Improved Separation of RNA Nucleotides, Nucleosides, and Nucleobases on Atlantis Premier BEH Z-HILIC Columns

Importance of the Topic

The accurate separation of RNA nucleobases, nucleosides, and nucleotides is crucial across food testing, metabolomics, and disease biomarker research. Highly polar analytes present challenges in reversed-phase chromatography, often requiring ion-pairing reagents that hinder mass spectrometric performance and prolong system equilibration. Hydrophilic interaction liquid chromatography (HILIC) offers a powerful alternative, enabling retention of small polar molecules using MS-friendly mobile phases and specialized stationary phases.

Study Objectives and Overview

This study aimed to evaluate and compare four HILIC stationary phases for the simultaneous separation of thirteen RNA components—including nucleobases, nucleosides, nucleotides, and ribose phosphate—within a single chromatographic run on short 2.1 × 50 mm columns. The goal was to identify a column chemistry providing optimal selectivity, resolution, and peak shape for structurally similar analytes.

Methodology

Sample preparation involved mixing standard solutions in acetonitrile:water with formic acid or KOH for guanine. A water–acetonitrile gradient with ammonium formate buffer (pH 3) was employed. Detection used scheduled multiple reaction monitoring (MRM) on a Xevo TQD triple quadrupole mass spectrometer. Comparative screening tested:

• ACQUITY UPLC BEH Amide
• Atlantis Premier BEH Z-HILIC
• Two commercial zwitterionic/mixed-mode HILIC phases (Competitor A and B)

Used Instrumentation

• ACQUITY Premier UPLC with Quaternary Solvent Manager
• Xevo TQD Triple Quadrupole MS
• MassLynx v4.2 software suite

Main Results and Discussion

Only the Atlantis Premier BEH Z-HILIC column achieved baseline resolution of all thirteen analytes. Other columns exhibited co-elution among structurally similar pairs. Z-HILIC’s unique combination of fully porous BEH 95 Å particles and zwitterionic ligands delivered distinct selectivity versus superficially porous or amide-bonded phases. Moreover, MaxPeak High Performance Surfaces hardware minimized metal–phosphate interactions, yielding 30–60 % narrower peak widths for nucleotides compared to alternatives.

Benefits and Practical Applications

• Complete resolution of closely related RNA components in under 4 minutes
• Enhanced sensitivity and peak shape for phosphorylated analytes
• Reduced analysis time and improved MS compatibility without ion-pairing reagents

Future Trends and Opportunities

Advancements in HILIC stationary phase design and surface treatments will further improve migration of polar metabolites. Integration with high-resolution mass spectrometry and automated sample preparation can extend applications to clinical diagnostics, environmental monitoring, and quality control in biopharmaceutical production.

Conclusion

The Atlantis Premier BEH Z-HILIC column, coupled with ACQUITY Premier hardware and MaxPeak surfaces, provides superior separation and peak performance for a comprehensive panel of RNA nucleobases, nucleosides, nucleotides, and ribose phosphate. This approach streamlines workflows in research and industrial laboratories by eliminating ion-pairing reagents and achieving robust MS-compatible separations.

References

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