| Objective:1. To develop a postmortem distribution model and a postmortem diffusion model of tramadol in rabbits; To establish a decomposition kinetics model of tramadol in preserved specimens of dogs.2. To study the postmortem distribution and postmortem diffusion of tramadol in rabbits and decomposition kinetics of tramadol in preserved specimens of dogs, and to provide a scientific evidence for the forensic identification of tramadol poisoning death.Methods:1. Postmortem distribution. Six rabbits were given an intragastric administration (ig) of tramadol with a dose of 10 LD50. As soon as the blood pressure, respiration and ECG disappeared, the rabbits were dissected, and the specimens such as heart blood, bile, vitreous humor, urine, right upper limb muscles, the right lower limb muscles, chest muscle, heart, liver, spleen, lung, kidney, brain, stomach were sampled immediately, in which the concentration of tramadol was detected qualitatively and quantitative by GC/MS and GC/NPD.2. Postmortem diffusion.24 male rabbits were allocated randomly to postmortem diffusion group (n= 15) and dose effected group (n= 9).Postmortem diffusion group:After being executed by an aeroembolism for 2 h,15 rabbits were given a 1/4LD5odose of tramadol by ig and placed at room temperature(20℃). Three animals were randomly dissected after postmortem administration of tramadol at 0.25h,0.5h,1h, h3 and 6h respectively and the specimen such as blood-heart, peripheral blood, bile, urine, vitreous humor, the right upper limb muscles, the right lower limb muscles, chest muscle, heart, liver, spleen, lung, kidney, brain, stomach, were sampled and stored at-20℃in refrigerator for detection, in which the tramadol was detected qualitatively and quantitative by GC/MS and GC/NPD.Dose affected group:After being executed by an aeroembolism for 2 h, three of nine rabbits were given a 1/8LD50, 1/4LD50 or 1/2LD50 dose of tramadol by ig and placed at room temperature(20℃). After the postmortem administration for 0.25h, the rabbits were dissected and the specimen such as blood-heart, peripheral blood, bile, urine, vitreous humor, the right upper limb muscles, the right lower limb muscles, chest muscle, heart, liver, spleen, lung, kidney, brain, stomach, were sampled and stored at-20℃in refrigerator for detection, in which the tramadol was detected qualitatively and quantitative by GC/MS and GC/NPD.3. Decomposition kinetics of tramadol in preserved specimen of dogs. After being given an intragastric administration of tramadol with a dose of 4LD50 for 2h,6 dogs were executed, and the heart-blood and liver of every dog were sampled and divided into for parts, and preserved at 20℃,-4℃,-20℃,20℃(NaF was added to 1% in blood and liver was fixed in 4% formaldehyde) respectively, in which tramadol was detected qualitatively and quantitative by GC/MS and GC/NPD on 0 h,7thd,27thd,53thd,99thd,156thd and 220thd after the preservation. The mean C-T data of tramadol in preserved blood and liver was manipulated by WinNorLin's pharmacokinetics software. The kinetic model was decided by AIC information criterion. The decomposition kinetic equation and parameter were estimated and calculated.4. Statistics Data was expressed as mean±SD model and analyzed statistically with SPSS 11.5 software by LSD t-test.Result:1. Postmortem distribution:After the administration of tramadol for 0.5±0.25h, some toxic symptoms were observed in rabbits and the death occurred in 2±0.5h later. The content of tramadol detected in poisoning death rabbits were as follows: kidney(162.96±15.27)>gaster(35.27±14.48), liver(130.22±53.65), spleen(112.48±14.16)> lungs (83.04±19.36), brain(73.92±32.20), heart(71.86±12.16), muscles of upper extremity (57.63±19.92), muscles of lower limb,54.77±17.40> urine (1.44±0.07), bile(0.72±0.13), heart blood flow(0.72±0.19), humor vitreous (0.38±0.05) (P<0.05).2. Postmortem diffusion. Except for in humor vitreous, bile, urine, brain, lung, muscles of upper extremity, muscles of lower limb of rabbits at 0.25h after the postmortem administration of 1/8 LD50, tramadol was detected in all other specimens sampled in postmortem diffusion group and dose effected group at 0.25-6h after the postmortem administration. Tramadol concentration detected in rabbits of