A method for a widely targeted analysis was developed for the metabolic profiling of yeast central metabolism. The widely targeted method consists of 2 analyses, namely, gas chromatography-quadrupole-mass spectrometry (GC-Q-MS) operated in selected ion monitoring mode with 25 m/z channels, and liquid chromatography triple-stage quadrupole (LC-QqQ)-MS operated in multiple reaction monitoring mode. This platform was set up to identify and quantify preselected 99 compounds, including sugars, sugar phosphates, organic adds, amino acids, and cofactors. The method showed good sensitivity and a wide dynamic range. For example, limits of detection for lactate and L-phenylalanine were 1.4 fmol and 2.0 fmol, respectively. The dynamic ranges for GC-Q-MS analysis and LC-QqQ-MS analysis were approximately 10(2)-10(5) and 10(3)-10(4), respectively. The metabolite profiles of 2 yeast strains, YPH499 and BY4741, under glucose-fermenting conditions were compared using the developed method. Although YPH499 and BY4741 were derived from an identical experimental strain, the profiling analysis successfully revealed a variation in metabolic phenotypes among experimental yeast strains demonstrating that the widely targeted method could be a robust and useful method for the investigation of metabolic phenotypes of Saccharomyces cerevisiae. (C) 2012, The Society for Biotechnology, Japan. All rights reserved.