RNA Nucleosides Using Accucore 150-Amide-HILIC

Significance of the Topic

Effective analysis of highly polar ribonucleosides and cationic surfactants such as cetylpyridinium in consumer products is essential for pharmaceutical quality control, regulatory compliance, and biochemical research. Traditional reversed-phase chromatography often fails to retain these analytes, driving the need for specialized hydrophilic interaction techniques.

Objectives and Study Overview

This study aimed to develop a robust HILIC-based method for simultaneous separation and detection of five common RNA nucleosides (adenosine, uridine, inosine, cytidine, guanosine) and cetylpyridinium in a mouthwash matrix. Key goals included achieving sharp peak shapes, baseline resolution, and interference-free quantification using dual detection modes.

Methodology

A hydrophilic interaction liquid chromatography (HILIC) approach was employed with a gradient of acetonitrile and aqueous ammonium acetate buffer (pH 5). The high organic content of the mobile phase enhanced retention of polar nucleosides, while controlled buffer strength improved peak symmetry. Separation was optimized by tuning gradient times, flow rate, and column temperature.

Instrumentation

• Column 1: Thermo Scientific Accucore 150-Amide-HILIC, 3.0 × 150 mm, 3.0 µm
• Column 2: Thermo Scientific Acclaim Surfactant Plus, 4.6 × 100 mm, 2.6 µm
• LC Systems: Thermo Scientific Dionex ICS-3000 IC and UltiMate 3000 RS
• Detectors: Diode Array Detector (200–400 nm, data shown at 220 nm) and Suppressed Conductivity Detector (CSRS 300, 2 mm)
• Injection Volume: 5 µL; Flow Rate: 1.00 mL/min; Column Temperature: 30 °C; Sample Temperature: 25 °C

Main Results and Discussion

The HILIC method provided baseline separation of all five nucleosides and cetylpyridinium, with retention times increasing in order of polarity. Peak shapes were sharp and symmetric under optimized buffer conditions. UV detection at 220 nm enabled sensitive quantification of nucleosides, while suppressed conductivity allowed interference-free measurement of the ionic surfactant.

Benefits and Practical Applications

• High retention and resolution of polar analytes not amenable to reversed-phase methods
• Dual detection enhances selectivity: UV for nucleosides, conductivity for ionic surfactants
• Robust, reproducible methodology suitable for routine QC of oral care and pharmaceutical formulations

Future Trends and Opportunities

Advancements may include coupling HILIC with mass spectrometry for enhanced structural confirmation, miniaturized column formats for high-throughput screening, and exploration of novel zwitterionic or mixed-mode stationary phases. On-line sample preparation and automation could further streamline analysis in industrial settings.

Conclusion

The presented HILIC method on the Accucore 150-Amide-HILIC column, combined with Acclaim Surfactant Plus and dual-mode detection, delivers reliable separation and quantification of RNA nucleosides and cetylpyridinium in mouthwash. This approach addresses key challenges in polarity and ionic interference, making it a valuable tool for analytical laboratories.

Reference

• Thermo Fisher Scientific Application Note PB20627_E, 2012
• Thermo Fisher Scientific Application Note PB20855_E, 2013