Development and Validation of a Stability-Indicating HPLC Method for the Determination of Tobramycin and Its Related Substances in an Ophthalmic Suspension

Abstract

An HPLC method was developed and subsequently validated for the quantitation of tobramycin and its related substances in an ophthalmic suspension. Current USP methodology for the assay of tobramycin involves pre-column derivatization with 2,4-dinitrofluorobenzene. The USP assay method was modified to encompass the determination of two known impurities (neamine and kanamycin) and a degradation product (nebramine). The method development involved evaluation of several factors including derivatization parameters (time, temperature, and acid concentration), mobile phase composition, column choice and temperature, wavelength evaluation, and response factors. The optimized conditions for tobramycin and its related substances include derivatizing standards and samples for 20 minutes at 70C with a 0.8 mM sulfuric acid. Chromatographic parameters include a mobile phase of acetonitrile/buffer (55/45; v/v) and a Nova-Pak C18, 3.9 × 150 mm column, maintained under ambient conditions. The wavelength of choice to maximize the detection of the related substances was 365 nm. Relative response factors for the impurities, neamine and kanamycin, and the degradation product, nebramine, were determined to be 1.00, 1.00, and 1.37 respectively. The described method is linear, reproducible, accurate, and selective over a range of 0.1%–150% of its label claim (3 mg/mL). The method precision, relative standard deviation (RSD), among 6 independent samples was not more than 0.9% for the assay and not more than 19.7% for the related substances. The intermediate precision was 1.0% (n=18) for the assay and 15.5% (n=11) for the related substances. The mean absolute recovery of tobramycin between 25 – 150% label claim was 99.6% with an RSD of 1.2% (n=6), while between 0.1 – 10% label claim was 100.7% with an RSD of 9.1% (n=5). Selectivity was evaluated by subjecting the ophthalmic suspension to thermal, acidic, basic, oxidative, and UV stress conditions. No interference in the analysis of degradation products and impurities was observed. Consequently, the validated method for the determination of tobramycin and its related substances in the ophthalmic suspension is regarded as stability-indicating.