Study On The Postmortem Distribution And Dynamic Distribution Of Ethanol And Its Metabolites In Rabbits

Objective:1.the establishment of rabbit blood,urine and tissue ethyl glucuronide(EtG),ethyl sulfate(EtS),fatty acid ethyl ester(FAEEs)headspace gas chromatography,high performance liquid chromatography Tandem mass spectrometry(LC-MS/MS),gas chromatography mass spectrometry(GC-MS/MS)detection methods;2.To study the distribution and dynamic distribution of ethanol and its metabolites in rabbits,the experimental basis for the forensic identification of ethanol-related cases was provided.Methods:1.post-mortem distribution model:New Zealand white rabbits 32,male,overnight fasting,were randomly divided into three groups(4LD50 group 8,2LD50 group 12,LD50 group12),oral administration to the corresponding volume diluted(2LD50 group,LD50 group 2 hours after CO2 sacrifice),the rapid death of the experimental animals(New Zealand white rabbits)after breathing,heartbeat and other vital signs disappeared(2LD50 group,LD50 group 2 hours after C02 sacrifice),the rapid development of Chinese white wine(diluted ethanol concentration of 50%),observed its performance,4LD50 Urine,brain,liver,body,liver,kidney,brain,left lower limb muscle,stomach,testis,sealed at-80 ? after the preservation of the test to be tested.2.the dynamic distribution model:New Zealand white rabbits 30,male,overnight fasting,during the experiment to add saline,experimental animals oral administration of Chinese liquor,the dose of 1/2LD50(LD5O = 6300mg/kg),after administration 0.5,1,2,4,8,12,24,48,72,96h after the death(at each time point of death 3),rapid dissection,extraction effort,peripheral blood(femoral vein),urine,Bile,heart,liver,spleen,lung,kidney,brain,left lower limb muscle,stomach,testis,sealed at-80 ? save to be checked.3.extraction detectionDetection of ethanol:100 ?L of the body to be measured in the headspace bottle,add the internal standard tert-butanol(40?g/mL)500?L,mix and take 1?L injection,headspace gas chromatograph detection,record the peak area,calculate the ethanol/Peak area ratio,the standard curve method to calculate the ethanol content.Take the test tissue 1g cut,add water 2mL homogenate for 3 minutes,3500rpm centrifugation for 10 minutes,take 100?L supernatant in the headspace bottle,add the internal standard tert-butanol(40?g/mL)500?L,mix and take 1?L Injection,headspace gas chromatograph detection,record the peak area,calculate the ethanol/internal standard peak area ratio,the standard curve method to calculate the ethanol content.EtG,EtS,:Taking a body fluid to be tested 100 ?L,was added 20 is ?Lof internal standard mixture(of EtG-d5 of,EtS-d5 of,25 ?g/mL),was added 100 ?L of water,add 800?L acetonitrile,vortexed 5min,bath sonication for 30 min 4? 13000 rpm centrifugation 10min,the supernatant into a glass tube,40 ? dry nitrogen,200 is ?L of methanol reconstitution,through the membrane,1?L injection.Tested tissues from 100 mg,cut into pieces,added to 20 is ?L of internal standard mixture(of EtG-d5 of,EtS-d5 of,25 ?g/mL),was added 100 ?L of water,add 800?L acetonitrile,vortexed 5min,water bath Ultrasound for 30min,4 ? 13000 rpm centrifugation 10min,the supernatant into a glass tube,40 ? dry nitrogen,200 is ?L of methanol reconstitution,through the membrane,.1 ?L injection.FAEEs:Suction fluid to be measured 200 is ?L,was added 50?g/mL internal standard heptadecane,ethyl ester 20 is ?L,was added 250 ?Lwater,add 1mL acetonitrile,vortex lmin,then add 5mL of ethyl acetate,vortex,ultrasound for 10min,3500rpm centrifugal 10min,the upper layer is evaporated to dryness,200 is ?L of methanol reconstitution,through the membrane,.1?Linjection.Tested tissues from 200mg cut into pieces,were added 50 ?g/mL internal standard heptadecane,ethyl ester 20 is ?L,was added 250?Lof water,was added 1mL of acetonitrile,vortexed lmin,was added 5mL of ethyl acetate,vortexed,ultrasonic 10min,3500 rpm for centrifugal 10min,the upper layer is evaporated to dryness,200 is ?L of methanol reconstitution,through the membrane,.1?L injection.4.data processing methods:WinNonlin pharmacokinetics software;GraphPad Prism 6 software;The t test and analysis of variance were used and the results expressed in x±s.Results:1.Detection of ethanol:Detection of ethanol:The lowest limit of detection(S/N = 3)and the working curve equation in blood,urine,and organ are:the minimum detection limits:1.1 mg/100 mL,1.0 mg/100 mL,1.0 mg/100 mL.The working curve equation:Y=0.04996X+0.0106,Y=0.05142X+0.0101,Y=0.05157X+0.0412.Recyc lingRate:100.27±2.34%?103.58±1.7%;intradayprecision:1.29%-1.95%;daytime precision:1.70%-2.34%;22.EtG,EtS detection:blood,urine,organ of EtG and EtS limit of detection(S/N =.3)and the working curve equation are:EtG detection limit:0.01 g/mL,0.02 g/mL,0.03 ?g/mL.Working curve equation:Y=7.844018E-004*X-0.00342,Y=4.020543E-004*X+0.058142,Y=7.703459E-004*X+0.1807.Recovery