Ultra-High-Performance Reversed-Phase Liquid Chromatography Hyphenated with ESI-Q-TOF-MS for the Analysis of Unmodified and Antisense Oligonucleotides

The reversed-phase ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry method was developed during the study and successfully applied to separation and analysis of unmodified and antisense oligonucleotides. It was shown that oligonucleotide separation strongly depends on the stationary phase type. The type, pH and salt content in the mobile phase influence also their chromatographic behavior and mass spectrometry sensitivity: increasing the apparent pH causes retention decrease and increase of sensitivity, while increasing the salt concentration produces greater oligonucleotide retention and lower sensitivity. Utilization of octadecyl stationary phase with aryl rings in reversed-phase mode allows successful separation of various modified and unmodified oligonucleotides, differing in length or sequence. The developed method has numerous advantages over current liquid chromatography methods: resolution is comparable to ion pair chromatography; oligonucleotides are analyzed with salts, which allow easy purification and separation without losing column performance, whereas columns run in the presence of ion pair reagents degrade more rapidly. Moreover, the differences in mass spectrometry sensitivity between reversed-phase liquid chromatography and ion pair chromatography were not significant for the studied compounds. The developed method is an effective analytical tool for the determination of oligonucleotide impurities.