Determination Of Skin Drug Content At The Point Of Injection Of Succinylcholine Rat By UHPLC-HRMS And Morphological Changes Of Target Organ Injury

Suxamethonium chloride is also known as scoline,chlorosuccinylcholine,succinylcholine chloride,and its chemical name is 2,2'-[?1,4-dioxo-1,4-Butylene?bis?oxy?]bis[N,N,N-trimethylethylamine]dihydrate,the molecular formula is C₁₄H₃₀Cl₂N₂O₄·2H₂O.It belongs to a depolarizing muscle relaxant.It is clinically used as a muscle relaxant during general anesthesia.It can cause adverse reactions such as bradycardia,arrhythmia,and cardiac arrest.The use of a ventilator can cause bronchospasm or anaphylactic shock death.It is a class B organic drama drug.In recent years,succinyl chloride has been used by darts to shoot poultry and livestock animals,and even more,Cases of succinyl chloride used as a killing tool by criminals have been reported frequently.According to the literature,Anhui,Zhejiang,Guangdong and other places are the high incidence areas of such cases.Forensic testing of succinylcholine chloride has become a new issue in judicial practice today.Because succinyl chloride is rapidly decomposed into succinic acid and choline in the body,about 2%is in the original form,and the rest is excreted from the urine in the form of metabolites.These two decomposition products exist in the body itself,which gives The test of succinylcholine chloride has increased the difficulty,seeking forensic pathology and physical and chemical characteristics after poisoning,and providing scientific reference for the qualitative case of succinylcholine chloride poisoning.Objective:1.To observe pathological changes of related organs or tissues in rats subjected to subcutaneous injection of succinylcholine chloride?SUC?.2.Determine SUC content in the injection point skin.Methodological validation of²H-SUC in kidney tissue of UPLC-MS/MS rats Methods:1.Rat was subcutaneously injected with SUC,and the pathological changes of muscle,heart,kidney,lung and trachea were observed under light microscope;The pathological changes of muscle,kidney and injection point skin were observed under electron microscope.2.SUC content in the injection point skin was determined by ultra-high-performance liquid chromatography-high resolution massspectrometry?UHPLC-HRMS?.Luna NH2 column?2mm×100mm,3ms?was used for the chromatography.The mobile phase was 0.1%formic acid aqueous solution-acetonitrile isometric elution.The flow rate was 0.2 ml/min,the injection volume of sample volume was 5?l,the column temperature was 37?,and the injection time was 5 min.Mass spectrometry was determined by quadrupole electrostatic field orbital trap high resolution mass spectrometer,heated electrospray ion source?HESI?and positive ion scanning mode.3.The UPLC-MS/MS method was used to determine ²H-SUC in the kidney;the chromatographic method was Luna NH2 chromatographic column?2 mm×100mm,3?m?,mobile phase was 0.1%formic acid aqueous solution-acetonitrile elution,flow rate was 0.2 positive ion scanning mode,multi-reaction monitoring mode,electrospray ionization source was detected;the linear relationship and detection limit,accuracy and precision,recovery rate,matrix effect and stability of the method were investigated.Results:1.SUC 1.5,3.0 and 6.0 mg/kg could induce obvious pathological and histologic damage in rat muscle,heart,kidney and lung;Mallory phosphotungstic acid histochemical staining showed that SUC caused muscle fiber loosening and gap enlargement in the rat heart.The protein expression of CD3,CD20,CD31,CD56 and neurosecretory granulosa membrane protein in rat muscle,heart,kidney,lung and bronchus was detected by using an immunohistochemical method.Only SUC could significantly increase the expression of CD3 in rat renal interstitium.Electron microscope examination showed that SUC could cause mitochondrial damage in renal cells and sarcomere and filamentous injury in skeletal muscle cells.2.There was no specific absorption peaks to be observed in SUC standard solution,rat serum adding SUC as well as serum of Suc-treated rats;SUC was detected in rat skin of the injection point by using UHPLC-HRMS method,and the SUC content increased in a dose-dependent manner.The linear range of suc is 0.2-10ng/ml,the recovery is 80.55%-90.44%,the relative standard deviation is less than 15%,and the precision and accuracy meet the requirements.3.Through UPLC-MS/MS method,the linear range of ²H-SUC in renal tissue was good at 0.5?100,the precision and accuracy of day and day were less than 15%,the recovery rate was between 85.30%?96.76%,and there was no obvious matrix effect in the treated samples.Conclusion:1.Subcutaneous injection with SUC led to obvious histopathological damages in rat heart,kidney,lung and muscle.2.UHPLC-HRMS is a reliable method for detecting SUC in rat skin of subcutaneous injection point.3.UPLC-MS/MS method can determine ²H-SUC,in renal tissue and establish a method for deuterated succinylcholine chloride in renal tissue.