Metabolomic Analysis via Reversed-Phase Ion-Pairing Liquid Chromatography Coupled to a Stand Alone Orbitrap Mass Spectrometer

We present a liquid chromatography−mass spectrometry (LC−MS) method that capitalizes on the mass-resolving power of the orbitrap to enable sensitive and specific measurement of known and unanticipated metabolites in parallel, with a focus on water-soluble species involved in core metabolism. The reversed phase LC method, with a cycle time 25 min, involves a water−methanol gradient on a C18 column with tributylamine as the ion pairing agent. The MS portion involves full scans from 85 to 1000 m/z at 1 Hz and 100 000 resolution in negative ion mode on a stand alone orbitrap (“Exactive”). The median limit of detection, across 80 metabolite standards, was 5 ng/mL with the linear range typically ≥100-fold. For both standards and a cellular extract from Saccharomyces cerevisiae (Baker’s yeast), the median inter-run relative standard deviation in peak intensity was 8%. In yeast exact, we detected 137 known compounds, whose 13C-labeling patterns could also be tracked to probe metabolic flux. In yeast engineered to...