Mass spectrometry (MS) based metabolic ux approaches, mainly utilizing 13C labeled tracer substrates, have emerged as a key technology in metabolic physiology and biotechnology.1-5 MS hereby plays a central role in providing accurate 13C labeling data of metabolites formed during cultivation of the studied organism on the tracer substrate. ese labeling data sensitively depend on the intracellular pathway uxes and are utilized for their estimation. e ux calculation from the labeling data is either performed by a global t of the unknown ux parameters6-10 or the calculation of local ux ratios in the network.11,12

Among dierent MS and NMR approaches available, GC-MS has turned out as the most popular technique for labeling measurement in metabolic ux studies. is is due to several advantageous characteristics of GC-MS such as high robustness, versatility, precision and sensitivity5 combined with an enormous separation capacity of the GC for the oen complex biological mixtures.13 Moreover, GC-MS provides labeling data with rich information content so that important ne structures of the metabolic network, e.g., uxes through parallel, bidirectional, or cyclic pathways, can be resolved. Combined with various simple one-pot protocols for sample derivatization GC-MS allows the labeling analysis of a variety of metabolites14 and is applicable for ux studies in many biological systems and cultivation conditions. is is underlined by recent examples from bacteria,10,11,15 yeasts,6,16,17 fungi,18 mammalian cells,19,20 or intact tissues.21,22 e most prominent ux approach is based on GC-MS labeling analysis of amino acids which generally have a high abundance in the studied cells23 and display an extensive set of labeling information. Knowing the precursor-amino acid relationships it is easy to deduce the labeling patterns of the precursor metabolites from labeling patterns of the amino acids.24 e analysis of amino acids from cell protein hydrolysates is a straightforward approach to analyze uxes during steady-state in chemostat culture or under conditions of balanced growth in batch culture.23,25 Moreover, also uxes in non-growing cells or uxes under dynamic conditions, e.g., in dierent phases of a production process, are accessible via amino acid labeling measurement, considering secreted compounds9 or free intracellular pools continuously adapting to the actual ux distribution.8