Humidity-Controlled NO+ Chemical Ionization of Aldehydes through Association-Enabled Water-Mediated Protonation

Protonated ions observed in chemical ionization (CI) are generally considered to arise from direct proton transfer from pre-protonated reagent ions to analytes, and proton affinity is therefore widely used to assess the thermodynamic feasibility of such reactions. Here, we identify a previously unrecognized association-enabled protonation pathway in NO+ CI, in which water serves as the proton source. Under 0.00% relative humidity (RH), ten aldehydes reacted with NO+ predominantly through hydride-ion abstraction to form [M−H]+ ions. Upon the introduction of only 0.67% RH, the dominant product ions switched from [M−H]+ to [M + H]+ and enhanced the overall ionization responses of aldehydes. Excess water promoted the gradual conversion of NO+ into hydrated proton cluster ions, thereby decreasing the aldehyde responses. Quantum chemical calculations using n-butyraldehyde as a model analyte showed that water acts as an active reactant: it associates with the NO+−aldehyde complex, substantially lowers the reaction barrier, and induces intracomplex proton transfer and structural rearrangement, thereby opening a previously unknown water-mediated protonation pathway that yields the protonated aldehyde and HONO. Replacing H2O with D2O completely converted [M + H]+ to [M + D]+, while the HONO/DONO signals increased with aldehyde concentration, indicating that water is the direct proton source and HONO is a concomitantly formed coproduct. This protonation pathway was further extended to diverse oxygen-, nitrogen-, and sulfur-containing organic compounds, as well as inorganic species, including CO2 and SO2. This work establishes an association-enabled, water-mediated protonation framework that provides a new reaction-chemical perspective for understanding atypical product-ion formation and cluster-mediated processes in complex CI sources.