A rapid cephradine USP assay method

Importance of the Topic

The accurate and rapid determination of cephradine in pharmaceutical formulations is critical for QA/QC in drug development and production. Modernization of USP monographs can significantly reduce analysis time, lower solvent consumption and waste, and improve compatibility with advanced detectors such as MS and CAD.

Study Objectives and Overview

This work aimed to update the USP monograph for cephradine (Chapter 621) by leveraging UHPLC technology to accelerate assay throughput while adhering to official acceptance criteria. A legacy method using a 250×4.6 mm, 10 µm column and complex mobile phase was replaced with a simplified, high‐efficiency protocol capable of analyzing fifty samples per hour.

Methodology

Calibration standards of cephalexin and cephradine were prepared at 0.1 mg/mL in water/acetonitrile (80:20). Mobile phase A comprised 25 mM ammonium acetate (pH 5.0) and mobile phase B was acetonitrile. Method optimization evaluated the effects of column temperature (40–60 °C) and flow rate (0.4–0.6 mL/min) to achieve capacity factors ≥2 and resolution ≥2. Method precision was assessed via 24 replicate injections at 50 °C and 0.6 mL/min.

Instrumentation

• Accucore aQ, 50×2.1 mm, 2.6 µm column
• Vanquish Flex Quaternary UHPLC system with SmartInject, Quaternary Pump F, Split Sampler FT, Column Compartment H, and Diode Array Detector HL with LightPipe flow cell
• Virtuoso Vial Identification System and 2 mL screw thread vials
• Chromeleon 7.2 SR4 software

Main Results and Discussion

The optimized UHPLC method reduced the retention time of cephradine from approximately 20 min to 1.2 min, while maintaining resolution (Rs > 4.0) and retention time reproducibility (RSD 0.14%). Higher temperatures and flow rates further accelerated analysis with only minor resolution loss. Daily throughput increased from three samples per hour to fifty samples per hour.

Benefits and Practical Applications

• Seventeen‐fold increase in sample throughput
• Thirty‐fold reduction in solvent use and waste generation per assay
• Simplified mobile phase preparation and compatibility with MS/CAD detectors
• Maintained USP‐specified acceptance criteria for resolution and precision

Future Trends and Opportunities

Further modernization across USP monographs can extend these gains to other antibiotic assays. Integration with high‐resolution MS, adoption of greener solvents, and AI‐driven method development promise continued efficiency improvements and environmental benefits in pharmaceutical analysis.

Conclusion

The presented UHPLC method for cephradine demonstrates that strategic column selection and method optimization within USP 621 guidelines can yield dramatic improvements in speed, cost, and sustainability, without compromising analytical quality.

References

• Accucore HPLC columns technical guide, Thermo Fisher Scientific.
• Agilent Application Note 5991-3356EN, transfer of traditional USP cephradine assay to Poroshell 120.