dose affected group showed a dose-dependent rise.Compared with tramadol detected in poisoning death rabbits at a IOLD50 dose, the highest concentration of tramadol detected in all tissue specimen of postmortem diffusion group rabbits sampled at 0.25-6h after the postmortem administration and was lower (P<0.05), while in body fluid was higher; Tramadol concentration detected in all tissue specimen of postmortem diffusion group rabbits given the 1/2LD50 dose was lower, while in body fluid was higher(P<0.05); Tramadol concentration detected in tissue and body fluid specimen of postmortem diffusion group rabbits given the 1/8LD50 dose was lower(P<0.05).3. Decomposition kinetics. The concentrations of tramadol detected in preserved blood and the liver descended in 220ds. On 156th day, the concentration of tramadol in stored blood was lower than at 0h(p<0.05). On 27th day,53th day,156th day,27th day, the concentration of tramadol in blood stored at 20℃,4℃,-20℃,20℃(4% formaldehyde) was lower than at Oh (p< 0.05). On 220th day, the concentrations of tramadol in stored blood and liver decreased to 41.9%(20℃),41.4%(4℃),50.0%(-20℃),63.7%(20℃,1% NaF) and 42.9%(20℃),49.5%(4℃), 49.4%(-20℃),55.6%(20℃,4% formaldehyde) of the initial content.4. The decomposition kinetics of tramadol in blood and liver met the one compartment open model with a first order kinetics, and could be expressed as CT=Coe-KeT. The decomposition half-lifes (t1/2) of in blood and livers of poisoned dogs, which were stored at and 20℃,4℃,-20℃, 20℃(1% NaF in blood or liver in 4% formaldehyde),were 200.5d,163.3d,190.9d,307.3d and 137.8d,175.2d,268.8d,241.3d. The observed values of tramadol content in preserved blood and liver were close to the predicted values calculated by CT=Coe-KeT.Conclusion:1. The postmortem distribution model (10LD50,ig,) of tramadol in rabbits, postmortem diffusion model (1/8,1/4,1/2LD50,ig) of tramadol in rabbits, and decomposition kinetics model (4LD50,ig) of tramadol in preserved samples of dogs have been developed, which can be applied to forensic identification and forensic toxicokinetics of tramadol poisoning death case.2. The order of tramadol contents detected in poisoning death rabbits was kidney, stomach, liver, spleen, lung, brain, heart, upper muscle, lower limb muscle, urine, bile, efforts, and vitreous humor. It was suggested that besides blood, gastric (stomach content), the kidney and liver should be sampled as specimen for analysis in the forensic identification of tramadol poisoning death case. The postmortem distribution can be used for estimating the tramadol in blood in the extreme case (example for dismembered body case).3. There was a dose-dependent postmortem diffusion of tramadol in rabbits. Only not in the early time after the postmortem administration of 1/8 LD50, was tramadol detected in brain and vitreous of rabbits. The highest concentration of tramadol detected in the body fluids specimen of rabbits from 0.25h to 6h after the postmortem administration of 1/4LD50, so was in the body fluids specimen of rabbits at 0.25h. It should be taken into consideration that the postmortem diffusion and postmortem redistribution can affect the analysis of tramadol in the forensic identification of tramadol poisoning death. The postmortem diffusion can be used for the identification of antemortem administration and postmortem administration of tramadol too.4. Tramadol in the blood and the liver can be decomposed. Low-temperature preservation of specimen can make the decomposition slow down. It suggests that the specimen for analysis of tramadol should be submitted within 1 month in the forensic identification of tramadol poisoning death case; otherwise, samples should be frozen, submitted and analyzed as soon as possible.5. The decomposition kinetics of tramadol in stored blood and the liver meets the one compartment open model with a first order kinetics. In the forensic identification of tramadol poisoning death, the equation as CT=Coe-KeT and decomposition kinetics parameter can be used to estimate the tramadol concentration in the specimens at sampled time, and provide a scientific evidence for the forensic identification of tramadol poisoning death case. |
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