Comprehending COVID-19: Using the Atlantis PREMIER BEH C18 AX Mixed-Mode Column for the Analysis of Umifenovir

Importance of the Topic

Mixed-mode chromatography has emerged as a versatile tool for separating ionizable drug molecules. In the context of the COVID-19 pandemic, repurposing antiviral drugs such as umifenovir (Arbidol®) demands robust analytical methods to support pharmacokinetic studies and quality control. The combination of reversed-phase and ion-exchange interactions in mixed-mode stationary phases can address challenges posed by basic analytes, delivering sharper peaks and improved analysis times compared to conventional reversed ‑phase columns.

Objectives and Study Overview

This application note investigates the performance of the Atlantis PREMIER BEH C18 AX mixed-mode column for the analysis of umifenovir, contrasting its behavior with a traditional C18 column (ACQUITY UPLC HSS T3). The study aims to demonstrate enhancements in peak shape, symmetry, and retention control when analyzing a protonated antiviral compound under isocratic conditions.

Methodology and Instrumentation

The analysis employed an ACQUITY UPLC H-Class Bio system with a PDA detector. Two columns were compared:

• Atlantis PREMIER BEH C18 AX, 1.7 μm, 2.1×50 mm
• ACQUITY UPLC HSS T3, 1.8 μm, 2.1×50 mm

Chromatographic conditions:

• Mobile phase: 10 mM ammonium formate, pH 3.0, 30% or 40% acetonitrile
• Flow rate: 0.3 mL/min
• Column temperature: 30 °C
• Sample: 100 μg/mL umifenovir in 0.1% formic acid/50% acetonitrile
• Injection volume: 1 μL; sample temperature: 12 °C

Key Results and Discussion

At pH 3.0, the C18 AX stationary phase carries a positive surface charge, causing ionic repulsion of protonated umifenovir. This reduced retention produced a retention factor similar to that of a conventional C18 column operated at higher organic content. Critical findings include:

• Peak width on Atlantis C18 AX: 6.9 s vs. 59 s on HSS T3 under identical mobile phase
• Retention factor adjustment on HSS T3 required 40% acetonitrile to approach the C18 AX k’ value
• USP tailing factor: 1.5 for C18 AX, >2.0 for HSS T3

These data confirm that mixed-mode interactions deliver narrower, more symmetric peaks for basic analytes without extensive mobile-phase modifications.

Benefits and Practical Applications

• Enhanced peak shape and symmetry for protonated drugs
• Faster elution and reduced analysis time
• Reduced method development effort by leveraging ionic and hydrophobic retention
• Improved loadability for high-capacity analyses

These advantages support routine QC assays and method screening in pharmaceutical research and development.

Future Trends and Opportunities

Mixed-mode stationary phases are expected to gain wider adoption for complex separations involving ionizable compounds. Future developments may include:

1. Tailored hybrid particles with optimized charge densities
2. Integration with high-throughput screening workflows
3. Expanded utility for bioanalysis of peptides and small polar metabolites

Such innovations will further streamline method development across life-science and industrial laboratories.

Conclusion

The Atlantis PREMIER BEH C18 AX column demonstrates superior performance for the analysis of umifenovir compared to a conventional C18 phase. By combining reversed-phase and anion-exchange mechanisms, this mixed-mode stationary phase offers sharper, more symmetric peaks, faster run times, and robust retention control for basic analytes. It represents a valuable addition to chromatographic toolkits in pharmaceutical analytics.

Reference

• Liu C. et al. Research and Development on Therapeutic Agents and Vaccines for COVID-19 and Related Human Coronavirus Diseases. ACS Central Science 2020;6(3):315–331.
• Walter TH. A New Mixed-Mode Reversed-Phase/Anion-Exchange Stationary Phase Based on Hybrid Particles. Waters Application Note 720006742EN.
• Iraneta PC, Wyndham KD, McCabe DR, Walter TH. A Review of Waters Hybrid Particle Technology. Part 3: Charged Surface Hybrid Technology and its Use in Liquid Chromatography. Waters White Paper 720003929EN, 2011.