Investigation And Control Of Impurity In Saxagliptin

Type 2 diabetes mellitus(T2DM)is a long-term metabolic disorder caused by insulin secretion and insulin activity disorder.As a dipeptidyl peptidase-4(DPP-4)inhibitor,Saxagliptin belongs to incretin.It is the first DPP-4 inhibitor approved by China for single-agent and combination therapy for T2 DM dual indications.Although Saxagliptin is listed slightly later,it is favored by doctors and has good research and development prospects because of its good therapeutic effect and less adverse reactions.In this paper,the related impurities of Saxagliptin were studied,and the isomers,related substances,residual solvent and genotoxic impurities of Saxagliptin were controlled by high performance liquid chromatography(HPLC)and gas chromatography(GC).The QbD guiding theory was used to carry out detailed design research on the preliminary development of the detection method,to determine the key parameters in the detection method,and to carry out detailed methodological verification work on the four methods to ensure accurate andreliable detection methods.This paper is mainly composed of the following four parts:The first part introduces the background and significance of the subject,including the pathogenesis of diabetes and the progress of drug research,the mechanism of action of Saxagliptin and market sales,the basic principles of HPLC,the origin of QbD and its application in chromatography development.The second part of the work mainly established the normal phase HPLC to control the enantiomer of Saxagliptin(impurity 1).Since there is no relevant pharmacopoeia quality standard as a reference,the method uses the starting material as a method to establish the basis,and uses the QbD concept to make corresponding research choices on the mobile phase,column and test sample concentration in the early stage of methoddevelopment,and finally to amylose.Tris(3,5-dimethylphenylcarbamate)column is a separation column,and n-hexane-isopropanol-methanol-diethylamine(65:20:15:0.1)is used as a mobile phase,which not only realizes impurities 1.Effective separation from Saxagliptin and other impurities,while ensuring the mutual solubility of n-hexane and methanol.The detection limits(S/N=3)and the limit of quantification(S/N=10)of impurity 1 were 0.50 μg/ml and 1.00 μg/ml,respectively;the linear equation was A=8 075.5c-1 611.8,r=0.999 9 The linear relationship is good;the RSD(n=6)of the peak area of the impurity1 is 0.38%,and the system precision is good;the sample recovery is between 101.0% and 107.0%,and the RSD(n=9)is 2.6%.The test sample can be stably stored for 12 hours at room temperature.Three batches of Saxagliptin bulk drug were tested by this method,and no impurity 1 was detected.The method has the advantages of simple operation,good separation degree,good reproducibility and accurate and reliable results.The third part is mainly to establish the reversed-phase liquid chromatography to control the impurities 2～6 of Saxagliptin.The method is based on the import drug registration standard and uses the QbD concept to optimize the mobile phase,elution method,column and dissolved solvent according to the characteristics of the self-developed product.An octadecylsilane bonded silica gel(ODS)column was used as a separation column,and a gradient elution with acetonitrile-water-phosphoric acid as a mobile phase was carried out to achieve effective separation of impurities2-6.The detection limits(S/N=3)of each impurity were 0.16 μg/ml,0.12μg/ml,0.12 μg/ml,0.10 μg/ml,0.10 μg/ml,respectively,and the limit of quantification(S/N=10)was 0.36 μg/ml,0.40 μg/ml,0.40 μg/ml,0.19μg/ml,0.20 μg/ml,the linear relationship is good;the correction factors of each impurity are 1.39,0.59,0.98,1.44,1.31;impurities 2～6 The RSD(n=6)of the peak area of the reference product was between 0.20% and1.20%,and the system precision was good.The recoveries of the spiked samples were between 98.0% and 104.0%,and the RSD was between 1.9%and 4.0%.The parameters of the micro chromatographic conditions change,the resolution of each impurity satisfies the requirements,and the method has good durability.Three batches of Saxagliptin bulk drug were tested according to the