Study On The Distribution Of The Toxic Amount Of Succinylcholine Chloride In Rats And The Determination Of Its Intermediate Metabolites

Background:Succinylcholine chloride（Suc）is a muscle relaxant used in general anesthesia.If injected in excess or not on a ventilator,the drug can cause cardiac arrest,respiratory paralysis and death.It used to be a class B organic drama drug.In recent years,succinylcholine chloride has been used by criminals in darts to kill poultry,livestock and even as a killing tool.Suc in under the action of butyryl cholinesterase rapidly into muscle relaxant effect is very weak succinylmonocholine chloride.Then,under the action of the enzyme continue to be metabolized without muscle relaxant effect of succinic acid and choline,only about 2%of the prototype drug exists in the body,and this two kinds of decomposition in the body exists,this gives succinylcholine chloride to death has increased the difficulty of the test.Therefore,the forensic examination of succinylcholine chloride has become a new problem in judicial practice.In the practice of judicial identification,the rapid and accurate qualitative and quantitative analysis of succinylcholine chloride in biological samples will be helpful to the investigation of cases.According to the literature and data,succinylcholine chloride is a quaternary ammonium compound with unstable high temperature and high ionization efficiency in electrospray mass spectrometry.UHPLC-MS/MS is an effective method for the analysis of compounds with high polarity and thermal instability.This method has a low detection limit,good reproducibility,and is accurate and reliable.It also eliminates the interference of biological matrix and is suitable for the detection of succinylcholine chloride.Objectives:In order to provide a reference for the forensic diagnosis of Suc poisoning and to play a social role as a deterrent to criminals,an appropriate UHPLC-MS/MS method was established for the qualitative and quantitative analysis of succinylcholine chloride and its metabolites in poisoned rats.Methods:The triple quadrupole tandem mass spectrometer,electrospray ion source（ESI）and positive ion scanning multi-reaction monitoring（MRM）were used to determine the parent ions of succinylcholine chloride as 145.1（m/z）,the subions as115.4（m/z）and 93.3（m/z）.The parent ions of deuterium succinylcholine chloride（²H-Suc）are 148.1（m/z）,and the daughter ions are 117.1（m/z）and 95.1（m/z）.The metabolites of succinylmonocholine chloride of succinylcholine chloride were 204.0（m/z）,106（m/z）and 85.2（m/z）.The former was qualitative while the latter was quantitative.Luna NH₂column（2 mm 100 mm,3 m）and UHPLC-MS/MS were used for the chromatography.The mobile phase A was 0.1%aqueous formic acid solution and the mobile phase B was 100%acetonitrile.The flow rate was 0.2 m L·min^-1,the gradient elution condition was 5 min,and the column temperature was 37 degrees.UHPLC-MS/MS method was established under the above conditions.The in vivo tracer experiment of subcutaneous injection of ²H-Suc was performed on rats,and then three doses of Suc were subcutaneously injected into rats.Three doses of Suc were injected subcutaneously in rats,and acetonitrile protein was precipitated in serum and main organ tissues of rats for 4 h,then purified by oversolid-phase extraction WCX column.Suc and metabolites were detected using UHPLC-MS/MS instrument.In vitro vessel diameter pressure meter was used for vascular experiment to determine the effect of Suc on vascular function.Results:A method for determination of ²H-Suc by UHPLC-MS/MS was established.A subcutaneous injection of ²H-Suc was performed to determine the main organs of Suc in rats.A UHPLC-MS/MS method for Suc detection was established.The detection limit of Suc was 0.01 ng·m L^-1,the recovery rate was 89.5%～95.4%,and the intra-day RSD was less than 15%.Suc was detected in serum,heart,liver,spleen,lung,kidney,brain,stomach,muscle and other tissues after subcutaneous injection of 0.75,1.5 and 3.0mg·kg^-1of Suc in rats.The Suc content in renal tissues was the highest,which was positively correlated with the dose of the drug.Smc,one of Suc metabolites,was detected in administered rats.Suc significantly attenuated kcl-induced cerebral basilar artery contraction and acetylcholine-induced diastolic response in rats.Suc significantly attenuated KCL-induced cerebral basilar artery contraction and acetylcholine-induced diastolic response in rats.Conclusions:Suc at poisoning dose was distributed in the serum,heart,liver,kidney and other tissues.The content in the kidney is the highest;UHPLC-MS/MS analysis is an effective and reliable method for detecting the drug content in Suc-poisoned rats;Using succinylmonocholine chloride as a method to detect Suc lethal rats in vivo can greatly improve the detection efficiency and accuracy;The vasoconstriction and diastolic reactions can be used to assist in the determination of Suc poisoning cases.