Analysis of Penicillin Sodium related substances

Importance of the topic

The analysis of penicillin sodium and its related impurities is critical to ensuring the safety, efficacy, and compliance of pharmaceutical products. Rigorous impurity profiling supports regulatory requirements, prevents adverse effects, and maintains consistent antibiotic potency in clinical and industrial settings.

Objectives and overview of the study

This application note describes the development and validation of a reversed‐phase liquid chromatography method using the Shim-pack GIST C18-AQ column. The aim is to achieve robust separation and quantification of penicillin sodium and its nine related impurities, including multiple isomeric forms, under a gradient elution program.

Methodology and instrumentation

A high‐performance liquid chromatography (HPLC) method was established with the following parameters:

• Column chemistry and dimensions: Shim-pack GIST C18-AQ, 250 mm × 4.6 mm I.D., 5 µm.
• Mobile phase A: Phosphoric buffer (10.6 g KH₂PO₄ in 1 L water) adjusted to pH 3.4 with KOH, mixed with methanol (72:14, v/v).
• Mobile phase B: Acetonitrile.
• Gradient program: 13.5% B (0–14 min) → 36% B (38–50 min) → 13.5% B (51–62 min).
• Flow rate: 1.0 mL/min.
• Column temperature: 34 °C.
• Injection volume: 20 µL.
• Detection: UV absorbance at 225 nm.

Instrumentation Used

The analysis was performed on a Shimadzu HPLC system equipped with a UV detector set at 225 nm. Sample vials were LabTotalTM vials for LC/LCMS.

Main results and discussion

System suitability testing demonstrated baseline separation of nine related substances: four isomers of Impurity E, single peaks for Impurity I and J, two isomers of Impurity F, and individual peaks for Impurities H, D, and G, followed by penicillin sodium. Chromatograms exhibited sharp, symmetrical peaks with resolution values exceeding regulatory thresholds. The method provided reproducible retention times and peak areas across replicate injections, confirming its suitability for routine quality control.

Benefits and practical applications of the method

The described method offers:

• Reliable separation of closely eluting β-lactam impurities.
• Reproducible performance for batch release and stability studies.
• Compatibility with standard QC laboratory equipment and workflows.

It supports pharmaceutical manufacturers and contract testing facilities in meeting regulatory impurity limits and ensuring product consistency.

Future trends and possibilities

Potential enhancements include coupling to mass spectrometry for increased sensitivity and structural identification, adoption of ultra-high-performance liquid chromatography (UHPLC) for reduced run times and solvent consumption, and automation of sample preparation for high‐throughput environments. The method could also be adapted for stability-indicating studies under stress conditions.

Conclusion

A robust reversed-phase HPLC method using Shim-pack GIST C18-AQ has been established for the analysis of penicillin sodium and its related impurities. The approach delivers clear separation, reproducibility, and practicality for routine pharmaceutical quality control.

Reference

• Shimadzu Corporation. Application Note ERAS-1000-0029, First Edition: March 2023.