In Vitro Identification and In Vivo Confirmation of Metabolites of High Molecular Weight Synthetic Phenolic Antioxidants

High molecular weight synthetic phenolic antioxidants (HMW SPAs) are widely used in various consumer products, resulting in a potential exposure risk. Studying human exposure can be most effectively performed through human biomonitoring. Since the parent compounds of HMW SPAs cannot be monitored in human urine, the detection of relevant metabolites is essential. Therefore, this study aimed to identify metabolites that can serve as human biomarkers for three HMW SPAs ubiquitously detected in environmental matrices: AO1076 (octadecyl-3-(3,5-ditert-butyl-4-hydroxyphenyl)propanoate), AO1024 (3-(3,5-ditert-butyl-4-hydroxyphenyl)-N'-[3-(3,5-ditert-butyl-4-hydroxyphenyl)propanoyl]propanehydrazide), and AO245 (2-[2-[2-[3-(3-tert-butyl-4-hydroxy-5-methylphenyl)propanoyloxy]ethoxy]ethoxy]ethyl-3-(3-tert-butyl-4-hydroxy-5-methylphenyl)propanoate). These antioxidants were exposed to pooled human plasma and in vitro hepatic assays, human liver microsomes and cytosol, to identify Phase I and Phase II metabolites, by liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (HPLC-QTOF). Furthermore, suspect screening and nontarget screening were employed to identify the metabolites. This led to the identification of three metabolites: fenozan acid (3-(3,5-ditert-butyl-4-hydroxyphenyl)propanoic acid) for AO1076 and AO1024, 3-[3,5-di(tert-butyl)-4-hydroxyphenyl]propanohydrazide for AO1024, and 3-(3-tert-butyl-4-hydroxy-5-methylphenyl) propanoic acid for AO245. Finally, the presence of these metabolites was evaluated in vivo, with fenozan acid confirmed as a metabolite due to its consistent presence in the urine of volunteers. Therefore, this study provides new data that can support future human biomonitoring studies focusing on HMW SPAs.