Isocratic Separation of RNA Nucleotide Triphosphates Including Pseudouridine using an Atlantis Premier BEH Z-HILIC Column

Importance of the Topic

High-quality nucleotide triphosphates are essential building blocks for synthetic RNA applications, including mRNA vaccines. Analytical methods capable of rapid, sensitive separation and detection of RNA nucleotides and their degradation products help ensure reagent integrity and support efficient process development in biopharmaceutical and research laboratories.

Study Objectives and Overview

This work presents the development of an isocratic hydrophilic interaction chromatography (HILIC) method to achieve baseline separation of the four canonical RNA nucleotide triphosphates (ATP, CTP, GTP, UTP) and the modified building block pseudouridine triphosphate (pUTP) in under 15 minutes, with improved peak shapes and compatibility with LC-UV and LC-MS detection.

Methodology and Instrumentation

A strategy of stepwise optimization was applied to a 2.1 x 100 mm Atlantis Premier BEH Z-HILIC column (2.5 µm) using the following:

• Mobile phase: 20 mM ammonium acetate pH 9.0 in acetonitrile:water (70:30 v/v).
• Column temperature: 20 °C; sample compartment: 10 °C; flow rate: optimized at 0.2 mL/min.
• Detection: UV monitoring at 260 nm; optional MS coupling for isobaric peak confirmation.
• Instrumentation: ACQUITY UPLC H-Class PLUS with Quaternary Solvent Manager, FTN sample manager, PDA detector; data processed in Empower 3 FR4.

Main Results and Discussion

The initial method, incorporating methanol in the mobile phase, achieved separation but exhibited poor peak shape for CTP, GTP and low UV response for pUTP. Removing methanol improved peak symmetry but increased retention. Raising aqueous content to 30% restored retention times while maintaining shape. Finally, reducing flow to 0.2 mL/min enhanced column efficiency, fully resolving pUTP from UTP and isolating minor diphosphate impurities. MaxPeak High Performance Surfaces technology minimized metal-induced peak tailing.

Benefits and Practical Applications

• Rapid isocratic analysis (<15 min) of key RNA nucleotides supports high-throughput quality control.
• Baseline resolution of isobaric species (pUTP vs. UTP) without ion-pair reagents.
• Simple mobile phase formulation compatible with UV and MS detection.
• Enhanced peak shape via advanced surface passivation technology, reducing secondary interactions.

Future Trends and Applications

HILIC methods may be extended to additional modified nucleotides and mixed phosphorylation states, integrated into automated QC workflows, or coupled with high-resolution MS for comprehensive impurity profiling. Further miniaturization and faster UHPLC formats could drive sub-10-minute assays, while advanced column chemistries will continue to improve robustness and sensitivity for next-generation RNA therapeutics development.

Conclusion

The optimized Atlantis Premier BEH Z-HILIC isocratic method delivers fast, robust separation of four RNA triphosphates and pseudouridine triphosphate with baseline resolution, simple mobile phase, and broad detector compatibility. This assay is well suited for quality control of nucleotide reagents in synthetic RNA workflows.

Reference

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