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.%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一串联质谱分析方法。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-10mmol／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
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