Enhanced Resolution for Longer Oligonucleotide Analytes With a MaxPeak™ Premier Oligonucleotide BEH C18 300 Å Column

Importance of the Topic

Analytical characterization of oligonucleotides is critical for the development and quality control of novel diagnostic assays and therapeutics. Ion-pairing reversed-phase liquid chromatography (IP-RPLC) combined with mass spectrometry (MS) offers sensitive detection and detailed molecular information. Choosing optimal stationary phase properties, such as pore size, is essential to achieve high resolution for a range of oligonucleotide lengths and support rigorous pharmacokinetic and bioanalysis studies.

Objectives and Study Overview

This study evaluated a novel ACQUITY Premier Oligonucleotide BEH C18 column with 300 Å pore size for its ability to separate single-stranded DNA ladders from 10 to 100 nucleotides. The goals were to compare performance to a conventional 130 Å pore column, assess compatibility with different ion-pairing reagents, and demonstrate online MS detection for intact mass confirmation.

Methodology and Instrumentation

The analysis employed two ACQUITY Premier Oligonucleotide BEH C18 columns (300 Å and 130 Å, 1.7 µm, 2.1×150 mm) on a BioAccord LC-MS System featuring a Premier BSM UPLC system, Tunable UV detector, and RDa mass detector. Mobile phases included TEA/HFIP and DIPEA/HFIP buffers optimized for negative-ion MS compatibility. Gradient methods were tailored for each ladder. Samples were ssDNA ladders (10–60-mer and 20–100-mer) diluted in water and analyzed in triplicate.

Main Results and Discussion

• The 300 Å column delivered superior resolution for oligonucleotides ≥70 nt without sacrificing performance for shorter species.
• Both TEA/HFIP and DIPEA/HFIP buffering systems provided robust separations, with DIPEA yielding slightly enhanced peak definition.
• Identical ladder components injected separately showed consistent retention times, supporting reliable peak tracking in complex workflows.
• Intact mass spectra deconvolution confirmed accurate mass measurements across the range (up to 100-mer) with mass errors <60 ppm.
• UV response reproducibility was high (RSD <3%), demonstrating system precision for quantitative applications.

Benefits and Practical Applications

• High-resolution separation of long-chain oligonucleotides facilitates impurity profiling and sequence confirmation in therapeutic development.
• Compatibility with standard ion-pairing reagents and MS detection streamlines method transfer and regulatory compliance.
• Consistent retention times enable reliable peak identification and batch-to-batch comparisons in QA/QC laboratories.

Future Trends and Applications

Advances in column technology and mobile phase design will continue to extend the range and throughput of oligonucleotide analysis. Potential directions include high-throughput screening of modified nucleic acid therapeutics, integration with automation platforms for large-scale biopharma QC, and coupling with top-down sequencing approaches for detailed structural elucidation.

Conclusion

The ACQUITY Premier Oligonucleotide BEH C18 300 Å column combined with the BioAccord LC-MS System offers a powerful platform for resolving and characterizing oligonucleotides up to 100 nt. Its enhanced resolution, reproducibility, and MS compatibility support critical stages of nucleic acid therapeutic and diagnostic development.

References

• Goyon A. et al. Journal of Pharmaceutical and Biomedical Analysis, 2020;182:113105.
• Demelenne A. et al. Journal of Chromatography A, 2021;1651:462283.
• Donegan M. et al. Journal of Chromatography A, 2022;1666:462860.
• Bagge J. et al. Journal of Chromatography A, 2020;1634:461653.