δ13C Determination of Benzoic Acid by High-Resolution Orbitrap MS: How Sample Introduction Strategy Governs Accuracy and Precision

Abstract Stable carbon isotope ratio measurements (δ13C) are traditionally performed by isotope ratio mass spectrometry (IRMS), but recent advances in high-resolution Orbitrap mass spectrometry (Orbitrap MS) enable molecular-level isotope ratio analyses without conversion to simple gases. Here, we systematically evaluate and compare three sample-introduction strategies for Orbitrap-MS-based δ13C determination of benzoic acid─dual-inlet (DI), single-inlet (SI), and automated HPLC flow-injection (column-free mode)─assessing how each governs measurement accuracy and precision. Analyses were performed using paired injections of an isotopically certified benzoic acid reference (IAEA-601) and a benzoic acid sample previously characterized by elemental analysis–IRMS (EA-IRMS), following a seven-block bracketing sequence (Ref-Smp-Ref-Smp-Ref-Smp-Ref) to minimize temporal drift and to generate within-run triplicates. Across all configurations, high analytical precision was achieved, within-block relative standard error (σRSE) consistently below approximately 0.6‰. Under DI conditions, replicate means closely approached the EA-IRMS benchmark (−27.77‰), with residuals of −0.40 to +0.50‰ on the Exploris 240 and −0.58 to −2.73‰ on the Q Exactive. SI analysis enabled the acquisition of a higher number of individual scans per block, contributing to lower σRSE (approximately 0.50–0.65‰) but exhibited larger accuracy offsets (residuals up to approximately 1–3‰), indicating greater susceptibility to systematic bias. Automated HPLC injection on the Exploris 240, optimized for concentration and AGC (50 μmol L–1 and 2.5 × 105, respectively), delivered δ13C values of 28.1‰, −27.19‰, and −27.79‰ (residuals of approximately 0.20–0.45‰) with no significant differences among replicate means (ANOVA/Tukey, 95% confidence level). Collectively, these findings confirm Orbitrap-MS as a reliable alternative to IRMS for δ13C analysis of benzoic acid, and reveal that the choice of injection strategy is the primary determinant of the accuracy–precision–throughput balance achievable with this platform.