Isotopic-labeling
experiments have been valuable to monitor the
flux of metabolic reactions in biological systems, which is crucial
to understand homeostatic alterations with disease. Experimental determination
of metabolic fluxes can be inferred from a characteristic rearrangement
of stable isotope tracers (e.g., 13C or 15N) that can be detected
by mass spectrometry (MS). Metabolites measured are generally members
of well-known metabolic pathways, and most of them can be detected
using both gas chromatography (GC)-MS and liquid chromatography (LC)-MS.
In here, we show that GC methods coupled to chemical ionization (CI)
MS have a clear advantage over alternative methodologies due to GC’s
superior chromatography separation efficiency and the fact that CI
is a soft ionization technique that yields identifiable protonated
molecular ion peaks. We tested diverse GC-CI-MS setups, including
methane and isobutane reagent gases, triple quadrupole (QqQ) MS in
SIM mode, or selected ion clusters using optimized narrow windows
(∼10 Da) in scan mode, and standard full scan methods using
high resolution GC-(q)TOF and GC-Orbitrap systems. Isobutane as a
reagent gas in combination with both low-resolution (LR) and high-resolution
(HR) MS showed the best performance, enabling precise detection of
isotopologues in most metabolic intermediates of central carbon metabolism.
Finally, with the aim of overcoming manual operations, we developed
an R-based tool called isoSCAN that automatically quantifies all isotopologues
of intermediate metabolites of glycolysis, TCA cycle, amino acids,
pentose phosphate pathway, and urea cycle, from LRMS and HRMS data.