| Objective1.To establish the analysis of the clozapine in living samples by a GC and a GC/MS.2.To develop a toxicokinetics,dynamic distribution,postmortem distribution,postmortem redistribution postmortem diffusion,decomposetion model of clozapine.3 . Study the toxicokinetics,dynamic distribution,postmortem distribution,postmortem redistribution postmortem diffusion,decomposition of clozapine,and provide a scientific evidence for the forensic identification of clozapine poisoning death.Methods1.GC/MS and GC: The samples including blood and tissues, were alkalified to pH10, and extracted with ether. The extract was analyzed qualitatively and quantitatively by GC/MS and GC respectively with an internal standard and working curve method.2.The rabbits were given intragastric administration of clozepine with a dose of 1/2LD50 and LD50. Blood samples were collected at different times after administration and extracted with ether. Analysis was performed by a GC equipped with a NPD and a GC/MS. Compartment model and toxicokinetic parameters of clozapine were assessed by WinNonlin program.3.The dynamic distribution study: The rats were administrated intragastricly with clozapine of 65mg/kg, the rats were put to death at different time points(1h,2h,3h,8h,12h,24h,48h,72h)after administration, and the specimens such as brain, heart, lung, liver, kidney, lien and blood were taken and detected. And do statistical treatment by the soft ware of SPSS11.5 and calculate the parameter by the WinNonLin soft fitting compartment model.4. The postmortem distribution of clozapine in rabbits: 6 rabbits were divided two teams at random and given clozapine through stomach with two doses: 270 mg/kg and 1080 mg/kg. The specimens such as heart, liver, spleen, lung, kidney, and brain of rats were collected at the time of death and analyzed by GC/MS and GC equipped with NPD.5.162 rats were made of 3 groups;the one was given an intragastric administration of clozapine with a therapeutic dose;the others were given an intragastric administration of clozapine with a dose of 1/2 LD50 (130 mg/kg).The rats were execute after 2 hours,and the group of 1/2 LD50 was removalled stomach. Rats were dissected and the specimens such as heart-blood at 0,2,4,8,12,24,48,72,96h.6.15 dead rabbits were given an intragastric administration of clozapine with a dose of LD50(270mg/kg)in 1h.3 rabbits were dissected and the specimens such as heart ,liver,spleen,lung,kidney, brain,left leg muscle, right leg muscle,heart-blood,peripheral blood,bile,urina,coporis vitre at 0.5d,1d,2d,4d,and 6d respectively. The sample was extracted with alkaline ether. Then the volume was metered with ethanol. The qualitative and quantitative analyze was carried out by GC.7.6 dogs were given an intragastric administration of clozapine with a dose of 4LD50(0.605/kg).At 2h 6 dogs were executed with bloodletting femoral artery and were dissected and the specimens such as liver and blood. The qualitative and quantitative analyze was carried out by GC.8.Statistics method:The datas was collected by software SPSS11.5 and expressed byResults1.The m/z of selected ion of clozapine was 243,256 by GC/MS. The linear range of clozapine in biomaterial by GC was 0.1μg to 48μg/mL orμg/g. The regression equation, linear range, correlation coefficient, recovery rate, limit of detection of clozapine in heart blood was Y = 33.396X + 0.9248 (μg/mL),(0.1 -48)μg/mL, 0.985,(92.40±1.0)%,0.1μg/mL.2.The mean blood concentration-time profile of clozapin was fitted to a one-compartment open model with first order kinetics. The toxicokinetic equation of clozapin was Ct(1/2LD50) = 3.397028(e - 2.57018t - e– 0.27963t) ; Ct(LD50) = 2.256018(e - 2.98751t - e– 0.33022t).3.The linear range of clozapine in blood was 0.1～20μg. The clozapine display that absorption show fast while absorption slows in rats body. The distribution of clozapine in rats are rather different, but brain, lien, liver and lung higher than