Identification of the Biotransformation Products of 2-Ethylhexyl 4-(N,N-Dimethylamino)benzoate

Abstract

Nowadays, 2-ethylhexyl 4-(N,N-dimethylamino)benzoate (EDP) is one of the most widely used UV filters in sunscreen cosmetics and other cosmetic products. However, undesirable processes such as percutaneous absorption and biological activity have been attributed to this compound. The in vitro metabolism of EDP was elucidated in the present work. First of all, the phase I biotransformation was studied in rat liver microsomes and two metabolites, N,N-dimethyl-p-aminobenzoic acid (DMP) and N-monomethyl-p-aminobenzoic acid (MMP), were identified by GC-MS analysis. Secondly, the phase II metabolism was investigated by means of LC-MS. The investigated reactions were acetylation and glucuronidation working with rat liver cytosol and with both human and rat liver microsomes, respectively. Analogue studies with p-aminobenzoic acid (PABA) were carried out in order to compare the well established metabolic pathway of PABA with the unknown biotransformation of EDP. In addition, a method for the determination of EDP and its two phase I metabolites in human urine was developed. The methodology requires a solid-phase extraction prior to LC-MS analysis. The method is based on standard addition quantification and has been fully validated. The repeatability of the method, expressed as relative standard deviation, was in the range 3.4-7.4% and the limit of detection for all quantified analytes was in the low ng mL(-1) range.

Fig. 1

Proposed metabolism pathway of EDP

Fig. 2

GC-MS chromatograms of (a) control incubation, in the absence of substrate solution, and (b) sample incubation, containing the substrate solution

Fig. 3

Extracted ion chromatogram of a fortified urine sample at 250 ng mL⁻¹ subjected to the described SPE-LC-MS method for (a) MMP (m/z = 152), DMP (m/z = 166) and (b) EDP (m/z = 278), I.S. (internal standard, 500 ng mL⁻¹, m/z = 270)

Fig. 4

Effect of the pH on the extraction of MMP (a), DMP (b) and EDP (c) from human urine (200 ng mL⁻¹ level). Error bars show standard deviation (n = 3)

Fig. 5

MS–MS spectra of acetylated PABA. Precursor ion 180.0 (m/z), collision-induced dissociation (CID) 0.55 V. The isolation window was 1 m/z