Analysis of Oligonucleotides Using an Ion-Pairing-Free Reversed-Phase Method with TOF LC/MS

Importance of the Topic

Oligonucleotide therapeutics and diagnostics demand robust analytical methods that deliver accurate mass measurement and high-resolution separation without environmentally harmful reagents. Traditional ion‐pairing reversed‐phase (IPRP) LC/MS methods pose challenges due to costly mobile phases, system contamination, and dedicated hardware needs. A non‐ion‐pairing reversed‐phase LC/MS approach with time‐of‐flight detection offers a sustainable and versatile alternative for routine analysis of a wide variety of synthetic and modified oligonucleotides.

Aims and Study Overview

This application note presents development and validation of a non‐ion‐pairing reversed‐phase LC/MS method on an Agilent TOF LC/MS platform. The goals were to:

• Eliminate ion‐pairing reagents and associated system maintenance burdens
• Achieve high‐resolution separation of unmodified and heavily modified oligonucleotides
• Demonstrate accurate mass determination across a broad length range (14–103 mer)
• Assess repeatability and stability under high‐temperature conditions

A panel of 13 oligonucleotide samples—including antisense, siRNA, sgRNA, and DNA up to 60 mer—was evaluated to illustrate method flexibility.

Methodology and Instrumentation

Sample Preparation:

• Resuspension in deionized water to 1–10 µM, stored at –80 °C
• Autosampler temperature 8 °C; samples held up to 2 days

Instrumentation:

• Agilent 6230B TOF LC/MS with Dual AJS ESI source
• Agilent MassHunter Acquisition 11 and BioConfirm 12.1 software
• Agilent 1290 Infinity II Bio Binary Pump, Multisampler, and Multicolumn Thermostat
• Agilent 1260 Infinity II Diode Array Detector HS

LC Conditions:

• Column: AdvanceBio Oligonucleotide RP, 2.1×50 mm, 2.7 µm
• Mobile Phase A: 20 mM ammonium bicarbonate in water (unadjusted pH)
• Mobile Phase B: Methanol
• Flow Rate: 0.8 mL/min; injection 2 µL; column at 75 °C
• Gradient: 10% B (0–0.5 min) to 50% B (5 min), ramp to 90% B (5.1 min), hold 6.0 min, re‐equilibrate

MS Source Parameters:

• Drying Gas 12 L/min, 300 °C; Sheath Gas 12 L/min, 400 °C; Nebulizer 30 psi
• Capillary 3,000 V; Nozzle 1,000 V; Fragmentor 180 V; Skimmer 65 V
• Mass Range: 750–3,000 m/z; acquisition 1.25 spectra/s

Main Results and Discussion

Mobile Phase Evaluation:
Comparison of 20 mM ammonium bicarbonate, acetate and formate (pH 8.5 for acetate/formate) showed:

• Ammonium bicarbonate delivered highest ionization efficiency and retention.
• Acetate and formate gave lower sensitivity under identical chromatographic conditions.

Separation Performance:

• 14–21 mer RNAs resolved with average UV resolution R≈1.47, approaching baseline for n–1 impurities.
• Sense and antisense strands of a 23 mer (Givosiran) separated cleanly in under 3 minutes.

Mass Accuracy and Repeatability:

• Across 13 analytes (14–103 mer), average mass differences were 2–12 ppm.
• Targeted Find‐by‐Formula processing in BioConfirm confirmed average error ≤8.6 ppm.
• 55 replicate injections of a challenging 20 mer ASO showed retention time %RSD 0.72 and area %RSD 1.74.

Benefits and Practical Applications

This non‐ion‐pairing method offers:

• Reduced system contamination and maintenance compared with IPRP LC.
• Cost savings by avoiding expensive ion‐pair reagents.
• Rapid, high‐throughput analysis of therapeutic oligonucleotides in QC and research labs.
• Compatibility with standard LC/MS platforms without hardware modifications.

Future Trends and Opportunities

Anticipated developments include:

• Integration with high‐resolution MS/MS workflows for impurity profiling.
• Automation of data processing and quantitation using advanced bioinformatics pipelines.
• Extension to longer oligonucleotides and conjugated constructs.
• Green chemistry advances in mobile phase buffer systems.

Conclusion

The described non‐ion‐pairing reversed‐phase TOF LC/MS method achieves accurate, repeatable analysis of diverse oligonucleotides without dedicated IPRP systems. It combines high mass accuracy, robust separation, and operational efficiency, providing an attractive alternative for pharmaceutical analytics and quality control environments.

Reference

1. Hayashi Y, Sun Y. Overcoming Challenges in Oligonucleotide Therapeutics Analysis: A Novel Nonion‐Pair Approach. J Am Soc Mass Spectrom. 2024;35(9). DOI:10.1021/jasms.4c00270