Study On Related Quality Control Of Menadione Sodium Bisulfite And Its Injection

Objective:To carry out the quality standard research of menadione sodium bisulfite and its injection.To establish HPLC method to investigate the related substances,and to confirm the structure of the impurity spectrum of the injection by LC-MS.To establish an HPLC method for the determination of menadione sodium bisulfite.To establish HPLC inspection of menadione sodium bisulfite sulfonate.To establish a GC method to determine the amount of ethanol residual solvent.To optimize identification?2?method.To provide experimental reference for improving the quality standard of menadione sodium bisulfite and its injection.Methods:Using welch xtimate C₁₈column?150mm×4.6mm,5?m?,0.02mol·L^-1ammonium acetate(containing 0.0015mol·L^-1tetrabutylammonium bromide)?adjust the p H of acetic acid to 4.8?-methanol?68:32?as mobile phase,isocratic elution,column temperature is 30?,Detection wavelength:Menadione is 251nm,the rest are 230nm,flow rate is 1.0m L·min^-1and injection volume is 20?L,Examining the related substances of menadione sodium bisulfite and its injection.Determine the relative retention time and detection limit of menadione sodium bisulfite injection and compare with the detection limit measured by the original pharmacopoeia method.?1?To establish LC-MS chromatographic system(using 0.02mol·L^-1ammonium acetate solution-methanol?92:8?as mobile phase).Using column switching technology,The impurity peak of the forced degradation test is directly switched to the MS system.According to the positive and negative ion spectrum and the HRMS results to determine the molecular weight and element composition of the impurities,according to the difference between the fragment ions to help structure analysis.At the same time,impurity 4 was synthesized by¹HNMR and organic synthesis.?2?The proportion of methanol organic phase was increased to 35%,and the content of the drug was determined by HPLC with other chromatographic conditions unchanged.?3?Some manufacturers of menadione sodium bisulfite raw materials use ethanol as solvent,so the GC method is used to determine the residual amount of ethanol in the menadione sodium bisulfite raw materials.The conditions are as follows:DB-624capillary chromatographic column?30m×0.53mm,3.0?m?,headspace flask equilibrium temperature is 80?,equilibrium time is 30min,programmed temperature rise:the initial temperature of chromatographic column is maintained at 40?for7min,and then the temperature rises to 230?at the rate of 50?per minute for12min,5:1 split injection 1000?L,detector is FID,detector temperature is 250?,inlet temperature is 200?,carrier gas is N₂,flow rate is 4m L·min^-1.?4?According to the pharmacopoeia general rules of sulfite?hydrogen?salt identification,the identification?2?was revised,and the HPLC method was updated.Results:The impurity spectrum of all batches of menadione sodium bisulfite raw materials are basically similar,and there are impurities 1?3 in the impurity spectrum of injection.The impurities in the injection are relatively large,but the batch difference is small.The impurity spectrum basically contains four main impurities?unstable,there is some mutual conversion?,that is,impurities 1 to 4 and a very small amount of menadione.The results of the forced degradation test show that light and oxidative damage have almost no effect on menadione sodium bisulfite.Menadione sodium bisulfite sulfonate is an important degradation product under thermal damage,which is related to heat.The destruction of acid and alkali can promote the precipitation of free menadione,meanwhile,At the same time,the destruction of alkali will increase the impurity 4 and menadione of the raw material,and the impurity 2 of the injection will increase suddenly,and the number of impurities will increase significantly.Then,by means of HPLC-PDA,UPLC-HRMS and¹HNMR,four important degradation impurities in the impurity spectrum of menadione sodium bisulfite injection were identified,and the impurity 4?i.e.menadione sodium bisulfite sulfonate,purity>99.0%?was successfully prepared.Relevant substance inspection by HPLC method confirmed that the detection limit of menadione sodium bisulfite sulfonate?relative retention time is about 3.5±0.3minute?was 0.0455?g·m L^-1,and the detection limit of Pharmacopeia method was 0.7mg·m L^-1,HPLC method is more sensitive.No ethanol was detected in three batches of raw materials from manufacturer B,while all three batches of raw materials from manufacturer A were detected.The linear range of ethanol is 2.0136?1006.8223?g·m L^-1,The r value is 0.9999,the average recovery rate is 105.4%,and the RSD?n=9?is 0.9%.The optimized identification?2?is more sensitive and accurate,and the new HPLC method has better specificity.The optimized identification?2?showed more sensitive experimental phenomena,more accurate results,and the newly established HPLC method has better specificity.Conclusion:The results show that the generation of impurity 1 is related to the addition of excessive stabilizer sodium bisulfite,Menadione di-sodium bisulfite of impurity 2 has the prodrug properties of menadione.There is an enol-ketone tautomerism equilibrium between impurity 3 and impurity 4,and there is tautomerism between MSB and impurity 4,which is irreversible.The heat destruction can aggravate the production of impurity 4,which indicates that the environmental temperature of medicine should not be too high during storage,transportation and shelf life.This study can be used for the inspection of related substances in sodium bisulfite menadione and its injection,at the same time,it provides a technical route for the preparation of menadione sodium bisulfite sulfonate?impurity 4?.Because of its simple operation,high sensitivity and strong specificity,HPLC can be used for the rapid determination and identification of menadione sodium bisulfite.The GC method can quickly and accurately determine the residual amount of ethanol in the menadione sodium bisulfite raw material.