Impurities test for Ofloxacin (EP-8.0 method)

Importance of the topic

Ofloxacin is a widely used fluoroquinolone antibiotic employed in the treatment of diverse bacterial infections. Ensuring the purity of pharmaceutical preparations is critical for patient safety, efficacy and regulatory compliance. Impurity profiling according to pharmacopeial standards helps to detect degradation products or synthetic by-products that may pose toxicological risks.

Objectives and overview

This work describes the European Pharmacopeia (EP-8.0) impurity test for ofloxacin. The primary goals are to develop and validate an HPLC method capable of separating and quantifying ofloxacin and its related impurity E under isocratic conditions, ensuring system suitability and compliance with specified resolution criteria.

Methodology

The procedure comprises two main phases:

• Sample preparation
  • Solvent mixture: Acetonitrile:Water (10:60 v/v).
  • Test solution: Dissolve 10 mg of ofloxacin in the solvent mixture and dilute to 50 mL.
  • Reference solution A: Serial dilutions of the test solution to achieve the required concentration.
  • Reference solution B: Preparation of impurity E standard and mixing with test solution, followed by further dilution steps.
• Chromatographic conditions
  • Column: Syncronis C18, 4.6 × 150 mm, 5 µm.
  • Mobile phase: A mixture of ammonium acetate, sodium perchlorate in water (pH 2.2 adjusted with phosphoric acid) and acetonitrile (ratio to give required strength).
  • Isocratic elution at 0.5 mL/min, column temperature 45 °C, injection volume 10 µL.
  • Detection: UV at 294 nm over a run time of 50 minutes.

Used instrumentation

• HPLC system: Thermo Scientific UltiMate 3000 LC.
• Column: Syncronis C18, part no. 97105-154630, lot 11811.
• Detector: UV absorbance set at 294 nm.

Main results and discussion

System suitability testing yielded a resolution of 3.26 between impurity E and the main ofloxacin peak, surpassing the EP requirement of not less than 2.0. Chromatograms demonstrated sharp, well-resolved peaks, stable baselines and reproducible retention times, confirming the method’s robustness and suitability for routine quality control.

Benefits and practical applications of the method

• Provides accurate identification and quantification of ofloxacin-related impurities.
• Supports quality assurance in pharmaceutical manufacturing and batch release.
• Aligns with pharmacopeial guidelines, facilitating regulatory approval and audit readiness.

Future trends and potential applications

• Coupling with mass spectrometry to enhance sensitivity and structural elucidation of unknown impurities.
• Automation of sample preparation to improve throughput and reduce manual errors.
• Adaptation to impurity profiling of other fluoroquinolone antibiotics within the same therapeutic class.

Conclusion

The EP-8.0 HPLC method for ofloxacin impurity testing delivers reliable separation, quantification and system suitability performance. Its adherence to pharmacopeial criteria makes it a valuable tool for ensuring product quality and compliance in pharmaceutical analysis.