Study On Toxicokinetics And Postmortem Distribution Of Higenamine And Higenamine Glucuronic Acid In Poisoned Rats

Objectives:Purpose:1.The liver microsome incubation system was established in vitro,higenamine glucuronic acid conjugate（Glu-HG）was obtained by incubating higenamine（HG）protomers.2.The conversion relationships between quantitative ions in higenamine and higenamine glucuronic acid conjugate was investigated by HPLC-MS/MS.Higenamine was obtained by hydrolysis of higenamine glucuronic acid conjugate,the quantitative ion peak area conversion relationship between them was explored to obtain the conversion factor.Semi-quantitative analysis of higenamine glucuronic acid conjugate was performed by conversion factors.3.The toxicokinetic animal models of higenamine and higenamine glucuronic acid conjugates in rats were established,to explore the metabolic rule of higenamine in rats and obtain the toxicokinetic equations and parameters of them.That might provide a theoretical basis for the determination of the poisoning time of Aconitine herbs.4.The postmortem distribution models of higenamine,higenamine glucuronic acid conjugate and diester aconitine alkaloids in rats were established,and comprehensively to explore the postmortem distribution rule of each substance in rats.It could provide significant guidings for the selection of relevant case samples.Method:1.Higenamine glucuronic acid binding in vitro liver microsomal incubation studies:A higenamine glucuronic acid-binding in vitro liver microsome incubation system was established:0.05 m M Tris-HCl buffer at p H=7.4,the incubation system volume was 1 m L,and the final concentrations of each substance were 5 m M Mg Cl₂·6H₂O,5 m M gluconate monolactone,5m M UDPGA,25mg/m L alamethicin,and Higenamine 1mM.The incubation solution was placed in a constant temperature incubator at 37℃,and the reaction was terminated at different times,which were 0h in the control group and 0.5 h,1 h,2 h and4 h in the experimental group,respectively,with four replicates in each group.200mL of the incubation solution was added to 800mL of acetonitrile to precipitate the protein,and the incubation reaction was terminated and then detected by HPLC-Q-TOF-MS and LC-MS/MS.2.The conversion factor experimentHigenamine and higenamine glucuronic acid conjugates were separated by high performance liquid chromatography to eliminate the interference of higenamine protomers on the conversion factor experiment.Higenamine glucuronic acid conjugate solution with a certain content isolated from the above liquid phase was placed in theβ-glucuronidase incubation system,which had 0.05 M sodium acetate buffer at p H=5.2,the incubation system volume was 400mL,and the final concentration of each substance was 5000 U/m Lβ-glucuronidase,respectively,and this incubation solution was placed in a thermostatic incubator at 37℃,and the reaction was terminated at 0.5 h for the control group and0.5h for the experimental group,with four replicates in each group.100mL of the incubation solution was added into 200mL of ice-cold acetonitrile containing internal standard dobutamine to terminate the incubation reaction,and after pretreatment,the conversion relationship of the peak area between higenamine and higenamine glucuronic acid conjugate quantitative ions was obtained by HPLC-MS/MS instrument detection,and the conversion rate of the quantitative ions of the two substances,that is,the conversion factor（γ）,was calculated,andγwas brought into the standard curve of higenamine,so that the higenamine glucuronic acid conjugate was semiquantitatively analyzed.3.To establish a toxicokinetic model of higenamine and higenamine glucuronic acid conjugates in ratsSix male SD rats were fed in the laboratory environment for a week,fasted before the experiment,could not help water for 12 h.Rats were intragastrically administered with higenamine standard(selected decoction of Radix Aconiti Kusnezoffii,when the content of aconitine was 1/2LD₅₀,the corresponding higenamine content in the decoction),and 800mL of orbital blood was collected at 5 min,10 min,30 min,1 h,2 h,4 h,6 h,12 h,24 h and 48 h.Centrifugation was performed at 13000 rpm/min for 10 min in a high-speed centrifuge,and the supernatant was stored in a-80℃freezer.After HPLC-MS/MS detection and data processing,the equations and parameters of Higenamine were obtained by DAS3.2.8pharmacokinetic software.4.To establish a postmortem in vivo distribution model of higenamine,higenamine glucuronic acid conjugate and diester aconitine alkaloids in rats exposed before deathSix male SD rats were fed in the laboratory environment for a week,fasted before the experiment,could not help water for 12 h.The rats were intragastrically administered with 1/2LD₅₀dose of aconitine in the decoction of Radix Aconiti Kusnezoffii,and after waiting for the disappearance of vital signs（rats whose vital signs did not disappear after 2 h of administration were sacrificed by cervical dislocation at 2 h）,the rats were immediately dissected,and heart blood,peripheral blood,heart,liver,spleen,lungs,kidneys,brain,left lower extremity muscles,and testicular tissues were taken and the test samples were stored in a-80℃refrigerator for examination.Results:1.The detection methods for higenamine,higenamine glucuronic acid conjugate and diester