Study On The Separation And Purification Process Of High-purity Troxerutin

Purpose:Troxerutin is a semi-synthetic product of rutin by hydroxyethylation,also known as trihydroxyethylrutin,which is stipulated in the Chinese Pharmacopoeia 2015 version,calculated as anhydrous,containing troxerutin should not be less than 80.0%（for oral use）and 88.0%（for injection）.Troxerutin can reduce the permeability of capillaries,and prevent thrombosis by inhibiting platelet coagulation.It is often used clinically to treat occlusive cerebrovascular disease.With the expansion of clinical application,reports on adverse reactions caused by troxerutin preparations are increasing day by day,and some literatures indicate that derivatives and impurities present in APIs may be important factors leading to adverse reactions.Therefore,developing a separation technology that can effectively improve the purity of troxerutin has certain clinical application value.The main purpose of this topic was to improve the purity of troxerutin.The column chromatography method was used to study the separation and purification process of troxerutin,and the optimal separation conditions were explored by screening of resin,studying of resin adsorption theory and optimization of chromatography conditions.Finally,the quality of troxerutin after purification was preliminarily evaluated.Methods:1.Screening of resinAccording to the polarity,specific surface area and pore size of different adsorption resins,three representative resin models were selected as the experimental objects of this subject.In order to select the most suitable resin for separation of troxerutin,the adsorption capacity and desorption rates of troxerutin on three resins were measured under static experimental conditions,and the separation efficiency of troxerutin on each resin were compared by column chromatography experiment.2.The static adsorption thermodynamics and kinetics study of troxerutin on SZ-3resinIn order to investigate the situation when troxerutin reached adsorption equilibrium on the resin,the adsorption isotherm of troxerutin on the resin was measured.Langmuir equation,Freundlich equation and Temkin equation were used to fit the adsorption isotherm data.In addition,in order to investigate the adsorption rate,equilibrium adsorption amount and control rate steps of adsorption process,the adsorption kinetic curve of troxerutin on the resin was determined,and the Pseudo-firth-order model,Pseudo-second-order model and Weber-Morris internal diffusion model were used to fit the kinetic data.3.Optimization of chromatography conditionsTaking troxerutin for injection（88%）as the separation object and SZ-3 resin as the chromatographic separation medium,the dynamic adsorption curve of troxerutin on the resin was measured.Then,we explored effect of the loading amount,concentration of eluent,the pH of eluent and the temperature of t elution on the separation efficiency,the purity of troxerutin as the main result of the investigation index to obtain the optimal separation conditions.The experiment was repeated three times under the optimal separation conditions to verify the stability of the separation process,and the average value of the purity and recovery of troxerutin was calculated.4.Regeneration efficiency of resinAccording to the optimal separation conditions,troxerutin was continuously adsorbed and eluted for 10 cycles,the adsorption capacity was calculated every 2 cycles,and the separation performance of troxerutin by the new resin and the resin after 10regenerations was compared.Taking the resin’s adsorption capacity and resolution（separation efficiency）of troxerutin as the judgment standard,the useful life of the resin was evaluated.5.Preliminary evaluation of the quality of the purified sampleA general identification test was conducted to investigate whether the appearance of the sample and the identification response were in accordance with the description of troxerutin in the 2015 edition of the Chinese Pharmacopoeia.Then,infrared spectroscopy was used to analyze whether the infrared absorption peak of the sample is consistent with the standard spectrum of troxerutin,and the compound composition of the sample was analyzed by mass spectrometry.Finally,high-performance liquid chromatography was used to analyze and detect,and the purity of the sample was calculated according to the area normalization method.6.Preliminary study on separation process of troxrutin impurity 1The macroporous adsorption resin HZ-806 was used as the separation medium to obtain the dynamic adsorption curve of troxerutin on the resin.Then,the purity of impurity 1 was used as the investigation index,the size of the chromatography column and the concentration of eluent were optimized to obtain