the other organ in the whole tendency.4.Postmortem distribution in rabbits: After the administration of clozapine, the rabbits showed less activities, lethargy state. The LD50 group rabbits died after the administration for3.24±1.17h, and the 4LD50 did for 1.33±0.25h. The order of clozapine detected in rabbits was as follows: (1) LD50 group: lung ( 108.63±39.55 ) >liver ( 46.38±9.59 ) ,brain( 44.79±38.57 ) ,spleen( 24.56±3.74 ),kidney( 24.14±2.39 ) ,bile(9.85±1.96 ) ,heart(9.70±4.40),heart blood(5.95±0.72),urine(5.02±0.57),vitreous fluid(4.92±0.55)>lower limb muscle(s4.00±1.28). (2) 4LD50 group: lung(273.16±131.24)>live(r269.03±120.75),brain(178.81±144.44),spleen(103.02±23.48),kidney(96.19±67.38),bile(36.53±25.92),heart(24.48±8.84),lower limb muscles(11.57±7.89),heart blood(10.26±2.91),vitreous fluid(7.85±4.84)>urine(5.35±0.88).5.The rats of 1/2 LD50 and removalled stomach found postmortem redistribution at postmortem 24h. The other group of 1/2 LD50 found postmortem redistribution at postmortem 2h.Rabbits of portal vein deligation found postmortem redistribution at postmortem 4h.Other rabbits was not find evident postmortem redistribution.6. After intragstric administration,the concentrations detected in heart,liver,spleen,lung,kidney,left leg muscle,right leg muscle,heart-blood,peripheral blood,corporis vitre ai 0.5d; the concentrations detected in brain and bile after 2d; it can not detected clozapine in urine at 6d. According to the results of this study, temperature,dose and irrigation stomach time can influence postmortem distribution speed.7.At different conditions, liver and blood of clozapine showed downtrend,and it was fitted to a one-compartment open model with first order kinetics.For 20℃,4℃,-20℃,20℃(NaF 1% ) ,the concentration of clozapine was 0.10%,12.14%,19.28%,62.20% of the initial concentration at 133d in blood. For 20℃,4℃,-20℃,20℃(10% formaldehyde),the concentration of clozapine was43.51%,41.27%,50.19%,10.90% of the initial concentration at 133d in liver.Conclusion1.The GC/MS and GC method is more selective, more sensitive, simpler, more accurate, and can be applied not only to the clinical diagnosis of clozapine poisoning, but also to forensic identification of clozapine poisoning death.2.Differences in Cmax, t1/2k10 and t1/2k01 have been revealed among rabbits receiving the different dose of drug ,which show that the dose of clozapine has an effect on the toxicokinetic of clozapine.3. The study has demonstrated that we should not only collect heart-blood as sample but also collect brain, lien, liver and lung when we happen to a clozapine poisoning case. According to the animal model established, we can infer to the dosage of administration following the content in heart-blood and main organ.4.The postmortem distribution of clozapine was not effected significantly by different dose. The concentration of clozapine in blood was lower than in other organs except for spleen and kidney. So, besides the blood, more organs of poisoning rats should be collected for samples in forensic identification of clozapine poisoning death.5.Postmortem redistribution took place. Stomach , portal vein deligation and different race easily affected postmortem redistribution .6.Spleen,lung,heart,liver,kidney is easily affected by postmortem diffusion.Left leg muscle, right leg muscle, coporis vitre, heart-blood , peripheral blood, brain, bile is not easily affected. Urina may be not affected. The postmortem distribution of clozapine in rats is in a maldistribution.It maybe owe to the proper nature of medicamentum,volume of blood flow ,the rate of protein binding,the distant and near of stomachal and so on.7.Temperature affected clozapine decomposition of liver and blood. Formaldehyde and NaF could evident affect clozapine decomposition in liver and blood. Formaldehyde can accelerate clozapine decomposition,and NaF can slow clozapine decomposition. |
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