液相色谱—串联质谱法在体内有毒动植物成分检测中的应用

发布时间：2018-08-14 08:41

【摘要】： 在我国民间,许多动植物作为药用和食用,有的甚至作为“滋补品”食用,这些动植物往往含有毒性成分,甚至是剧毒成分,其治疗量与中毒量接近,因用药不当、盲目和超量服用可造成中毒和死亡。在司法鉴定实践中,由此导致的各类误服、自杀、投毒、医疗纠纷等的案件时有发生。由于有毒动植物所含成分复杂多样、毒性大、含量低以及在人体内代谢迅速、且中毒无特异性临床表现等问题,使复杂生物体系中有毒动植物成分的鉴定成为长期以来未能有效解决的技术难题,对有毒动植物中毒的法医学鉴定、临床诊治以及食品安全监测缺乏有效的检测手段和科学的评判依据。现代分析技术的发展使得复杂生物体系中有毒动植物成分的检测和中毒鉴定成为可能。近年来,液相色谱—质谱联用法在法医和临床毒物分析领域的应用得到迅速发展。具有液相色谱高效的在线分离能力和质谱的高选择性、高灵敏度的检测能力的液相色谱-质谱联用法,可以同时得到化合物的保留时间、分子量以及特征结构碎片等丰富的信息,尤其是串联质谱可从复杂基质中确认目标物,采用选择离子监测或多反应监测模式,更可大大提高分析的专一性和灵敏度,完全有望解决复杂生物体系中痕量有毒动植物成分的检测,并在分析目标物不明确或系统中可能存在数种不同性质的目标物时,用一个分析系统同时检测多种不同性质的目标物。同时液相色谱-质谱联用法分析目标物范围广,前处理简单,不需衍生化,体现出比气相色谱-质谱联用法更灵敏、有效的检测手段,从而对复杂生物体系中的有毒动植物成分的鉴定和确认提供科学依据。本课题以血液、尿液和组织为主要生物检材,研究、探索、建立了22种常见有毒生物碱成分系统筛选方法,乌头碱、新乌头碱、次乌头碱、秋水仙碱和河豚毒素的定性定量方法,并对乌头碱、新乌头碱、次乌头碱和秋水仙碱的定量分析方法进行了全面、系统的方法有效性验证,解决了LC-MS/MS分析中的样品处理、色谱分离及优化、质谱条件的选择与优化等关键技术问题,同时对它们在急性中毒豚鼠的体内分布进行探讨,并成功地将所建方法应用于司法鉴定实践,为涉毒案件的审理提供了科学证据。具体研究内容如下: 一、建立了基于LC-MS/MS-MRM技术的血液中22种有毒生物碱成分的筛选体系和确证方法。目标物涵盖了在实际检案工作中常见的有毒生物碱。所建方法简便、快速、灵敏度高、专一性强,色谱分离采用两根不同性能的色谱柱,使目标物的分离、检测灵敏度、检测所需时间等方面得到互补,整个过程可在16min内完成。最低检测限除羟基喜树碱稍高(20ng/mL)外,绝大多数的LOD足以满足法医毒物学和临床药物学中中毒或治疗浓度目标物的检测要求。二、乌头生物碱的中毒、检测及应用 1.建立了测定血液、尿液和组织中乌头碱、新乌头碱和次乌头碱的LC-MS/MS-MRM分析方法,并对其定量方法进行了全面系统的方法有效性验证,内容包括考察方法的线性范围、最低检出限、精密度、回收率、准确度、基质效应、稳定性等,取得了满意的结果。所建方法简便、快速、灵敏,适用于体内痕量乌头碱、新乌头碱和次乌头碱的检测。 2.研究了乌头碱在急性中毒豚鼠的体液和组织中的分布。取豚鼠8只,以1.0-4.0mg/kg剂量灌胃给药致豚鼠乌头碱急性中毒,解剖后取体液和组织进行乌头碱测定。乌头碱在体液和组织中的含量从大到小依次为:胆汁＞血液＞尿液＞胃＞肺＞心＞脾＞肾＞胰＞肝。血液可作为乌头碱中毒、死亡分析的首选生物检材。 3.将所建方法成功地应用于鉴定实践,对生物检材中痕量乌头生物碱的成分进行定性定量分析,并对多起乌头植物中毒尸体中体液和组织中的乌头碱、新乌头碱和次乌头碱的含量进行测定,首次报道了新乌头碱和次乌头碱在尸体组织中分布,为涉及乌头中毒案件的死因判定和案件的审理提供了明确的方向和科学的证据。三、秋水仙碱的中毒、检测及应用 1.建立了测定血液、尿液和组织中秋水仙碱的LC-MS/MS-MRM分析方法,并对其定量方法进行了全面系统的方法学验证,内容包括方法的线性范围、最低检出限、精密度、回收率、准确度、基质效应、稳定性等,所得结果符合法医毒物分析的要求。所建方法简便、快速、灵敏,最低检出限可低至0.05ng/mL,能够满足法医毒物分析和临床毒物分析的要求。 2.研究了秋水仙碱在急性中毒豚鼠体内的分布。取豚鼠5只,以4.0mg/kg剂量灌胃给药致豚鼠秋水仙碱急性中毒,解剖后取体液和组织进行秋水仙碱含量测定。秋水仙碱在体液和组织中的含量从大到小依次为:胆汁＞尿液＞脾＞胃＞肺＞肾＞心＞胰＞肝＞肾上腺＞睾丸,心血中秋水仙碱的含量最低。尿液可作为秋水仙碱分析的首选生物检材。 3.将所建方法成功地应用于鉴定实践,对中毒尸体的生物检材及野生植物中秋水仙碱的成分进行定性定量分析,为涉及有毒植物中毒案件的死因判定提供了明确的方向和证据。四、生物检材中河豚毒素的检测及应用建立了测定血液、尿液和组织中河豚毒素的LC-MS/MS-MRM测定方法,考察了方法的线性范围及最低检出限。并将所建方法应用于食用河豚鱼中毒的实际案例中,取得了满意的效果。本研究的创新点及研究的意义如下: 1.建立的有毒生物碱成分筛选分析方法简便、灵敏,避免了生物检材中该类物质用传统方法检测可能出现的漏检、错检现象,整个分析可在16min内完成。筛选目标物涵盖了司法鉴定中常见的有毒生物碱成分,且LC-MS/MS筛选体系可随着目标物的增加而不断扩充,从而实现对更多的目标物进行筛选。该方法为首创方法。 2.全面系统地阐述了生物检材中定量方法的有效性验证,规范了方法的评价指标,通过对生物检材中乌头碱、新乌头碱、次乌头碱和秋水仙碱的检测方法的有效性验证,证明了所建方法准确、灵敏、专属、重现和可靠。 3.建立了生物检材中乌头生物碱LC-MS/MS-MRM分析方法。首次报道了乌头中毒尸体中与乌头碱共存的新乌头碱和次乌头碱的体内分布,为涉及有毒植物中毒案件的死因判定提供了明确的方向和证据。 4.建立了生物检材中秋水仙碱LC-MS/MS-MRM分析方法。所建方法足够灵敏,最低检出限可低至0.05ng/mL,能够满足法医毒物分析和临床毒物分析的要求。 5.建立了生物检材中河豚毒素的LC-MS/MS-MRM测定方法,并首次报道了河豚鱼中毒尸体中河豚毒素在各组织中的分布情况。研究成果填补了体内有毒动植物检测及评判的空白,使我国的复杂生物体系中常见有毒动植物成分分析技术跃升到国际先进水平,将为有毒动植物的科学研究、中毒鉴定、临床诊断及救治、食品安全监测等提供技术平台。
