Abstract
A normal phase high-performance liquid chromatographic (HPLC) method has been developed for the assay of neomycin sulfate. The method involves precolumn derivatization with 2-naphthalenesulfonyl chloride (NSCI) followed by extraction in chloroform and chromatography using a normal phase silica column with detection at 254 nm. The standard curve for the HPLC assay of neomycin sulfate is linear with a correlation coefficient of 0.9996 over the range of 0.02 to 0.4 mg/ml. Neomycins B, and C, and neamine can be separated and quantified isocratically with relative standard deviations of 0.92% and 1.4% for neomycin (B + 1/2C) and neamine, respectively. Prednisolone is used as an internal standard to aid in quantification. Mass spectrometric data confirms that neomycin is derivatized at all the six primary amines on the neomycin molecule. Eight lots of neomycin sulfate were used to compare the HPLC [NSCI and 1-fluoro-2,4-dinitrobenzene (DNFB)], gas-liquid chromatographic and microbiological assay methods. The average results of the NSCIHPLC method fell between those of the microbiological and DNFB-HPLC methods. Also, good correlation of the neomycin C contents in neomycin were obtained between the NSCl-HPLC and DNFB-HPLC methods.
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