Dveloping A Method For The Determination Of Esreboxetine Succinate By High Performance Liquidchromatography

Depression is a kind of unhappy experience of mind,depressive disorder is a clinical symptom caused by various causes and highly morbid.The incidence of depression worldwide is 11%,China's prevalence rate is about 3-5%.Esreboxetine is a selective noradrenaline reuptake inhibitors,the most potent enantiomer of all four steroisomers of the antidepressant reboxetine,it shows efficacy and tolerability in both short-and long-term treatment of depression.This study combined the international and domestical research of methods for determination of reboxetine and the actual synthetic route of our company,established a method for determination of esreboxetine succinate by HPLC and a method for chiral analysis of esreboxetine succinate.Firstly,the separation HPLC conditions for seven compounds were selected by changing the liquid phase additives,buffer salts,chromatographic columns and elution conditions.Optimal HPLC conditions were selected with separation degree,symmetry factor and plate number as target parameters.The study made a validation for the HPLC method we established for the determination of esreboxetine succinate.The results showed that the method was accurate and feasible.The separation of esreboxetine succinate and its R Isomer by different mobile phases and chromatographic columns in positive phase high performance liquid chromatography(HPLC)and reverse phase high performance liquid chromatography(RHPLC)was studied.The establishment of high performance liquid chromatography and chiral analysis methods for esreboxetine succinate can be used for monitoring the reaction,purity,content and optical purity.This thesis is divided into six chapters,the main contents include:The first chapter introduces the background of depression and the classification of antidepressants.It also introduces the current research status of esreboxetine succinate,and the objectives and main contents of this study.In chapter 2,the HPLC method of esreboxetine succinate was established.Firstly,the effects of different acids on the resolution and peak shape were investigated by adding formic acid,acetic acid,trifluoroacetic acid and phosphoric acid to the mobile phase;then compared the effects of different phosphate buffers with different pH values on the resolution and peak shape;next,examined the effect of adding different concentrations of triethylamine to the mobile phase on the peak shape,the types of additives for the mobile phase were determined by these three experiments;then the concentration of mobile phase additive,column temperature and flow rate were determined by orthogonal experiments;finally testing different columns determined the best column,and compared different elution gradient,established the HPLC method of esreboxetine succinate.In chapter 3,the study made a validation for the HPLC method we established for the determination of esreboxetine succinate.Destruction test under high temperature,acid,alkali,oxidation,light conditions,the result shows that esreboxetine succinate is stable under high temperature,acid,alkali and light conditions,and generates a small amount of impurities under oxidizing conditions.The generated impurities are completely separated from the main peak,and the resolution is greater than 1.5,which does not interfere with the determination of the main peak.The limit of detection(LOD)of intermediate D,intermediate E,intermediate F,and esreboxetine succinate was between 4.96 and 5.01 ng,the lower limit of quantification(LLOQ)was between 9.81 and 15.03 ng.The correlation coefficient of four compounds were all bigger than 0.999;the average recovery rate were between 98.10% and 100.99%(n=9);The stability of the esreboxetine succinate solution was stable within 24 hours,and the repeatability test and intermediate precision were good.The method is fast and accurate,and can be used to test the purity and content of esreboxetine succinate.In chapter 4,a chiral analysis method was established for the separation of esreboxetine succinate and its R Isomer.The effects on the separation of mobile phase with formic acid,trifluoroacetic acid and ammonia respectively were compared in a reversed phase high performance liquid phase system,used four columns;The acetic acid-ethanol-hexane mobile phase system,acetic acid-isopropanol-hexane mobile phase system,diethylamine-ethanol-hexane mobile phase system and diethylamine-isopropylamine-hexane mobile phase system were compared in the normal phase liquid-phase system,used four columns in this systems.From the comparison of the experimental results,the final conditions for chiral separation of esreboxetine succinate were determined as follows: The column was ChiralPak OD-H(150 mm×4.6 mm,5 ?m).The mobile phase A was n-hexane and the mobile phase B was an isopropanol solution containing 0.1% diethylamine.The isocratic elution ratio was MPA:MPB.= 90:10,flow rate 1.0 ml/min,column temperature 30 °C,wavelength 210 nm.In chapter 5,summarized the whole thesis and looked into the prospect of the further research work.