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 240nm was used for analysis. The mobile phase consisted of phosphate buffer (pH6) and acetonitrile 50:50 (v/v) and was eluted at a flow rate of 1.2mL/min. Separation took place on a reversed-phase Kinetex C18 column (150×4.60mm; 5μm i.d.). Column temperature was set at 40°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.