Ag（Ⅲ）化学发光分析新方法建立及其在法医毒物分析中的应用研究
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摘要
化学发光分析是近年来迅速发展的一种高灵敏的微量和痕量分析技术,以其灵敏度高、线性范围宽、仪器设备简单和容易实现自动化等优点,而成为分析化学中一个十分活跃的研究热点。化学发光检测方法与流动注射技术、毛细管电泳分离技术相结合,可用于环境与生物样本中微量组分的的分离与测定。近年来,化学发光技术已成为毒物与药物分析领域的研究热点之一。在前期的研究工作中,我们首次报道了Ag(III)配合物-鲁米诺化学发光新体系。本文对Ag(III)配合物-鲁米诺化学发光新体系在法医毒物分析中的应用进行了探讨,主要内容如下：
第一部分、 Ag(III)化学发光-流动注射分析新方法的建立。
1)根据多巴胺在碱性溶液中具有显著增强Ag(III)配合物-鲁米诺体系化学发光信号的特性,建立了流动注射-化学发光法测定注射液中多巴胺的新方法。在优化的实验条件下,多巴胺检测限达到3.0×10-11M,线性范围1.0×10-10～4.0×10-8M。对1.0×10-8M的多巴胺平行测定13次,相对标准偏差为2.3%。方法成功用于市售多巴胺注射液的测定。通过对化学发光光谱的研究和自由基捕获实验,对该化学发光反应的机理进行了研究。
2)基于氯胺酮在碱性溶液中具有显著增强Ag(III)配合物-鲁米诺体系化学发光信号的特性,建立了流动注射-化学发光法测定注射液中氯胺酮的新方法。在优化的实验条件下,氯胺酮检测限达到0.05mg／L,线性范围0.1～2.5mg／L。对浓度为0.5mg／L的氯胺酮溶液进行11次平行测定的相对标准偏差为1.9%。方法成功用于氯胺酮注射液的测定。
第二部分、Ag(III)化学发光-毛细管电泳分离分析新方法的建立。
1)尿液中儿茶酚胺类神经递质的含量在法医学上对于反映创伤性应激具有重要意义。基于肾上腺素、去甲肾上腺素和多巴胺在碱性介质中对Ag(III)配合物-鲁米诺体系化学发光信号的增强效应,建立了毛细管电泳—化学发光法分离检测尿液中3种儿茶酚胺类神经递质的新方法。分离所用缓冲溶液为20.0mM的硼砂和1.0mM鲁米诺,8min内实现3种物质的基线分离。在优化的实验条件下,肾上腺素、去甲肾上腺素和多巴胺的检出限分别达到7.9×10-8M,1.0×10-7M和6.9×10-8M。RSD为4.7-5.4%(n=5)。方法成功用于26位嗜铬细胞瘤患者尿液中和12名正常人尿液中儿茶酚胺类神经递质的检测。
2)基于氯胺酮在碱性介质中对Ag(III)配合物-鲁米诺体系化学发光信号的增强效应,建立了固相萃取-毛细管电泳—化学发光法分离检测尿液中氯胺酮的新方法。分离所用缓冲溶液为5.0mM的硼砂和2.0mM鲁米诺。在优化的实验条件下,氯胺酮检出限为1×10-7M。方法成功用于尿液中氯胺酮的检测。
3)基于沙丁胺醇、硫酸特布他林和莱克多巴胺在碱性介质中对Ag(III)配合物-鲁米诺体系化学发光信号的增强效应,建立了固相萃取-毛细管电泳—化学发光法分离检测这3种“瘦肉精”的新方法。分离所用缓冲溶液为1.0mM硼砂+15mMNaOH+2ppm SDS+1.OmM鲁米诺,8min内实现3种物质的基线分离。在优化的实验条件下,沙丁胺醇、硫酸特布他林和莱克多巴胺的检出限分别达到2.5mg/L,0.75mg/L和5.0mg/L,RSD为3.28%～4.5%(n=11)。方法成功用于动物饲料添加剂和兔尿中沙丁胺醇、硫酸特布他林和莱克多巴胺的检测。
4)在碱性介质中,罂粟碱能显著增强Ag(III)-鲁米诺化学发光体系的化学发光,且发光强度与样品浓度在一定范围内呈良好的线性关系。在优化的化学发光和电泳条件下,罂粟碱最低检测限为30.0mg／L。线性范围0.4～8.0g/L。对6.0g／L的罂粟碱进行平行测定5次,相对标准偏差(RSD)为4.3%(n=5)。方法成功用于盐酸罂粟碱注射液和强力枇杷露中罂粟碱含量的测定。
第三部分、氯胺酮急性致死大鼠组织样品预处理及体内分布研究初探。
取雄性Wistar大鼠16只,随机分成两组,分别以2LD50(894mg/kg)和4LD50(1788mg/kg)剂量给大鼠灌胃,建立大鼠氯胺酮灌胃给药致死模型,并考察氯胺酮致死大鼠体内死后的分布规律。待呼吸和心跳全部消失时,迅速解剖大鼠,取外周血、心脏、肝、脾、肺、肾、脑、肌肉组织冷冻保存,以高效液相色谱法测定氯胺酮含量。测得2LD50组氯胺酮含量为：脑>肝>肾>肺、脾、肌肉、心、外周血；测得4LD50组氯胺酮含量为：脑>肾>肝>肺、脾、外周血、心、肌肉。实验结果显示氯胺酮广泛分布于死后大鼠体内和血液中,脑、肝和肾含量最高,提示法医鉴定时应合理采取检材进行毒物分析。
Chemiluminescence (CL) has become one of the focuses in the area of analytical chemistry due to its advantages such as higher sensitivity, wider linear range, simpler instrumentation, versatility of use and the easy automation. Combined with flow-injection analysis (FIA) or with capillary electrophoresis (CE) technique, the CL method can be utilized in environmental and biological samples analysis. Previously, we firstly reported a new CL system, i.e., Ag (III) complex-luminol system. In this study, we discussed its application in forensic toxicological analysis. The main contents are shown as follows:
Part1Flow-injection analysis combined with luminol-Ag(III) complex chemiluminescence detection.
