High-throughput Ion-Pairing Free Reversed Phase Analysis of Oligonucleotides using RapidFire Quadrupole Time-of-Flight Mass Spectrometry

Importance of the Topic

Oligonucleotide therapeutics and research demand fast, reliable, and contamination-free analytical methods. Traditional ion-pair reverse-phase protocols can introduce persistent reagents that interfere with subsequent analyses. An ion-pairing-free reversed-phase approach streamlines workflows, minimizes carryover, and enhances mass spectrometry compatibility.

Objectives and Study Overview

This work presents the development of a high-throughput, ion-pairing-free LC-MS method for oligonucleotide characterization. The method leverages an automated solid-phase extraction platform coupled to a high-resolution quadrupole time-of-flight mass spectrometer to achieve rapid cycle times and broad applicability to various sequence lengths and chemical modifications.

Methodology

Online solid-phase extraction is performed with a PLRP-S cartridge on the RapidFire 400 system. Desalting uses 30 mM ammonium bicarbonate with 2% acetonitrile, and elution employs a 50:50 mixture of 30 mM ammonium bicarbonate and acetonitrile. Each sample cycle—loading, elution, and re-equilibration—completes in 12 seconds. MS data are acquired in positive-ion mode over an m/z range of 400–3200 at four spectra per second.

Used Instrumentation

• Agilent RapidFire 400 automated SPE system
• Agilent 6545XT AdvanceBio LC/Q-TOF
• Dual Jet Stream electrospray ionization source
• PLRP-S cartridge (30 µm, 1000 Å, 4 µL bed volume)

Main Results and Discussion

The method was applied to oligonucleotides from 18 to 60 bases, including unmodified and heavily modified sequences (phosphorothioate, 2’-O-methyl, 2’-fluoro). Raw spectra and deconvoluted mass profiles show clear charge distributions and accurate mass assignments for all analytes. Ion intensity decreases modestly with increased length, but signal quality remains sufficient for confident identification. Twelve replicate injections of an 18mer antisense oligo demonstrated consistent total ion chromatograms and deconvoluted peak heights, confirming excellent method reproducibility.

Benefits and Practical Applications

• Eliminates ion-pair reagents, reducing system contamination and carryover
• Achieves high sample throughput with 12-second cycle times
• Compatible with a wide range of oligonucleotide lengths and chemical modifications
• Generates high-quality positive-ion MS data for quality control, pharmacokinetic studies, and therapeutic development

Future Trends and Opportunities

Integration with fully automated sample preparation and data processing workflows will further enhance throughput. Adaptation to larger nucleic acids such as siRNA and mRNA is anticipated. Advances in cartridge chemistry and high-speed mass analyzers, along with machine learning–driven data analysis, promise deeper impurity profiling and real-time decision support in drug development.

Conclusion

An ion-pairing-free reversed-phase LC-MS workflow using Agilent RapidFire and Q-TOF provides a rapid, reproducible, and versatile platform for oligonucleotide analysis. This approach simplifies analytical routines and meets the evolving demands of research laboratories and biopharmaceutical quality control.

References

1. Rye P, Yang Y. Agilent Technologies application note 5994-3753EN, 2022.
2. Rye P. Agilent Technologies application note 5994-4945EN, 2022.