建立了一种快速、准确的同时测定大肠杆菌(E.coli)24种胞内代谢中间产物的液相色谱-串联质谱分析方法。样品经过淬灭、洗涤和提取后得到胞内代谢物,高速冷冻离心去除杂质。经Waters Atlantis HILIC silica色谱柱分离,以乙腈-10 mmol/L乙酸铵水溶液为流动相进行梯度洗脱,流速为250μL/min。电喷雾负离子(ESI-)模式电离,多反映监测模式(MRM)检测,基质标准溶液法进行定量。结果表明:24种代谢物在0.10～100μmol/L范围内呈良好的线性关系,相关系数均大于0.99;24种代谢物的检出限(S/N>3)为0.05～0.34μmol/L,3个添加水平的回收率为83.7%～108.1%,相对标准偏差(RSD)均小于10%。该方法具有简便、灵敏度高、定性准确等特点。

An high performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS) method was established for the determination of 24 intracellular metabolites in E.coli.After quenching,washing and extraction,the samples were centrifuged to remove impurities,and then separated on a Waters Atlantis HILIC silica column.The mobile phases were acetonitrile and water(containing 10 mmol/L ammonium acetate) at a flow rate of 250 μL/min.The electrospray was operated in the negative ionization mode and the 24 metabolites were identified by multiple reaction monitoring(MRM) mode.The method of matrix-matched standard solution was adopted as the quantitative method.The calibration curves showed good linearity within the concentrations of 0.10~100 μmol/L with the correlation coefficients r >0.99.The limits of detection of the 24 metabolites were 0.05~0.34 μmol/L.The recoveries of the 24 metabolites were 83.7%~108.1% at the 3 spiked levels.The relative standard deviations were less than 10%.This method is simple,sensitive and accurate in the determination of 24 metabolites.