(1) A novel flow injection analysis-direct chemiluminescence (FI-CL) method has been developed for determinations of trace amounts of dopamine (DA) based on the enhancing effect of DA on the CL reaction of luminol with an Ag(III) complex in alkaline solution. Under optimum conditions, CL intensities are proportional to the concentration of DA in the range of1.0x10-10-4.0x10-8mol·L-1. The detection limit is3.0x10-11mol·L-1for DA (3s), with a relative standard deviation (n=13) of2.3%for1.0x10-8mol·L-1DA. This method has also been applied for the determination of DA in commercial pharmaceutical injection samples. On the basis of the CL spectra and the results of the free-radical trapping experiment of this work, a reaction mechanism for this CL reaction is proposed and discussed.
(2) A novel flow injection analysis-direct chemiluminescence(CL) method is proposed for the determination of ketamine, based on the enhancing effect of ketamine on the CL reaction of luminol with Ag (III) complex in alkaline solution. Under optimum conditions, CL intensities were proportional to the concentration of ketamine in the0.1-2.5mg/L range. The detection limit was0.05mg/L, with a relative standard deviation (n=11) of1.9%. This method has been applied for the determination of ketamine in injection.
Part2Capillary electrophoresis analysis coupled with luminol-Ag(III) complex chemiluminescence detection.
(1) A sensitive, simple method is presented for the determination of three major catecholamines in human urine by capillary electrophoresis (CE) with on-line chemiluminescence (CL) detection. This was also the first time that the luminol-Ag(III) complex CL system was used for CE detections. This method was based on the enhancing effect of epinephrine (EP), norepinephrine (NE) and dopamine (DA) on the CL reaction between luminol and the Ag(III) complex in alkaline solution. The separations and determinations were performed with an electrophoretic buffer consisting of20.0mM sodium borate and1.0mM luminol. Under optimized conditions, the three catecholamines were baseline separated and detected in less than8min. Detection limits of7.9×10-8M,1.0×10-7M and6.9×10-8M were observed for EP, NE and DA, respectively. RSD values for the peak height were4.7-5.4%(n=5). Our proposed method was applied to the determinations of the catecholamines in urine samples from12healthy individuals and26pheochromocytoma patients. Our results suggest that this method might be useful to monitor the catecholamine levels in routine screening and to diagnose pheochromocytoma.
(2) A capillary electrophoresis instrument coupled with chemilumine-scence detection was designed for the determination of ketamine in urine samples. An important enhancement of the CL emission of luminol with Ag(III) complex was observed in the presence of ketamine; so this system was selected for its detection after CE separation. Under optimal conditions, the limits of detection (LODs, S/N=3) for ketamine is1.0×10-7M, with linear range of2.0x10-7-1.0×10-5M. RSD value for the peak height was4.3%(n=5). The proposed method was tested by analyzing ketamine in urines. The results demonstrate the suitability of this approach to analyze trace amount of ketamine in urine.
