Analysis Of Plant And Animal Toxicants In Biological Specimens By LC-MS/MS

In China,many plant and animals are often used for food and medicine,some of which are even used for nutritional supplements.However,these plant and animals often contain toxins or extremely toxicants whose therapeutic dose nearly equals to toxic dose so that many people were poisoned or killed because of misusing or overdose taking.In forensic science practice,many cases about mistaking,suicide, poison spreading and medical dispute are caused by these plant and animal toxicants. Because of complicate and various components,high toxicity and low concentration in toxic plant and animal,rapid metabolism and lack of specific clinical features,the identification of herb and animal's toxic components in complicate biological system has become a technical problem which is very difficult to effectively solve and there is still no effective identification method and scientific evaluation basis to forensic identification and clinical therapy caused by plant and animal toxicants and food safety monitoring.The development of modem analytical technology has made it possible for the detection of the in plant and animal toxicants and identification of poisoning.In recent years,the application of LC-MS/MS in the field of forensic and clinical toxicological analysis has been developed rapidly.Combined with LC's high performance online separation ability and MS's high selective and sensitive detection ability the LC-MS/MS system can get rich information including the compounds' retention time, molecular weight and characteristic structure fragment etc.at the same time, especially that the system can confirm the target compounds from complicated matrix and highly enhance the specificity and sensitivity with SIM and MRM mode so that detect trace amount of the plant and animal toxicants in complicated biological system. In addition,the system can also determine various target compounds with different characters at the same time when the target compounds can not be confirm or various target compounds with different characters exist in the system.With wide range of targets,easy pretreatment and no derivatization the LC-MS/MS system,more sensitive and effective than GC-MS~n system,can provide scientific basis for determination and identification of plant and animals toxicants in complicated biological system.This study has researched,explored and established a screening method for 22 kinds of common toxic alkaloid in blood and urine.It also set up a determination and quantification method for aconite alkaloids,colchicine and tetrodotoxin(TTX),give a all-around and systematic validation on the method and solve the key technical problem on sample pretreatment,chromatographic separation and mass spectrometric condition selecting and optimizing during LC-MS/MS analytical procedure.In addition,this study have successfully applied these methods on forensic science case and provided a clear direction for further trial.Main content is as follows:1.A screening and determination method for 22 kinds of toxic alkaloids was established which are very common in routine cases in blood by LC-MS/MS in MRM mode.The method was approved simple,fast,sensitive and specified.With two different chromatographic columns,the separation,detection sensitivity and cycle time received complementation and the whole procedure could be finished in 16 minutes.Except the LOD of hydroxycamptothecin(20 ng/ml) was a little bit high, most of the LODs were low enough to reach the determine desire of target compounds toxic and clinical concentration in forensic toxicology and clinical pharmacology.2.The determination,poisoning,evaluation and analytical method application of aconite alkaloids2.1 A determination method for aconitine,mesaconitine and hypaconitine in blood, urine and tissue by LC-MS/MS in MRM mode was established.An all-around and systematic validation was given to the method including linear range,LOD,precision, recovery,accuracy,matrix effect,and stability and a satisfied result was gotten.The simple,fast and sensitive method can be applied on the determination of aconitine, mesaconitine and hypaconitine in vivo.2.2 The distribution of aconitine in poisoning guinea pigs' biological fluid and tissue was studied.After given aconitine at the dose of 1～4mg/kg through intragastric administration 8 guinea pigs got death and then their biological fluid and tissue were taken out for determination of the aconitine.The concentration of aconitine in fluid and tissue was in the decreased sequence of bile,blood,urine,stomach,lung,heart, spleen,kidney,pancreas,liver.The results showed that blood can be the first choice for aconitine poisoning judgment and death analysis.2.3 The method was successfully applied on the identification case including the determination and quantification of aconite alkaloids in biological samples and quantification of aconitine,mesaconitine and hypaconitine in the biological fluid and tissue of whom was killed by aconitum.The distribution of mesaconitine and hypaconitine in the tissue were reported for the first time which provided clear direction and scientific evidence for the death reason and trial of toxic plant and animal caused poisoning cases.3.The determination,poisoning,evaluation and analytical method application of colchicine3.1 A determination method for colchicine in blood,urine and tissue by LC-MS/MS in MRM mode were established.An all-around and systematic validation was given to the method including linear range,LOD,precision,recovery,accuracy,matrix effect,and stability and the result reached the desire of forensic toxicants analysis. The simple,fast and sensitive(LOD was as low as 0.05ng/mg) method could be applied on the determination of colchicine and reached the desire of forensic and clinical toxicological analysis.3.2 The distribution of colchicine in poisoning guinea pigs' biological fluid and tissue was studied.After given colchicine at the dose of 4mg/kg through intragastric administration,5 guinea pigs got death and then their body fluid and tissue were taken out for determination of the toxicants.The concentration of colchicine in fluid and tissue was in the decreased sequence of bile,urine,spleen,stomach,lung,kidney, heart,pancreas,liver,adrenal,didymus,The concentration in blood is the lowest.The results showed that urine can be the first choice for colchicine poisoning judgment and death analysis3.3 The method was successfully applied on the identification case including the determination and quantification of colchicine in biological samples and quantification of colchicine in the biological fluid and tissue of whom was killed by colchicine.The establishment of the method also provided clear direction and evidence for the death reason and trial of toxic plant and animals caused poisoning cases.4.The determination and analytical method application of TTX in biological sample A determination method for TTX in blood,urine and tissue by LC-MS/MS in MRM mode were established.The method included linear range,LOD.After applyed on the poisoning cases of eating puffer fish of mistake,the method got a satisfied result.The innovations and significations of the study are as follows:1.The screening method of toxic alkaloid was simple,sensitive and it also could prevent mistake and omission which often happened by traditional determine method and be finished in 16 minutes.The target compounds of the method included the common toxic alkaloids.In addition,with the extension of the amount of the target compounds,the method could be used for more compounds. The method was an innovation.2.This study gave an all-round and systematic explain on validation of the quantitation method in biological samples and standardized the relating information about the method validation.After validating,the method on the determination of aconitine,mesaconitine,hypaconitine and colchicine were improved to be accurate,sensitive,specified,productive and credible.3.A determination method for aconitine,mesaconitine and hypaconitine in blood, urine and tissue by LC-MS/MS in MRM mode were established.The distribution of mesaconitine and hypaconitine in the postmortem toxicology were reported for the first time which provided clear direction and evidence for the death reason and trial of toxic plant and animal caused poisoning cases.4.A determination method for colchicine in blood,urine and tissue by LC-MS/MS in MRM mod were established.The simple,fast and sensitive(LOD was as low as 0.05ng/mg) method could be applied on the determination of colchicine and reached the desire of forensic and clinical toxicological analysis.5.A determination method for TTX in blood,urine and tissue by LC-MS/MS in MRM mode was established and the distribution of TTX in human's tissue who was killed by TTX was reported for the first time.The achievement filled the gap of the determination and evaluation of plant and animal toxicants in vivo,which advanced the analytical technology of common toxic plant and animal in complicated biological system to international level.In addition, this achievement would provide technological platform for the scientific research, poisoning judgement,clinical diagnose and rescue and food safety monitoring on toxic plant and animals.