實驗室移至現(xiàn)場的服務(wù)路線,將為盡快檢測出染毒物質(zhì)、挽救人民生命,為搶救病人贏得時間、節(jié)省大量人力物力提供簡便、實用的方法。該方法特別適合在基層推廣,使大量刑事染毒案件的取證檢驗工作可以在基層完成檢測。 2.氟離子檢測芯片與其他常見的F-測定方法如離子色譜法、分光光度法等比較,此檢測芯片價格低,操作簡便,抗干擾能力強,能夠快速檢測樣品中F-含量,可以廣泛應(yīng)用于環(huán)境樣品、體液、食品中F-含量的檢測,是初篩實際樣品中F-含量的首選方法。氟離子芯片直接給出樣品氟離子濃度,響應(yīng)速度快,較易實現(xiàn)連續(xù)測定與自動監(jiān)控,有利于發(fā)展成為日常檢測氟含量的一種方法。 3.本文建立了生物檢材中氟乙酸和氟檸檬酸HPLC-MS/MS檢測方法,回收率高,靈敏度高,重現(xiàn)性好,方法準確、可靠,操作簡便,樣品不需要衍生化處理,色譜柱壽命長,適用于生物檢材中氟乙酰胺代謝產(chǎn)物的快速檢測。 4.氟乙酰胺中毒的代謝產(chǎn)物氟乙酸和氟檸檬酸廣泛存在家兔體內(nèi),血液中氟檸檬酸含量最高,氟乙酸胃內(nèi)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.
【學位授予單位】：山西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：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.
【學位授予單位】：山西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：D919
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相關(guān)期刊論文 前10條
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