Evaluating HILIC Stationary Phases for Oligonucleotide Separation by LC/MS

Significance of the topic

The separation and characterization of oligonucleotides play a critical role in biopharmaceutical research and quality control of therapeutic nucleic acids. Hydrophilic interaction chromatography (HILIC) offers an alternative to ion-pair reversed-phase liquid chromatography (IP-RPLC) and ion-exchange methods by providing MS-compatible mobile phases and complementary selectivity for intact and modified oligonucleotides.

Objectives and study overview

This work aimed to evaluate five distinct HILIC stationary phases under varying mobile phase pH conditions to identify optimal retention, resolution, and mass spectrometric response for DNA and RNA oligonucleotides ranging from 14 to 40 nucleotides, as well as a heavily modified 18-mer antisense oligonucleotide (ASO). The study compared columns based on bare silica, poly-hydroxy fructan, zwitterionic, neutral amide, and mixed-mode amide chemistries.

Methodology and instrumentation

Sample Preparation and Columns:

• Standards: DNA ladder and RNA resolution kits and a pharmaceutically relevant 18-mer ASO with 2ʹ-methoxyethoxy modifications.
• Columns Evaluated:
  
  • Poroshell 120 HILIC (bare silica)
  • Poroshell 120 HILIC-OH5 (poly-hydroxy fructan)
  • Poroshell 120 HILIC-Z (zwitterionic)
  • AdvanceBio Glycan Mapping (neutral amide)
  • AdvanceBio Amide HILIC (mixed-mode amide/ion-exchange)
• Mobile Phases: 10 mM ammonium acetate buffers at pH 4.4, 6.8, and 9.0 mixed with acetonitrile in HILIC gradients.

Used instrumentation

• Agilent 1290 Infinity II LC System including high-speed pump, multisampler with thermostat, multicolumn thermostat, and diode array detector.
• Agilent 6545XT AdvanceBio LC/Q-TOF mass spectrometer with Jet Stream electrospray source in negative ion mode.

Main results and discussion

• Mobile phase pH influenced chromatographic selectivity, peak shape, and electrospray charge state distribution, with neutral to basic pH (6.8–9.0) generally improving resolution and MS signal for longer oligonucleotides.
• The Glycan Mapping column at pH 6.8 and the HILIC-Z column at pH 9.0 provided the best separation of DNA and RNA standards, including critical n–1 impurity resolution.
• Charge state distributions shifted toward fewer, sharper charge states at higher pH, facilitating accurate mass measurement and spectral deconvolution.
• Both selected columns produced sharp, high-sensitivity peaks for an 18-mer ASO, with Glycan Mapping exhibiting broader charge state coverage and improved signal intensity.
• The Glycan Mapping column demonstrated excellent long-term stability over 1,000 injections with <3% RSD in retention times.

Benefits and practical applications of the method

HILIC-LC/MS methods developed here enable robust, ion-pair free analysis of therapeutic oligonucleotides, simplifying instrument workflows and allowing sequence confirmation of intact and modified species. The approach is applicable to quality control of DNA, RNA, antisense oligonucleotides, siRNA, and other emerging oligonucleotide modalities.

Future trends and opportunities

Continued expansion of HILIC phases tailored for nucleic acids may support high-throughput profiling of complex oligonucleotide therapeutics. Integration with high-resolution tandem MS will further enable detailed mapping of sequence variants and modifications. Advances in stationary phase chemistries and mobile-phase additives promise enhanced selectivity for novel oligonucleotide designs.

Conclusion

This systematic evaluation confirms that carefully chosen HILIC stationary phases and mobile phase pH yield superior separation and detection of oligonucleotides by LC/MS. The optimized Glycan Mapping and HILIC-Z methods deliver high resolution, reproducibility, and spectral quality, offering valuable alternatives to ion-pair and ion-exchange chromatography for biopharma analytics.

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