In-vitro metabolites characterization of 1,3-diphenylguanidine and 1,3-di-o-tolylguanidine by high-resolution mass spectrometry and urinary profiling

Abstract

The discovery of the hazardous effects associated with the polymer additives 1,3-diphenyl guanidine (DPG) and 1,3-di-o-tolylguanidine (DTG) has prompted the need for biomonitoring studies to detect human exposure. However, limited information is available about their metabolism. To address this gap, this study investigates the Phase I and II in-vitro biotransformation of both chemicals using human liver microsomes and cytosol. The samples were analyzed using liquid chromatography coupled to high resolution-(tandem) mass spectrometry through suspect (of in-silico predicted metabolites) and non-target screening. The analysis revealed four Phase I and two Phase II metabolic products for both DPG and DTG. Hydroxylation of the benzene ring led to the tentative identification of mono- and di-hydroxylated metabolites. Subsequent Phase I deamination followed by oxidation resulted in the formation of hydroxy-phenylurea and an intramolecular cyclization resulted in the formation of hydroxy-cyclic products. Furthermore, N-glucuronidation and O-glucuronidation products were identified for the first time. After performing urinalysis, DPG and DTG could be quantified in the 0.02–0.23 μg L−1 range, and DPG-227 (mono-hydroxylated DPG) was estimated to be present at ca. 0.01–0.10 μg L−1 range, using DPG response as quantification surrogate. Finally, toxicity assessment using an in-silico tool indicated the need to consider these human metabolites in (eco)toxicological assessments, as they may have the same or even greater effects on humans and the environment.