In-operando electrospray ionization mass spectrometry (ESI-MS) is a powerful tool for investigating reaction mechanisms by monitoring ions directly from the reacting solution in real time. However, observations based on m/z alone often do not constrain mechanistic assignments uniquely. Here we examine the interpretive limits of in-operando ESI-MS using copper-mediated carboxylation chemistry as a case study. Complementary characterization by collision-induced dissociation (CID), threshold CID energetics, infrared multiple-photon dissociation spectroscopy, and density-functional theory reveals that the dominant ion corresponds to an N-bound Cu–benzoxazole resting adduct rather than the Cu–C2 intermediate crystallographically characterized under stoichiometric conditions. Reinterpretation of the observed ions in light of these constraints establishes that the N-bound adduct is the genuine entry point into the productive catalytic cycle, and that the Cu–C2 intermediate─while chemically competent─does not accumulate under catalytic conditions, consistent with the principle that the most readily isolable intermediate is not necessarily the kinetically relevant one. This case study demonstrates that structural and energetic constraints are essential when using in-operando ESI-MS to investigate reaction mechanisms.
Ogilvy et al. (2026) studied this question.