Modernizing the USP Ceftizoxime Sodium HPLC Method Following the Revised USP <621> Guidelines

Significance of the Topic

The modernization of United States Pharmacopeia (USP) monograph methods is critical for pharmaceutical quality control laboratories seeking to enhance throughput, reduce solvent usage, and maintain compliance. By leveraging recent USP <621> guidelines, analysts can update legacy HPLC assays—such as the Ceftizoxime Sodium assay—using smaller particle size columns without full method revalidation.

Objectives and Study Overview

This study aimed to adapt the USP Ceftizoxime Sodium isocratic HPLC method—originally developed on 4.6 × 300 mm, 10 µm columns—to newer column technologies: totally porous particle (TPP) columns with 5 and 3.5 µm particles (Agilent ZORBAX Eclipse Plus C18) and superficially porous particle (SPP) columns with 4.0 and 2.7 µm particles (Agilent InfinityLab Poroshell 120 EC-C18). Key goals included meeting system suitability criteria, shortening run time, and lowering mobile phase consumption without requiring revalidation.

Instrumentation Used

• Agilent 1260 Infinity II LC system with binary pump, multisampler, multicolumn thermostat, diode array detector (DAD) WR
• Agilent InfinityLab Poroshell 120 EC-C18 columns (various dimensions)
• Agilent ZORBAX Eclipse Plus C18 columns (various dimensions)
• OpenLab CDS Version 2.8 software
• ELGA PURELAB Chorus water purification system

Methodology and Instrumentation

Sample preparation followed USP monograph instructions: internal standard salicylic acid and analyte ceftizoxime sodium in pH 7.0 buffer (0.02 mg/mL and 0.3 mg/mL, respectively). Mobile phase comprised pH 3.6 citrate/phosphate buffer and acetonitrile (9:1). The original method’s flow rate (2.0 mL/min) and injection volume (10 µL) were adjusted using USP equations to maintain the column length–to–particle diameter (L/dp) ratio within –25 % to +50 % and keep system pressures below instrument limits. For smaller internal diameter columns, linear velocity adjustments and extracolumn volume minimization (0.12 mm id tubing throughout) were implemented. Temperature was raised to 40 °C for certain SPP columns to reduce backpressure within allowable USP ranges.

Main Results and Discussion

• TPP Columns: Three ZORBAX Eclipse Plus C18 column configurations (4.6 × 150 mm, 5 µm; 3.0 × 150 mm, 3.5 µm; 2.1 × 100 mm, 3.5 µm) delivered plate numbers (N) between 3,551 and 12,256, tailing factors below 1.25, resolution above 11, and RSDs < 2 %, meeting USP <621> criteria.
• SPP Columns: InfiniteLab Poroshell 120 EC-C18 columns of varying dimensions (4.6 × 100 mm, 4 µm; 4.6 × 50 mm, 4 µm; 3 × 75 mm, 2.7 µm; 3 × 50 mm, 2.7 µm) mostly fulfilled system suitability except the 4.6 × 50 mm column (plate number too low) and required temperature adjustment for the 3 × 75 mm column.
• Run Time and Solvent Savings: Compared to 13 min and 26 mL per injection on the original column, the optimized methods achieved run times from 1.1 to 6.5 min and solvent use from 2.3 to 16.9 mL, representing up to 92 % time and 91 % solvent savings.

Benefits and Practical Applications

• Significant reduction in analysis duration enhances sample throughput in quality control laboratories.
• Lower mobile phase consumption supports cost savings and greener laboratory practices.
• Method transfers between HPLC and UHPLC platforms become straightforward due to column scalability.
• No revalidation required when following USP <621> modernization rules, accelerating implementation.

Future Trends and Opportunities

• Greater adoption of sub-2 µm particles and ultrahigh-pressure LC systems for further speed gains.
• Integration of green solvents and water-rich mobile phases to minimize environmental impact.
• Real-time QC monitoring using online HPLC-UHPLC hyphenation and mass detection.
• Expansion of USP guidelines to encompass automated method adjustment tools.

Conclusion

This application note demonstrates that legacy USP Ceftizoxime Sodium HPLC assays can be modernized using smaller particle TPP and SPP columns per USP <621> without revalidation. The updated methods satisfy system suitability, drastically reduce run times and solvent use, and enable rapid analytical throughput improvements in pharmaceutical quality control.

References

1. USP Harmonized Standards Home Page. Supplement USP Stage 4 Harmonization, Official, December 1, 2022.
2. USP Monographs Ceftizoxime Sodium, United States Pharmacopeia: 2024.
3. Fu, R.; Grover, M.; Freeman, R.; Long, W. Understanding the Latest Revisions to USP <621>. Agilent Technologies White Paper, Publication 5994-6618EN, 2023.