Simultaneous Quantification of Neomycin and Bacitracin by LC-ELSD

The first simultaneous quantification of neomycin and bacitracin using liquid chromatography evaporative light scattering detection as an alternative to MS detection and pre-/post-column derivatisation, respectively, was the aim of this study. The developed method was validated for two strength of neomycin and one strength of bacitracin in sterile pharmaceutical formulation and is a fast and efficient tool for content uniformity tests in quality control. With this method the separation of neomycin from sulfate and the base line separation of the four major components of bacitracin (bacitracin A, B1, B2 and B3) was achieved. These four components are responsible for 96% of the microbiological activity. A Phenomenex Synergi POLAR analytical column (250 mm × 4.6 mm, 4 μm I.D.) in combination with 0.5% perfluoropropionic acid and acetonitrile in gradient mode, the peaks of interest could be separated with high efficiency within 14 min. The calibration was performed using a second order regression with an R ² = 0.9999 for neomycin (B and C) sulfate and R ² = 0.9996 for bacitracin A, B1, B2 and B3. The results of the accuracy evaluation were 99.2 and 99.7%, respectively, for neomycin and 100.8% for bacitracin. Injection precision results are 0.4–1.5 RSD% recorded for six injections. The established method has a high potential for routine high-throughput analyses in the pharmaceutical industry.