Analysis of Nucleoside and Nucleic Acid Bas

Significance of the Topic

The accurate separation and quantification of nucleosides and nucleic acid bases are critical in fields such as pharmaceutical development, clinical diagnostics, and molecular biology.
This type of analysis supports quality control of therapeutic oligonucleotides, metabolic profiling, and purity assessment of raw materials.

Objectives and Study Overview

This application note outlines a reversed-phase HPLC method using a Shim-pack GIST C18 column for the simultaneous separation of ten nucleoside and nucleic acid base analytes.
The goal is to achieve baseline resolution under isocratic conditions with reliable retention times and reproducible quantitation.

Methodology

An isocratic mobile phase composed of 0.1 M KH2PO4 and 0.2 M NaClO4 adjusted to pH 2.03 was selected to optimize retention and peak shape.
The flow rate was set at 1.0 mL/min and the column temperature maintained at 40 °C to ensure consistent chromatographic performance.
The detection wavelength of 260 nm allows sensitive UV detection of all target compounds.

Instrumentation Used

Column: Shim-pack GIST C18, 150 mm L. × 4.6 mm I.D., 5 µm (P/N: 227-30017-07)
Mobile phase: 0.1 M KH2PO4, 0.2 M NaClO4 (pH 2.03)
Flow rate: 1.0 mL/min
Column temperature: 40 °C
Detection: UV at 260 nm
Injection volume: 1 µL

Main Results and Discussion

The method achieved clear separation of cytosine, uracil, guanine, adenine, cytidine, uridine, thymine, adenosine, guanosine, and thymidine in a single run.
Baseline resolution was observed for all analyte pairs, demonstrating the column’s selectivity under acidic conditions.
Retention times were consistent across replicate injections, confirming method precision.

Benefits and Practical Applications of the Method

This protocol offers a rapid, robust approach for routine analysis in quality control laboratories.
Its isocratic nature simplifies method transfer and reduces equilibration time, while the wide applicability to both nucleobases and nucleosides makes it versatile for diverse sample matrices.

Future Trends and Opportunities

Integration with mass spectrometric detection could extend sensitivity and specificity for trace-level impurities.
Development of shorter particle columns or UHPLC formats may further reduce analysis time while maintaining resolution.
Automation of sample preparation and robotic injection will support high-throughput screening in metabolomic and pharmacokinetic studies.

Conclusion

The described HPLC method on a Shim-pack GIST C18 column provides reliable separation and quantitation of key nucleosides and nucleic acid bases.
Its simplicity, reproducibility, and suitability for routine QC make it a valuable tool in analytical laboratories.

Reference

Shimadzu Corporation. Application Note ERAS-1000-0219A: Analysis of Nucleoside and Nucleic Acid Base with Shim-pack GIST C18. First Edition December 2021, © Shimadzu Corporation 2024.