High-throughput, Ion-Pairing-Free, HILIC Analysis of Oligonucleotides Using Agilent RapidFire Coupled to Quadrupole Time-of-Flight Mass Spectrometry

Importance of the Topic

Oligonucleotide analysis is central to pharmaceutical development, biotechnology research, and quality control laboratories. Conventional ion-pairing reverse-phase methods can impair mass spectrometry performance and require extensive flushing when switching ion modes. An ion-pairing-free hydrophilic interaction chromatography (HILIC) approach coupled with high-throughput mass spectrometry addresses these limitations, delivering rapid and robust characterization of diverse oligonucleotide chemistries.

Objectives and Study Overview

This study presents an optimized HILIC method using the Agilent RapidFire 365 system paired with a 6545XT Q-TOF mass spectrometer. The goals were to achieve a 12-second analysis cycle, detect low-level impurities (<0.5% relative abundance), attain single-digit nanomolar detection limits, and maintain linearity over more than three orders of magnitude. Nine oligonucleotides, including unmodified DNA, antisense constructs, and aptamers, were evaluated to demonstrate method versatility.

Methodology and Instrumentation

The workflow combines an Agilent RapidFire 365 high-throughput MS system with an Agilent 6545XT AdvanceBio Q-TOF LC/MS. A HILIC-Z cartridge (4 µL bed volume) is used for online solid-phase extraction. Mobile phases consist of acetonitrile (50–85%) and 15 mM ammonium acetate, eliminating ion-pairing agents. Each 12-second cycle involves sample aspiration, desalting, elution, and re-equilibration. Data are acquired in negative ion mode at 4 spectra/s and processed with MassHunter BioConfirm for automatic deconvolution and impurity assignment.

Key Results and Discussion

Compared to ion-pairing reverse-phase methods, HILIC produced narrower charge-state distributions and a predictable shift of predominant charge states with increasing oligo length. Sensitivity tests on a 20-mer poly-dT showed detection limits near 10 nM with signal-to-noise ratios above 4. Linearity was confirmed from 9.7 to 1250 nM (R² = 0.9988). Twenty-four replicate injections of a 21-mer demonstrated excellent reproducibility of both total ion chromatograms and deconvoluted spectra. Impurities such as n-1 truncations and depurinations at levels below 1% were readily identified. The method successfully characterized all nine test oligonucleotides, including heavily modified antisense and aptamer sequences.

Benefits and Practical Applications

• High throughput at 12 seconds per sample without hardware changes
• Ion-pairing-free mobile phases enable positive-mode analyses and minimize carryover
• Robust detection of impurities down to 0.5% relative abundance
• Single-digit nanomolar sensitivity with a wide dynamic range
• Applicable to unmodified, modified, antisense, and aptamer oligonucleotides

Future Trends and Applications

Future work will extend HILIC RapidFire/Q-TOF to larger oligonucleotides beyond 60-mers, refine ionization conditions for low-abundance species, and explore novel cartridge chemistries. Integration with automated sample handling and artificial intelligence–driven data analysis could further accelerate oligonucleotide development in therapeutic and diagnostic contexts.

Conclusion

The ion-pairing-free HILIC RapidFire/Q-TOF method delivers rapid, reproducible, and sensitive characterization of a broad spectrum of oligonucleotide chemistries. Its minimal system maintenance, low carryover, and robust impurity profiling make it an effective solution for high-throughput research, development, and quality control.

Reference

1. Rye P. et al. High-throughput Mass Spectrometry of Synthetic Oligonucleotides: A Comparison of Data from Fast LC and RapidFire Methods. ASMS 2020.
2. Lobue P.A. et al. Oligonucleotide Analysis by HILIC-MS in the Absence of Ion-Pair Reagents. J. Chromatogr. A 2019;1595:39–48.
3. Huang M. et al. Analytical Characterization of DNA and RNA Oligonucleotides by HILIC-MS/MS. J. Chromatogr. A 2021;1648:46–2184.