Analysis of Oligonucleotides Using Ion-Pairing Alternatives on the Agilent Pro iQ Plus

Importance of the Topic

The analysis of synthetic oligonucleotides is essential for advancing nucleic acid therapeutics, ensuring correct sequence synthesis, confirming molecular weight, and supporting quality control in research and biopharma production.

Goals and Study Overview

This application demonstrates an ion-pair-free LC/MS approach using ammonium bicarbonate on the Agilent InfinityLab Pro iQ Plus system, aiming for robust, high-throughput confirmation of various oligonucleotides, including antisense and siRNA sequences. The study evaluates chromatographic performance, mass accuracy, reproducibility, and data processing using Agilent OpenLab CDS deconvolution.

Methodology and Instrumentation

Sample Preparation:

• Oligonucleotides diluted to 50 μM in deionized water, stored at –80 °C.
• Transferred to temperature-controlled autosampler vials at 8 °C and analyzed within 48 hours.

Used Instrumentation:

• Agilent InfinityLab Pro iQ Plus LC/MS system with Jet Stream ESI source.
• Agilent Infinity II 1290 bio binary pump, multisampler, column compartment, and 1260 diode array detector HS.

Chromatography and MS Parameters:

• Column: Agilent AdvanceBio oligonucleotide, 2.1×50 mm, 2.7 μm at 75 °C.
• Mobile phases: 20 mM ammonium bicarbonate (A) and methanol (B), gradient 5–40 %B in 3 minutes at 0.7 mL/min.
• ESI in positive mode, m/z range 700–2800, optimized for lower charge states.
• Automated deconvolution in OpenLab CDS with unit mass settings, MW range 3000–10000 Da, curve-fit algorithm.

Results and Discussion

The ammonium bicarbonate method achieved strong retention and sharp peaks for antisense oligos and siRNA without toxic reagents. Twenty replicate injections of three antisense sequences showed mass accuracy within 0.6 Da and relative standard deviations below 0.1%. siRNA duplex separation required slight gradient adjustment to 45 %B and reduced fragmentor voltage to preserve conjugates. Charge state distributions shifted to lower states, reducing spectral overlap. Automated deconvolution reliably identified full-length products, common impurities (depurination, sodium adducts), and potential conjugate losses.

Benefits and Practical Applications

• Eliminates the need for toxic ion-pairing agents and dedicated negative-mode systems.
• Reduces buffer preparation time and instrument maintenance.
• Enables high-throughput, reproducible molecular weight confirmation for quality control.
• Simplifies data analysis with minimal optimization, supporting automated workflows.

Future Trends and Opportunities

The method can be extended to high-resolution MS for detailed impurity profiling and sequence confirmation. Further automation of sample handling and data processing may enable real-time monitoring. Exploration of alternative volatile buffers and solvent systems could improve sensitivity. Integration with regulatory-compliant software will facilitate broader adoption in pharmaceutical development.

Conclusion

The presented ammonium bicarbonate LC/MS workflow on the Agilent Pro iQ Plus offers a practical, robust, and environmentally friendly solution for oligonucleotide molecular confirmation. It delivers accurate mass data, reliable chromatographic performance, and streamlined analysis, supporting medium- to high-throughput laboratory operations.

Reference

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