Evaluation of Different Ion-Pairing Reagents for LC/UV and LC/MS Analysis of Oligonucleotides

Significance of the Topic

The reliable analysis of synthetic oligonucleotides underpins the development, quality assurance, and therapeutic application of modern nucleic acid drugs. Ion-pair liquid chromatography coupled with UV or mass spectrometric detection is widely used to separate and characterize oligonucleotide products, identify process-related impurities, and support sequence confirmation. Optimizing mobile phase additives enhances separation efficiency, detection sensitivity, and reduces analysis cost.

Objectives and Study Overview

This work evaluates the impact of various ion-pairing reagents—triethylamine, n-butylamine, dibutylamine, and hexylamine—combined with acetate or hexafluoroisopropanol (HFIP) counterions on reversed-phase LC/UV and LC/MS performance for oligonucleotides. Chromatographic retention, resolution, mass spectral quality, charge-state distribution, and adduct formation were compared using DNA and RNA ladder standards as well as therapeutic oligonucleotides.

Methodology and Instrumentation

Experimental conditions included an Agilent AdvanceBio oligonucleotide column (2.1×50 mm, 2.7 µm) on an Agilent 1290 Infinity II LC system with diode-array detection and an Agilent 6530 LC/Q-TOF MS with Jet Stream ESI. Mobile phases comprised equimolar amine/acetic acid buffers (100 mM) or mixtures of 15 mM amine with HFIP (25–400 mM) in water, eluted with shallow gradients of acetonitrile or methanol at 65 °C.

Main Results and Discussion

• Amine-acetate buffers: Hexylamine and dibutylamine improved retention and peak resolution relative to triethylamine for both DNA and RNA standards under LC/UV detection.
• HFIP-based phases: Combining triethylamine with HFIP dramatically increased MS sensitivity and shifted charge envelopes to higher states, facilitating cleaner spectra.
• HFIP concentration: Lowering HFIP from 400 mM to 25–100 mM maintained or improved chromatographic resolution while reducing reagent cost and adduct formation.
• Therapeutic samples: Alternative mobile phases enhanced separation of sense/antisense strands, reduced co-elution of shortmers, and improved mass spectral clarity.

Benefits and Practical Applications

Optimized ion-pair reagent selection allows tailored separations for diverse oligonucleotide chemistries, improves impurity resolution, and boosts MS detection while lowering consumable usage. Reduced adduct formation enhances confidence in mass assignments, supporting robust sequence verification and impurity profiling in QA/QC and research environments.

Future Trends and Opportunities

Advances may include novel pairing agents with lower toxicity, integration of multidimensional LC workflows, greener volatile counterions, and enhanced high-resolution MS platforms. Automated method development algorithms could expedite reagent screening, and routine applications may target larger constructs such as long RNAs and conjugated oligonucleotides.

Conclusion

The study demonstrates that careful selection of ion-pairing amines and counterions, coupled with gradient optimization, yields significant gains in chromatographic performance and MS sensitivity for oligonucleotide analysis, offering cost and quality advantages over traditional formulations.

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