Postmortem Redistribution Study Of Remifentanil And Its Metabolites

Objective:1.Develop a high-performance liquid chromatography-tandem mass spectrometry（HPLC-MS/MS）method for the detection of remifentanil and its two major metabolites,remifentanil acid and norcarfentanil,in biological specimens.2.The distribution model of remifentanil and its metabolites was established to provide experimental basis for the extraction of samples in related cases.3.To establish a homographic model of the postmortem redistribution of remifentanil and its metabolites,explore the postmortem redistribution law of remifentanil and its metabolites,and study the main mechanism of its postmortem redistribution,so as to provide technical support and court evidence for forensic identification of related cases.Methods:1.Establishment of sample pretreatment and detection methods200μL of the test blood and urine were extracted,and 200 mg of tissue homogenate was weighed and added with internal standard Remifentanil -¹³C₆ after being acidified with citric acid.The protein was precipitated with acetonitrile and centrifuged at high speed for 10 minutes.The supernatant was dried under a nitrogen flow at 35℃ and reconstituted with 200μL of the initial mobile phase.After passing through a 0.22μm organic filter membrane,the sample was analyzed using HPLC-MS/MS.2.Establishment of animal modelsSix male Bama miniature pigs with a body weight of 20kg±2kg were adaptively fed for one week and fasted for 12 hours before the experiment.The therapeutic dose of remifentanil hydrochloride（60μg/kg,converted based on the body surface area of humans and pigs）was injected via ear vein,followed by euthanasia under hypoxia.Distribution group（n=6）: Blood,urine,heart,liver,spleen,lung,kidney,gastric wall,brain,right lower limb muscle,and testes were collected immediately after death.Postmortem redistribution group: The deceased Bama miniature pigs were randomly divided into room temperature group and 4℃ group（n=3）and placed in a supine position in climate boxes maintained at room temperature（20～24℃）and 4℃,respectively.Samples were collected at 0h,6h,12 h,1d,2d,3d,4d,5d,6d,7d,10 d,and15d after death（same as postmortem distribution group）.The concentration of remifentanil and its metabolites in each specimen was detected using the established method mentioned above.Results:1.Detection and analysis of remifentanil and its metabolites by HPLC-MS/MS:The concentrations of remifentanil in blood,urine,and liver showed good linear correlation in the range of 0.1～100ng/mL,with detection limits of 0.03ng/mL,0.01ng/mL,and 0.02ng/mL,respectively,and quantification limits of 0.1ng/mL,0.03ng/mL,and 0.05ng/mL,respectively.The concentrations of remifentanil acid in blood,urine,and liver were linearly related within the range of 0.2～200ng/mL,with detection limits of 0.015ng/mL,0.01ng/mL,and 0.02ng/mL,and quantification limits of 0.1ng/mL,0.04ng/mL,and0.05ng/mL,respectively.The concentrations of norcarfentanil in blood,urine,and liver were linearly related within the range of 0.1～10ng/mL,with detection limits of 0.0125ng/mL,0.01ng/mL,and0.02ng/mL,and quantification limits of 0.05ng/mL,0.05ng/mL,and 0.05ng/mL,respectively.The correlation coefficients（R²）for the concentration of remifentanil and its metabolites were all >0.999.The intra-day and inter-day precision were both <13.67%.The extraction recoveries were all >80%.The matrix effects were all in the range of3.69%～14.56%.The stability of the samples in the auto-sampler showed a relative deviation of within 12.5%.2.The distribution of remifentanil and its metabolites in the body of Bama miniature pigsAfter ear vein injection of remifentanil hydrochloride in Bama miniature pigs,the above method was used to detect remifentanil and its metabolites in various specimens.According to statistical analysis,the distribution of remifentanil in the body of pigs was:spleen,kidney > cardiac blood,peripheral blood,heart,liver,lung,stomach,brain,muscle,testes,urine,P<0.05;the distribution of remifentanil acid in the body of pigs was:kidney,lung,cardiac blood,brain,heart,liver,spleen,peripheral blood> muscle,testes,stomach,urine,P<0.05;the distribution of norcarfentanil in the body of pigs was: liver,kidney> cardiac blood,peripheral blood,heart,spleen,lung,stomach,brain,muscle,testes,P<0.05,all with statistical significance.3.The postmortem redistribution of remifentanil and its metabolites in the body of Bama miniature pigsUnder ambient temperature storage conditions,the concentration of remifentanil in the spleen,kidney,and brain decreased with the prolongation of death time（P<0.05）;while the concentration of remifentanil acid in the spleen,lung,and brain increased with the prolongation of death time（P<0.05）.The concentration of norcarfentanil in the heart and gastric wall changed significantly（P<0.05）.Under 4℃ storage conditions,the trend of changes in remifentanil and its metabolites in various organs and tissues was consistent with that under ambient temperature conditions.The original data of the concentrations of remifentanil and its metabolites in various biological samples,as well as the study of the ratio of C_{postmortem time/}C_{at the time of death},demonstrate that postmortem redistribution of remifentanil and its metabolites occurs in Bama miniature pigs when the ratio of C_{postmortem time}/C_{at the time of death} is ≥1.15 or ≤0.85.Conclusion:1.This study established a sample preparation method and an HPLC-MS/MS qualitative and quantitative method for simultaneously detecting the opioid analgesic,remifentanil,and its metabolites,remifentanil acid and norcarfentanil,in biological samples.The linear range,sensitivity,accuracy,precision,and detection limit of the method all meet the requirements of sample analysis in the field of forensic toxicology.This method can provide technical support for clinical drug concentration monitoring and forensic identification of related cases.2.This study established a distribution model of remifentanil in Bama miniature pigs and found that remifentanil and its metabolites have higher concentrations in the spleen,kidneys,and heart,indicating that in cases related to remifentanil,besides collecting routine specimens such as blood and urine,toxicological analysis can also be performed on biological specimens such as kidneys,spleen,and heart.3.Established a postmortem redistribution model of remifentanil in Bama miniature pigs,and found that remifentanil and its metabolites exhibited postmortem redistribution in the body of the pigs at both room temperature and 4℃ storage conditions.The trend of concentration changes of remifentanil and its metabolites with the prolonged postmortem interval was basically consistent,and the influence of redistribution on muscle,gastric wall,and testis was relatively small.Therefore,in the forensic identification of remifentanil,in addition to analyzing the concentration of remifentanil and its metabolites in the heart and peripheral blood,qualitative and quantitative analysis of muscle,gastric wall,and testis can also be used as an auxiliary criterion,fully considering the influence of postmortem interval on the concentration of remifentanil and its metabolites,and the influence of postmortem redistribution on toxicological analysis results,to make scientific and objective forensic identification conclusions.