Developing a Novel, Integrated LC-MS Workflow for High-resolution Monitoring and Characterization of Oligonucleotides

Importance of the Topic

Therapeutic oligonucleotides demand precise structural confirmation, molecular weight verification, and impurity profiling to ensure safety and efficacy. High-resolution mass spectrometry coupled with liquid chromatography and UV detection offers a comprehensive platform for identity confirmation, impurity screening, and full-sequence verification, supporting drug development and quality control.

Objectives and Study Overview

This application note presents the development of an integrated LC-MS workflow for oligonucleotide analysis, combining reversed-phase UPLC, tunable UV detection, high-resolution time-of-flight MS, and automated data processing. The key aims were to optimize chromatographic separation, minimize salt adduct formation, achieve low detection limits, and enable high-throughput impurity profiling and MS/MS ladder sequencing.

Methodology and Instrumentation

The workflow employs:

• ACQUITY UPLC H-Class Bio System with Waters Oligonucleotide BEH C18 column at 60 °C
• ACQUITY UPLC Tunable UV Detector set at 260 nm
• Xevo G2-XS QTof Mass Spectrometer in negative-ion ESI mode
• MassLynx v4.1 for instrument control and data acquisition
• ProMass HR integrated with MassLynx for spectral deconvolution and automated reporting

Mobile phases consist of 15 mM triethylamine and 400 mM HFIP in water or methanol, prepared gravimetrically. MS settings: 400–3000 Da range, cone voltage 80 V for MS, 120 V for MS/MS, desolvation at 500 °C.

Key Results and Discussion

• Chromatographic separation of polyT standards (15–35 nt) achieved baseline resolution of full-length and truncation products.
• MS spectra exhibited minimal sodium adducts (< 5%) and clear isotopic envelopes, with average monoisotopic mass accuracy of ~1.25 ppm.
• Limit of detection for the polyT standard was determined at 20 fmol on-column, with UV detection as the limiting factor.
• Automated batch processing of 48 samples demonstrated clear identification of target oligonucleotides, single-base deletion/insertion impurities, and color-coded HTML reporting.
• MS/MS ladder sequencing of a 21-nt siRNA strand provided full C and Y ion series coverage, confirming sequence identity through deconvoluted fragment matching.

Benefits and Practical Applications

This integrated workflow streamlines oligonucleotide analysis by combining high-resolution mass accuracy, robust chromatography, and automated data processing. It supports high-throughput impurity screening, precise quantification via UV absorbance, and full-sequence MS/MS confirmation, making it suitable for R&D labs, quality control, and biopharmaceutical production.

Future Trends and Opportunities

Advances may include deeper integration with laboratory information management systems, enhanced algorithms for post-translational and chemical modification mapping, application to increasingly complex oligonucleotide formats (e.g., locked nucleic acids, conjugates), and expanded high-throughput capabilities for large library screening.

Conclusion

The described LC-MS workflow offers a robust, sensitive, and fully automated approach for comprehensive oligonucleotide characterization. Its high resolution, low detection limits, and advanced informatics enable reliable impurity profiling and full-sequence verification, addressing critical needs in therapeutic oligonucleotide development and quality control.

References

1. Waters Application Note 720005557EN. Routine LC-MS Analysis of Intact Antibodies.
2. Waters Application Note 720005378EN. High Sensitivity Intact Mass Analysis of Antibodies (IgG1) Using ionKey/MS.
3. Waters Application Note 720005399EN. A Streamlined Data Dependent Acquisition Peptide Mapping Workflow.
4. Waters Application Note 720004619EN. Biopharmaceutical Platform Solution with UNIFI for Glycan Analysis.
5. Waters Application Note 720005632EN. Adding Mass Detection to Synthetic Oligonucleotide Analyses with the ACQUITY QDa Detector.
6. Waters Application Note 720005681EN. High-throughput Screening of Oligonucleotides for Identity and Purity Assessment.