Bioanalytical HILIC LC-MS Compatibility Using the GTxResolve™ Premier Amide Column and OligoWorks™ SPE Sample Preparation Solution

Importance of the Topic

This summary addresses the growing use of hydrophilic interaction liquid chromatography (HILIC) for oligonucleotide analysis by mass spectrometry. HILIC methods eliminate the need for ion-pairing reagents, reducing toxicity and cost compared with ion-pair reversed-phase approaches. Starting gradients at high organic content also improves compatibility with bioanalytical samples, minimizes non-specific adsorption of polar analytes and enhances solubility of oligonucleotides.

Objectives and Study Overview

The primary goal of this study was to evaluate the compatibility of the OligoWorks™ SPE bioanalytical sample preparation solution with HILIC LC-MS workflows. Key aspects included:

• Comparing HILIC separation on the GTxResolve™ Premier Amide Column with traditional IP-RP-LC for a panel of unmodified and conjugated oligonucleotides.
• Testing injection volumes from 1 to 10 µL of SPE eluates diluted in acetonitrile.
• Assessing chromatographic performance, retention behavior and mass spectrometric response.

Methodology and Used Instrumentation

Sample Preparation:

• Oligonucleotide standards prepared in RNase-free water or OligoWorks SPE Eluent diluted 1:1 or 1:2 with MS-grade acetonitrile.

Chromatographic Conditions:

• HILIC mobile phase A: 25 mM ammonium acetate (pH 6.8); mobile phase B: 100 % acetonitrile.
• IP-RP mobile phase A: 0.1 % DIPEA/1 % HFIP in water; mobile phase B: 0.0375 % DIPEA/0.75 % HFIP in 65:35 acetonitrile:water.
• Gradients and column formats followed protocols optimized for each separation mode.

Instrumentation:

• Waters ACQUITY UPLC I-Class PLUS System with Flow-Through Needle sample manager.
• Waters ACQUITY Premier System with Fixed-Loop sample manager and partial-loop pressure assist injection.
• GTxResolve Premier BEH Amide Column (300 Å, 1.7 µm, 2.1 ×50 mm).
• BioAccord™ HRMS with RDa detector and TQ Absolute MS for quantitation.

Main Results and Discussion

Chromatographic Comparison:

• IP-RP-LC retention correlated with the hydrophobicity of conjugated oligonucleotides via ion-pairing interactions.
• HILIC retention increased for polar, unmodified oligonucleotides, while more hydrophobic conjugates eluted earlier.

Injection Volume Tolerance:

• Dilutions of SPE eluate in acetonitrile (1:1 and 1:2) enabled up to 10 µL injections without peak distortion for both polar and lipid-conjugated ASOs.
• Partial-loop pressure assist mode further improved reproducibility at higher volumes.

Mass Spectrometric Response:

• Polar oligonucleotides exhibited enhanced MS signal across the injection volume range, demonstrating the method’s suitability for quantitative bioanalysis.

Benefits and Practical Applications

The combined OligoWorks SPE/HILIC LC-MS workflow offers:

• Avoidance of toxic ion-pairing agents, reducing user exposure and disposal costs.
• Improved sample compatibility through high-organic starting conditions that minimize analyte precipitation and adsorption.
• Enhanced sensitivity via larger injection volumes enabled by optimized dilution and injection modes.
• Seamless integration on existing UPLC-MS platforms for routine bioanalytical support of oligonucleotide therapeutics.

Future Trends and Potential Applications

Emerging directions include:

• Automation of online SPE-HILIC workflows for high-throughput oligonucleotide quantitation.
• Extension of HILIC-MS methods to other classes of polar biomolecules and conjugates.
• Development of tailored stationary phases to further refine selectivity for modified oligonucleotides.
• Integration with multi-omic platforms to enable comprehensive biomarker profiling in complex matrices.

Conclusion

This study confirms that the OligoWorks SPE solution is fully compatible with HILIC-MS using the GTxResolve Premier Amide Column. Minor gradient and injection adjustments allow injection volumes up to 10 µL without compromising chromatographic integrity or MS sensitivity, supporting robust bioanalytical workflows for diverse oligonucleotide therapeutics.

Reference

Araya M., Trudeau M., Addepalli B., Lauber M. Bioanalytical HILIC LC-MS Compatibility Using the GTxResolve Premier Amide Column and OligoWorks SPE Sample Preparation Solution. Waters Corporation, September 15, 2025.