Analysis of pharmaceutical drug oligomers by selective comprehensive two-dimensional liquid chromatography-high resolution mass spectrometry

Abstract The presence of oligomeric impurities in drugs is often overlooked and less studied due to conventional small molecule analytical methods which are often not capable of capturing these oligomers. In the present work, the oligomer species of a lipophilic active pharmaceutical ingredient (API) containing an azetidine ring was investigated. No separation was observed by reversed-phase liquid chromatography (RPLC) methods, and only a partial separation of oligomer peaks was achieved by a size exclusion chromatography (SEC) method. To improve the resolution of the different oligomers species and understand the root cause of the formation of the oligomers, a selective comprehensive two-dimensional liquid chromatography (2D-LC) method was developed by coupling SEC to RPLC. The selective comprehensive SEC × RPLC method allowed the separation of 16 species and evidenced four main groups of oligomer impurities. The contour plots of different API lots helped to visualize the oligomer profiles and quickly compare the difference between these lots. Finally, the different oligomer peaks separated by 2D-LC were identified by high-resolution mass spectrometry (HRMS) using a Q Exactive mass spectrometer. The developed 2D-LC/HRMS workflow provides a fast and generic screening approach to quickly examine and visualize the oligomeric impurities in API materials, and direct the impurity control strategy during process development.