Evaluation of Alternative Ion-pairing Reagents in the Analysis of Oligonucleotides with the ACQUITY QDa Detector

Importance of the Topic

This summary covers the evaluation of alternative ion-pairing reagents for reversed-phase UPLC–MS analysis of oligonucleotides. Traditional buffers using triethylamine (TEA) and HFIP deliver high sensitivity but may not suit all new base modifications. Exploring butylamine (BA) and dibutylamine (DBA) offers potential cost and performance advantages.

Objectives and Study Overview

The study compared TEA, BA, and DBA as ion-pairing reagents for oligonucleotide separations on an ACQUITY UPLC H-Class system with ACQUITY QDa detection. It assessed chromatographic selectivity, MS response, column longevity under elevated pH, and impurity profiling using polyT standards (15–35 nt) and a 21 nt ssRNA.

Methodology and Instrumentation

• Mobile phases: 15 mM TEA/400 mM HFIP (pH 8.0), 15 mM BA/50 mM HFIP (pH 9.0), 15 mM DBA/25 mM HFIP (pH 9.5)
• Chromatography: ACQUITY UPLC H-Class, Oligonucleotide BEH C18 column, 60 °C, gradient elution
• Detection: ACQUITY UPLC TUV at 260 nm and ACQUITY QDa (negative mode, 410–1250 Da)
• Software: MassLynx SCN 9.25 with MaxEnt deconvolution

Main Results and Discussion

• Adapted gradient slopes enabled BA and DBA to match TEA selectivity by compensating for amine hydrophobicity.
• BA:HFIP produced equivalent or improved MS signal compared to TEA; DBA:HFIP showed a twofold signal reduction but still detected critical impurity peaks.
• Reduced HFIP requirements cut reagent costs by up to 20× versus TEA:HFIP.
• Column performance under pH 9.0 and 60 °C remained stable over 400 injections (retention time RSD ≤1.66%, peak width ≤0.08 min, RSD ≤1.49%).
• High-resolution impurity profiling of a 21 nt ssRNA separated N-1 and N+1 species with comparable MS response (1.68×10⁸ vs. 1.43×10⁸ counts for TEA vs. BA).

Benefits and Practical Applications

• Customizable IP reagents address diverse oligonucleotide chemistries while preserving chromatographic and MS performance.
• Significant cost savings through reduced HFIP consumption.
• Robust, high-throughput methods for QC and therapeutic oligonucleotide workflows.

Future Trends and Opportunities

Continued development of alternative ion-pairing agents and column chemistries, integration with high-resolution detectors, automated method optimization, and greener solvents will enhance sensitivity, selectivity, and sustainability in oligonucleotide analysis.

Conclusion

Butylamine and dibutylamine represent cost-effective, robust alternatives to TEA for IP-RPLC/MS of oligonucleotides on the ACQUITY QDa platform, delivering comparable separations, stable column lifetime, and reliable MS detection.

References

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• Gong L. et al. Rapid Commun. Mass Spectrom. 2014, 28, 339–350