A highly selective and sensitive method for identification and quantification of intracellular metabolites involved in central carbon metabolism (including glycolysis, pentose phosphate pathway and tricarboxylic acid cycle) by means of liquid chromatography-tandem quadrupole mass spectrometry (LC-MS/MS) was developed. The volatile ion pair modifier tributylammonium acetate (TBAA) was employed in the mobile phase for simultaneously separation of 29 negatively charged compounds including sugar phosphates, nucleotides, and carboxylic acids on a common C18 reversed-phase column. Method validation results displayed that limits of detection (LODs) calculated according to DIN (German Institute for Standardization) 32645 are mostly below 60 nM, only with the exception of pyruvate and malate. The calibration curves showed excellent linearity mainly over three orders of magnitude with correlation coefficients R(2)>0.9982. This LC-MS/MS method was successfully applied to determine these metabolites in cell extracts of Escherichia coli. Most of the intracellular metabolites were found within the detection range and the relative standard deviations of the measurements were smaller than 5.65% (n=5) for a cell extract sample.