Impact of BHT Additive on the Optical and Reactive Behavior of Fluorescent Peroxynitrite Probes

Peroxynitrite (ONOO^-) is a reactive oxygen and nitrogen species that is central to oxidative stress and pathophysiological signaling. Fluorescent small-molecule chemicals have been widely employed for detecting peroxynitrite, also known as peroxynitrite-responsive probes (PRPs). However, the influence of common solvent stabilizers on sensor performance remains insufficiently examined. Here, we report the impact of a ubiquitous stabilizer, trace amounts of butylated hydroxytoluene (BHT), a frequently used antioxidant in organic solvents (i.e., tetrahydrofuran, diethyl ether, and isopropyl ether), which significantly alters the baseline optical properties and the apparent reactivity of model fluorescent probes with peroxynitrite. Utilizing UV-visible spectroscopy, fluorescence spectroscopy, and nuclear magnetic resonance (NMR), our results demonstrate that BHT has an impact at concentrations as low as 0.25 μM, which is ∼10³-10⁴-fold lower than the typical 900-1800 μM found in standard 4 L solvent bottles. The stabilizer (1) induces absorption baseline elevation near 310 nm, (2) partially distorts the probe's emission band, and results in a (3) loss of phenolic proton signal consistent with oxidation products in CD₃CN after exposure to ONOO^-. These effects lead to an artificially suppressed apparent sensor response, corresponding to a 98% decrease in the expected signal. Our findings demonstrate that the presence of BHT in commercial solvents can compromise analytical peroxynitrite sensor assays, especially those reliant on short-wavelength (≤350 nm) excitations. This further emphasizes the need to consider stabilizer impurities when fabricating PRPs for both fundamental studies and translational applications.