Study On Triptreygium Wilfordii Hook.f., Nerium Oleander And Toxic Alkaloids Poisoning, Determination And Evaluation

Plants are widely distributed in nature, and are closely related to people’s life. However, the chemical compositions of plants are complex, many of which are poisonous. Poisoning cases often occur as the results of incorrect processing and use, unintentional ingestion, homicide, or accident. The toxic compositions in plants are complicated, toxic, at low level in biological specimenss, rapidly metabolited, and with non-specific clinical manifestations of poisoning, making the detection and intepretation of relevant target compounds in complex biological systems a technical problem for toxicological analysis. Triptreygium wilfordii Hook.f. is an important kind of natural medicine resource in our country, and is one of the commonest Chinese herbal medicine related to poisoning incidents for nearly half a century; its toxicity ranks third among the traditional Chinese herbal medicine. As the clinical application of Triptreygium wilfordii Hook.f. becomes wider, its side effects have attracted more and more attentions. Triptolide is a diterpenoid epoxide isolated from Triptreygium wilfordii Hook.f.. It is one of the main effective components of Triptreygium wilfordii Hook.f. and also the main component responsible for the side effeces. Wilforlide A is one kind of triterpenoid found in Triptreygium wilfordii Hook.f. with obvious side effects. Nerium oleander is a Chinese herbal medicine with ornamental value, and it is famous for its toxicity. Oleandrin is the main toxic component found in Nerium oleander. It is a delayed cardiac glycoside with strong vomiting effects. Due to the potent toxicity of Triptreygium wilfordii Hook.f. and Nerium oleander, poisoning cases in humans have been frequently reported. In addition, poisoning cases of unknown toxic alkaloids occurred sometimes in the practice of forensic medicine. Nevertheless, related research is not sufficient enough; only a few reports are available. The existing methods for determination could hardly meet the demands of trace amount, rapid and simultaneous analysis for multiple targets in biological specimenss, and the studies on toxicokinetics, evaluation and inference of analysis results are still limited and not comprehensive. Therefore, it is important to develop reliable, accurate, rapid, and sensitive methods for screening and determining related compounds in biological specimenss, and study the related toxicokinetic characteristics, in order to identify and interpret the results in forensic investigation.The present research is aimed to develop simple, sensitive and accurate analytical methods for determination of the components in Triptreygium wilfordii Hook.f., Nerium oleander, and common toxic alkaloids in biological samples by liquid chromatography with tandem mass spectrometry(LC-MS/MS) respectively and to study on toxicokinetics of triptolide in rats. These methods can be applied to the analysis of actual biological samples and provide directions and theoretical basis for clinical diagnosis and treatment, sample collection, and interpretation of the results of forensic toxicological analysis.This paper consists of four chapters.The first chapter is the study on Triptreygium wilfordii Hook.f. poisoning, determination and toxicokinetics. This chapter divides into two parts. The first research part proposed a method for the simultaneous determination of triptolide and wilforlide A in blood, urine and liver by liquid-liquid extraction(LLE) and LC-MS/MS. Naloxone was chosen as the internal standard(IS), and the total run time was 10 min per injection. Triptolide, wilforlide A, and naloxone were well separated under the liquid chromatographic condition. The target compounds exhibited excellent linearity in the range of 5-500 ng/mL(ng/g) in blood, urine and liver, with a linear correlation coefficients no less than 0.9950. The limits of detection were all 2 ng/mL(ng/g). The extraction recoveries were greater than 60%; the matrix effects were between 82.06%-107.43%; the inter-day and intra-day accuracy were between 90.61%-105.80%; the relative standard deviations(RSD) in inter-day and intra-day precision study were all less than 15%. This method could qualitatively and quantitatively analyze trace amount of triptolide and wilforlide A in biological samples, and was suitable for determination of Triptreygium wilfordii Hook.f. poisoning in forensic toxicological analysis and therapeutic drug monitoring. The second part of this study established the animal experiment model of triptolide poisoning, and investigated the characteristics of concentration-time curve, tissue distribution, and excretion after intragastrically administration