Sequencing of Phosphorodiamidate\nMorpholino Oligomers\nby Hydrophilic Interaction Chromatography Coupled to Tandem Mass Spectrometry

Sequencing of phosphorodiamidate morpholino oligomers\n(PMOs) by\nhydrophilic interaction chromatography (HILIC) coupled to tandem mass\nspectrometry (MS/MS) is reported. The MS/MS analysis was performed\nusing a quadrupole/time-of-flight (Q-ToF) mass analyzer and collision\ninduced dissociation (CID) in negative ion mode. To improve MS sensitivity\nin negative ion mode, HILIC conditions, including the separation column,\nmobile phases, and MS parameters, were optimized. Using the developed\nHILIC–CID-MS/MS method, 100% sequence coverage was achieved\nfor PMOs ranging from 18-mer to 25-mer. Additionally, the method was\nsuccessfully applied to identifying positional isomers of <i>n</i> – 1 deletion impurities present in PMO drug substances.