[Abstract]:In our country, many animals and plants are used as medicinal and edible, and some even as "tonic" food. These animals and plants often contain toxic ingredients, even highly toxic ingredients. Their treatment dose is close to that of poisoning. Drug, suicide, poisoning, medical disputes and other cases occur from time to time. The identification of toxic animal and plant components in complex biological systems has long been an unsolved technical problem due to the complex and diverse components, high toxicity, low content, rapid metabolism in the human body, and no specific clinical manifestations of poisoning. There is no effective means and scientific basis for forensic identification, clinical diagnosis, treatment and food safety monitoring of toxic animal and plant poisoning.
The development of modern analytical techniques makes it possible to detect and identify toxic plant and animal components in complex biological systems. In recent years, the application of liquid chromatography-mass spectrometry (LC-MS) in forensic and clinical toxicological analysis has been developed rapidly. It has high performance in on-line separation of liquid chromatography and high selectivity and high sensitivity in mass spectrometry. Liquid chromatography-mass spectrometry (LC-MS) can simultaneously obtain abundant information such as retention time, molecular weight and characteristic structure fragments of compounds. Especially, tandem mass spectrometry can identify the target from complex matrices, and selective ion monitoring or multi-reaction monitoring mode can greatly improve the specificity and sensitivity of the analysis. It is hopeful to solve the problem of detecting trace toxic animal and plant components in complex biological systems, and to use an analytical system for simultaneous detection of a variety of different kinds of targets when the target is not clear or there may be several different kinds of targets in the system. Simple and non-derivatization methods are more sensitive and effective than gas chromatography-mass spectrometry, thus providing a scientific basis for the identification and identification of toxic animal and plant components in complex biological systems.
A systematic screening method for 22 common toxic alkaloids, including aconitine, neoaconitine, hypoaconitine, colchicine and tetrodotoxin, was established. The quantitative analysis methods of aconitine, neoaconitine, hypoaconitine and colchicine were also studied. All-round and systematic validation of the method has solved the key technical problems in LC-MS/MS analysis, such as sample processing, chromatographic separation and optimization, and the selection and optimization of mass spectrometry conditions. At the same time, the distribution of the methods in acute poisoning guinea pigs has been discussed, and the established methods have been successfully applied in forensic practice for the trial of drug-related cases. Li provides scientific evidence.
