First, we report the application of stable isotope dilution theory in metabolome characterization of aerobic glucose limited chemostat culture of S. cerevisiae CEN.PK 113-7D using liquid chromatography - electrospray ionization MS/MS (LC-ESI-MS/MS). A glucose-limited chemostat culture of S. cerevisiae was grown to steady state at a specific growth rate (mu) = 0.05 h(-1) in a medium containing only naturally labeled (99% U-C-12, 1% U-C-13) carbon source. Upon reaching steady state, defined as 5 volume changes, the culture medium was switched to chemically identical medium except that the carbon source was replaced with 100% uniformly (U) C-13 labeled stable carbon isotope, fed for 4 h, with sampling every hour. We observed that within a period of 1 h similar to80% of the measured glycolytic metabolites were U-C-13-labeled. Surprisingly, during the next 3 h no significant increase of the U-C-13-labeled metabolites occurred. Second, we demonstrate for the first time the LC-ESI-MS/MS-based quantification of intracellular metabolite concentrations using U-C-13-labeled metabolite extracts from chemostat cultivated S. cerevisiae cells, harvested after 4 h of feeding with 100% U-C-13-labeled medium, as internal standard. This method is hereby termed "Mass Isotopomer Ratio Analysis of U-C-13 Labeled Extracts" (MIRACLE). With this method each metabolite concentration is quantified relative to the concentration of its U-C-13-labeled equivalent, thereby eliminating drawbacks of LC-ESI-MS/MS analysis such as nonlinear response and matrix effects and thus leads to a significant reduction of experimental error and work load (i.e., no spiking and standard additions). By coextracting a known amount of U-C-13 labeled cells with the unlabeled samples, metabolite losses occurring during the sample extraction procedure are corrected for. (C) 2004 Wiley Periodicals, Inc.