rate:85.2±7.46%‘93.34±7.92%;intraday precision:4.36%?5.24%;daytime precision:2.86%?4.87%.EtS detection limit:0.04?g/mL,0.02?g/mL,0.05 ?g/mL.Working curve equation:Y=0.005504*X-0.205045,Y=0.002367*X-0.086097,Y=0.002366*X+0.059167.Recovery rate:96.02±.77%?99.01 ±0.71%;intraday precision:2.93%?3.01%;daytime precision:0.43%?4.18%3.FAEEs detection:The lowest limit of detection of ethyl oleate and ethyl palmitate in blood,urine,and organ(S/N = 3)and the working curve equation are:the minimum detection limit of ethyl oleate:0.16?g/mL,0.05 ?g/mL,0.02 ?g/mL.The working curve equation:Y=3.963856E-005*X+0.004181,Y=4.101344E-005*X-0.001519,Y=3.665950E-005*X+0.220228.Recovery rate:94.83±1.37%?99.01 ±0.71%;intraday precision:0.68%?3.42%;daytime preci si on:3.63%-4.24%.The minimum detection limits of ethyl palmitate:0.02?g/mL,0.01 ?g/mL,0.05?g/mL.Working curve equation:Y=4.968302E-004*X-0.005896,Y=4.521989E-004-X+0.044525,Y=5.021595E-004*X-0.057003.Recovery rate:98.24=10.52%?105.85±3.76%;intraday precision:4.97%-5.39%;daytime precision:5.66%?6.76%.4.postmortem Distribution:Death rabbits each organ and its metabolites in body fluids ethanol content were respectively(LSD t-test,P<0.05)LD50 group:Ethanol content:bile,heart blood,vitreous humor,peripheral blood>urine>stomach,lung,kidney,testis,heart,liver,brain>left lower limb muscle;EtG:liver>kidney,urine>peripheral blood,stomach wall>testis,heart,lung,heart blood,left lower limb muscle,brain,bile,vitreous humor;EtS content:urine>kidney,bile,lung,liver>heart,heart blood,stomach wall,peripheral blood,testis,left lower limb muscle,brain,vitreous fluid;Ethyl oleate content:heart>liver,blood,peripheral blood,Kidney,urine,bile;Ethyl palmitate:heart>liver>lung,kidney,brain,testis>stomach wall,blood,peripheral blood,left lower limb muscle>bile,urine,vitreous humor.2LD50 group:Ethanol content:blood,vitreous humor,peripheral blood,bile>stomach wall,urine>lung,brain,heart,testis,liver,kidney>left lower limb muscle;EtG content:kidney,liver>lung,heart,Blood,stomach,peripheral blood,testis,left lower extremity muscle,urine,bile,brain,vitreous fluid;EtS content:kidney>liver,lung,heart,bile>stomach wall,blood,peripheral blood,testis,urine>brain>Left lower limb muscle,vitreous fluid;ethyl oleate content:heart>liver,stomach wall,blood,testis,peripheral blood,kidney,brain,bile,urine;Ethyl palmitate content:heart>liver>lung,kidney,testicle,brain,stomach>blood,peripheral blood,left lower limb muscle,bile,urine,vitreous humor.4LD50 group:Ethanol content:stomach>blood,bile>peripheral blood,vitreous humor>lung,liver,brain,heart,kidney,urine,testis,left lower limb muscle;EtG content:liver>kidney>heart blood,stomach wall,lung,Heart,testis,left lower limb muscle,peripheral blood,bile,brain,urine,vitreous humor;EtS content:liver,heart,lung,kidney>bile,blood,peripheral blood,urine,stomach wall,testis>left lower limb muscle,Vitreous fluid,brain;Ethyl oleate content:heart>liver,blood,stomach,testis,kidney,lung,brain,peripheral blood,bile,urine,vitreous humor;Ethyl palmitate content:heart,liver>kidney,Lungs,Testis,Brain,Stomach>Blood,left lower extremity muscle,peripheral blood,bile,urine,vitreous humor.5.Dynamic distribution:0.5,1,2,4,8h after intragastric administration,B can be detected in rabbit fluid and tissue Alcohol,after 8 hours,no ethanol content was detected in all organs of rabbits,and it was only detectable in body fluids such as blood,peripheral blood,and urine;the concentrations of ethanol and its metabolites in blood,liver,and kidney were high,EtG In the liver after 0.5 to 8 hours after intragastric administration,the concentration in the liver is much higher than that of other organs,which may be related to the liver as the main site of ethanol metabolism.Conclusion:1.Qualitative and quantitative high performance liquid chromatography tandem mass spectrometry(LC-MS/MS)and triple quadrupole mass spectrometry(GC-MS/MS)detection methods for the determination of EtG,EtS and FAEEs in biological samples established in this paper.With good reproducibility,high recovery,high specificity and high sensitivity,it can be used to provide experimental evidence for the forensic identification of ethanol-related cases.2.Ethanol and its metabolites are not uniformly distributed in the infected rabbits,and the content of ethanol in body fluids is significantly higher than that in the organs;EtG,EtS are higher in the liver and kidney,and the content in brain and vitreous humor is low.Ethyl oleate was the highest in the heart,followed by the liver,and little in the body fluids of the other organs;fatty acid ethyl esters were higher in the organs than in the body fluids.3.Comparison of the dynamic distribution of ethanol and its metabolites,suggesting that ethanol metabolites can be used as a marker for ethanol administration,provide experimental evidence for forensic medical identification in cases of ethanol poisoning(death).