Position-Specific Hydrogen Isotope Analysis of Palmitic Acid by GC-Orbitrap Mass Spectrometry

The hydrogen isotope composition (δ2H) of fatty acids is a promising proxy for tracing cellular metabolic pathways and rates and for cancer diagnosis. However, conventional methods based on gas chromatography-isotope ratio mass spectrometry (GC-IRMS) could only measure molecular-average isotopic values, obscuring position-specific variations that could be more sensitive to metabolisms. To perform position-specific hydrogen isotope analysis (PSIA) of fatty acids, we used a GC-Orbitrap (Exploris 240 GC) to acquire isotopologue ratios of two fragment ions, 12C42H1H616O2+/12C41H716O2+ and 12C52H1H816O2+/12C51H916O2+, in methyl esters of palmitic acids. To allow longer acquisition of isotopologue ratios of each sample analysis, we added a custom peak-broadening system composed of two four-port switch-valves and a passivated sample loop. This peak broadener is installed between the chromatographic column and the MS transfer line and enables precise capture and release control of target compounds, allowing 12 min stable release of the eluate. Each analysis consumes only 7.6 nmol of sample and reaches experimental precision of 5.4‰ for C4H7O2+ and 8.8‰ for C5H9O2+ (1 standard deviation of 5 analyses). Using palmitic acid labeled with one 2H atom in the 12th position, we found negligible hydrogen scrambling. Also, label-addition experiments allow us to compare whole-molecule δ2H measurements using GC-IRMS with those from GC-Orbitrap, and we obtained a near-precise match with a correlation slope of 1.044 across a composition range of −223–418‰. We establish a method for position-specific δ2H measurement of fatty acids on nanomolar sample quantities, opening possibilities for new geological, environmental, and medical applications.