Identification of di-2-ethylhexyl terephthalate (DEHTP) metabolites using human liver microsomes for biomonitoring applications

Abstract

Di-2-ethylhexyl terephthalate (DEHTP), a structural isomer of the plasticizer di-2-ethylhexyl phthalate (DEHP), is used in food packaging and medical devices, among other applications, and is a potential replacement for DEHP and other ortho-phthalate plasticizers. Identifying sensitive and specific biomarkers of DEHTP is necessary to assess humans' background exposure to DEHTP. Using mass spectrometry, we investigated the metabolism of DEHTP by human liver microsomes to identify in vitro DEHTP metabolites. We unequivocally identified terephthalic acid (TPA) and mono-2-ethylhydroxyhexyl terephthalate (MEHHTP), using authentic standards, and tentatively identified mono-2-ethylhexyl terephthalate (MEHTP) and two other oxidative metabolites of DEHTP: mono-2-ethyloxohexyl terephthalate (MEOHTP), and mono-2-ethyl-5-carboxypentyl terephthalate (MECPTP) from their mass spectrometry fragmentation patterns. We also evaluated the formation of in vitro metabolites of DEHP. DEHTP and DEHP produced similar metabolites, but their metabolite profiles differed considerably. DEHTP metabolized to form TPA, a metabolite of several terephthalates, as the major in vitro metabolite, followed by MEHTP, MEHHTP, MEOHTP and MECPTP. MEHTP, MEHHTP, MEOHTP and MECPTP, which are specific metabolites of DEHTP, may be suitable biomarkers for assessing exposure to DEHTP. Nonetheless, data on the urinary excretion fraction and temporal stability of these metabolites, among other considerations, are needed to demonstrate their utility as exposure biomarkers.

Keywords: Biomonitoring; DEHTP; Di-2-ethylhexyl terephthalate; Environmental exposure; Oxidative metabolites.

Fig. 1

Metabolic products tentatively identified in the supernatant of the human liver microsome suspension after incubating with di-2-ethylhexyl terephthalate for 5 h. *The structures shown for MEOHTP and MEHHTP are for one isomer only.

Fig. 2

Chromatographic separation of di-2-ethylhexyl phthalate and di-2-ethylhexyl terephthalate metabolites detected in the supernatant of the human liver microsomes suspension of DEHTP after 5 h incubation at 37 °C and spiked with DEHP metabolites.

Fig. 3

Comparison of mass spectrometric fragmentation of hydrolytic metabolites of DEHTP and DEHP: TPA and PA (A), MEHTP and MEHP (B).

Fig. 4

Mass spectrometric fragmentation of oxidative metabolites of DEHTP. MEHHTP and MEHHP (A), MEHHTP standard (A′), MECPTP and MECPP (B), and MEOHP and MEOHTP (C).

Fig. 5

In vitro metabolism of DEHP (A) and DEHTP (B) with human liver microsomes. Error bars represent standard deviation. N = 3, TPA, MECPTP, MEHTP and MEOHTP were quantified using analogous phthalate metabolites.