去痛片及其代谢物在大鼠体内的分布研究

发布时间：2018-02-08 23:11

本文关键词： 去痛片代谢物 GC-MS/MS 分布　出处：《山西医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:1.建立SLE固相支撑液液萃取、GC-MS/MS同时检测去痛片及其代谢物的方法;2.观察去痛片及其代谢物在大鼠体内的分布;3.观察去痛片及其代谢物在中毒死亡者体内的死后分布。方法:1.方法建立利用去痛片及其代谢物标准品,建立SLE固相支撑液液萃取,GC-MS/MS法同时检测去痛片及其代谢物。2.标本采集16只雄性SD大鼠,分为两组,每组8只。禁食12h后,经口灌服去痛片950mg/kg,A组于1.5h脱臼处死,取心血肝素抗凝、心肌、肺、肝脏、脾、肾、上肢肌肉、下肢肌肉、脑组织置于-20℃待检。B组于24h脱臼处死,取心血肝素抗凝,并收集24h尿液,置于-20℃待检。中毒案例样品2例,来源于山西医科大学司法鉴定中心。3.提取检测待检生物样品中添加烯丙基异丙基巴比妥(50μg/m L),氨基比林-13C2(25μg/m L)混合内标工作液80μL,经SLE固相支撑液液萃取,GC-MS/MS检测,MRM模式扫描,利用保留时间、定量离子与定性离子对比例定性,内标法和工作曲线法定量。4.数据处理数据用SPSS 20.0进行正态性检验和方差分析。各脏器间含量用Graphpad 6.0进行Kruskal-Wallis H检验,各组间用Dunn's检验多重比较得出统计学结果,P0.05时,认为各组间有统计学差异。结果:1.空白血液加入去痛片及其代谢物标准品,经SLE分别用正己烷、二氯甲烷、乙醚、甲基叔丁基醚、乙酸乙酯2m L淋洗,结果乙酸乙酯同时提取去痛片及其代谢物的提取回收率MAA为37.57%±0.62%,AAA为41.99%±1.21%,其余化合物均在58.19%±1.41%~95.87%±1.79%之间,相较其他溶剂有较高的提取回收率。2.GC-MS/MS法可同时检测去痛片及其代谢物,血液、尿液、肝脏加标样品在0.125μg/m L~16.00μg/m L呈线性关系,血液样品工作曲线相关系数r为0.9896~0.9997,最低检测限为0.23ng/m L~14.48ng/m L;尿液样品工作曲线相关系数r为0.995~0.9997,最低检测限为0.08ng/m L~4.71ng/m L;肝脏样品工作曲线相关系数r为0.9953~0.9997,最低检测限为0.16ng/m L~11.18ng/m L。方法准确度为79.64%~122.91%,日内精密度为0.99%~7.43%,日间精密度为2.19%~10.60%,3.经口灌服950mg/kg去痛片,1.5小时后去痛片及其代谢物在大鼠体内的分布:氨基比林血液含量上、下肢肌肉含量,P0.05。MAA血液、肝脏含量下肢肌肉含量,P0.05。AA血液、上、下肢肌肉、肝脏、肾脏、心肌含量胃壁含量,P0.05。FAA和AAA各脏器间含量无统计学差异。非那西丁血液、肺脏、脾脏含量肝脏含量,P0.05。APAP血液、下肢肌肉、心肌、肾脏、肝脏、脾脏含量胃壁含量,P0.05。咖啡因血液、肝脏含量上、下肢肌肉、脑组织含量,P0.05。17X、37X、13X各脏器间含量无统计学差异。苯巴比妥肝脏含量脾脏、脑、上、下肢肌肉含量,P0.05。经口灌服去痛片24h后,血液中MAA、AA含量高于FAA,P0.05。非那西丁和APAP,咖啡因和17X、37X、13X间含量均无统计学差异。24小时尿液中AAA含量高于AM、MAA、FAA,APAP含量高于非那西丁,P0.05。咖啡因和其代谢物含量无统计学差异。结论:1.经SLE固相支撑液液萃取,同时提取生物样品中去痛片及其代谢产物,乙酸乙酯较正己烷、二氯甲烷、乙醚和甲基叔丁基醚有较高的提取回收率。2.GC-MS/MS法可同时检测生物样品中去痛片及其代谢产物,利用保留时间和二级质谱图定量离子和定性离子对比例定性,利用内标物与目标物峰面积比值,内标法和工作曲线法定量。方法回收率高、专属性强、特异性高、内源性物质对目标物检测干扰小,方法灵敏度高、准确度高,可满足去痛片中毒案例定性、定量的要求,可应用于去痛片相关案例的法医学鉴定实践中。3.去痛片及其代谢物在大鼠体内的分布不均匀,口服去痛片中毒案例中优先选择胃内容物和胃壁作为检材,还可提取肝、肾、心血作为检材。针对氨基比林、非那西丁、咖啡因和苯巴比妥单一药物或复方制剂中毒的案例,应根据药物的分布特点,科学全面地选取检材,且应该考虑到药物所处吸收时相或消除时相时对各脏器分布的影响,以便为中毒案件提供科学的依据。
[Abstract]:Objective: to establish 1. SLE supported by solid liquid liquid extraction, and detection method of Compound Aminopyrine Phenacetin Tablets and its metabolite GC-MS/MS; 2. were Compound Aminopyrine Phenacetin Tablets and its metabolite distribution in rat; 3. were Compound Aminopyrine Phenacetin Tablets and its metabolites in poisoning deaths postportem distribution. Methods: 1. methods to set up by Compound Aminopyrine Phenacetin Tablets and its metabolite standard, the establishment of SLE solid support liquid liquid extraction, and acquisition of 16 male SD rats and its metabolite.2. were Compound Aminopyrine Phenacetin Tablets GC-MS/MS, divided into two groups, 8 rats in each group. After fasting 12h, oral gavage of Compound Aminopyrine Phenacetin Tablets 950mg/kg, A in 1.5h group were sacrificed by dislocation and taken blood heparin, myocardium, lung, liver, spleen, kidney, upper limb the lower limb muscles, muscle, brain tissue at -20 DEG C to be detected in the.B group 24h were sacrificed by dislocation and taken blood heparin, and 24h urine collection, at -20 DEG C to be detected. 2 cases were poisoning, from the judicial identification center of Shanxi Medical University.3 . extraction to add isopropyl allyl barbituric detection in biological samples (50 g/m L), amidopyrin -13C2 (25 g/m L) 80 L internal standard working solution mixed by SLE, supported by solid liquid liquid extraction, GC-MS/MS detection, MRM scanning mode, the retention time, qualitative and quantitative and qualitative ion the ion ratio, internal standard method and the working curve method, quantitative.4. data were analyzed by SPSS 20 test of normality and variance. The organs between content of Graphpad with 6 Kruskal-Wallis H test between groups using Dunn's test multiple comparison statistical results, P0.05, that there were significant differences between the two groups. Results: 1. join Compound Aminopyrine Phenacetin Tablets and blank blood metabolite standard, respectively by SLE with n-hexane, dichloromethane, ethyl