The application of MALDI-TOF MS for the quantification of lysine, alanine, and glucose is described. The method is based on using stable isotopes as internal standards and allows fast, sensitive, and reproducible quantification of these compounds. It is demonstrated for aqueous standard solutions with concentrations of the analytes between 10 muM and 100 mM. The mean standard deviations from five replicates each were 4.3% (lysine), 3.7% (alanine), and 3.2% (glucose). In addition, sucrose could be measured by MALDI-TOF MS, but was not quantified due to lack of an internal standard. The method developed can be applied to quantify metabolites in cultivations of C. glutamicum ATCC 21253, without processing of the sample except a 1:5 dilution. Excellent agreement of the data with conventional techniques like HPLC or enzyme assay was found. (C) 2001 John Wiley & Sons, Inc.