High Speed Analysis of Phenytoin in accordance with chapter 621 in USP 39

Importance of the Topic

High-performance liquid chromatography (HPLC) remains a cornerstone technique in pharmaceutical analysis, providing robust separation and quantification of active pharmaceutical ingredients. Recent demands for higher laboratory throughput and efficiency have driven the adoption of ultra-high-performance LC and sub-2-micron columns. Aligning HPLC methods with the flexibility offered by USP General Chapter <621> ensures both regulatory compliance and accelerated workflows.

Study Objectives and Overview

This application note demonstrates an optimized isocratic HPLC protocol for the assay of phenytoin, an anticonvulsant drug, in full compliance with USP 39 Chapter <621>. The goal is to reduce analysis time significantly while preserving chromatographic resolution and system suitability.

Methodology and Instrumentation

Allowed adjustments under USP <621> include column length, internal diameter, particle size, flow rate, injection volume, and column temperature within defined limits. Key experimental choices:

• Original USP method: 150 × 4.6 mm ID, 3 µm column; 1.0 mL/min; 20 µL injection; 40 °C.
• Fast method: 100 × 2.1 mm ID, 2 µm column; 0.4 mL/min; 10 µL injection; 40 °C.
• Mobile phase: 0.05 M KH₂PO₄ buffer (pH 2.5) and methanol:acetonitrile (60:40 v/v) in a 60:40 ratio.
• Instrumentation: Shimadzu LC-2040C 3D with Shim-pack GIS C18 columns and PDA detector (190–800 nm).

Main Findings and Discussion

The fast method reduced the retention time of phenytoin from 14.7 min to 5.3 min (–64%) while meeting system suitability criteria. Both tailing factor (<1.5) and RSD (<0.73%) remained well within USP limits and closely matched or improved upon the original method’s performance.

Benefits and Practical Applications

This accelerated USP-compliant method offers:

• 64 % shorter run times, enabling greater sample throughput.
• Approximately 60 % lower solvent consumption, reducing operational costs and environmental impact.
• Maintained resolution and reproducibility in routine quality control settings.

Future Trends and Applications

Advancements likely to further enhance HPLC performance include:

• Wider adoption of core–shell and sub-2 µm particles for faster separations at moderate backpressures.
• Integration of higher-pressure systems for seamless UHPLC workflows.
• Automated method optimization driven by AI and machine learning to reduce development time.
• Miniaturized and microfluidic LC formats for portable, on-site analysis.

Conclusion

The described fast USP method for phenytoin analysis leverages allowable parameter adjustments to achieve a rapid, cost-effective, and fully compliant HPLC assay. Implementation of this protocol can substantially enhance laboratory productivity without compromising analytical quality.