ether, methyl tert butyl ether, ethyl acetate and 2m L leaching, the extraction recovery of ethyl acetate extraction of Compound Aminopyrine Phenacetin Tablets and its metabolites rate MAA 37.57% + 0.62%, 41.99% + 1.21% AAA, the remaining compounds were between 58.19% + 1.41%~95.87% + 1.79%, compared with other solvent extraction recovery rate is higher in.2.GC-MS/MS method for simultaneous determination of Compound Aminopyrine Phenacetin Tablets and its metabolites, blood, urine, liver samples with a linear relationship in the 0.125 g/m L~ 16 g/m L, blood sample curve correlation coefficient r is 0.9896~0.9997, the minimum detection limit is 0.23ng/m L~14.48ng/m L; urine sample curve correlation coefficient r is 0.995~0.9997, the minimum detection limit is 0.08ng/m L~4.71ng/m L; liver samples curve correlation coefficient r is 0.9953~0.9997, the minimum detection limit is 0.16ng/m L~11.18ng/m L. the accuracy of 79.64%~122.91%, the within day precision is 0.99%~7.43%. The inter day precision is 2.19%~10.60%, 3. oral gavage of 950mg/kg 1.5 hours after Compound Aminopyrine Phenacetin Tablets, Compound Aminopyrine Phenacetin Tablets and its metabolite distribution in rat: aminopyrine The content of blood, muscle content, lower limb blood P0.05.MAA content in liver, lower limb muscle P0.05.AA content, blood, liver, kidney, muscle, myocardial content of gastric content, no significant difference between P0.05.FAA and AAA in various organs. Phenacetin blood, lung, spleen in liver P0.05.APAP content, blood, lower limb muscle, myocardium the liver, spleen, kidney, stomach contents of caffeine content, P0.05. content of blood, liver, muscle, brain tissue content, P0.05.17X, 37X, 13X were no significant difference between the content of benzene. Pakistan than spleen, liver, brain with content, lower limb muscle content, P0.05. oral gavage Compound Aminopyrine Phenacetin Tablets 24h, blood in MAA, the content of AA was higher than FAA P0.05., phenacetin and caffeine and APAP, 17X, 37X, 13X were no statistically significant differences between the content of AAA in the urine.24 hours is higher than AM, MAA, FAA, APAP were higher than that of phenacetin, caffeine and its P0.05. There was no significant difference in the contents of metabolites. Conclusion: 1. by SLE supported by solid liquid liquid extraction, simultaneous extraction of Compound Aminopyrine Phenacetin Tablets and its metabolites in biological samples, ethyl acetate with hexane, dichloromethane, ethyl ether and methyl tert butyl ether extract has a higher recovery rate of.2.GC-MS/MS method for simultaneous determination of Compound Aminopyrine Phenacetin Tablets and its metabolites in biological samples, with retention time and two quantitative and qualitative ion ion mass spectra on the proportion of qualitative, the internal standard and the target using the peak area ratio, internal standard method and the working curve method. The recovery rate is high, strong specificity, high specificity, endogenous substances on the target detection of small interference, high sensitivity, high accuracy, can be meet Compound Aminopyrine Phenacetin Tablets poisoning cases qualitative and quantitative requirements, and can be applied to the Compound Aminopyrine Phenacetin Tablets case forensic practice in Compound Aminopyrine Phenacetin Tablets.3. and its metabolites in uneven distribution of rats, oral In the case of poisoning Compound Aminopyrine Phenacetin Tablets preferred gastric contents and gastric wall as samples, can be extracted from the liver, kidney, blood samples. As for aminopyrine, phenacetin, caffeine and phenobarbital single drug or compound poisoning case, according to the distribution of drugs, scientifically selected samples, and should be considered drug absorption phase or eliminate phase influence on the distribution of various organs, in order to provide scientific basis for poisoning cases.

【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：D919

【参考文献】