MS/MS Oligonucleotide Sequencing Using LC/Q-TOF with HILIC Chromatography

Significance of topic

Liquid chromatographic separation and mass spectrometric sequencing of oligonucleotides are essential for the characterization of synthetic DNA and RNA therapeutics. The widespread use of ion-pairing agents in reversed-phase LC/MS, while effective, can contaminate analytical systems and complicate multipurpose workflows. A non-ion-pairing HILIC approach reduces carryover and streamlines analysis of diverse oligo chemistries.

Study objectives and overview

This work demonstrates high-resolution MS/MS sequencing of various oligonucleotides under HILIC conditions using an Agilent LC/Q-TOF platform. Key aims include:

• Confirming complete sequence coverage for five distinct oligonucleotides
• Evaluating chromatographic performance, retention stability, and throughput
• Defining optimized charge-state targeting and collision energy schemes

Methodology and instrumentation

Sample preparation involved direct dissolution of synthetic oligos in water, with injections of 100 pmol for DNA and 25 pmol for modified oligos.

• LC conditions
  
  • Agilent 1290 Infinity II with Poroshell 120 HILIC-Z column (2.1×50 mm, 2.7 µm) at 30 °C
  • Mobile phases: A) 90% acetonitrile/10% water + 15 mM ammonium acetate; B) 10% acetonitrile/90% water + 15 mM ammonium acetate
  • Gradient from 25% to 75% B over 5 min, flow 0.4 mL/min, injection 1 µL, autosampler at 4 °C
• MS conditions
  
  • Agilent 6545XT AdvanceBio LC/Q-TOF in dual AJS negative mode
  • MS1 range m/z 400–3200 at 4 spectra/sec; MS/MS range m/z 100–3200 at 1 spectra/sec
  • Fragmentor 180 V; skimmer 65 V; octopole RF 750 Vpp
  • Targeted MS/MS on selected charge states with multiple collision energies
• Data analysis with Agilent MassHunter BioConfirm 12.0 using 15 ppm mass tolerance, theoretical abundance ≥20%, two-scan averaging

Key results and discussion

Five oligonucleotides—20- and 30-mer DNA, 18-mer antisense with phosphorothioate and methoxyethoxy modifications, 28-mer aptamer, and 21-mer RNA—showed consistent retention times (1.5–3.5 min) and bimodal charge distributions. Sequence coverage was achieved by targeting the most abundant and the next higher charge states in duplicate or triplicate runs. Higher charge precursors generally improved coverage for longer strands. A simplified collision energy schema was defined:

• For precursors m/z <1500: 12, 15, 18, and 20 V
• For precursors m/z >1500: 15, 20, 25, and 30 V

Benefits and practical applications

The described HILIC MS/MS workflow:

• Avoids ion-pair reagents and minimizes system contamination
• Delivers 100% sequence confirmation across varied oligo chemistries
• Supports rapid, routine analysis in QA/QC and therapeutic development

Future trends and opportunities

Integration of automated charge-state selection and dynamic collision energy adjustment could further enhance throughput. Expanding this HILIC strategy to larger or more heavily modified oligos and coupling with advanced informatics will enable real-time quality assessment in complex biopharma settings.

Conclusion

A robust, non-ion-pairing HILIC LC/Q-TOF method with targeted MS/MS and simplified collision energy settings enables complete sequence coverage for a broad array of oligonucleotides. This streamlined approach enhances efficiency and data quality for research and production workflows.

References

1. Rye P; Schwarzer C. MS1 Oligonucleotide Characterization Using LC/Q-TOF with HILIC. Agilent application note 5994-5631EN, 2023.
2. Rye P. High-Throughput, Ion-Pairing-Free, HILIC Analysis of Oligonucleotides Using Agilent RapidFire Coupled to Quadrupole Time-of-Flight Mass Spectrometry. Agilent application note 5994-4945EN, 2022.