Scaling USP Methods to Smaller Particle Columns on the Alliance HPLC System

Importance of Topic

High-performance liquid chromatography (HPLC) method modernization through geometric scaling to smaller particle columns enables pharmaceutical laboratories to reduce analysis time and solvent use while maintaining assay accuracy and impurity resolution. Optimizing column dimensions addresses industry demands for higher throughput and cost savings without compromising regulatory compliance.

Objectives and Study Overview

This work demonstrates scaling of United States Pharmacopeia (USP) monograph methods for quetiapine fumarate assay (isocratic) and impurity profiling (gradient) on a Waters Alliance e2695 HPLC system. Original 5 µm, 4.6×250 mm and 3.5 µm, 4.6×150 mm columns were geometrically scaled to 3.5 µm, 3.0×150 mm and 2.5 µm, 3.0×100 mm columns, respectively, using the Waters Columns Calculator. Performance metrics—resolution, tailing factor, retention time and area reproducibility, and quantitative recovery—were compared between original and scaled formats.

Methodology and Instruments

Instrument configuration:

• Alliance e2695 separations module with 100 µL sample syringe and passive preheater
• 2998 PDA detector
• Empower 3 chromatography data software

Assay method (isocratic):

• Mobile phase: methanol/acetonitrile/buffer (54:7:39)
• Buffer: 2.6 g/L dibasic ammonium phosphate, pH 6.5
• Detection at 230 nm

Impurities method (gradient):

• Solution A: acetonitrile/ammonium acetate buffer (25:75), pH ≥9.2
• Solution B: acetonitrile
• Detection at 250 nm
• Gradient scaled by column volume ratios

Main Results and Discussion

Isocratic assay scaling achieved a 57% reduction in runtime and 66% less solvent without exceeding system pressure limits when adjusting flow from 0.79 to 0.60 mL/min. Resolution, tailing, and relative standard deviations (RSDs) for retention time and peak area remained within USP criteria. Quantitative recovery for quetiapine fumarate in an unknown sample was 109.5% (original) versus 110.1% (scaled).
Gradient impurity profiling runtime decreased by 51% and solvent consumption by 71% at 0.89 mL/min. Critical resolutions and RSDs met monograph requirements, and total impurities in the test sample were 0.17% (original) versus 0.20% (scaled).

Benefits and Practical Applications

• Higher sample throughput and lower operating costs
• Preserved chromatographic performance for USP-compliant assays
• Reduced environmental impact via solvent savings
• Straightforward method transfer using column scaling tools

Future Trends and Potential Applications

Continued evolution of sub-2 µm UHPLC columns and instrumentation with lower extra-column dispersion will further enhance speed and efficiency. Integration with automation and real-time data analytics may streamline method development, validation, and lifecycle management in regulated environments.

Conclusion

Geometric scaling of USP methods to smaller particle columns on a conventional HPLC platform is feasible without compromising assay accuracy or impurity resolution. Using the Waters Columns Calculator and mindful adjustment of flow rates under pressure constraints, laboratories can significantly cut run times and solvent use while meeting stringent pharmacopeial standards.

Reference

• USP 40-NF35 S1. Quetiapine Fumarate Monograph. United States Pharmacopeia; 2017.
• Dlugasch AB, Simeone J, McConville PR. Scaling USP Assay for Quetiapine Fumarate Across LC Systems. Waters Application Note; 2018.
• Dlugasch AB, Simeone J, McConville PR. Scaling USP Gradient Method on ACQUITY Arc System. Waters Application Note; 2018.
• Hong P, McConville P. Dwell Volume and Extra-Column Volume. Waters Application Note; 2018.