The Impact of Adducts and Strategies to Control Them in IP-RPLC Based Oligonucleotide Analyses

Importance of the Topic

Oligonucleotide therapeutics and diagnostics rely on precise characterization. Ion-pair reversed-phase liquid chromatography (IP-RPLC) coupled with mass spectrometry is widely used due to high separation efficiency based on charge interactions. However, trace alkali metal salt adducts accumulate over time, compromising retention times, peak shapes, and MS sensitivity. Effective adduct control is essential for consistent assay performance and reliable results in analytical and QA/QC environments.

Study Objectives and Overview

This brief evaluates the impact of salt adduct accumulation in IP-RPLC analyses of a 21-mer single-stranded RNA and demonstrates a rapid mitigation strategy. The goal is to maintain chromatographic and MS performance over extended runs with minimal downtime, improving productivity and reproducibility.

Methodology

• Sample: 21-mer ssRNA (5′-UCGUCAAGCGAUUACAAGGTT-3′).
• System: Waters ACQUITY UPLC H-Class Bio with ACQUITY QDa mass detector in-line post-UV.
• Column: Oligonucleotide BEH C18 (130 Å, 1.7 µm, 2.1 mm × 50 mm).
• Mobile phase: 15 mM triethylamine (TEA), 400 mM hexafluoro-2-isopropanol (HFIP), pH 8.0.
• Time study: Eight-hour continuous injections with a four-minute gradient (18% to 20% B).
• Adduct monitoring: Deconvoluted mass spectra to track Na+ and K+ adduct formation.

Instrumentation Used

• Waters ACQUITY UPLC H-Class Bio system.
• Waters ACQUITY QDa mass detector.
• Oligonucleotide BEH C18 column.

Main Results and Discussion

• Without mitigation, adducts increased from 6% to 63% over eight hours, causing a 0.2 min retention shift and peak shape deterioration.
• High pH reconditioning was insufficient to remove adsorbed metal cations, leading to ongoing performance decline.
• A low-pH regeneration step (0.1% formic acid, one minute) effectively displaced metal adducts in each cycle.
• Using the low-pH method, the neutral target peak maintained >92.5% spectral purity and stable retention (mean 2.44 min, RSD 0.57%).

Benefits and Practical Applications

The rapid low-pH regeneration strategy can be integrated directly into IP-RPLC methods to:

• Minimize system downtime compared to offline chelation cleaning.
• Ensure consistent chromatographic retention and peak shape over long sequences of injections.
• Maintain high MS sensitivity and reproducibility for oligonucleotide analysis.

Future Trends and Opportunities

• Implementation of automated low-pH regeneration in routine workflows for therapeutics and PCR product analysis.
• Development of novel stationary phases with reduced metal-binding sites to further lower adduct formation.
• Exploration of alternative ion-pair agents and buffer systems to improve robustness against trace salt contamination.

Conclusion

Controlling alkali metal adducts is critical for reliable IP-RPLC-MS analysis of oligonucleotides. The incorporation of a short low-pH regeneration step maintains chromatographic and MS performance over extended runs with minimal productivity impact. This strategy supports high-throughput and reproducible analysis in research and quality control laboratories.

References

• Birdsall RE, et al. Reduction of metal adducts in oligonucleotide mass spectra in ion-pair reversed-phase chromatography/mass spectrometry analysis. Rapid Commun. Mass Spectrom. 2016;30(14):1667.