(3) This method was based on the enhancing effect of salbutamol, terbutaline and ractopamine on the CL reaction of luminol with an Ag(III) complex in alkaline solution. Under optimum conditions, the three (32-agonists were separated on baseline and detected in less than10minutes. The detection limits for salbutamol, terbutaline and ractopamine were determined to be2.5mg/L,0.75mg/L and5.0mg/L, respectively. RSD values for the peak height were3.28-6.1%(n=5).This method was also applied successfully to the analyses of the three β2-agonists contents in some animals'forages and urine samples.
(4) Under alkaline conditions, papaverine can significantly enhance the chemiluminescence of the luminol-Ag(III) complex CL system, and the chemiluminescent intensity show good linearity to the concentration of the analytes within a certain range. Based on this fact, a novel method for the analysis of papaverine was developed. The detection limits (S/N=3) for papaverine was0.03g/L, with a linear range of4.0-80.0g/L. RSD value for the peak height was4.3%for6.0g/L of papaverine (n=5). Finally, the presented method was used for the analysis of papaverine in papaverine hydrochloride injection and traditional Chinese medicine preparation Qiangli Pipa Syrup successfully.
Part3Pretreatment of tissue and distribution of ketamine in rats poisoned to death.
Sixteen male Wistar rats were divided into two groups at random and given intragastric administration of ketamine respectively. Group I was given with the dcse of894mg/kg (twice as much as LD50) and group II was given with the dose of1788mg/kg (four times as much as LD50). The poisoning rats were rapidly dissected after breathing and heartbeat all disappeared, in order to stablish death model of rats died of intragastric administration of ketamine arid to explore the distribution of ketamine in rats died of ketamine poisoning. Peripheral blood, heart, liver, spleen, lung, kidney and brain, muscle tissue were taken and saved in fridge at low temperature. The content of ketamine was determined by high performance liquid chromatographic. Ketamine content order of group Ⅰ was as follows:brain> liver> kidney> lung, spleen, muscle, heart, and peripheral blood. Ketamine content order of group Ⅰ was as follows:brain>kidney> liver> lung, spleen, heart, and peripheral blood muscles. The results indicated that ketamine widely distributed in poisoning rats in vivo and blood, the highest content of ketamine appeared in brain, liver and kidney. It indicates that the inspection material for toxicological analysis should be selected reasonablely in the forensic identification.
第一部分、 Ag(III)化学发光-流动注射分析新方法的建立。
1)根据多巴胺在碱性溶液中具有显著增强Ag(III)配合物-鲁米诺体系化学发光信号的特性,建立了流动注射-化学发光法测定注射液中多巴胺的新方法。在优化的实验条件下,多巴胺检测限达到3.0×10-11M,线性范围1.0×10-10～4.0×10-8M。对1.0×10-8M的多巴胺平行测定13次,相对标准偏差为2.3%。方法成功用于市售多巴胺注射液的测定。通过对化学发光光谱的研究和自由基捕获实验,对该化学发光反应的机理进行了研究。
