Systematic Evaluation of Hydrophilic Interaction Liquid Chromatography Stationary Phases for Oligonucleotide Characterization by LC/MS

Significance of the Topic

Oligonucleotide therapeutics and diagnostics demand reliable liquid chromatography–mass spectrometry (LC-MS) methods. Traditional ion-pair reversed-phase (IP-RPLC) and ion exchange (IEX) techniques have limitations such as ion-pair reagent carryover and MS incompatibility. Hydrophilic interaction liquid chromatography (HILIC) offers MS-friendly mobile phases and versatile selectivity, making it a promising alternative for characterizing native and modified oligonucleotides.

Objectives and Study Overview

This study systematically evaluates five HILIC stationary phases for oligonucleotide separation and LC-MS analysis. Major goals include:

• Characterizing retention mechanisms using the Tanaka test
• Comparing separation performance of Poroshell 120 HILIC, HILIC-OH5, HILIC-Z, AdvanceBio Glycan Mapping, and Amide HILIC phases
• Assessing the impact of mobile phase pH and column temperature on DNA, RNA, and modified antisense oligonucleotides (ASOs)

Methodology and Instrumentation

The experimental workflow comprised:

1. Tanaka Test Screening to quantify hydrophilicity, hydrophobicity, steric selectivity, hydrogen bonding, ion exchange, and acidic/basic interactions of each HILIC phase
2. LC-MS separations of oligo standards using Agilent 1260 Infinity Bioinert quaternary pump, 1260 diode array detector, 1290 thermostatted column compartment, and 6545XT Q-TOF
3. Mobile phases based on 100 mM ammonium acetate adjusted to pH 4.4, 6.8, or 9.0; solvent A was 10% buffer/90% water and solvent B was 10% buffer/90% acetonitrile

Main Results and Discussion

The Tanaka test revealed distinct selectivity profiles: Poroshell 120 HILIC and HILIC-OH5 showed strong hydrophilicity, HILIC-Z offered notable ion-exchange character, Glycan Mapping provided balanced interactions, and Amide HILIC excelled in hydrogen bonding. Real-world separations of 15 – 40 mer DNA and RNA standards confirmed these trends under gradients optimized for each phase.

Adjusting mobile phase pH from 6.8 to 9.0 shifted retention times and increased sodium/potassium adduct formation. Column temperatures up to 60 °C improved peak shape and resolution, particularly for modified ASOs analyzed on the HILIC-Z column. Two custom 21-mer ASO sequences with MOE modifications were baseline-resolved at elevated pH and temperature.

Benefits and Practical Applications

HILIC-LC-MS offers:

• Compatibility with MS detection without ion-pair reagents
• Flexible use on standard LC systems for both native and modified oligos
• Enhanced selectivity by tuning stationary phase chemistry, pH, and temperature

Future Trends and Opportunities

Potential developments include:

• Further buffer optimization to minimize metal adducts and improve sensitivity
• Refinement of MS source parameters for robust oligo fragment analysis
• Expansion of HILIC phases with novel functional groups for enhanced separation of challenging modifications

Conclusion

This work demonstrates that choosing the appropriate HILIC phase and optimizing mobile phase pH and temperature are key to high-performance LC-MS analysis of oligonucleotides. HILIC emerges as a versatile tool for characterizing therapeutic and diagnostic oligos without the drawbacks of ion-pairing reagents.

Reference

• B Buszewski and S Noga. Hydrophilic interaction liquid chromatography – a powerful separation technique Anal Bioanal Chem 402 231-247 2012
• Y Kawachi et al. Chromatographic characterization of hydrophilic interaction liquid chromatography stationary phases J Chromatogr A 1218 5903-5919 2011
• H Lardeux D Guillarme VD D’Atri Comprehensive evaluation of zwitterionic hydrophilic liquid chromatography stationary phases for oligonucleotide characterization J Chromatogr A 1690 463785 2023