Study On Isolation Of Minor Impurities In Drugs With Recycling Chromatography

The content of impurities in drugs is closely related to the safety of drugs,and even minor impurities may affect the quality of drugs and the safety of patients.Preparative liquid chromatography is usually used to isolate impurities in drugs.However,a single chromatographic run might be insufficient to separate minor impurities completely and thus several separation steps are required,resulting in high dilution of the product and high energy consumption for evaporation.Recycling chromatography can significantly improve the separation by increasing the effective length of the column,but because of the band broadening effect,the number of cycles and the amount of feed are limited.In order to suppress the adverse effects of band broadening on separation,two recycling strategies were developed for the separation of minor impurities in two different drug systems.One is a pharmaceutical intermediate that is soluble in methanol and acetonitrile,a reverse phase system;the other is an antioxidant that is easily soluble in n-hexane,a normal phase system.For the reverse phase system,a twin-column recycling chromatography with solvent gradient was employed,which introduced solvent gradient based on conventional recycling chromatography.In reversed phase chromatography,the elution strength of the mobile phase decreases with the water content.Thereby,solvent gradient is created by adding water between the upstream and downstream columns of the recycling chromatography to make the elution strength of the upstream stronger than the downstream.In this way,the tail of the solute band migrates faster than the front of the band,resulting in an compression effect on the band to offset the band broadening.Neither methanol nor acetonitrile could isolate all impurities by a single step.It is necessary to use two steps to for the removal of all the impurities.The recycling chromatography with solvent gradient could integrate the two-step separation into one step:A portion of the impurities were separated with methanol-water,and then the main component was not eluted out of the system,and the eluent was directly replaced with acetonitrile-water to continue to separate the remaining impurities.The effects of mobile phase composition,solvent gradient size and feed amount on the separation process were investigated.After optimization,suitable separation conditions were obtained:two columns(1cm×20cm,C8)were used,the concentration of the raw material solution was 2 mg/mL,and the feed amount was 60mL.The eluents in the first stage and the second stage were respectively methanol/water(65/35,v/v)and acetonitrile/water(55/45,v/v),the amount of water is 0.2mL/min.Under this condition,the qualified product liquid concentration was 3.8 mg/mL,and the yield was 94.7%.For the normal phase system,if the twin-column recycling chromatography with solvent gradient is employed,the amount of modifier will be 10 times that of the reverse phase system,which obviously increases the consumption of organic solvents.In addition,different from the reversed-phase system,once the solvent gradient was introduced in the normal phase system,the downstream flow rate will be much greater than the upstream flow rate,thus offsetting the band compression formed by the solvent gradient.Therefore,it is not advisable to use the twin-column recycling chromatography with solvent gradient.However,the impurity to be removed is less retained,and hence it can be removed by the displacement effect.The amount of feed in conventional recycling chromatography is small,and the band will broaden to decrease in height during the cycle so that the displacement effect is weakened to make against the removal of impurities.By increasing the number of columns to form an eight-column recycling chromatography,the feed amount can be increased to delay the reduction of the band height,and the positive effect of the displacement effect on the separation of impurities can be fully exploited accordingly.The effects of feed volume,elution volume,number of cycles,feed concentration and eluent composition on the separation process were investigated.The suitable separating conditions were obtained:the feed concentration was 115 mg/mL,the feed volume was 40 mL,and the eluent was n-hexane/ethyl acetate(96/4,v/v).The elution volume was 36 mL,the number of cycles was 7,the recovery was 92.1%,the yield was 67.46 g/(L·h),and the solvent consumption was 0.085 L/g.