Can we determine superoxide ion reliably? – Automatic flow system for the measurement of the superoxide lifetime in varied aqueous media

ABSTRACT There is a quest of advanced analytical methodologies for reliable determination of trace level concentrations of superoxide anion radical in biological and environmental matrices, yet the validity of the current calibration approaches is questionable on account of the extremely short lifetime of the radical in aqueous media. To address this limitation, an automatic flow system using potassium superoxide dissolved in dimethylsulfoxide (DMSO) is herein proposed as a reliable calibration source. In DMSO, the superoxide stability was significantly ameliorated as compared to that in aqueous matrices with a half-life ( t 1/2 ) of 29.6 ± 1.5 min following first-order decay kinetics. The flow setup capitalizes on a visible-light chemiluminescent Cypridina luciferin analog MCLA for highly sensitive detection of the target, obtaining a limit of detection (LOD) of 126 fmol·L -1 and a linearity up to 6.4 nmol·L -1 with cross-validated recoveries ranging from 88 to 108 %. Reliable kinetic measurements of the superoxide radical generated via the xanthine/xanthine oxidase system were performed in different aqueous matrices, including marine culture medium, groundwater, seawater and HEPES buffer. The aim behind is to calculate t 1/2 in every matrix as a crucial parameter for the determination of superoxide in water media. The calculated t 1/2 values ranged from 2.2 s in seawater to 8.3 s in the marine culture medium, with variations attributed to differences in ionic strength and trace metal content. This work provides invaluable data on superoxide decay kinetics in environmental and biological aqueous systems, serving as an unrivalled calibration benchmark for accurate quantification of superoxide in real-world scenarios.