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

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.