Hydrogen peroxide, antioxidant compounds and biological targets: An in vitro approach for determination of scavenging capacity using fluorimetric multisyringe flow injection analysis

In the present work, an automatic method based on multisyringe flow injection analysis (MSFIA) was developed for determination of scavenging capacity against H2O2 using a non-enzymatic fluorimetric assay for H2O2 detection based on the formation of europium-tetracycline-H2O2 complex. The MSFIA-fluorimetric methodology was developed to perform in-line the scavenging reaction of H2O2 prior to reaction of the remaining H2O2 with the fluorescent probe, using conditions close to those found in vivo regarding pH (6.9), temperature (37 °C) and H2O2 concentration (25 μM). This approach allowed the evaluation of scavenging capacity against H2O2 in a non-competitive (antioxidant + H2O2) or a competitive (antioxidant + H2O2 + biological target) scheme. Using the first strategy, IC50 values determined for the antioxidant compounds glutathione reduced (1191 ± 46 μM) and pyruvate (446 ± 49 μM) were lower than those obtained for biological targets such as cysteine (2616 ± 182 μM), taurine (359 ± 38 mM) and adenine (2224 ± 214 μM), indicating that reactivity towards H2O2 was higher for antioxidant compounds than for biological targets. However, when a competitive scheme was applied, the scavenging effectiveness against H2O2 depended on the biological molecule present, showing that antioxidant assessment should also take into consideration the concomitant reactivity of biological molecules or structures that are prone to oxidative damage.