The specific research contents are as follows:
Firstly, a screening system and confirmation method of 22 toxic alkaloids in blood based on LC-MS/MS-MRM technology were established. The target substances covered the common toxic alkaloids in the actual detection work. The method was simple, rapid, sensitive and specific. Two chromatographic columns with different performance were used for chromatographic separation and detection of the target substances. The detection sensitivity and time required are complementary, and the whole process can be completed within 16 minutes. Except for the slightly higher hydroxycamptothecin (20ng/mL), the vast majority of LOD is sufficient to meet the requirements of forensic toxicology and clinical pharmacology for the detection of poisoning or therapeutic concentration targets.
Two, poisoning and detection and application of Aconitum alkaloids
1. A LC-MS/MS-MRM method for the determination of aconitine, neoaconitine and hypaconitine in blood, urine and tissues was established, and its quantitative method was validated by a comprehensive and systematic method, including the linear range, minimum detection limit, precision, recovery rate, accuracy, matrix effect, stability and so on. The method is simple, rapid and sensitive for the determination of trace aconitine, neoaconitine and hypaconitine in vivo.
2. The distribution of aconitine in body fluid and tissues of acute poisoning guinea pigs was studied. Eight guinea pigs were given aconitine by intragastric administration at a dose of 1.0-4.0 mg/kg. After dissection, the contents of aconitine in body fluid and tissues were determined. The order of aconitine content in body fluid and tissues was: bile > blood > urine > stomach > lung > Blood can be used as the first choice of biological analysis for death analysis.
3. The established method was successfully applied to the identification practice. The contents of trace aconitine in biological samples were qualitatively and quantitatively analyzed. The contents of aconitine, neoaconitine and hypaconitine in body fluids and tissues of several aconitine poisoned corpses were determined. The contents of neoaconitine and hypaconitine in corpse tissues were reported for the first time. The distribution provides clear direction and scientific evidence for the determination of the cause of death and the trial of cases involving Aconitum poisoning.
Three, colchicine poisoning, detection and Application
1. A LC-MS/MS-MRM method for the determination of colchicine in blood, urine and tissues was established, and its quantitative method was systematically and systematically validated, including linear range, minimum detection limit, precision, recovery rate, accuracy, matrix effect, stability and so on. The method is simple, rapid, sensitive and the minimum detection limit can be as low as 0.05ng/mL. It can meet the requirements of forensic toxicology analysis and clinical toxicology analysis.
2. The distribution of colchicine in acute poisoned guinea pigs was studied. Five guinea pigs were given colchicine by intragastric administration at a dose of 4.0 mg/kg. The contents of colchicine in body fluid and tissues were measured after dissection. The order of colchicine content in body fluid and tissues was bile > urine > spleen > stomach > lung > kidney. The content of colchicine in heart blood is the lowest. Urine can be used as the first choice for colchicine analysis.
3. The method has been successfully applied to the identification of poisoned plants. The qualitative and quantitative analysis of colchicine in the biological samples of poisoned corpses and wild plants has provided clear direction and evidence for the determination of the cause of death in poisoned plants.
Four, the detection and application of tetrodotoxin in biological samples.
A method for the determination of tetrodotoxin in blood, urine and tissues by LC-MS/MS-MRM was established. The linear range and the minimum detection limit of the method were investigated. The method was applied to the practical cases of tetrodotoxin poisoning in edible puffer fish and satisfactory results were obtained.
The innovation and significance of this study are as follows:
1. The established screening and analysis method for toxic alkaloids is simple and sensitive, avoiding the missed and mistaken detection phenomena that may occur in biological samples by traditional methods. The whole analysis can be completed within 16 minutes. The screening target covers the common toxic alkaloids in forensic identification, and the LC-MS/MS screening system can follow the target. With the increase and expansion of objects, more targets can be screened. This method is a pioneering method.
2. The validity of the quantitative method in biological samples was expounded systematically and comprehensively, and the evaluation index of the method was standardized. The validity of the method for the determination of aconitine, neoaconitine, hypoaconitine and colchicine in biological samples was proved to be accurate, sensitive, exclusive, reproducible and reliable.
3. A LC-MS/MS-MRM method for the determination of aconitine in biological samples was established. The distribution of neoaconitine and hypaconitine in aconitine-poisoned corpses was reported for the first time, which provided clear direction and evidence for the determination of the cause of death in poisoned plants.
4. A LC-MS/MS-MRM method for the determination of colchicine in biological samples was established. The method is sensitive enough and the detection limit can be as low as 0.05 ng/mL. It can meet the requirements of forensic toxicology analysis and clinical toxicology analysis.
5. A method for the determination of tetrodotoxin in biological samples by LC-MS/MS-MRM was established. The distribution of tetrodotoxin in various tissues of tetrodotoxin poisoned corpses was reported for the first time.
The research results fill the blank of toxic animal and plant detection and evaluation in vivo, and make the component analysis technology of common toxic animal and plant in complex biological system of our country leap to the international advanced level. It will provide a technical platform for the scientific research of toxic animal and plant, poisoning identification, clinical diagnosis and treatment, food safety monitoring and so on.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：D919.1

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