Eupenicillium sp.UN88胞内活性物质的鉴定及含量测定

Identification and Determination of an Active Metabolite from Fermentation of Eupenicillium sp.UN88

采用液相色谱-质谱联用技术对Eupenicillium sp.UN88胞内活性物质进行定性鉴别,以高效液相色谱法测定其含量。色谱条件:色谱柱为Kromail C18(4.6mm×250mm,5μm),以甲醇∶水(60∶40,V/V)为流动相,流速1.0mL/min,检测波长230nm,柱温30℃。采用ESI-MS总离子流跟踪分析HPLC的洗脱液,并用低能量CID-MS-MS及CID-MS-MS-MS进一步确定目标离子峰。结果表明Eupenicillium sp.UN88胞内主要产布雷菲德菌素A(BFA)。BFA在0.1—5.0mg/mL浓度范围内呈良好的线性关系(r=0.9999,n=6),平均回收率为100.6%,RSD为1.3%(n=9),检出限为0.3μg/mL。因此,Eupenicillium sp.UN88发酵产生具有活性的胞内次级代谢产物与BFA同质;建立检测BFA的RP-HPLC快速、简便、准确可靠,为筛选BFA的高产菌和质量监测提供可靠依据。

The active metabolite produced by Eupenicilliium sp.UN88 was identified qualitatively by liquid chromatography-mass spectrometry（LC-MS） and determined by high-performance liquid chromatography（HPLC）.HPLC was performed at 30℃ using Kromail C18 column（4.6mm×250mm,5μm）,with the mobile phase of methanol-water（60∶40,V/V） at a flow rate of 1.0mL/min,and detection wavelength of 230nm.Electrospray ionization-mass spectrometry（ESI-MS） was used to monitor the eluent,and the target ion peak was identified by further collision-induced dissociation（CID）-MS-MS and CID-MS-MS-MS analysis.The results showed that Eupenicillium sp.UN88 mainly produced brefeldin A（BFA）,and most of BFA existed in mycelia.The calibration curve was linear in the range of 0.1—5.0mg/mL with r=0.9999（n=6）,and the average recovery of BFA was 100.6% with RSD of 1.3%（n=9） and LOD of 0.3μg/mL.Therefore,the active secondary metabolite from fermentation of Eupenicillium sp.UN88 and BFA are homogeneous.The established RP-HPLC is rapid,simple,accurate and reliable,and can provide reliable basis for the screening of high-yield strains and quality monitoring of BFA.