Application Of Reversed Phase Chromatography Inanalysis Of Neutral And Ionization Samples

High Performance Liquid Chromatography（HPLC）is one method of all kind chromatograghy regarding separate and analyzing for chemical mixture.After continuous improvement of several decades,the HPLC method is close to be a kind of completed method.Since HPLC method was collected by China Pharmacopeia,the application in drug products increased rapidly.It became a popular way in analysis and monitor for pharmaceutical quality controls,and among it the reverse phase chromatography（RP-HPLC）is the most widespread application.In this article,we focus on two kinds of Active Pharmaceutical Ingredients（API）,Rivaroxaban and Entecavir which are excluded in the China Pharmacopeia（Version 2015）,to study the impact from different chemical character samples during RP-HPLC analysis method development.And then guide the method development better and to establish test method with a more effective way.The first section in the article is“Rivaroxaban（Neutral sample）in process control method development”.Rivaroxaban is an oral oxazolidinone-based anticoagulant medication researched and developed by Bayer Germany,is a potent,selective direct inhibitor of factor Xa that is used in the prevention of venous thromboembolism in adult patients.This experiment applies RP-HPLC method with the column of C18,take advantage of the sample without positive electric charge and negative electric charge and focus on the starting material,another two impurities produced in process and drug substance to develop the test method,so as to monitor the product quality quickly and accurately during the production process.It is shown that is this method shows good specificity for Rivaroxaban and other three impurities.The linearity range of main peak of Rivaroxaban is from 0.054 to 2.718μg/ml,for the starting material of C₄H₇NO₂ is from0.053 to 2.670μg/ml,for impurityⅠis from 0.051 to 2.548μg/ml and for impurityⅡis from 0.051 to 2.571μg/ml.All relative factors are above 0.999.So it is proved that the linearity is good and at same time the corrective factors for three impurities are established.The LOQ&LOD,precision and accuracy are satisfied.And the method is sensitivity and robust method with the characters of simple operation.It meets all requirements we expected.The other section in this article is“Entecavir（Ionization sample）test method development for assay of API and impurities”.Entecaviracts as a new potent anti-HBV drug researched and developed by Bristol-Myers Squibb USA,in the clinical trial in animal experiments it shows strong efficiency for the virus of HBV.Currently,Entecavir is widespread use in treatment of chronic hepatitis B,and it is shown good safety.During the method development,through the study of concentration of buffer solution,the wave length of UV,proportion of gradient of mobile phase and column temperature,to evaluate the theoretical plate numbers,resolution,talor factor and capability factor to select the appropriate parameter.Finally,the experiment was performed with the RP-HPLC method of gradient proportion of mobile phase to separate the Entecavir,isomer including ET-d1,ET-d2 and ET-d3,and related impurities including impurity A,B and C,and to test the assay of Entecavir.The test method was executed by the column of C18,the mobile phase are trifluoroacetic acid with water（mobile phase A）and acetonitrile（mobile phase B）to test sample at 254nm under 30C of column temperature.The linearity range of main peak of Entecavir is from 0.061 to 0.61μg/ml,for ET-d1 it is from 0.063 to 0.627μg/ml,for ET-d2 it is from 0.058 to 0.582μg/ml,for ET-d3 it is from 0.060 to 0.597μg/ml,for impurity A it is from 0.061to 0.606μg/ml,for impurity B it is from 0.061 to 0.606μg/ml and for impurity C it is from 0.060 to 0.603μg/ml.The method has good linearity with the peak area respectively.All of related factors（R）are above 0.9999respectively.The average recoveries are 98.3%,95.3%,96.8%,94.6%,100.7%,101.0%and 99.1%.The RSD%is less than 2%（n=6）.The method has good specificity,highly accuracy and repeatable.The study provided sufficient data for method establishment.