微生物发酵法生产去氢表雄酮检测方法的建立

摘要

建立了一个微生物发酵生产去氢表雄酮(DHEA)的检测方法,采用气相色谱法同时测定发酵液中雄甾-4-烯-3,17-二酮(4-AD)、去氢表雄酮甲氧基甲基醚和DHEA含量.发酵液经乙酸乙酯萃取,可直接进样分析.气相色谱条件为:FID检测器,DB-1型(30 m×0.53 mm ×0.25 m)毛细管柱;柱温280℃,进样口280℃,检测器310℃;载气为高纯N2,流速1 mL/min.在实验室条件下,发酵液中各主要成分分离情况良好.结果表明:4-AD、去氢表雄酮甲氧基甲基醚和DHEA的浓度线性范围分别为:4-AD为0.2～1.0 g/L(R2=0.996);去氢表雄酮甲氧基甲基醚为0.1 ～0.9 g/L(R2 =0.992);DHEA为0.2～1.0 g/L(R2=0.998),平均回收率依次为(97.88±0.74)％,(99.39±1.07)％和(98.91±1.09)％.该方法具有步骤简单,重现性好,准确度和灵敏度高的特点.%A gas chromatography (GC) method for the simultaneous determination of Androst-4-ene-3,17-dione (4-AD),3β-methoxymethyl-ether-dehydrepiandrosterone and dehydroepiandrosterone (DHEA) from a culture of microbial fermentation for the production of transformable 3β-methoxymethyl-ether-phytosterols was described.The samples were quantitated by internal standard method,and they were separated by GC with DB-1 capillary column and detected by FID.The column and the injector were operated at 280 ℃,the detector was set as 310 ℃ and the carrier gas was nitrogen,flowing at a rate of 1 mL min-1.The samples were quantitated by internal standard method.Under the conditions,all components in culture were well separated and rectilinear responses were obtained in the range of 0.2 ～ 1.0 g/L (4-AD),0.1 ～ 0.9 g/L (3β-methoxymethyl-ether-dehydrepiandrosterone),and 0.2 ～ 1.0 g/L (DHEA).The correlation coefficients (R2) are 0.996,0.992 and 0.996 respectively.Recoveries of 4-AD,3β-methoxymethyl-ether-dehydrepiandrosterone and DHEA added to culture were (97.88 ± 0.74) ％,(99.39 ± 1.07) ％and (98.91 ± 1.09)％ respectively.The method is accurate,rapid and suitable for routine analysis.