| Genotoxic impurities are DNA reactive substances with direct DNA damage potential.And the presence of DNA reactive impurities in API products has brought great problems to drug regulatory agencies and industries.In order to ensure people’s medication safety,at present,the content of genotoxic impurities is strictly controlled at home and abroad.Sigliptin phosphate is an oral,potent and highly selective dipeptidyl peptidase-4(DPP-4)inhibitor,which is an effective and generally well-tolerated treatment option for patients with type 2 diabetes.Therefore,the drug has a broad market prospect.Based on the potential genotoxic impurities possibly introduced in the synthesis of sitagliptin phosphate,it should be comprehensively studied.According to the current regulatory guidelines for genotoxic impurities,analytical methods should be formulated to meet the toxicological concern threshold of individual impurities(TTC,1.5 μg/d).The daily recommended dose is 100 mg based on sitagliptin,and the maximum residual amount of genotoxic impurities in this API is 11.45 ppm,so as to avoid the carcinogenic and mutagenic threat to patients.Therefore,it is necessary to develop an analytical method at an allowable level.Methods The specific research contents are as follows:(1)Detection of potential genotoxic impurities meldrum’s acid: In this chapter,the test method,chromatographic column,mobile phase,flow rate and mass spectrometry conditions were optimized,and a simple and direct high performance liquid chromatography-tandem mass spectrometry(LC-MS/MS)was established to determine the content of genotoxic impurities meldrum’s acid in sitagliptin phosphate,and the methodology was verified.The results showed that the concentration range of meldrum’s acid was 0.60 ng/m L～36.03 ng/m L,and there was a good linear relationship.Under three different concentrations,the average recovery rate of meldrum’s acid was 97.91%,RSD was 2.46%.In the repeatability and precision tests,the RSD is5.40% and 4.86%,and the detection limit and quantitative limit are 0.19 and 0.60 ppm,respectively.The retention time is ahead,the detection speed is fast(the sample runs for 6 min),and the analysis time is shortened.This method has the advantages of simple pretreatment,no derivatization of samples,accurate quantification,high sensitivity and strong anti-interference ability.It is a good quality control tool for quantitative determination of very low level of meldrum’s acid in the production process.(2)Detection of potential genotoxic impurity 5-[1-hydroxy-2-(2,4,5-trifluorophenyl)ethylidene]-2,2-dimethyl-1,3-dioxane-4,6-dione: In this chapter,the chromatographic column,column temperature,mobile phase,blank solvent and mass spectrometry conditions were optimized to establish a sensitive The results show that the impurity concentration is in the range of 0.11 ng/m L～32.08 ng/m L,and there is a good linear relationship.Under three different levels of concentration,the average recovery rate was 100.33% and RSD was 1.46%.In the repeatability and precision test,RSD is 2.17% and 1.86%,respectively.The limit of quantitation was 1.07 ng/m L,and the limit of detection was 0.36 ng/m L.The method has strong specificity,high sensitivity and short analysis time,which can provide help for the detection of the impurity residue in sitagliptin phosphate bulk drug and make up the gap of its detection method.(3)Detection of potential genotoxic impurity methyl(3R)-3-acetamido-4-(2,4,5-trifluophenyl)-butanoate: In this chapter,the chromatographic column,mobile phase,flow rate and mass spectrometry conditions were optimized,and an effective and highly selective high performance liquid chromatography-tandem mass spectrometry(LC-MS/MS)was established for the determination of genotoxic impurities in sitagliptin phosphate,which improved the sensitivity and sensitivity.The results show that the impurity concentration is in the range of 0.26ng/m L～32.21 ng/m L,and there is a good linear relationship.Under three different levels of concentration,the average recovery rate was 100.89% and RSD was 3.06%.In the repeatability and precision test,RSD is 4.83% and 4.90%,respectively.The quantitative limit was 3.85 ng/m L,and the detection limit was 1.29 ng/m L.The newly developed detection method of methyl(3R)-3-acetamido-4-(2,4,5-trifluorophenyl)-butanoate has the advantages of simple experimental steps,strong specificity,high accuracy and short analysis time,and can accurately quantify the trace impurity in sitagliptin phosphate.(4)Detection of genotoxic impurity chloroacetic acid: In this chapter,chloroacetic acid was derivatized by dimethyl sulfate,and the chromatographic column,extraction solvent and mass spectrometry conditions were optimized.A gas chromatography-tandem mass spectrometry(GC-MS/MS)was established to determine the content of genotoxic impurity chloroacetic acid in sitagliptin phosphate,and and the methodology was verified.The results showed that chloroacetic acid had a good linear relationship in the concentration range of 176.56ng/m L～706.23 ng/m L.Under three different concentrations,the average recovery rate of chloroacetic acid was 97.15% with RSD of 4.29%.In the repeatability and precision test,RSD is6.00% and 3.69%,respectively.The limit of quantitation was 176.56 ng/m L,and the limit of detection was 52.97 ng/m L.The method is scientific,accurate and durable,and can provide technical support for the detection of residual chloroacetic acid in sitagliptin phosphate. |
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