Analysis of oligonucleotides by ion-pairing hydrophilic interaction liquid chromatography/electrospray ionization mass spectrometry.

Rationale: Hydrophilic interaction liquid chromatography/electrospray ionization mass spectrometry (HILIC-LC/ESI-MS) has been proved to be useful for the quality control of oligonucleotides. However, the lack of separation for some oligonucleotides using HILIC-LC/MS has proved problematic. This study aimed to improve the resolving ability of HILIC-LC/MS.

Methods: The study was performed on a Waters UPLC® system coupled to a Waters LCT premier XE ESI-TOF mass spectrometer using a Zorbax® RRHD HILIC column (2.1 mm × 100 mm, 1.8 μm). Buffer systems contained triethylammonium acetate (TEAA) and acetonitrile. The effects of the concentration of TEAA and the type of organic modifiers on the separation of oligonucleotides were investigated.

Results: The results showed that the optimum concentration of TEAA is 10 mM and acetonitrile is a better organic solvent than methanol. The addition of TEAA in the HILIC mobile phase improved the separation of N from N + A significantly compared to the HILIC method buffered with ammonium acetate. The IP-HILIC chromatography has demonstrated that the separation of oligonucleotides is sequence dependent. In addition, the IP-HILIC method produces a much simpler mass spectrum of an oligonucleotide with very efficient desalting.

Conclusions: The HILIC-LC/MS method with the addition of TEAA at a MS-compatible concentration has improved the separation of oligonucleotides. The IP-HILIC-LC/MS method also produces very simple mass spectra with high desalting efficiency.