GC-QTOFMS with a low-energy electron ionization source for advancing isotopologue analysis in ¹³C-based metabolic...

For the study of different levels of (intra)cellular regulation and condition-dependent insight into metabolic activities, fluxomics experiments based on stable isotope tracer experiments using ¹³C have become a well-established approach. The experimentally obtained non-naturally distributed ¹³C labeling patterns of metabolite pools can be measured by mass spectrometric detection with front-end separation and can be consequently incorporated into biochemical network models. Here, despite a tedious derivatization step, gas chromatographic separation of polar metabolites is favorable because of the wide coverage range and high isomer separation efficiency. However, the typically employed electron ionization energy of 70 eV leads to significant fragmentation and consequently only low-abundant ions with an intact carbon backbone. Since these ions are considered a prerequisite for the analysis of the non-naturally distributed labeling patterns and further integration into modeling strategies, a softer ionization technique is needed. In the present work, a novel low energy electron ionization source is optimized for the analysis of primary metabolites and compared with a chemical ionization approach in terms of trueness, precision, and sensitivity.