USP Assay Method Transfer for Cephradine from a Traditional 5 μm Column to Agilent InfinityLab Poroshell 120 Columns

Significance of the Topic

Modernizing compendial HPLC assays is essential to improve laboratory efficiency and sustainability. Traditional USP monographs often rely on older totally porous particle (TPP) columns, resulting in lengthy run times and high solvent use. Adapting these methods to superficially porous particle (SPP) columns under the revised USP <621> guidelines enables laboratories to maintain pharmacopoeial compliance while reducing analysis time and solvent consumption.

Objectives and Study Overview

This application note describes the transfer of the USP assay method for cephradine from a conventional 4.6 × 250 mm, 5 µm ZORBAX Eclipse Plus C18 column to various formats of 2.7 µm Agilent InfinityLab Poroshell 120 EC-C18 columns. The goal was to meet all system suitability criteria without requiring revalidation, and to quantify reductions in analysis time and mobile phase usage.

Methodology and Instrumentation

The assay employed isocratic separation with a mobile phase composed of water, methanol, 0.5 M sodium acetate, and 0.7 N acetic acid (782:200:15:3). Key steps included:

• Injection volume and flow rate adjustments calculated via USP <621> equations to maintain equivalent linear velocity and column loading.
• System: Agilent 1260 Infinity II LC equipped with quaternary pump, vial sampler, multicolumn thermostat, and diode array detector (DAD) set at 254 nm.
• Columns evaluated: ZORBAX Eclipse Plus C18 (4.6 × 250 mm, 5 µm) and InfinityLab Poroshell 120 EC-C18 columns at 4.6 × 100 mm, 4.6 × 75 mm, and 3 × 100 mm dimensions.
• System suitability metrics included plate number (N), symmetry factor (As), resolution (R), and peak area relative standard deviation (RSD) for cephradine and internal standard cephalexin.

Main Results and Discussion

All modernized methods satisfied USP system suitability requirements. Compared to the original TPP column:

• Retention and resolution remained equivalent, with plate counts within ±25 % of the USP-prescribed values.
• Analysis time was reduced by 62–86 % depending on column format and flow rate.
• Mobile phase consumption decreased by 62–75 %, enhancing sustainability and cost efficiency.

Practical Benefits and Applications

The study demonstrates that transitioning to SPP columns can be achieved rapidly and without full revalidation, preserving assay performance while delivering substantial savings in time and solvents. This approach supports green analytical initiatives and high-throughput quality control in pharmaceutical manufacturing.

Future Trends and Possibilities

Adoption of superficially porous and core-shell technologies is expected to grow, with further integration into gradient methods, UHPLC, and automated workflows. Continued refinement of USP <621> guidelines will facilitate broader modernization of legacy assays across various drug substances.

Conclusion

Applying USP <621> transfer criteria enabled seamless migration of a cephradine assay from traditional TPP to modern SPP columns. The InfinityLab Poroshell 120 EC-C18 columns provided equal chromatographic quality with significant reductions in run times and solvent use, underscoring the value of updated column technologies in pharmacopeial method modernization.

References

• USP General Chapter <621> Chromatography, United States Pharmacopeia, December 2022.
• United States Pharmacopeia Monograph: Cephradine, USP, 2024.