Abstract
Stable isotope analysis is a vital tool across chemistry, geology, and environmental science, but conventional Isotope Ratio Mass Spectrometry (IRMS) techniques have limited capabilities for site-specific or multiply substituted ("clumped") isotope analyses, and are particularly limited for analyses of complex mixtures without prior analyte purification. This study addresses this gap by employing a high-resolution Orbitrap mass spectrometer to directly measure the (13)C/(12)C ratio in a model naphthenic acid (1,2,3,4-tetrahydro-2-naphthoic acid, THN) within complex organic matrices. We applied a "zero-enrichment" experimental design to evaluate accuracy and precision by comparing pure standards to the same compound in synthetic samples resembling natural waters. Complementary experiments using low-molecular-weight organic acids and natural rumen fluid were conducted to define the method's limits under controlled and severe ion-suppression conditions. The results demonstrated that matrix effects and ion statistics can substantially degrade both accuracy and precision under certain conditions. At very low analyte concentrations, incomplete ion accumulation led to heightened δ(13)C variability, a condition analogous to a "blank effect". Paradoxically, adding 1% NH(4)OH improved the precision of (13)C/(12)C measurements (reducing the relative standard error from ∼0.80‰ to ∼0.63‰ at 0.1 μM THN), despite a reduced signal, by promoting more stable deprotonation and minimizing ion suppression. We also identified that coaccumulated ions, even when baseline-resolved, such as a matrix-derived fragment at m/z 177, degrade precision by perturbing the space-charge balance. Removing this interference fully restored precision, underscoring the need to control coaccumulating ions. Crucially, experiments with small organic acids demonstrated that moderate ion suppression does not lead to isotopic bias, which emerges only when severe suppression reduces analyte ion counts below a critical statistical threshold. Finally, we identified an "isotopic stability plateau"─an optimal signal range where δ(13)C measurements are most precise and accurate, poised between noise-dominated and space-charge-distorted regimes. This work demonstrates that Orbitrap-MS can perform reliable isotope analysis in complex organic mixtures when instrumental and chemical parameters are carefully optimized, opening new applications in petroleum geochemistry, environmental forensics, and other topics.