Assay Analysis for Cefalexin (EP- method)

Importance of the Topic

The accurate assay of cefalexin, a widely used β-lactam antibiotic, is essential for ensuring product quality and patient safety. Reliable quantification methods support compliance with pharmacopeial standards and maintain consistency across production batches.

Objectives and Study Overview

This work presents a European Pharmacopoeia (EP) compliant assay for cefalexin monohydrate. The study aims to establish a robust chromatographic procedure capable of separating cefalexin from its related compound, cefradine, and to demonstrate system suitability parameters that meet pharmacopeial criteria.

Methodology and Instrumentation

The method involves straightforward sample preparation followed by high-performance liquid chromatography under isocratic conditions.

• Sample preparation
  • Reference solution A: 50 mg cefalexin monohydrate dissolved in water, diluted to 100 mL.
  • Reference solution B: 10 mg cefradine added to 20 mL of solution A, then diluted to 100 mL.
• Chromatographic conditions
  • Instrument: UltiMate 3000 LC system with UV detection at 254 nm.
  • Column: Acclaim 120-C18, 4.6 × 250 mm, 5 μm.
  • Mobile phase: Methanol : Acetonitrile : 0.0136 M potassium dihydrogen phosphate : Water (2 : 5 : 10 : 83, v/v/v/v).
  • Isocratic separation at 25 °C, flow rate 1.5 mL/min, injection volume 20 μL, run time 40 min.
• System suitability
  • Resolution between cefalexin and cefradine required ≥ 4.0; obtained value = 14.12, indicating excellent separation.

Main Results and Discussion

The assay achieved baseline separation of cefalexin and cefradine well above the EP resolution criterion. The extended run time of 40 minutes ensures peak shape consistency and reproducibility. No significant interference was observed, confirming the selectivity of the method.

Benefits and Practical Applications

This EP method offers several advantages for quality control laboratories:

• High resolution and selectivity facilitate accurate quantitation of cefalexin in the presence of related substances.
• Simple sample preparation and isocratic elution enhance reproducibility and ease of use.
• Compliance with pharmacopeial standards supports regulatory submissions and routine batch release testing.

Future Trends and Applications

Advancements may include:

• Transition to ultrahigh-performance liquid chromatography (UHPLC) to reduce analysis time and solvent consumption.
• Incorporation of mass spectrometric detection for improved sensitivity and identification of trace impurities.
• Automated sample preparation systems to increase throughput and minimize manual handling errors.

Conclusion

The EP-compliant assay for cefalexin on an UltiMate 3000 LC system demonstrates robust separation, high reproducibility, and full compliance with pharmacopeial suitability criteria. It is well suited for routine quality control in pharmaceutical manufacturing environments.

Used Instrumentation

• UltiMate 3000 liquid chromatograph with UV detector (254 nm)
• Acclaim 120-C18 column, 4.6 × 250 mm, 5 μm
• Standard laboratory glassware and analytical balances for sample preparation