Gas chromatographic–mass spectrometric analysis of di(2-ethylhexyl) phthalate and its metabolites in hepatic microsomal incubations

Abstract

A method is reported for the determination of di(2-ethylhexyl) phthalate (DEHP) and its metabolites in in vitro metabolism studies. Gas chromatography–mass spectrometry (GC–MS) analysis allows separation of 18 by-products of DEHP metabolism. On the basis of retention time and specific mass spectra m/ z values, three classes of compounds can be identified: (i) alcohols as hydrolysis product; (ii) acids produced by alcohol oxidation; (iii) compounds retaining phthalic moiety. The chromatogram can also be acquired in SIM mode at m/ z 149 resulting in 13 well-separated chromatographic peaks: from retention time and mass spectra it can be inferred that the main peaks correspond to mono(2-ethylhexyl) phthalate (MEHP) and (ω-1)-hydroxyl-MEHP. The kinetics of DEHP metabolism was studied using an S9 Aroclor-induced liver fraction as in vitro model and following incubation assay after 20, 40, 60 and 90 min. The composition of incubation mixtures can be quantitatively evaluated from selected ion monitoring chromatograms at m/ z 149: the by-product concentration increases during the incubation time, as a consequence of DEHP degradation. During the incubation test a significant conversion of DEHP into MEHP is observed: a conversion yield of 10, 13, 16 and 20% of the original DEHP is obtained after 20, 40, 60 and 90 min, respectively. The metabolic conversion of DEHP to MEHP explains the endocrine-disrupting activity of the original DEHP; moreover, it has been demonstrated that MEHP and its (ω-1)-oxidation metabolite induce peroxisome proliferation. This result strengthens the suggestion that the study of DEHP metabolic pathway is fundamental to better understanding its toxicological behavior.