| ObjectivesTo optimize a qualitative and quantitative method for determination of estazolam in biological samples by high-performance liquid chromatography. To develop an animal model for the toxicokinetics, distribution,postmortem redistribution of estazolam. Study the toxicokinetics, distribution, postmortem redistribution of estazolam in rats and stability of estazolam in dogs.Methods1.HPLC: After adding diazepam (Internal Standard, IS), the biological samples were alkalized and extracted with aether.,The extraction were analyzed qualitatively and quantitatively by high-performance liquid chromatography with mobile phase (methanol:water=75:25).2.Toxicokinetics and dynamic distribution of estazolam in rats: The rats of experimental group (experimental group 40 and control group 8) were given an intragastric administration of a LD50 dose of estazolam(2.85mg/g). The heart blood,heart,liver,spleen,lung,kidney,brain and stomach were collected at 0.5h, 1h, 2h, 5h, 12h, 24h, 48h, 72h after administration. The biological samples were extracted with aether, the extraction were analyzed by high-performance liquid chromatography.3. Distribution of estazolam in rats: The rats of experimental group (experimental group 10 and control group 2)were given an intragastric administration of estazolam with a dose of LD50 (2.85mg/g)and 2 LD50 (5.70mg/g). The rats were executed after 1h and the specimens such as heart blood, heart,liver, spleen, lung, kidney, brain, stomach and muscle were sampled immediately. Concentration of estazolam was determined qualitatively and quantitatively by high-performance liquid chromatography after samples being extracted.4. Postmortem redistribution of estazolam in rats: After being given an intragastric administration of estazolam with a dose of 0.108mg/g for 3h, the rats of experimental group (experimental group 35 and control group 5)were executed and preserved at 20℃. Specimens such as heart blood, heart, liver, spleen, lung, kidney, brain and muscle were sampled at 0h , 2h, 4h, 8h, 12h, 24 h, 48h after rats death, respectively. Concentration of estazolam was determined qualitatively and quantitatively by high-performance liquid chromatography after samples being extracted. 5. Stability of estazolam in preserved biological samples: Five dogs were intragastric administration of estazolam with a dose of 37.6mg/kg.When the dogs were executed after administration of estazolam three hours, the heart-blood and liver of every dog were sampled and divided equally four parts respectively. The heart-blood and liver were preserved at -20℃, 4℃, 20℃and 20℃(1% sodium fluoride solution adding in blood). The estazolam in preserved samples was extracted and determined by high-performance liquid chromatography at 0d, 1d, 10d, 30d, 90d, 150d and 210d.6. Stability of estazolam in tissues of dogs preserved in formaldehyde solution: A dog was intragastric administration of estazolam with a dose of 37.6mg/kg.When the dog was executed after administration of estazolam three hours, heart,liver,kidney and brain of the dogs were cut up into 1g and preserved in 4% formaldehyde solution respectively. The estazolam in preserved samples was extracted and determined by high-performance liquid chromatography at 0d, 1d, 7d, 14d, 21d, 28d ,35d,42d,49d,56dand 63d.Results1. The mean serum concentration-time profile of estazolam after intragastric administration of 2.85mg/g to rats was fitted to two-compartment model with a first kinetics. Tmax =5.570h, K10= 0.046h-1, t1/2k10=30.250h, V=450.974L/kg, AUC=3347.159, Cmax=45.286μg/mL.2. The estazolam's contents in stomach, kidney and heart were more than other tissues from 3h after a LD50 dose.The order of the estazolam's contents detected in poisoned rats after a LD50 dose was stomac(h58.212±4.649μg/g)﹥splee(n24.211±2.310μg/g)﹥live(r23.284±3.217μg/g)﹥heart(18.453±6.240μg/g)﹥lung(17.833±1.942μg/g)﹥brain(15.442±6.994μg/g)﹥kidney(14.975±3.378μg/g)﹥heart blood(11.061±2.012μg/g) at 1h. The order of the estazolam's contents detected in poisoned rats after a 2 LD50 dose was stomach(288.230±54.067μg/g)﹥(109.258±19.445μg/g)﹥liver(67.990±30.607μg/g)﹥heart(59.877±33.427μg/g)﹥spleen(37.528±30.270μg/g)﹥lung(35.332±17.134μg/g)﹥brain(33.534±20.257μg/g)﹥kidney(25.509±8.162μg/g)at 1h.3. The contents of estazolam in heart blood,liver,kidney of dead rats stored at 20℃significantly increased at 12h after death(P<0.05)and then decreased with time prolonging.The content of estazolam in spleen was significantly increased(P<0.05)and significantly decreased in heart and muscle at 8h after death(P<0.05). The content of estazolam in lung was significantly decreased at 4h(P<0.05)and in brain significantly decreased at 2h after death(P<0.05).4. The contents of estazolam in blood of poisoned dogs stored at 1d,10d,30d,90d,150d and 210d(4℃,20℃and -20℃)were no significant difference compare with that at 0d (P>0.05). When livers of poisoned dogs were respectively stored at 4℃and -20℃, the contents of estazolam in livers stored at1d,10d,30d,90d,150d and 210d were no significant difference compare with that at 0d (P>0.05).However,the contents of estazolam in tissues fixed in 4% formaldehyde(20℃) were significantly decreased from 1st day(P<0.05).Conclusion1. An animal model for the toxicokinetics, distribution , postmortem redistribution of estazolam have been developed, which can be applied to the study on forensic toxicokinetics of estazolam.2. Toxicokinetics of estazolam in rats fitted to two-compartment model with a first kinetics. The eliminate of estazolam in rats is slower.3. The distribution of estazolam in rats at diffirent time is different. Estazolam distributions with different dose of estazolam in rats have different trends.4. There is a postmortem redistribution of estazolam in poisoned rats. The content of estazolam in heart blood, liver, kidney and spleen significantly increased after death then decreased with time prolonging. The content of estazolam in heart, lung, brain and muscle has a trend of decrease. It suggests that postmortem redistribution should be taken into consideration in the forensic identification of estazolam poisoned death and the anatomy should be carried out within postmortem 8h.5. Estazolam in blood stored at 4℃,20℃and -20℃is stable, the tempareture can not influence the stability of estazolam. Estazolam in livers stored at 4℃and -20℃is also stable ,but when tissues were fixed in 4% formaldehyde(20℃),the content of estazolam was significantly decreased. So samples for determination of estazolam can not be fixed in formaldehyde.
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