better separation conditions.Results:1.The results of static adsorption experiments showed that the absorption of troxerutin on SZ-3 was 132 mg/g resin,which was twice that of HZ-806 and HZ-816.The results of column chromatography experiments showed that SZ-3 resin had the best chromatographic separation efficiency for troxerutin.Therefore,SZ-3 resin was selected to be the most suitable separation medium for the separation of troxerutin among the three selected adsorption resins.2.The adsorption isotherm fitting results showed that the Langmuir equation could fit the adsorption isotherm data of troxerutin on the resin at 20℃,and the correlation coefficient was 0.9934,indicating that troxerutin favored monolayer adsorption.The adsorption kinetics fitting results showed that the Pseudo-second-order model has a good fitting effect on the adsorption kinetics data at various temperatures,and the correlation coefficients are all greater than 0.99.Then,the initial adsorption rate V₀ and equilibrium adsorption amount Q_e were calculated from the fitting equation of the model.The calculation results showed that the initial adsorption rate of troxerutin on SZ-3 resin was the largest at 20℃,and the experimental value of equilibrium adsorption amount was basically consistent with the calculated value.The fitting result of the Weber-Morris internal diffusion model showed that the adsorption rate was mainly controlled by the external diffusion of the particles（membrane diffusion）and the internal diffusion of the particles.3.The chromatography conditions of troxerutin was optimized to obtain the best separation process:one-time chromatography,the loading amount was 90 mg/g resin,the eluent was 25%methanol,and the elution volume was 30 BV（bed volumes）,the adsorption and elution temperature was 20℃,and the adsorption and elution flow rate was 0.8 mL/min.Secondary chromatography:crude solution a as loading solution,the eluent was 25%methanol,and the elution volume was 20 BV;crude solution b as loading solution,the eluent was 40%methanol,and the elution volume was 10 BV.According to the experimental results of separation process verification,the purity of troxerutin after one-step chromatography separation could reach 96.4%,and the recovery rate was 39%.The purity achieved after two-step chromatography separation was 98.6%,and the recovery rate was 12%.The purity of eluent that had not reached more than 95%could be increased to more than 96%after secondary chromatography.Compared with the average value of 3 experiments,the purity of troxerutin obtained by this separation process was stable,but the stability of the yield was relatively poor.The results of resin regeneration experiments showed that SZ-3 resin could be used for at least10 cycles in the separation and purification experiment of troxerutin.4.A preliminary evaluation of the quality of the purified sample,the appearance of the sample and the experimental results of the identification reaction were in line with the description of troxerutin in the 2015 edition of the Chinese Pharmacopoeia.The infrared spectroscopy analysis results showed that the infrared absorption peaks of the sample were consistent with the standard spectrum of troxerutin.Mass spectrometry analysis also showed that the sample contained troxerutin,tetrahydroxyethylrutin（impurity 1）,dihydroxyethylrutin and its isomer（impurity 2）.The results of the sample purity were calculated according to the area normalization method,the content of troxerutin in sample a and sample b were 96.28%and 98.30%,the total impurity content were 3.7%and 1.7%,respectively.5.The HZ-806 resin could be used as separation medium to separate impurity 1（tetrahydroxyethylrutin）with higher purity.The chromatographic conditions were:the loading solution was 25 mL of a concentrated solution rich in impurity 1,the eluent was8%methanol,the adsorption elution flow rate was 1 mL/min,finally,the content of impurity 1 in the second to third BV effluent could reach about 97%.Conclusion:In view of the current difficulty of separation of troxerutin,this paper used the adsorption capacity and desorption rate of troxerutin and the purity of troxerutin as the judgment indicators,SZ-3 was screened to separate and purify troxerutin from 3 different types of resin,and the chromatographic conditions were optimized.Finally,a separation and purification process that could significantly improve the purity of troxerutin was successfully established.Compared with the traditional purification process,the process had higher purity and fewer purification steps.The separation process of troxrutin impurity 1 explored the effect of chromatography column size and eluent concentration on separation efficiency,the purity of impurity 1 finally reached about 97%.