Scaling and Migration of a Hydrophilic Interaction Liquid Chromatography (HILIC) Method for Related Compounds of Ribavirin to Modern HPLC Systems

Importance of the Topic

Consistent analytical methods for antiviral compounds like ribavirin are essential in pharmaceutical development and quality control to ensure product safety and regulatory compliance. Method scaling and migration across different chromatography platforms allow laboratories with varied instrumentation to produce equivalent results, maintaining data integrity and accelerating method implementation.

Objectives and Study Overview

This study aimed to adapt a hydrophilic interaction liquid chromatography (HILIC) assay for ribavirin and its related compounds from high-pressure systems with sub-2 µm columns to larger particle columns (2.5 µm and 3.5 µm), and to transfer the method between modern Waters UHPLC and HPLC platforms. Performance metrics including separation quality, system suitability, and assay accuracy were compared across systems.

Methodology and Instrumentation

A HILIC method originally developed on a 1.7 µm, 2.1×50 mm column was geometrically scaled to 2.5 µm (3.0×75 mm) and 3.5 µm (4.6×100 mm) columns using the Waters Columns Calculator to maintain a constant L/dp ratio. The method employed a ternary mobile phase (acetonitrile, water, ammonium bicarbonate buffer at pH 10), a column temperature of 30 °C, UV detection at 220 nm, and temperature-controlled autosampler conditions.

• ACQUITY UPLC H-Class PLUS System
• ACQUITY Arc and Arc HPLC Systems
• Alliance iS HPLC System
• Empower 3 Chromatography Data System

Gradient SmartStart technology in Empower was used to automatically adjust gradient delay based on each system's dwell volume.

Main Results and Discussion

Geometric scaling preserved chromatographic resolution and retention times across columns of different particle sizes, maintaining an L/dp ratio near 30 000. The use of Gradient SmartStart effectively compensated for dwell volume differences (0.375 mL to 1.5 mL), yielding equivalent separation profiles on all systems. System suitability tests (n=6) demonstrated consistent retention time and peak area repeatability. Assay measurements of ribavirin and impurities showed comparable purity and relative retention times across platforms.

Benefits and Practical Applications

• Reliable migration of HILIC assays between UHPLC and legacy HPLC instruments
• Automated gradient adjustment reduces manual method revalidation
• Columns Calculator streamlines scaling of flow rate, gradient, and injection volume
• Maintains regulatory compliance by ensuring equivalent assay performance

Future Trends and Potential Applications

Continued advancement in instrument automation and software-driven method transfer is expected to simplify cross-platform chromatography. Integration of predictive modeling and AI for method development could further reduce transfer times. Expansion to other therapeutic compounds and adoption of universal scaling tools will enhance laboratory agility and ensure harmonized analytical workflows.

Conclusion

The HILIC method for ribavirin and related compounds was successfully scaled to larger particle columns and migrated across multiple Waters chromatographic systems with equivalent analytical performance. Consideration of column scaling parameters and system dwell volume is critical for preserving method robustness during platform transitions.

References

1. Dong MW. Ultrahigh-Pressure Liquid Chromatography, Part III: Potential Issues. LC-GC North America, 2017.
2. USP General Chapter <1224>, Transfer of Analytical Procedures. USP 43-NF 38, 2013.
3. Hong H, McConville PR. Dwell Volume and Extra-Column Volume: Impact on Method Transfer. Waters White Paper, 2018.
4. Berthelette KD et al. Comprehending COVID-19: Fast Analysis of Ribavirin and Related Compounds Using HILIC. Waters Application Note, 2020.
5. MedlinePlus. Ribavirin Drug Information.
6. Waters Columns Calculator. Waters Corporation.
7. Waters Corp. Protocol for Gradient Delay Measurement. Applications Notebook, 2013.