2)基于氯胺酮在碱性溶液中具有显著增强Ag(III)配合物-鲁米诺体系化学发光信号的特性,建立了流动注射-化学发光法测定注射液中氯胺酮的新方法。在优化的实验条件下,氯胺酮检测限达到0.05mg／L,线性范围0.1～2.5mg／L。对浓度为0.5mg／L的氯胺酮溶液进行11次平行测定的相对标准偏差为1.9%。方法成功用于氯胺酮注射液的测定。
第二部分、Ag(III)化学发光-毛细管电泳分离分析新方法的建立。
1)尿液中儿茶酚胺类神经递质的含量在法医学上对于反映创伤性应激具有重要意义。基于肾上腺素、去甲肾上腺素和多巴胺在碱性介质中对Ag(III)配合物-鲁米诺体系化学发光信号的增强效应,建立了毛细管电泳—化学发光法分离检测尿液中3种儿茶酚胺类神经递质的新方法。分离所用缓冲溶液为20.0mM的硼砂和1.0mM鲁米诺,8min内实现3种物质的基线分离。在优化的实验条件下,肾上腺素、去甲肾上腺素和多巴胺的检出限分别达到7.9×10-8M,1.0×10-7M和6.9×10-8M。RSD为4.7-5.4%(n=5)。方法成功用于26位嗜铬细胞瘤患者尿液中和12名正常人尿液中儿茶酚胺类神经递质的检测。
2)基于氯胺酮在碱性介质中对Ag(III)配合物-鲁米诺体系化学发光信号的增强效应,建立了固相萃取-毛细管电泳—化学发光法分离检测尿液中氯胺酮的新方法。分离所用缓冲溶液为5.0mM的硼砂和2.0mM鲁米诺。在优化的实验条件下,氯胺酮检出限为1×10-7M。方法成功用于尿液中氯胺酮的检测。
3)基于沙丁胺醇、硫酸特布他林和莱克多巴胺在碱性介质中对Ag(III)配合物-鲁米诺体系化学发光信号的增强效应,建立了固相萃取-毛细管电泳—化学发光法分离检测这3种“瘦肉精”的新方法。分离所用缓冲溶液为1.0mM硼砂+15mMNaOH+2ppm SDS+1.OmM鲁米诺,8min内实现3种物质的基线分离。在优化的实验条件下,沙丁胺醇、硫酸特布他林和莱克多巴胺的检出限分别达到2.5mg/L,0.75mg/L和5.0mg/L,RSD为3.28%～4.5%(n=11)。方法成功用于动物饲料添加剂和兔尿中沙丁胺醇、硫酸特布他林和莱克多巴胺的检测。
4)在碱性介质中,罂粟碱能显著增强Ag(III)-鲁米诺化学发光体系的化学发光,且发光强度与样品浓度在一定范围内呈良好的线性关系。在优化的化学发光和电泳条件下,罂粟碱最低检测限为30.0mg／L。线性范围0.4～8.0g/L。对6.0g／L的罂粟碱进行平行测定5次,相对标准偏差(RSD)为4.3%(n=5)。方法成功用于盐酸罂粟碱注射液和强力枇杷露中罂粟碱含量的测定。
第三部分、氯胺酮急性致死大鼠组织样品预处理及体内分布研究初探。
取雄性Wistar大鼠16只,随机分成两组,分别以2LD50(894mg/kg)和4LD50(1788mg/kg)剂量给大鼠灌胃,建立大鼠氯胺酮灌胃给药致死模型,并考察氯胺酮致死大鼠体内死后的分布规律。待呼吸和心跳全部消失时,迅速解剖大鼠,取外周血、心脏、肝、脾、肺、肾、脑、肌肉组织冷冻保存,以高效液相色谱法测定氯胺酮含量。测得2LD50组氯胺酮含量为：脑>肝>肾>肺、脾、肌肉、心、外周血；测得4LD50组氯胺酮含量为：脑>肾>肝>肺、脾、外周血、心、肌肉。实验结果显示氯胺酮广泛分布于死后大鼠体内和血液中,脑、肝和肾含量最高,提示法医鉴定时应合理采取检材进行毒物分析。
Chemiluminescence (CL) has become one of the focuses in the area of analytical chemistry due to its advantages such as higher sensitivity, wider linear range, simpler instrumentation, versatility of use and the easy automation. Combined with flow-injection analysis (FIA) or with capillary electrophoresis (CE) technique, the CL method can be utilized in environmental and biological samples analysis. Previously, we firstly reported a new CL system, i.e., Ag (III) complex-luminol system. In this study, we discussed its application in forensic toxicological analysis. The main contents are shown as follows:
Part1Flow-injection analysis combined with luminol-Ag(III) complex chemiluminescence detection.
(1) A novel flow injection analysis-direct chemiluminescence (FI-CL) method has been developed for determinations of trace amounts of dopamine (DA) based on the enhancing effect of DA on the CL reaction of luminol with an Ag(III) complex in alkaline solution. Under optimum conditions, CL intensities are proportional to the concentration of DA in the range of1.0x10-10-4.0x10-8mol·L-1. The detection limit is3.0x10-11mol·L-1for DA (3s), with a relative standard deviation (n=13) of2.3%for1.0x10-8mol·L-1DA. This method has also been applied for the determination of DA in commercial pharmaceutical injection samples. On the basis of the CL spectra and the results of the free-radical trapping experiment of this work, a reaction mechanism for this CL reaction is proposed and discussed.
