Capillary flow ion pair reversed-phase separation for very sensitive oligonucleotide LC-HRMS analysis and characterisation

Significance of the topic

Due to the increasing use of oligonucleotide therapeutics including small antisense oligonucleotides and large mRNA vaccines, highly sensitive and efficient analytical methods are required for sequence characterization and impurity profiling. Ion-pair reversed-phase chromatography coupled with high-resolution mass spectrometry (IP-RP LC-HRMS) is a key tool in QC/QA environments, but conventional methods consume large sample volumes and suffer from adduct formation.

Objectives and Study Overview

This study presents a novel capillary-flow IP-RP LC-HRMS workflow employing a 300 µm ID monodisperse DNAPac RP column on a Vanquish Neo UHPLC system coupled to an Orbitrap Exploris 480 MS. The goals were to improve sensitivity, reduce sample and solvent consumption by 100-fold compared to standard microflow methods, and deliver clean mass spectra free of metal or ion-pair adducts for oligonucleotide sizes ranging from 10 mer to 55 mer as well as duplexes.

Methodology

• Chromatography: DNAPac RP (300 µm ID, 50 mm) capillary column; gradient flow at 2–5 µL/min; buffers of 20 mM triethylamine and 60 mM HFIP (A: water; B: methanol).
• UHPLC: Vanquish Neo system with inert flow path enabling low-nl/min to 100 µL/min gradients.
• Mass Spectrometry: Orbitrap Exploris 480 with EASY-Spray low-flow ion source; negative electrospray; resolution 120 000; optimized to minimize in-source fragmentation and adduct formation.
• Software: Data acquired via Xcalibur or Chromeleon CDS; deconvolution and impurity identification performed in BioPharma Finder and Chromeleon.

Main Results and Discussion

• Excellent separation of oligonucleotides 10 mer to 55 mer in under 4 minutes at 4 µL/min with robust peak shapes and baseline resolution.
• Detection sensitivity increased by at least 100-fold versus microflow methods due to reduced dilution and optimized source conditions.
• No detectable metal adducts or in-source base loss, achieved by inert flow path, purified reagents, and optimized UHPLC cleaning.
• Intact deconvolution with isotopic sliding windows provided accurate retention times and mass assignments for full-length products and N-1/N+1 impurities.
• Satisfactory separation of sense/antisense duplex in 7 minutes, demonstrating the method’s capability for complex mixtures.

Benefits and Practical Applications

• Significant reduction in sample and solvent consumption, enabling analysis of limited-quantity samples such as IVT mRNA at 100 µg scales.
• High-throughput compatible with automated data processing and reporting in Chromeleon CDS, including direct impurity matching via delta mass.
• Enhanced sensitivity and clean spectra support reliable QC workflows in pharmaceutical development and manufacturing.

Future Trends and Potential Applications

Further miniaturization of flow rates and integration with front-end sample preparation could drive sensitivity even higher. Expansion of impurity databases and incorporation of advanced MS/MS fragmentation will facilitate comprehensive sequence mapping of modified oligonucleotides and mRNA constructs. Coupling with AI-based data analysis tools can streamline impurity identification and accelerate method development for emerging modalities.

Conclusion

The capillary-flow IP-RP LC-HRMS method using DNAPac RP column and Vanquish Neo/Orbitrap Exploris platform delivers outstanding sensitivity, resolution, and robustness for oligonucleotide analysis while minimizing sample and reagent consumption. This workflow addresses analytic challenges posed by small and large nucleic acid therapeutics, enabling routine high-throughput characterization and impurity monitoring in a compliant environment.

Used Instrumentation

• Vanquish Neo UHPLC System (Thermo Scientific)
• DNAPac RP Capillary Column (300 µm ID, 50 mm; Thermo Scientific)
• Orbitrap Exploris 480 Mass Spectrometer with EASY-Spray Low-Flow Ion Source
• Chromeleon CDS v7.3.2 and BioPharma Finder v5.2

References

1. Vanhinsbergh CJ, Criscuolo A, Sutton JN, Murphy K, Williamson AJ, Cook K, Dickman MJ. Characterization and Sequence Mapping of Large RNA and mRNA Therapeutics Using Mass Spectrometry. Analytical Chemistry. 2022;94(17):7339–7349.
2. Rentel C, Gaus H, Bradley K, Luu N, Kolkey K, Mai B, Madsen M, Pearce M, Bock B, Capaldi D. Assay, Purity, and Impurity Profile of Phosphorothioate Oligonucleotide Therapeutics by Ion Pair–HPLC–MS. Nucleic Acid Therapeutics. 2022;32(3):206–220.