aconitine alkaloids were determined.The pretreatment process of solid phase extraction enrichment was established.The linear range of higenamine,higenamine glucuronic acid conjugate and diester aconitine alkaloids in rat plasma was determined to be 0.5～500 ng/m L,the lowest limit of detection（LOD）was 0.01～0.05 ng/m L,and the lowest limit of quantification（LOQ）was 0.1～0.2 ng/m L;in rat liver,the linear range was 0.01～0.05 ng/g,the LOD was 0.03～1.07 ng/g,and the LOQ was 0.1～0.2 ng/g;the correlation coefficients（R²）of higenamine,higenamine glucuronic acid conjugate and diester aconitine alkaloids in rat plasma and liver were greater than 0.99,the intra-day and inter-day precisions were less than 20%,the extraction recoveries were greater than 50%,and the matrix effects were within 50%～120%.2.Incubation experiment of higenamine glucuronic acid conjugate:Compared the 0h control group to the 0.5 h,1 h,2 h and 4 h experimental groups,the ions of higenamine glucuronic acid conjugate with a mass-to-charge ratio of4481.576 were found in the experimental group in the full scan mode by HPLC-Q-TOF-MS and HPLC-MS/MS,and the differences in the theoretical mass-to-charge ratios calculated by Agilent Mass Hunter Qualitative Analysis Navigator B.08.00 software were within 20 ppm.After collecting the secondary mass spectrometric information of higenamine glucuronic acid conjugate,we found that the mass-to-charge ratio of higenamine protomer was 272.1259 in the product ion,which further verified the incubation study of higenamine glucuronic acid conjugate.3.Conversion factor experiment:The conversion factor was calculated as follows:γ=△Glu-HG/HG（where Glu-HG=peak area without hydrolysis/internal standard peak area-peak area after hydrolysis/internal standard peak area,△HG=peak area after hydrolysis/internal standard peak area-peak area without hydrolysis/internal standard peak area）,and the finalγvalue was 0.211,andγwas brought into the standard curve of higenamine for semi-quantitative analysis of Glu-HG.4.Toxicokinetic studies:After oral gavage administration of higenamine,plasma was taken for detection,and it was found that HG protomers were not detected in plasma but Glu-HG could be detected,and Glu-HG was not detected in plasma extracted without treatment.Glu-HG was detected in the plasma of rats at 5 min after intragastric administration,with a mean level of 0.53 ng/m L,and its time to peak was between 1 h and 3 h.After peak,it showed a continuous decreasing trend,and Glu-HG was not detected until 24 h later.higenamine glucuronic acid conjugates in plasma were characterized by a one-compartment model of drug metabolism with a fitted equation of C=16.068(e^-0.759t-e^-0.13t).4.Postmortem distribution studies of higenamine,higenamine glucuronic acid conjugates and diester aconitine alkaloidsStatistical analysis（non-parametric test,P<0.05）showed that the postmortem distribution of higenamine,higenamine glucuronic acid conjugate and diester aconitine alkaloids in exposed rats was as follows:HG:liver>kidney>muscle,spleen,lung,testis and brain;Glu-HG:heart blood,peripheral blood>liver,kidney,spleen>lung,testis,brain and muscle;AC:heart blood>liver,peripheral blood>heart,very organ,lung>muscle,brain and testis;MA:liver,peripheral blood>lung,heart,kidney,heart>brain,muscle and testis;HA:liver,heart blood,peripheral blood>lung,kidney,heart,spleen>muscle,brain and testis.Conclusion:1.A method for the determination of higenamine,higenamine glucuronic acid conjugate and diester aconitine alkaloids by HPLC-MS/MS was established.The sample pretreatment process was simple,rapid,sensitive and had a high recovery rate,and could be used for detection in related experiments.2.In this study,semiquantitative conversion factor（γ）of higenamine glucuronic acid conjugate was obtained by incubation of liver microsomes in vitro,and semiquantitative analysis of higenamine glucuronic acid conjugate was performed.3.In this study,we established a rat model of toxicokinetics of higenamine glucuronic acid conjugate and obtained its toxicokinetic equation and parameters.It was found that higenamine glucuronic acid conjugate could be detected in plasma,while higenamine protomer could not be detected in plasma,which was considered to be caused by the accumulation of higenamine in organs.The toxicokinetic model and equation of higenamine glucuronic acid conjugate could provide theoretical basis for inferring the poisoning time of aconitine poisoning cases.4.For the suspected cases related to the death of Aconitum poisoning caused by traditional Chinese medicine,it was necessary to carefully inquire the history of medication and medical history before death,and the selection of examination materials should be considered in turn:organs with rich blood flow such as blood,kidney,liver,lung and spleen,gastric tissues containing gastric contents,and relevant traditional Chinese medicines taken by the deceased before death.It should be noted that the whole process of submission ensures cryopreservation and timely submission for inspection.For the suspected cases of Aconitum poisoning,the detection of HG,Glu-HG and DDAs was comprehensively investigated,and the cases in which Glu-HG components were detected and DDAs and HG were detected at the same time could be judged as taking poison before death.