Study On The Postmortem Distribution And Redistribution Of Propofol And Its Metabolites

Objective:1.To establish a high performance liquid chromatography-tandem mass spectrometry and gas chromatography-tandem mass spectrometry analysis method for propofol and its metabolites in biological samples;2.To establish animal models of postmortem distribution and redistribution of propofol and its metabolites;3.To study the postmortem distribution and redistribution of propofol and its metabolites in experimental animals,and provide experimental evidence of forensic toxicological results interpretation associate with propofol anesthesia cases.Methods:1.Sample preprocessing:The concentration of propofol and 4-hydroxy propofol were detected by gas chromatography-tandem mass spectrometry.The concentration of propofol glucuronide was analyzed by high performance liquid chromatography-tandem mass spectrometry.The thymol was used as internal standard and added in the sample after homogenization,.The samples were extracted by cyclohexane and concentrated to dryness under nitrogen at water bath.Then the residue was dissolved in methanol for GC/MS analysis.The samples were extracted by acetonitrile after homogenization and concentrated to dryness under nitrogen at water bath..The residue was reacted 30 min with pyridine and MSTFA at 50°C water bath..The reaction solution was used for GC/MS analysis after cooling down to room temperature.;The samples were extracted by acetonitrile after homogenization and concentrated to dryness under nitrogen at water bath.The residue was dissolved in methanol for LC/MS analysis.The all above mentioned compounds were analyzed by MRM mode.The qualitative was used by retention time and ion ratio.The standard curve was used to calculate the concentration of compounds in samples.2.Animal model:Animal model of postmortem distribution:Male Sprague-Dawley rats were injected propofol via the tail vein with clinical therapeutically maximal doses（6.3 x 2 mg/kg）and LD₅₀ doses（42 mg/kg）.the heart blood,heart muscle,liver,spleen,lung,kidney,brain tissue,left lower extremity muscle,and testicles were collected after death.The samples were placed at-20°C until analysis.Animal model of postmortem redistribution:Bama minipigs were injected clinical therapy dose propofol（1.1 x 2mg/kg）through jugular vein and sacrificed by asphyxia,The heart blood,peripheral blood,myocardium,liver,lung,kidney,brain tissue,and left lower extremity muscle were collected at different postmortem interval time（6h,12h,24h,48h,72h,96h,120h,144h,168h）.The samples were placed at-20°C until analysis.3.Statistical analysis:Statistical analyses were performed with the SPSS program13.0.Descriptive analysis of the data（mean,median,standard deviation and range）were determined and expressed as mean±standard deviation（SD）.Statistical dependence analysis between variables was analyzed using normal distribution and homogeneity of variance.A value of P≤0.05 was considered statistically significant.Results:1.Method validation of propofol and its metabolitesA calibration curve for each biological matrix was generated.The limit of detection（LOD）of propofol in blood and liver was found to be at 2 ng/m L or ng/g level using a signal-to-noise（S/N）ratio of 3:1,and the limit of quantitation（LOQ）was 8 ng/m L or ng/g using an S/N ratio of 10:1.The calibration curves for propofol ranged from 0.0625μg/m L orμg/g to 10μg/mL orμg/g;The limit of detection（LOD）of 4-hydroxy propofol in blood and liver was found to be at 1 ng/mL or ng/g,and the limit of quantitation（LOQ）was 4 ng/mL or ng/g.The calibration curves for 4-hydroxy propofol ranged from0.0625μg/m L orμg/g to 10μg/mL orμg/g;The limit of detection（LOD）of glucuronidol in blood and liver was found to be at 0.25 ng/m L and 0.5 ng/g,and the limit of quantitation（LOQ）was 1 ng/m L and 2 ng/g.The calibration curves for glucuronidol ranged from 0.03125μg/mL orμg/g to 5μg/m L orμg/g.The correlation coefficient（R²）>0.990.99;Method accuracy（represented by relative error of the mean REM）and precision（represented by relative standard deviation RSD）were determined based on the quality control data.The intra-day%REM and%RSD were less than 9.14%.The inter-day%REM and%RSD were less than 9.1%.And the recovery was from 92.11%to109.4%.2.Postmortem distribution of propofol and its metabolitesThe level of propofol in rats which was administrated LD₅₀ dose of propofol by intravenously was following:blood>heart>brain>kidney>liver>lung>muscle>testis>spleen;the level of 4-hydroxy propofol was following:Lung>Kidney>Liver>Heart>Blood>Spleen>Testis>Muscle>Brain;the level of glucuronide propofol was following:Liver>Kidney>Blood>Testis>Lung>Spleen>Heart>Muscle>Brain.The level of propofol in rats which was administrated clinical dose of propofol by intravenously was following:liver>kidney>brain>heart>testis>lung>muscle>blood>spleen;the level of 4-hydroxy propofol was following:kidney>lung>blood>muscle>testis>liver>heart>spleen>brain;the level of glucuronide propofol was following:liver>lung>kidney>Testis>Blood>Spleen>Heart>Muscle>Brain.The distribution of propofol and its metabolites in rats after the death showed differences in dose and tissue.The postmortem distribution of propofol was related to administered dose.There seemed no related with administered dose at postmortem distribution of propofol metabolites.3.Postmortem redistribution of propofol and its metabolitesThe postmortem redistribution of propofol and its metabolites was observed at room temperature,4°C and-20°C storage conditions after administrated propofol in Bama minipigs,.at room temperature,the concentration of propofol in blood is stable after 6 hours of death;There was no significant change with the time of death in muscle and brain of 4-hydroxy-propofol;the concentration of glucuronidol in the heart and muscle did not change significantly with the time of death.At 4°C,the concentration of propofol in blood did not change significantly with the time of death;the concentration of 4-hydroxy-propofol in muscle and brain was stable with the time of death;the concentration of glucuronidol in the heart and muscle was not changed at postmortem interval time.At-20°C,the concentration of propofol in blood was relatively stable at0-48h and 96-168h.The concentration of 4-hydroxy-propofol in heart and blood did not change significantly with the time of death.The glucosaldehyde in the heart and muscle was stable.Conclusion:1.This study established a high performance liquid chromatography-tandem mass spectrometry and gas chromatography-tandem mass spectrometry analysis method of propofol and its metabolites in biological samples.The method can be used with forensic toxicological results interpretation of cases involving propofol anesthesia.2.This study established a postmortem distribution and redistribution animal model of administrated propofol.The models could be used for the study of forensic toxicology studies involving propofol anesthesia cases.3.The postmortem distribution is very different between the administrated LD₅₀ and clinically dose.There was not observed obvious different in propofol’s metabolites.4.The postmortem redistribution was found in Bpa minipigs after administrated propofol at room temperature,4°C,and-20°C,respectively.The samples including blood,heart,muscle and brain should be collected in cases about propofol.