of toxic doses in rats. The results show that the trend of the blood concentration for triptolide with time conformed to two compartment model of first order kinetics elimination. The concentration-dose relationship presented linear dynamic properties across three toxic doses, and the concentration of triptolide in blood increased as the dose increased. The concentration changes of each organization at different time points are all 5 min﹥15 min﹥60 min. The concentration in liver and kidney are higher than in other tissues, so liver and kidney could be selected as proper biological specimenss. A large portion of the analytes excreted from faeces in the unchanged form. There was interindividual variation of triptolide absorbance by rats, while no significant difference between the absorbance by different gender was observed. These results can provide reference for identification and evaluation of triptolide poisoning.The second chapter described the development and validation of a determination method of oleandrin in biological samples by LLE-LC-MS/MS. External standard method was used in this method. Oleandrin exhibited excellent linearity in the range of 5-500 ng/mL and 20-2000 ng/g in blood and liver, respectively, the correlation coefficients of which were greater than 0.9950. The limits of detection were 1 ng/mL and 2 ng/g in blood and liver for oleandrin. The recoveries were greater than 70%. The matrix effects were between 91% and 106%. The inter-day and intra-day accuracy were between 91%-112%, and the inter-day and intra-day imprecision was less than 12%. Using the method described in this study, a fatal case of oleandrin poisoning was successfully analyzed. Meanwhile through the ion pair obtained by the existing literature and determination of molecular formula by the high resolution mass spectrometry(LTQ-Orbitrap) technology, the metabolite of oleandrin-oleandrigenin was detected. Oleandrin and oleandrigenin got a good separation by this method. This method was sensitive, selective, easy, and rapid, which was feasible for forensic analysis.In the third chapter, a method for simultaneous screening for 45 poisonous alkaloids in blood by LLE-LC-MS/MS was proposed and validated. SKF525 A was used as the IS, and the total run time was 12 min per injection. The limits of detection ranged from 0.05 ng/mL to 25 ng/mL. The linear correlation coefficients were more than 0.9950. The inter-day and intra-day accuracy were between 85.90% and 114.95%, while the inter-day and intra-day imprecisions were less than 15%. This method was of good selectivity, sensitivity, and the sample preparation was effective and simple, making it a applicable choice for the screening and determination of unknown alkaloids in forensic and clinical toxicology.The fourth chapter summarized is the conclusion of the present research including a discussion on the innovative aspects of the research, and proposes the direction of further study, which lays the foundation for deeper study of screening, identification and interpretation of common poisonous components from plant such as Triptreygium wilfordii Hook.f.. The significance and innovation of the present paper is as follows:1. The method for simultaneous determination of triptolide and wilforlide A in blood, urine and liver by LC-MS/MS was established for the first time. The method was simple, rapid, and is suitable for forensic investigation and clinical diagnosis and treatment. A systematical study on toxicokinetics, tissue distribution, and excretion of triptolide in rats was carried out.2. An LC-MS/MS analysis method of determining oleandrin in complex biological samples was created. The method is sensitive, rapid, and highly sensitive. This method was successfully applied to a fatal case of oleandrin poisoning. It was the first time that the distribution of oleandrin in various human tissues was discussed thoroughly. The molecular structure of suspicious metabolite, oleandrigenin was detected along with oleandrin in various tissues, which added more information to the practice of related forensic toxicology.3. An LC-MS/MS method for simultaneous screening for 45 poisonous alkaloids in blood, simple, sensitive, rapid, reliable was developed and validated. This method could meet the request of simultaneous analysis of multiple alkaloids in poisoing cases, and was successfully applied to several cases. Thus, more comprehensive information was obtained for alkaloid poisoning from both forensic and clinical aspects.This research partly filled the gaps of identification and evaluation for Triptreygium wilfordii Hook.f., Nerium oleander and common alkaloids poisoning in the field of forensic toxicology, and could provide a scientific support for forensic and clinical practice.