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

Objective1. To establish the simultaneous analysis of diazepam and its metabolites in living samples by SPE-HPLC.2. To establish the dynamic distribution and postmortem diffusion models of diazepam in rabbits.3. To study the dynamic distribution and postmortem diffusion of diazepam and its metabolites in rabbit. To provide a scientific evidence for the forensic identification of diazepam poisoning death cases.Methods1. Extraction and analysis method:Estazolam (internal standard), diazepam, nordiazepam, oxazepam, oxazepam glucuronide, temazepam glucuronide were added in specimens. Solid phase extraction (SPE) and HPLC were used to extract and analyse diazepam in specimens. Based on extraction rate, SPE column was choosen. The qualitative analysis was based on retention time in the chromatographic system. The quantitative analysis was based on an internal standard method and calibration curve.2. The dynamic distribution:After being administrated intragastricly with a diazepam of58.3mg/kg, the rabbits were put to death at8h,12h,24h, and the specimens such as brain, heart, lung, liver, kidney, spleen, urine, muscle, bile, vitreous humor, heart-blood and peripheral blood were collected. Diazepam and its metabolites in all specimens were detected by a SPE-HPLC.3. The postmortem diffusion: Group1:Fitteen dead rabbits were given a postmortem intragastric administration of diazepam with a dose of53.8mg/kg at1h after death. Three rabbits were dissected and the specimens such as brain, heart, lung, liver, kidney, spleen,urine, muscle, bile, vitreous humor, heart-blood and peripheral blood were taken at2h,6h,12h,24h,48h, and96h after the postmortem administration respectively. Diazepam and its metabolites in all specimens were detected by a SPE-HPLC.Group2:Nine dead rabbits were given a postmortem intragastric administration of diazepam with a dose of53.8mg/kg at1h after death. Three out of them were put at room temperature,4℃and-20℃respectively. Rabbits were dissected and the specimens as group1’s were collected at48h after the postmortem administration. Diazepam and its metabolites in all specimens were detected by a SPE-HPLC.Group3:Nine dead rabbits were put at room temperature, three out of which were given an intragastric administration of diazepam with a dose of53.8mg/kg,29.2mg/kg and14.6mg/kg respectively at1h after the death. Rabbits were dissected and the specimens as group1’s were collected at48h after the postmortem administration. Diazepam and its metabolites in all specimens were detected by a SPE-HPLC.Results1. Compared with three solid phase column, C18column was selected for extraction. The diazepam, nordiazepam, oxazepam, estazolam, oxazepam glucuronide, temazepam glucuronide could be detected simultaneously by a SPE-HPLC. The limit of detection was1ng for diazepam, nordiazepam and oxazepam, and2ng for oxazepam glucuronide and temazepam glucuronide respectively. The limit of quantification was30ng/mL for diazepam, nordiazepam and oxazepam, and60ng/mL for oxazepam glucuronide and temazepam glucuronide, the linearity was over the range of30-2500ng/mL and60-2500ng/mL respectively.2. Dynamic distribution:At8,12,24h after the postmortem administration, diazepam, nordiazepam and oxazepam were detected in all collected body fluid and tissue of rabbits. Except for in humor vitreous, brain and muscles, oxazepam glucuronide and temazepam glucuronide were detected in all collected body fluid and tissue of rabbits. The concentrations of diazepam and its metabolites were higher in blood, liver, kidney, lung, spleen and brain. The concentrations of metabolites were higher in urine. At8,12,24h after the postmortem administration, the ratio of the concentration of diazepam to the concentration of nordiazepam (Cdiazepam/Cnordiazepam) in peripheral blood werel.37,0.53,0.3respectively. At8,12,24h after the postmortem administration, the ratio of the concentration of oxazepam to the concentration of oxazepam glucuronide (Coxazepam/Coxazepam glucuronide) in urine were0.5,0.7,0.74respectively.3. Postmortem diffusion:At2h after the postmortem administration of diazepam, diazepam was only detected in spleen of rabbits. At48h after the postmortem administration of diazepam, diazepam was detected in all specimens of rabbits. Time, temperature and dose showed an affection on the postmortem diffusion. All metabolites of diazepam could not be detected in all collected specimens.Conclusion1. A simultaneous analysis of diazepam and its metabolites in living samples by SPE-HPLC was established, which can be used for the detection of diazepam and its metabolites in the study on the forensic toxicokinetics and the forensic identification of diazepam poisoning death.2. The dynamic distribution and postmortem diffusion models of diazepam in rabbits were established, which could be applied to studies on the forensic toxicokinetics of diazepam. 3. There were a maldistribution of diazepam and its metabolites in rabbit. The concentrations of diazepam and its metabolites in blood, liver, kidney, brai n were higher and the concentrations of metabolites in urine were higher. Thes e can provide experimental evidence for the specimen collection, judging admi nistration route and death time.4. There was a postmortem diffusion of diazepam in rabbits, which related to the time after postmortem administration, the storing temperature of cadaver and administration dose. Diazepam had been detected in some specimens of rabbits at96h after the postmortem administration, but no metabolites of diazepam had been detected in all collected specimens.5. The dynamic distribution and postmortem diffusion of diazepam and its metabolites in rabbits indicated that metabolites of diazepam, especially oxazepam glucuronide and temazepam glucuronide, can be used as the markers of antemortem administration of diazepam, and provide experimental evidence for identifying antemortem taken diazepam and postmortem given diazepam.