Position-Specific Carbon Isotope Analysis of Glucose at Natural Isotope Abundance by Electrospray-Ionization Orbitrap...

The isotopic composition of glucose carries the signature of the environmental and metabolic processes that act on it, but most conventional isotope analytical methods cannot resolve its intramolecular isotopic structure. Here, we present a new method for position-specific isotope analysis (PSIA) of carbon in glucose using electrospray ionization-Orbitrap (ESI-Orbitrap) mass spectrometry. This method measures δ¹³C values at five unique intramolecular sites in glucose at natural isotope abundance and requires <50 μg of glucose per sample, over 3 orders of magnitude less than similar measurements by nuclear magnetic resonance (NMR). By oxidizing glucose to gluconate to improve both ionization yield and fragmentation behavior and measuring with ESI-Orbitrap, we resolve the isotopic composition of the molecular ion and four fragment ions with analytical precision of 0.5-0.8‰ (2 SE). Using a positionally labeled glucose standard, we demonstrate the accuracy of the measurement for both molecular average and position-specific carbon isotope composition. Our method reproduces intramolecular δ¹³C patterns previously demonstrated for natural sugars formed through C₃ and C₄ photosynthetic pathways while enabling substantially higher throughput and sensitivity. This is the first application of Orbitrap-PSIA to a carbohydrate, and it enables the tracing of sugar fluxes in environmental, biomedical, and ecological systems. Future developments could extend the method to include oxygen and hydrogen isotopes, further enhancing its value for investigating glucose dynamics across many natural settings.