MS1 Oligonucleotide Characterization Using LC/Q‑TOF with HILIC Chromatography

Importance of the Topic

The characterization of synthetic oligonucleotides is critical in pharmaceutical research, quality assurance, and quality control. High-performance liquid chromatography coupled with high-resolution mass spectrometry (LC/Q-TOF) provides both separation and accurate mass identification, enabling the detection of product variants and impurities without extensive sample preparation or contamination by ion-pairing reagents.

Objectives and Study Overview

This work demonstrates a hydrophilic interaction chromatography (HILIC) approach for intact mass and impurity analysis of diverse oligonucleotides. The method evaluates chromatographic performance, mass spectral quality, retention time reproducibility, ability to preserve higher-order structures such as siRNA duplexes, and detection of low-abundance impurities across DNA and RNA oligos ranging from 18- to 100-mers.

Methodology and Instrumentation

Ultrapure mobile phases based on ammonium acetate (15 mM, pH 7) and acetonitrile were employed with an Agilent InfinityLab Poroshell 120 HILIC-Z column (2.1×100 mm, 1.9 μm). Separation was performed on an Agilent 1290 Infinity II LC system and online MS detection used an Agilent 6545XT AdvanceBio Q-TOF with dual-spray JetStream source. Key conditions included:

• Gradient: 15–60% B over 13 min; flow rate 0.4 mL/min; column at 30 °C.
• Injection volume: 2–5 μL; autosampler at 4 °C.
• MS settings: negative polarity, 350 °C gas temp, 400 °C sheath gas, m/z 400–3200, 1 spectrum/s.

Main Results and Discussion

• Baseline separation of 20, 40, 60, 80, and 100-mers was achieved in a 15 min gradient, with efficient deconvolution of intact masses and identification of low-level depurination/depyrimidination products.
• Charge state distributions under HILIC were narrow and shifted to lower charge, consistent with native-like conditions. Larger oligos showed reduced ion intensities, highlighting the need for extended m/z range and optimized desolvation.
• Retention time reproducibility was excellent (<0.1% RSD) after two preconditioning injections, even with short (5–10 min) re-equilibration times.
• The method preserved higher-order structures: siRNA duplexes remained intact throughout analysis, and deconvoluted mass peaks matched duplex and individual strand masses.
• Low-abundance impurities down to <0.1% of full-length product were detected using untargeted and targeted deconvolution workflows.
• Complex mixtures with mixed bases at terminal positions were resolved by mass, even when chromatographic coelution occurred.

Benefits and Practical Applications

• Avoidance of ion-pairing reagents reduces instrument cleaning and enables rapid polarity switching.
• Robust performance for a broad range of oligonucleotide sizes, chemistries, and structures.
• High dynamic range MS detection supports comprehensive impurity profiling and relative quantitation.
• Fast throughput with reproducible retention times and minimal equilibration requirements.

Future Trends and Potential Applications

• Extension to longer RNA strands and heavily modified oligos for therapeutic development.
• Automation of HILIC-MS workflows for high-throughput quality control in oligonucleotide manufacturing.
• Integration with real-time data analysis and artificial intelligence for rapid impurity screening.
• Exploration of alternative zwitterionic and polymeric HILIC phases to further improve selectivity and robustness.

Conclusion

The described HILIC-LC/Q-TOF method offers a versatile, fast, and sensitive platform for intact mass analysis and impurity characterization of synthetic oligonucleotides. It combines excellent chromatographic resolution, native-like preservation of secondary structures, and high dynamic range mass detection, making it well suited for research and quality control applications.

Reference

1. Rye P., Schwarzer C. MS1 Oligonucleotide Characterization Using LC/Q-TOF with HILIC Chromatography. Agilent Technologies, Application Note 2023.
2. Lobue P. et al. Oligonucleotide Analysis by HILIC-MS without Ion-Pair Reagents. J. Chromatogr. A 2019, 1595, 39–48.
3. Guimaraes R. et al. Impact of Mobile Phase pH in Oligonucleotide IP-LC-MS. Future Sci. OA 2021, FSO753.
4. Guo H. et al. Secondary Structural Characterization of Oligonucleotides by ESI-MS. Nucleic Acids Res. 2005, 33(11), 3659–3666.