HPLC-UV method development and validation to monitor difluprednate synthesis

Abstract

During the synthesis of active pharmaceutical ingredients (APIs) there is a need for the development and validation of a simple and rapid high performance liquid chromatography (HPLC) method for the determination and quantification of the synthesized product and related by-products. An HPLC method gives a better understanding of how a synthesis is proceeding. A rapid and easy to use HPLC-UV (ultraviolet) method for the determination of difluprednate and monitoring of impurities generated during synthesis was developed and validated. A Shimadzu VP Series HPLC equipped with a LabSolutions software and UV detector set at 240 nm was used for analysis. The mobile phase consisted of phosphate buffer (pH 6) and acetonitrile 50:50 (v/v) and was eluted at a flow rate of 1.2 mL/min. Separation took place on a reversed-phase Kinetex C18 column (150 脳 4.60 mm; 5 渭m i.d.). Column temperature was set at 40掳C. The developed method was found to have good linearity and acceptable accuracy and precision. The developed method may be effectively applied to determine products and by-products formed during synthetic reactions of steroids and to calculate the yield of the products obtained during each step of the synthesis.