The Study On Chromatographic Analysis Methods Of Sitagliptin And Related Substances

Sitagliptin phosphate is the first dipeptidyi peptidase-IV(DPP-4)inhibitor approved by the FDA for the treatment of type 2 diabetes.It is safe,well tolerated and has fewer adverse effects.At present,although the method of detection of the Sitagliptin content is reported,these methods are not systematic.There is also no report on the separation and detection of Sitagliptin and its related substances.In this paper,we mainly use high-performance liquid chromatography,first of all,reverse the study of the Sitagliptin USP discussion paper to determine the best method for the determination of Sitagliptin in practical application.Secondly,the method of separation and purification of impurities and degradation impurities in the synthetic route was established,and the method was validated.In addition,the method for the detection of the Sitagliptin isomer is validated,which can provide the reference for the further production,detection and use of the drug.Specifically divided into the following three parts:The first part introduces the general situation of diabetes and DPP-4 inhibitors,summarizes the application of HPLC in medicine,and summarizes the status quo of analysis of-Sitagliptin.In the second part,the chromatographic conditions of the Sitagliptin in practical application were determined by reverse analysis of the analytical method in the Sitagliptin USP discussion paper.The chromatographic conditions were:Agilent ZORBAX SB-CN 5 ?m 150×4.6 mm column,30 ?,the flow rate was 1 mL·min-1 and the detection wavelength was 210 nm.The isocratic elution was performed by equilibration elution with acetonitrile acetonitrile-buffer(15%-85%)(v/v)for 20 min.The method was proved to be stable,accurate and reproducible.The linear regression equation of sitagliptin in the range of 0.37 mg·mL-1 to 0.60 mg·mL-1 was shown by the method:y = 250.44x-3.2639(R2 = 0.9939),with good linearity.In the third part of the experiment,the chromatographic method of the related substances of the Sitagliptin was established and the method was validated:(1)The chromatographic conditions for the degradation of impurity were as follows:Agilent ZORBAX SB-CN 5 ?m 150×4.6 mm,column temperature 30 ?,flow rate 1 mL·min-1,detection wavelength 210 nm,gradient elution.The elution was carried out in equal volumes of acetonitrile-buffer(15%-85%)(v/v)for 18 min.The acetonitrile was raised to 37%in 2 min and the isocratic elution was completed at 35 min.The method was proved to be stable,accurate and reproducible.(2)The chromatographic conditions of a raw material and intermediate were as follows:Phenomenex Luna 250×4.6 mm 5 ?m C18 column,column temperature 30 ?,flow rate 1 mL·min-1,detection wavelength 210 nm,isocratic elution was performed by equilibration elution with acetonitrile-buffer(40%-60%)(v/v)for 30 min.The method was proved to be stable,accurate and reproducible.(3)The chromatographic conditions of two raw material and intermediate were as follows:Phenomenex Luna 250×4.6mm 5 ?m C18 column,column temperature 30 ?,flow rate 1 mL min-1,detection wavelength 210 nm,the gradient elution method was as follows:acetonitrile-buffer(18%-82%)(v/v)isocratic elution for 6 min,1 min acetonitrile content to 31%isocratic to 18 min,acetonitrile content will be raised to 70%,continuous elution for 10 minutes,30 min the end of the test.The method was proved to be stable,accurate and reproducible.(4)The method for the determination of the The Sitagliptin isomer was validated.The separation between the main peak and the isomer was 4.01;R2was 0.9982 in the range of 3.00?12.00 ?g mL-1,and the linearity was good.The results showed that the method was stable,accurate and reproducible.