Experimental determination of fluxes by 13C-tracers relies on detection of 13C-patterns in metabolites or by-products. In the field of 13C metabolic flux analysis, the most recent developments point toward recording labeling patterns by liquid chromatography (LC)-mass spectrometry (MS)/MS directly in intermediates in central carbon metabolism (CCM) to increase temporal resolution. Surprisingly, the flux studies published so far with LC-MS measurements were based on intact metabolic intermediatesthus neglected the potential benefits of using positional information to improve flux estimation. For the first time, we exploit collisional fragmentation to obtain more fine-grained 13C-data on intermediates of CCM and investigate their impact in 13C metabolic flux analysis. For the case study of Bacillus subtilis grown in mineral medium with 13C-labeled glucose, we compare the flux estimates obtained by iterative isotopologue balancing of 13C-data obtained either by LC-MS/MS for solely intact intermediates or LC-MS/MS for intact and fragmented intermediates of CCM. We show that with LC-MS/MS data, fragment information leads to more precise estimates of fluxes in pentose phosphate pathway, glycolysis, and to the tricarboxylic acid cycle. Additionally, we present an efficient analytical strategy to rapidly acquire large sets of 13C-patterns by tandem MS, and an in-depth analysis of the collisional fragmentation of primary intermediates. In the future, this catalogue will enable comprehensive in silico calculability analyses to identify the most sensitive measurements and direct experimental design. Biotechnol. Bioeng. 2012; 109:763771. (C) 2011 Wiley Periodicals, Inc.