Study On The Forensic Toxicokinetics Of Diazepam Poisoning Biomarkers

Objective: 1.To establish a method for preparation of glucuronide conjugates,II phase metabolites of diazepam;2.To observe the decomposition of glucuronide conjugates,II phase metabolites of diazepam,to clarify the way and time limit of conservation;3.To screen diazepam poisoning biomarkers with non-target metabolomics,and provide basics for the study of forensic toxicokinetics.Methods: 1.Preparation of glucuronide conjugates,II phase metabolites of diazepam After incubation of oxazepam with human liver microsomes(HLM)and extraction of oxazepam glucuronide and temazepam glucuronide in urine,high performance liquid phase tandem mass spectrometry(HPLC-MS/MS)was used to detect the glucuronides of oxazepam and temazepam.After compared with the standard of oxazepam glucuronide and temazepam glucuronide,the information of metabolites could be obtained.2.Decomposition of glucuronide conjugates,II phase metabolites of diazepam Fore 100 m L blood(urine)was collected from elbow vein of volunteers(three men and three women)at 24h(48h)after administration,packed in heparin sodium tube(centrifuge tube)at 20℃,4℃,-20℃and sodium fluoride tube at 20℃,respectively.Another twelve 100 μL blood(urine)was added respectively on filter paper,after dried for at least 2 h,I and I? phase metabolites of diazepam on dried blood(urine)spots(DBS/DUS)at different time points were studied with HPLC-MS/MS.3.Application of non-target metabolomics in the screening of diazepam poisoning markers and its kinetics Data preprocessing,principal component analysis(PCA)and differential compound analysis were employed to analyze the mass data to discriminate the benzodiazepines(diazepam,oxazepam,clonazepam,nitrazepam,estazolam,alprazolam)poisoning rats from control rats.Then screening diazepam poisoning biomarkers through the metabolic fingerprints and pathways comparison of each benzodiazepine.Results: 1.Preparation of glucuronide conjugates,II phase metabolites of diazepam Mass data errors between metabolite in incubation and oxazepam glucuronide,between metabolites in urine extraction and oxazepam glucuronide,temazepam glucuronide were within the maximum allowable relative error range(25%),then it could be determined that the corresponding glucuronic acid conjugates could be prepared by either method.2.Decomposition of glucuronide conjugates,II phase metabolites of diazepam II phase metabolites of diazepam increased both in blood and urine,which were not related to the condition of conservation.However,their results in DBS and DUS showed a greater difference: temazepam glucuronide was stable in DBS,and not related to the condition of conservation;In DUS,TG was still stable at 20℃(1%Na F),4℃ and-20℃,but it decreased slowly at 20℃.Oxazepam glucuronide degraded in DBS in all condition of conservation,while increased in DUS,which were not related to the condition of conservation as well.3.Application of non-target metabolomics in the screening of diazepam poisoning markers and its kinetics The results of diazepam metabolomics showed that biomarkers identified in blood were Pantothenic acid and 5-oxoproline,those were citric acid,malic acid,succinic acid,Succinic semialdehyde,L-threonine,and 4-guanidine butyric acid in urine.Compared with other benzodiazepines,the concentration of 5-oxoproline in blood changed obviously,it involved mainly in glutathione metabolic pathway.Conclusion: 1.The study have obtained glucuronide conjugates of diazepam through incubation with liver microsome and extraction with urine of diazepam poisoners,which could be applied in studies of glucuronide conjugates of diazepam;2.The study established a method of detecting diazepam and its I and I? phase metabolites simultaneously in DBS and DUS with HPLC-MS-MS,the limiter of quantitation(LOQ),linear range and recovery could meet the requirements of decomposition study,it could be used for the study of decomposition kinetics of diazepam and its metabolites in DBS and DUS.3.The stability of diazepam and its metabolites could be affected by many factors.In which temazepam glucuronide in DBS and DUS conserved at 20℃Na F,4℃ or-20℃ were stable relatively.4.Based on the technology of non-target metabolomics,the study discussed initially the potential biomarkers in plasma and urine of diazepam poisoning rabbits.As a result,5-oxoproline in blood was selected as biomarker in diazepam poisoning rats,which involved in Glutathione metabolism,it provided basics for the further study of target metabolomics and forensic toxicokinetics.