Sequencing of Phosphorodiamidate Morpholino Oligomers by Hydrophilic Interaction Chromatography Coupled to Tandem Mass Spectrometry

• Published in: Journal of the American Society for Mass Spectrometry Vol. 35; no. 10; pp. 2490 - 2498
• Main Authors: Wang, Mingming, O’Day, Brian, Michaels, Brian, Jurayj, Jurjus, Cai, Bao Zhong, Wei, Tao
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 02.10.2024
• Subjects: Chromatography, Liquid - methods; Hydrophobic and Hydrophilic Interactions; Morpholinos - chemistry; Tandem Mass Spectrometry - methods; phosphorodiamidate morpholino oligomer (PMO); tandem mass spectrometry (MS/MS); collision induced dissociation (CID); hydrophilic interaction chromatography (HILIC); impurity characterization; sequencing
• ISSN: 1044-0305; 1879-1123; 1879-1123
• DOI: 10.1021/jasms.4c00281

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