N-去甲基万古霉素的分离分析与鉴定

SEPARATION AND IDENTIFICATION OF N-DEMETHYLVANCOMYCIN

在制备万古霉素国家标准品时,发现国产万古霉素(以下简称Ⅰ)的效价比万古霉素国际标准品高,超出理论值约10%。为此对其组分进行了分离,分析,并对其主要组分的化学结构进行了测定,证明其分子结构为N-去甲基万古霉素。

More than ten years have passed, since Chinese vancomyein was produced by pharmaceutical factories. Comparing with typical vancomycin, however, Chinese vancomycin showes some differences in antibacterial activity and chromatographic behaviour. Reverse phase high-performance liquid chromatography was employed for the analysis of vancomycin. The two samples of vancomycin (commercial preparation of vancomycin Eli Lilly, USA and domestic preparation) were resolved by means of an ODS small particle (10 μg)column and a mobile phrase of methanol-ammonium phosphate dibasic. According to the chromatogramexamined, domestic vaneomycin, which has been identified as N-demethylvancomycin, was different from vancomycin(Eli Lilly, USA). By using vitamin B_(12) as an internal standard, both vancomycin and the commercial preparation (Eli Lilly, USA) were determined. The CV of the quantitative determination of domestic vancomycin was found to be 1.01%. A gradient column Chromatography equipped with CM-sephadex C-50 for separation and purification of the components of domestic vancomycin was developed. The fractionation patterns of components were plotted. The main component was isolated and recrystallized. A single component was obtained and the purity was found to be 99.05%. The retention time of the main component of domestic vancomycin was found to be different from that of typical vancomycin. Based on the data of mielear magnetic resonance (~1HNMR and ^(13)CNMR), fast atom bombardment mass spectrum and its hydrolized products, it was confirmed that there is a leucine rather than N-methyl-leucine in the molecule of Chinese vancomycin, but all the other residues are the same as that of vancomycin. Consequently, Chinese vancomycin is actually N-demetlaylvancomycin.