A Pilot Study On The Relationship Between Ammonia,Polyamines And Postmortem Submersion Interval And Research On The Detection Method Of Urea

Objective: The estimation of postmortem interval of submersed corpses is different from that of corpses on land.Chemical substances produced by corruption in the body are less affected by the external environment,which can be used to assist in the estimation of postmortem interval.After death,under the action of bacteria,protein and other nitrogencontaining substances can be decomposed into ammonia,putrescine and cadaverine.With the passage of postmortem interval,these small molecules of nitrogen-containing substances continue to increase,and have a certain correlation with postmortem submersion interval.This study aims to use derivatization methods to detect ammonia and polyamines in the liver of submersed rats after death,and explore their relationships with postmortem submersion interval;And on the basis of ammonia detection,a gas chromatography-mass spectrometry detection method combing enzymatic method and derivatization method was developed to supplement the methods of urea determination.Methods: This research was divided into three parts.Part Ⅰ: Took healthy SD rats.After being sacrificed by cervical dislocation,they were submersed in river water(28.1°C)and a water tank filled with room temperature river water(25°C).Rat liver tissue were taken at different times and pre-processing operations were performed.Gas chromatographymass spectrometry was used to determine the ammonia content in the rat liver,and Origin2018 was used to fit the ammonia content in the liver at different postmortem submersion interval time points to obtain a fitting curve.Part Ⅱ: Took healthy SD rats.After being sacrificed by cervical dislocation,they were submersed separately in natural river water(summer: 28.1°C,autumn: 13.3°C)and a water tank filled with room temperature river water(summer: 25°C,autumn:15°C).The rat liver was taken at different time points,after derivatization,the content of polyamines in the liver was detected by gas chromatography-mass spectrometry,and Origin 2018 was used to fit the content of polyamines in rat liver at different postmortem submersed time points,and the fitting curve was obtained.Part Ⅲ:Urea in the sample was decomposed into ammonia by urease,and then the ammonia molecule was combined with the derivative reagent molecule.Through the standard control method,gas chromatography-mass spectrometry was used to detect the content of ammonia derivative,then urea content in the sample was calculated.Results: Part of the method optimization: 1.The derivatization of ammonia and polyamines with Heptafluorobutyryl chloride was the best in p H 14 sodium hydroxide solution.2.The derivatization temperature should be controlled at about 40 ° C to make the reaction more complete.3.The ratio of ethyl acetate to n-hexane was 1:1,which can solve the problem of emulsification,and the extraction effect was good.4.The derivatization reaction of Heptafluorobutyryl chloride with ammonia and polyamines was rapid and complete in 5 minutes.Part of the animal model: 1.The content of ammonia in the liver of rat increased with the prolongation of postmortem submersion interval.The content of ammonia in the liver of rat in the room temperature submersed group and the river water submersed group showed similar changes,showing a trend of first fast and then slow and then fast.2.The contents of putrescine and cadaverine in rat liver increased with the prolongation of postmortem submersion interval,while the contents of spermidine and spermine decreased slowly with time.3.The higher the temperature of the submerged environment,the faster the content of ammonia,putrescine and cadaverine increased with postmortem submersion interval in the rat liver.Conclusion: 1.The changes of ammonia and polyamines contents in the liver of rat after death are correlated with postmortem submersion interval,which is expected to provide an auxiliary role for the estimation of postmortem submersion interval.2.Higher temperature can promote the production of ammonia,putrescine and cadaverine in postmortem submersed corpse liver,while lower temperature can slow down the production of the three,which may be related to the proliferation of microorganisms.3.The combination of enzyme and chemical methods and the gas chromatography-mass spectrometry has high sensitivity and specificity,simple operation and good linearity,which is expected to solve the problem of difficult qualitative and quantitative of urea in forensic identification.