method,and the content of impurity 3 was 0.06%,0.09%and 0.08%,respectively,and impurities 2,4,5 and 6 were not detected.The method is simple and convenient,high in sensitivity,accurate and reliable,but since the impurities 5 and 6 are easily degraded,the test sample needs to be freshly prepared or stored in a low temperature injection chamber(5°C).The work of the fourth part is mainly to establish a gas chromatography(GC)method to control the residual solvent of Saxagliptin.The nine organic solvents used in the Saxagliptin route were controlled according to the relevant provisions of the Chinese Pharmacopoeia’s 2015 edition of the four general rules for GC and residual solvent determination.With 6% cyanopropylbenzene 94% dimethyl siloxane as the separation column,the headspace conditions,temperature rising procedure and column temperature were selected and optimized respectively to achieve effective separation of nine organic solvents.The detection limits(S/N=3)of each organic solvent were 0.295 μg/ml,0.497 μg/ml,0.088 μg/ml,0.320μg/ml,0.200 μg/ml,0.072 μg/ml,0.086 μg/ml,2.718 μg/ml,0.067 μg/ml,the limit of quantification(S/N=10)was 1.970 μg/ml,0.995 μg/ml,4.383μg/ml,0.800 μg/ml,0.333 μg/ml,0.144 μg/ml,respectively.0.171 μg/ml,13.590 μg/ml,0.166 μg/ml,the linear relationship is good;the RSD(n=6)of the peak area of the reference substance of the nine organic solvents is between 1.6% and 3.6%,and the system precision is good;The recoveries of the spiked samples were between 96.0% and 109.0%,and the RSD was between 0.3% and 10.2%.The parameters of the micro chromatographic conditions were investigated,and the resolution of each impurity was satisfactory.The method was durable.Three batches of Saxagliptin bulk drug were tested according to the method,and the methylene chloride contents were 0.01%,0.01% and 0.03%,respectively,and the ethyl acetate contents were 0.31%,0.11% and 0.38%,respectively,and the organic solvents were not detected.The method is simple and convenient,high in sensitivity,accurate and reliable.The fifth part is mainly to establish GC method to control the genotoxic impurities of Saxagliptin.Genotoxic impurities are a hot topic in the research of bulk drug impurities in recent years.Because methanesulfonic acid,methanol and isopropanol are used in the Saxagliptin synthesis route,methyl methanesulfonate,ethyl methanesulfonate,and A can be produced.Isopropyl sulfonate,which has been clearly shown to be genotoxic,is therefore necessary for the control of these three solvents.This experiment refers to the toxicological attention threshold to evaluate the limit of genotoxic impurities in the drug substance,with 1.5 μg/day as the acceptable threshold,and the limit of the three genotoxic impurities according to the recommended dose of Saxagliptin 5 mg/day.It is set at 0.03%.6% cyanopropylbenzene 94%dimethyl siloxane was used as the separation column,and the direct injection method was adopted to ensure the sensitivity of the detection method and the effective separation of the genotoxic impurities.The detection limits(S/N=3)of each genotoxic impurity were 0.0012 μg/ml,0.0005 μg/ml,and 0.0006 μg/ml,respectively,and the limit of quantification(S/N=10)was 0.003 μg/ml and 0.001 μg/,respectively.Ml,0.0012 μg/ml,the linear relationship is good;the control and the test solution can remain stable for 10 hours at room temperature.Three batches of Saxagliptin bulk drug were tested according to this method,and none of the three genotoxic impurities were detected.The method is simple and convenient,and has high sensitivity.Based on the registration standards for imported drugs,this paper establishes a method for detecting isomers(impurity 1).This method has not been mentioned in any literature.Paying attention to research and control of popular genotoxic impurities in recent years,some related substances detection methods have been optimized accordingly,and the Saxagliptin impurity detection method has been improved.The market share of Saxagliptin preparation is gradually increasing,and the further optimization of the impurity determination method provides a reliable impurity control means for the research and development of the drug substance and the preparation.