HPLC법에 의한 2,4-디니트로플루오로벤젠을 유도체화제로 한 Stretoalloteichus hindustanus ATCC 31218 변이균의 배양액 중 네브라마이신 펙터 2,4,5,5`,6, 가나마이신 A 분석

Determination of Nebramycin Factor 2,4,5,5'',6 and Kanamycin A inFermentation Broth of Streptoalloteichus hindustanus ATCC 31218 Mutant Using 2,4-Dinitrofluorobenzene(DNFB) as a Derivatizing Agent by High Performance Liquid Chromatography

A procedure for the high-performance liquid chromatographic determination of Nebramycin factors in fermentation broth of Streptoalloteichus hindustanus ATCC 31218 mutant was investigated using pre-column derivatization and UV detection. The method is based on pre-column derivatization of Nebramycin factors with 2,4-dinitrofluorobenzene(DNFB) in the presence of Tris(hydroxymethyl)aminoethane. The chromatographic separation of derivatives of Nebramycin factors and unknown impurities is achieved using reversed-phase column (NOVA-PAK C18, Waters Co.) and AcCN : H20 : AcOH (53.0: 46.5: 0.5) as a mobile phase. The mixture of these derivatives were separated within 35 minutes and the optimum wavelength (lamdamax) of the UV detector was 353 nm. The linearity of response for derivatives of Nebramycin factors is demonstrated for concentrations up to 500 mcg/ml and the relative standard deviation is less than 0.79%. Detection limit was 1.67 ng for the 10 mcl sample volume employed.