Study On The Isolation And Preparation Of Drug Impurities By Twin-column Recycling Chromatography With A Solvent Gradient

Impurities,refering to substances in active pharmaceutical ingredients(APIs)that affect the purity of active ingredients,are closely related to drug quality and clinical safety.Impurity reference substances are chemical standard substances used for impurity identification,quantitative analysis,impurity control,etc.,and play an important role in impurity research.The purity requirements on the reference substance is generally above than 95%,and the synthetic method is always too difficult to meet the requirement.Thereby,separation and purification of impurity reference substances from the APIs by chromatography has become an appropriate method.As the impurities have physical and chemical properties close to the active components,the separation by conventional chromatography is unsatisfactory.Recycling chromatography technology can improve the separation,but the band broadening,caused by non-ideal factors and nonlinear adsorption,has a negative impact on the separation of minor impurities.In this work,twin-column recycling chromatography with a solvent gradient was used to solve the above problem.A modifier was constantly injected between the two columns to decrease the eluotropic strength of the downstream solvent,so that the migratory velocity of the band front was slower than the tail.Thus the band compression mechanism was established to effectively prevent the band broadening and improve the concentration of impurity products.Firstly,a key pre-impurity in Orlistat was selected as the target component to investigate the effects of different operating conditions on the separation.Compared with isometric recycling chromatography,solvent gradient can load more raw materials,significantly increase product concentration and improve separation efficiency.The larger the gradient,the higher the product concentration,and the more obvious the improvement effect within a certain range.In addition,the separation can be improved by appropriately increasing the water content of the eluent,prolonging the cycle period and increasing the cycle times.The optimum conditions were as follows:mobile phase of methanol/water(87/13,v/v),solvent gradient of 1.5%,cycle period of 29.1 min and 10 cycle times.Finally,the key impurity with content of 0.7%in the raw material was purified to 96%,and the concentration was increased by 8.4 times.Secondly,two impurities c1 and c2 in crude paclitaxel were selected as the target components to verify the feasibility of solvent gradient recycling chromatography in complex mixture system.The experiment was divided into two steps.The first step was preliminary enrichment,and methanol/water was used as eluent to separate the two impurities.In the second step,with acetonitrile/water as eluent,the two impurities were further purified.The influence factors such as water content of eluent,flow rate of modifier,cycle period and cycle times were investigated to obtain the optimal enrichment and purification operating conditions.Finally,the impurities c1 and c2 with a total content of no more than 1.8%in the raw materials were purified to 95.3%and 99.1%,respectively.The total yield of the two target impurities was 36.3%,and the total concentration was 1.5 times that of the raw material solution.The results showed twin-column recycling chromatography with a solvent gradient can simultaneously purify and enrich the minor impurities from the APIs.The separation is much better than isocratic recycling chromatography.