Unveiling an unexpected superoxide-mediated photooxidation mechanism of squalene monohydroperoxides to squalene hydroperoxy cyclic peroxides through ESR and LC–MS/MS analyses

Lipid cyclic peroxides are a rarely reported and documented class of compounds in the human organism. Recently, we reported the formation of squalene (SQ) hydroperoxy cyclic peroxides derived from SQ monohydroperoxide isomers (SQ-OOHs) for the first time. Notably, we successfully detected and quantified cis-2-OOH-3-(1,2-dioxane)-SQ in the human skin. Nevertheless, the underlying mechanism governing the formation of these compounds remained elusive. Therefore, in the current study, we set to determine the reaction’s mechanism. To this end, a comprehensive analysis of the precise conditions involved in the onset and propagation of this conversion was carried out by oxidizing total SQ-OOHs under different conditions, including singlet oxygen ((1)O(2)), thermal, and photoinduced oxidations monitored by quantifying the generated 2-OOH-3-(1,2-dioxane)-SQ using liquid chromatography-tandem mass spectrometry (LC–MS/MS). Radical intermediates were thoroughly investigated using Electron Spin Resonance (ESR) with the aid of spin traps and radical references. Moreover, calculations of SQ-OOHs’ electrostatic charges were performed on Spartan 18 software. We found that the reaction is ideally induced and favored under photooxidation in the presence of (3)O(2) in hexane, and that superoxide radical (O(2)(•−)) is the first key intermediate in this mechanism, whereas peroxyl radicals were the major species observed throughout the oxidation. Chemical calculations provided an explanation for the targeting of tertiary SQ-OOHs by this reaction and gave further evidence on the proposed heterolytic cleavage initiating the reaction. The novel oxidation mechanism suggested herein offers new insights into understanding lipid secondary oxidation and is a promising finding for further studying lipid cyclic peroxides in general.