Normal-phase high-performance liquid chromatographic determination of neomycin sulfate derivatized with 1-fluoro-2,4-dinitrobenzene.

A high-performance liquid chromatographic (HPLC) assay method has been developed for quantifying neomycin sulfate powders and ointments containing neomycin. A dinitrobenzene derivative of neomycin is formed and then chromatographed isocratically on a normal-phase system. Neomycin B and C and be separated and quantified with a relative standard deviation of approximately 1%. When monitored at 350 nm, the method is sensitive to about 1 ng of neomycin base per column injected. Gradient elution and isocratic HPLC methods for the analysis of derivatized neamine have also been developed. The relative standard deviation is less than 1% for the isocratic method. The HPLC method is applicable to the analysis of other aminoglucoside antibiotics.