Comprehensive and Integrated Workflow for Oligonucleotide Sequence Confirmation by Agilent High-Resolution LC/Q-TOF

Comprehensive and Integrated Workflow for Oligonucleotide Sequence Confirmation by High-Resolution LC/Q-TOF

Significance of the topic

Oligonucleotide therapeutics and research reagents—including siRNA, antisense oligonucleotides, aptamers and CRISPR guides—demand precise mass, purity and sequence information to ensure efficacy and safety. High-resolution liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (LC/Q-TOF) integrated with automated data analysis accelerates confidence in sequence confirmation and impurity profiling.

Objectives and overview of the application note

This application note outlines a rapid, end-to-end workflow using the Agilent 1290 Infinity II LC and 6545XT AdvanceBio LC/Q-TOF, combined with MassHunter BioConfirm 12.0 software. The goal is to deliver complete sequence confirmation of synthetic oligonucleotides—including modified, isomeric and truncated species—through a single injection and automated MS/MS fragment matching in under one minute of data processing.

Methodology and instrumentation

• Chromatography: Agilent 1290 Infinity II LC with AdvanceBio oligonucleotide column (2.1×50 mm, 2.7 µm), gradient from 10 % to 95 % methanol in 15 mM TEA/400 mM HFIP, flow rate 0.5 mL/min, column at 65 °C.
• Mass spectrometry: Agilent 6545XT AdvanceBio LC/Q-TOF with Dual AJS ESI source in negative ion mode; gas temperatures and flows optimized; mass range m/z 350–3000; targeted MS/MS acquisition 100–3000 m/z at up to 2 spectra/s.
• Data analysis: MassHunter BioConfirm 12.0 software uses theoretical isotope pattern matching and a fragment confirmation ladder. Users can group MS/MS scans by charge state or collision energy to optimize sequence coverage.

Main results and discussion

• Resolution standards (14-, 17-, 20- and 21-mer RNA) achieved baseline chromatographic separation of single-nucleotide variants, sub-ppm mass accuracy and 100 % sequence coverage from single injections.
• Isomeric and base-swap pairs were distinguished solely by MS/MS fragment ions, confirming single-nucleotide differences with full coverage.
• Double-stranded DNA duplexes co-eluting under LC conditions were each sequenced independently by targeted MS/MS, yielding complete coverage for both strands.
• A heavily modified 18-mer antisense oligonucleotide bearing 2’-methoxyethoxy groups generated clear high-resolution MS/MS spectra and full fragment ladders at optimized collision energies.
• Low-abundance n-1 and n-2 truncated impurities of a 21-mer were detected (<5 % relative abundance) and fully sequenced, demonstrating high sensitivity for minor species.

Benefits and practical applications

• Rapid, single-injection analysis and automated MS/MS processing reduce turnaround to under one minute per sample.
• High-resolution MS1 and targeted MS/MS deliver high-confidence sequence assignments, including for modified, isomeric and impurity species.
• Automated fragment confirmation ladder in BioConfirm standardizes data interpretation and minimizes manual review.

Future trends and potential applications

As oligonucleotide modalities expand, demand will grow for scalable, automated sequence verification. Future directions include integration of machine learning for impurity prediction, expanded support for longer and more complex constructs, multiplexed analyses, and cloud-based data sharing to facilitate cross-laboratory standardization.

Conclusion

The Agilent 1290 Infinity II LC/6545XT AdvanceBio LC/Q-TOF system combined with MassHunter BioConfirm 12.0 offers a robust, high-throughput workflow for oligonucleotide sequence confirmation and impurity profiling. Its speed, accuracy and automation support accelerated development and stringent quality control of oligonucleotide-based therapeutics and research reagents.

References

1. Wong D.L.; Rye P. An Integrated Workflow for the Analysis of Oligonucleotides and Their Impurities by Agilent High-Resolution LC/(Q-)TOF Mass Spectrometry. Agilent Technologies Application Note 5994-4817EN, 2022.
2. McLuckey S.; et al. Tandem Mass Spectrometry of Small, Multiply Charged Oligonucleotides. Journal of the American Society for Mass Spectrometry, 1992, 3(1), 60–70.
3. McCloskey J.; et al. Interpretation of Oligonucleotide Mass Spectra for Determination of Sequence Using Electrospray Ionization and Tandem Mass Spectrometry. Analytical Chemistry, 1996, 68(1), 1989–1999.
4. Bahal R.; et al. Antisense Oligonucleotides: An Emerging Area in Drug Discovery and Development. Journal of Clinical Medicine, 2020, 9, 2004.