Development Sensitive And Specific UPLC-MS/MS Methods For Ultra-trace Level Determination Genotoxic Impurities In Mezlocillin And Sulbactam

Objective:Ultra-performance liquid chromatography coupled with triple quadrupole mass spectrometry（UPLC-QqQ-MS/MS）was used to establish an ultra-trace analysis method with sensitivity,specificity and accuracy for acyl chloride genotoxic impurities in mezlocillin and two brominated genotoxic impurities in sulbactam.Methods:（1）The silico tools were used to predict the mutagenic and carcinogenicity of compounds containing the alerting structure of genotoxicity in mezlocillin and sulbactan,and to confirm the genotoxic impurities and control limits that need to be studied.（2）According to the characteristics of the genotoxic impurity of acyl chloride in mezlocillin,the derivative route was formulated to prepare the derivative standard,and the LC-HRMS/MS,NMR and IR were utilized to determine the precise structure and purity.（3）The trace analysis methods of UPLC-QqQ-MS/MS acyl chloride and brominated genotoxic impurities was established and optimized by the Qb D concept and BBD method.（4）The sensitivity and reliability of the methods were proved by the methodology validation.Results:（1）IthasbeenidentifiedthattheCMI（1-Chlorocarbonyl-3-Methanesulfonyl-2-Imidazolidinone）used for the synthesis of meloxicillin and the impurity C and impurity D of sulbactam are mutagenic and carcinogenic substances by in silico tools（Tox Tree and VEGA）based on rule-based system and statistical model.（2）By optimizing the derivatization conditions of the mezlocillin chloride impurity,the best derivatization condition was determined by the reaction of the acyl chloride impurity with methanol at 35℃for 45min.（3）Establish mezlocillin chloride genotoxic impurity detection method,and the chromatographic column C18（2.1×100mm,2.6μm）were used.The column temperature was set at 30℃.The auto-sampler temperature was held at 8℃for sample stability.The mobile phase consisted of 0.1%formic acid within H₂O（v/v）（Mobile Phase A）and acetonitrile（Mobile Phase B）.The flow rate was held constant at 0.2 ml/min with an injection volume of 20μl.Electrospray Ionization（ESI）was used in positive mode,collision energy:23V,sheath gas:23arb,vaporizer temperature:100℃,the specified ion pairs:223.0 m/z→79.0 m/z.（4）Establish sulbactam brominated genotoxic impurities and the chromatographic column C18（4.6×250mm,5μm）.The auto-sampler temperature was held at 8℃for sample stability.The mobile phase consisted of 0.1%formic acid within H₂O（v/v）（Mobile Phase A）and acetonitrile（Mobile Phase B）.The flow rate was held constant at 1.0 ml/min with an injection volume of 20μl.Electrospray Ionization（ESI）was used in negative mode,collision energy:24V,sheath gas:50arb,vaporizer temperature:216℃,SBT-C specified ion pairs:309.9 m/z→78.9 m/z,SBT-D specified ion pair:277.9 m/z→136.9 m/z.（5）Methodological validations of the two established methods were carried out.The detection limit（LOD）and the quantitative limit（LOQ）for acyl chloride genotoxic impurities in mezlocillin reached 14ppb and 20ppb.The LOD and LOQ for brominated genotoxic impurities in sulbactam were 5ppb and 25ppb,respectively.The results from the validation studies exhibited good linearity,specificity,accuracy,precision,and stability.Conlclusion:Two sensitive and specific ultra-trace level methods of UPLC-MS/MS have been developed to quantify acyl chloride potential genotoxic impurity in mezlocillin and brominated genotoxic impurities in sulbactam,respectively.It is concluded that the method elucidated here could be useful for accurate quantification of potential genotoxic impurity during the manufacture of mezlocillin and sulbactam,as well as provide valuable reference for the quality standards.