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Protocol for Oligonucleotides Characterization Using Hydrophilic Interaction Chromatography.

Davy Guillarme1,2, Szabolcs Fekete3, Sylwia Studzińska4

  • 1School of Pharmaceutical Sciences, University of Geneva, Geneva, Switzerland.

Journal of Separation Science
|January 29, 2025
PubMed
Summary

This protocol details hydrophilic interaction chromatography (HILIC) for analyzing oligonucleotides (ONs), offering an alternative to ion pairing reverse-phase liquid chromatography (IP-RPLC) for improved mass spectrometry coupling.

Keywords:
adsorptionhydrophilic interaction chromatographymass spectrometrymobile phaseprotocolstationary phase

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Area of Science:

  • Analytical Chemistry
  • Chromatography
  • Mass Spectrometry

Background:

  • Oligonucleotides (ONs) are crucial therapeutics, especially for genetic diseases.
  • Analyzing ONs is challenging due to their hydrophilic nature and synthesis impurities.
  • Current methods like IP-RPLC have limitations for mass spectrometry (MS) coupling.

Purpose of the Study:

  • To provide a protocol for using hydrophilic interaction chromatography (HILIC) for ONs analysis.
  • To establish HILIC as a viable alternative to IP-RPLC for ON characterization.
  • To guide researchers in implementing HILIC for ON analysis, including MS coupling.

Main Methods:

  • Exploration of HILIC principles for ON separation.
  • Optimization of stationary and mobile phases for HILIC.
  • Development of gradient conditions and MS coupling strategies for ON analysis.

Main Results:

  • HILIC presents a promising alternative for ONs analysis.
  • Detailed considerations for HILIC method development are provided.
  • The protocol addresses sample preparation and key analytical parameters.

Conclusions:

  • HILIC offers advantages for ONs analysis, particularly for MS coupling.
  • This protocol facilitates the adoption of HILIC in oligonucleotide characterization.
  • HILIC has the potential to become a universal solution for ON analysis.