Probing reactive oxygen species formation in electrospray ionization microdroplets

Electrospray ionization (ESI) is conventionally regarded as an ionization process governed primarily by charged-residue and ion-evaporation mechanisms. However, increasing evidence suggests that ESI microdroplets may also provide a chemically active environment capable of promoting redox reactions and generating reactive oxygen species (ROS). The origin of such reactivity remains controversial because electrochemical reactions at metal emitters, strong interfacial electric fields, and electrical discharge may all contribute to the observed chemistry. Therefore, this study systematically investigates whether ROS-related oxidation occurs during ESI and how operating parameters influence this process.

3,3',5,5'-Tetramethylbenzidine (TMB), 5,5-dimethyl-1-pyrroline N-oxide (DMPO), and ascorbic acid (AA) were employed as complementary probes of oxidation and radical-related chemistry. Without added catalysts during ESI-MS analysis, TMB oxidation generated a peak at m/z 240, assigned to its radical-cation intermediate, and this signal was strongly suppressed by AA, demonstrating its redox-sensitive nature. The empirical intensity ratio I₂₄₀/I₂₄₁ increased as the orifice voltage changed from -1500 to -4500 V but decreased at higher voltage magnitudes, accompanied by an increase in ion current from ∼5 nA at -4500 V to ∼740 nA at -6000 V, suggesting possible corona-discharge involvement. Lower sample flow rates enhanced I₂₄₀/I₂₄₁, while oxidation of the spin-trapping reagent DMPO provided additional evidence for ROS formation. TMB oxidation persisted with an insulating capillary without direct electrical contact, excluding metal-emitter electrochemistry as the primary origin of the observed oxidation.

These results provide systematic evidence that ESI can function as a chemically active microenvironment, in which ROS-related oxidation occurs and is strongly dependent on operating conditions.