Research On The Related Substances In Levamisole Hydrochloride Tablets And Improvement Of Its Quality Standard

Objective:To analyze the structure of the impurities in levamisole hydrochloride tablets by UPLC-QTOF-MS.To predict the toxicological parameters of this impurities by ADMET Predictor 8.5,and analyze the risk of possible adverse reactions from this impurities.To establish an HPLC method for the determination of the related substances in levamisole hydrochloride tablets,and research the distributions and the sources of them.To investigate the content of dextrose impurities in the sample by a chiral column HPLC,and analyze the source of it.To evaluate the rationality of packaging and storage conditions by the sample stability study,and evaluate the rationality of the prescription by observing the compatibility of the raw material and accessories in levamisole hydrochloride tablets.To establish a HPLC method for determination of content of levamisole hydrochloride tablets,in order to provide the information for the quality control.Methods:?1?The method of analyze the structure of the impurities:using the HPLC with a C₁₈ column?2.1 mm×150 mm,2.7?m?,while the mobile phase was 50 mmol/L ammonium acetate solution-acetonitrile?80:20?,and the flow rate was 0.4 ml/min,and the column temperature was 40?.Under positive ion mode,first-phase full-scan mass spectrometry is used to obtain the accurate relative molecular mass and elemental information of each impurity,and then fragment ion information is obtained according to second-level full-scan mass spectrometry.Finally,unknown impurities are identified by structure.Predicting the toxicological parameters of these impurities by ADMET Predictor 8.5 with the structural characteristics of impurities.?2?The method for the determination of the related substances:using the HPLC with a C₁₈ column?4.6 mm×250 mm,5?m?,while the mobile phase A was 0.5%ammonium dihydrogen phosphate solution?adjusted to p H 6.5 with triethylamine?,the mobile phase B is acetonitrile,with gradient elution.The flow rate was 1.0 ml/min,and the column temperature was 30?,with the detection wavelength at 215 nm.?3?The method for the determination of the dextrose impurities:using the HPLC with a chiral column?4.6 mm×250 mm,5?m?,while the mobile phase A was 50mmol/L Potassium dihydrogen phosphate solution-acetonitrile?75:25?,and the flow rate was 1.0 ml/min,and the detection wavelength at 214 nm,with the column temperature was 40?.?4?The method for the determination of the Content:using the HPLC with a C₁₈ column?4.6 mm×250 mm,5?m?,while the mobile phase was 0.5%ammonium dihydrogen phosphate solution?adjusted to p H 6.5 with triethylamine?-acetonitrile?80:20?,and the flow rate was 1.0 ml/min,the detection wavelength at 215 nm,with the column temperature was 30?.?5?According to Guidance Principles for Stability Testing of Pharmaceutical Preparations requirements,the stability testing of levamisole hydrochloride tablets was carried out,which included stress testing,accelerated testing and long-term testing,and evaluate the rationality of packing and storage conditions by examining the changes in impurities and content in the sample.?6?Evaluate the rationality of the prescription by mixing the raw materials and excipients at a certain ratio.?7?The method for the determination of dissolution of levamisole hydrochloride tablets:the dissolution medium was 900 ml of water,and the rotation speed was 50revolutions per minute.When the dissolution time is 30 minutes,determinationed the test solution by HPLC.Results:?1?Preliminary identified structures of fourteen components in levamisole hydrochloride tablets.According to the results of toxicity risk prediction,we can known that:14 impurities without cardiotoxicity and acute toxicity;It is possible that the impurities A?C?D?1?2?4?5 and 6 have liver toxicity,the impurities 4?5 and 6 have carcinogenic toxicity,the impurities C?D and 1 have chromosome mutation toxicity,the impurities C?D?E?1?2?3?5?6?8 and 9 have reproductive toxicity,the impurities C and 1 have mutagenic risk.Finally,the toxicity risk assessment showed that the highest risk was impurities A?C?2?5 and 6.?2?The linear range of impurities A?B?C?D and E were 0.5?10?g/ml?r>0.999?.Levamisole hydrochloride and its relates substance could be well separated.The average recoveries of impurities A?B?C?D and E were 99.2%?98.9%?98.4%?97.6%and 97.9%respectively.?3?The linear range of dextrose impurities was 0.1?5?g/ml?r=0.999 7?,and the average recovery was 102.2%.The detection limit was 0.013?g/ml.?4?The linear range of levamisole hydrochloride was 20?500?g/ml?r=0.999 6?,and the average recovery was99.5%.The detection limit was 0.051?g/ml.?5?According to the stability testing,the levamisole hydrochloride tablets are unstable under strong light,and sensitive to temperature.?6?The impurity C is significantly increased in the mixture of levamisole hydrochloride and silica,it shows that incompatibility between the raw material and accessories in levamisole hydrochloride tablets.?7?A HPLC method for the determination of dissolution of levamisole hydrochloride tablets was established.The dissolution of 24batches of samples was more than 80%.Conclusion?s?:Set up and verified the HPLC method for the determination of the related substances?dextrose impurities?content determination and dissolution,and preliminary identified structures of fourteen components in levamisole hydrochloride tablets.Analyzing the risk of possible adverse reactions from impurities,we can know that most impurities have toxicity risks.It is recommended that production enterprises avoid using as much as possible,because silica and raw materials have compatibility problems.Finally,a more scientific?more suitable method for the requirements of modern analytical methodologies was provided,which provides a favorable scientific theoretical basis for improving the quality of levamisole hydrochloride tablets.