MODERNIZATION OF AN ANTIVIRAL IMPURITIES MONOGRAPH USING THE GLOBALLY HARMONIZED PHARMACOPEIAL GUIDANCE FOR LIQUID CHROMATOGRAPHY

Significance of the Topic

The modernization of antiviral impurity monographs using the globally harmonized USP <621> guidance supports reliable quality assessment in pharmaceutical analysis. By standardizing gradient method adjustments, laboratories can ensure regulatory compliance, streamline workflows, and improve efficiency in detecting critical impurities in antiviral drugs.

Objectives and Study Overview

This study applies the updated USP <621> Chromatography chapter to the Abacavir sulfate antiviral impurity monograph. The goals were to mathematically adjust flow rate, injection volume, and gradient timing to transfer a legacy 3.9×150 mm, 5 µm HPLC method to modern 2.1×75 mm, 2.5 µm UHPLC columns and various intermediate dimensions, while maintaining system suitability criteria.

Methodology and Instrumentation

• Adjustment formulas from USP <621> were used: (1) flow and injection volume scaling to preserve linear velocity and analyte loading; (2) gradient time correction based on column dimensions; (3) an offset formula for dwell volume differences when moving from HPLC to UHPLC.
• Manual calculations were verified with Waters Preparative OBD and Columns Calculator 2.0 online tools to confirm gradient slopes and backpressure limits.

Instrumentation Used

• Columns: Symmetry C18 (3.9×150 mm, 5 µm; 4.6×150 mm, 5 µm; 4.6×100 mm, 3.5 µm), XBridge C18 (4.6×100 mm, 3.5 µm), XSelect HSS T3 (4.6×150 mm, 3.5 µm), Symmetry C18 and XSelect/XBridge BEH (2.1×100 mm, 3.5 µm; 2.1×75 mm, 2.5 µm).
• Instruments: Waters Alliance HPLC and Alliance iS systems; ACQUITY Arc, UPLC I-Class and I-Class PLUS UHPLC systems.
• Software: Empower Chromatography Data System (CDS).

Main Results and Discussion

All modernized column configurations achieved the critical impurity pair resolution of ≥1.5. Transitioning to shorter, smaller-particle columns reduced run times from 50 minutes down to 12 minutes on UHPLC. Solvent consumption dropped from 71 mL to 8 mL per run, and injection volume decreased from 27.8 µL to 2.9 µL. Throughput increased from 1.2 to 5 runs per hour, demonstrating substantial efficiency gains without sacrificing chromatographic performance.

Practical Benefits and Applications

• Faster analysis times enable higher sample throughput and rapid batch release.
• Lower mobile phase and sample consumption reduce operational costs and environmental impact.
• Consistent method performance across HPLC and UHPLC platforms improves lab flexibility and resource utilization.

Conclusions

The harmonized USP <621> guidance allows reliable mathematical method adjustments for modern column hardware and instrumentation. Using established formulas and online calculators ensures maintained system suitability and efficient method transfer from HPLC to UHPLC, delivering robust impurity analysis for antiviral compounds.

Future Trends and Opportunities

• Integration of automated method adjustment within CDS platforms could further streamline modernization workflows.
• Expansion of harmonized guidance to additional chromatographic modes (e.g., chiral, ion chromatography) may broaden application scope.
• Continued miniaturization and high-throughput platforms will drive further reductions in analysis time and solvent use.

References

1. Layton CE, Rainville PD. Achieving Method Modernization with the New Liquid Chromatographic Gradient Allowances Provided by USP General Chapter <621> Chromatography and the Alliance iS HPLC System. Waters Corporation; 2023.
2. United States Pharmacopeia. General Chapter <621> Chromatography. Official Date: 01-Dec-2022.
3. United States Pharmacopeia–National Formulary. Abacavir Sulfate Monograph. Official Date: 01-May-2020.
4. Waters Corporation. Preparative OBD Column Calculator. Accessed Jan 15, 2023.
5. Waters Corporation. Columns Calculator 2.0. Accessed Jan 15, 2023.