(2) A novel flow injection analysis-direct chemiluminescence(CL) method is proposed for the determination of ketamine, based on the enhancing effect of ketamine on the CL reaction of luminol with Ag (III) complex in alkaline solution. Under optimum conditions, CL intensities were proportional to the concentration of ketamine in the0.1-2.5mg/L range. The detection limit was0.05mg/L, with a relative standard deviation (n=11) of1.9%. This method has been applied for the determination of ketamine in injection.
Part2Capillary electrophoresis analysis coupled with luminol-Ag(III) complex chemiluminescence detection.
(1) A sensitive, simple method is presented for the determination of three major catecholamines in human urine by capillary electrophoresis (CE) with on-line chemiluminescence (CL) detection. This was also the first time that the luminol-Ag(III) complex CL system was used for CE detections. This method was based on the enhancing effect of epinephrine (EP), norepinephrine (NE) and dopamine (DA) on the CL reaction between luminol and the Ag(III) complex in alkaline solution. The separations and determinations were performed with an electrophoretic buffer consisting of20.0mM sodium borate and1.0mM luminol. Under optimized conditions, the three catecholamines were baseline separated and detected in less than8min. Detection limits of7.9×10-8M,1.0×10-7M and6.9×10-8M were observed for EP, NE and DA, respectively. RSD values for the peak height were4.7-5.4%(n=5). Our proposed method was applied to the determinations of the catecholamines in urine samples from12healthy individuals and26pheochromocytoma patients. Our results suggest that this method might be useful to monitor the catecholamine levels in routine screening and to diagnose pheochromocytoma.
(2) A capillary electrophoresis instrument coupled with chemilumine-scence detection was designed for the determination of ketamine in urine samples. An important enhancement of the CL emission of luminol with Ag(III) complex was observed in the presence of ketamine; so this system was selected for its detection after CE separation. Under optimal conditions, the limits of detection (LODs, S/N=3) for ketamine is1.0×10-7M, with linear range of2.0x10-7-1.0×10-5M. RSD value for the peak height was4.3%(n=5). The proposed method was tested by analyzing ketamine in urines. The results demonstrate the suitability of this approach to analyze trace amount of ketamine in urine.
(3) This method was based on the enhancing effect of salbutamol, terbutaline and ractopamine on the CL reaction of luminol with an Ag(III) complex in alkaline solution. Under optimum conditions, the three (32-agonists were separated on baseline and detected in less than10minutes. The detection limits for salbutamol, terbutaline and ractopamine were determined to be2.5mg/L,0.75mg/L and5.0mg/L, respectively. RSD values for the peak height were3.28-6.1%(n=5).This method was also applied successfully to the analyses of the three β2-agonists contents in some animals'forages and urine samples.
(4) Under alkaline conditions, papaverine can significantly enhance the chemiluminescence of the luminol-Ag(III) complex CL system, and the chemiluminescent intensity show good linearity to the concentration of the analytes within a certain range. Based on this fact, a novel method for the analysis of papaverine was developed. The detection limits (S/N=3) for papaverine was0.03g/L, with a linear range of4.0-80.0g/L. RSD value for the peak height was4.3%for6.0g/L of papaverine (n=5). Finally, the presented method was used for the analysis of papaverine in papaverine hydrochloride injection and traditional Chinese medicine preparation Qiangli Pipa Syrup successfully.
Part3Pretreatment of tissue and distribution of ketamine in rats poisoned to death.
Sixteen male Wistar rats were divided into two groups at random and given intragastric administration of ketamine respectively. Group I was given with the dcse of894mg/kg (twice as much as LD50) and group II was given with the dose of1788mg/kg (four times as much as LD50). The poisoning rats were rapidly dissected after breathing and heartbeat all disappeared, in order to stablish death model of rats died of intragastric administration of ketamine arid to explore the distribution of ketamine in rats died of ketamine poisoning. Peripheral blood, heart, liver, spleen, lung, kidney and brain, muscle tissue were taken and saved in fridge at low temperature. The content of ketamine was determined by high performance liquid chromatographic. Ketamine content order of group Ⅰ was as follows:brain> liver> kidney> lung, spleen, muscle, heart, and peripheral blood. Ketamine content order of group Ⅰ was as follows:brain>kidney> liver> lung, spleen, heart, and peripheral blood muscles. The results indicated that ketamine widely distributed in poisoning rats in vivo and blood, the highest content of ketamine appeared in brain, liver and kidney. It indicates that the inspection material for toxicological analysis should be selected reasonablely in the forensic identification.
引文
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