High-resolution mass spectrometry provides novel

Anal Bioanal Chem DOI 10.1007/s00216-015-8466-z

RESEARCH PAPER

High-resolution mass spectrometry provides novel insights into products of human metabolism of organophosphate and brominated flame retardants Mohamed Abou-Elwafa Abdallah & Jinkang Zhang & Gopal Pawar & Mark R. Viant & J. Kevin Chipman & Kyle D’Silva & Maciej Bromirski & Stuart Harrad

Received: 14 November 2014 / Revised: 27 December 2014 / Accepted: 5 January 2015 # Springer-Verlag Berlin Heidelberg 2015

Abstract The high resolution, accurate mass, and fast scanning features of the OrbitrapTM mass spectrometer, combined with the separation power of ultrahigh-performance liquid chromatography were applied for the first time to study the metabolic profiles of several organic flame retardants (FRs) present in indoor dust. To mimic real-life exposure, in vitro cultured HepG2 human hepatocyte cell lines were exposed simultaneously to various FRs in an indoor dust extract for 24 h. Target parent FRs, hexabromocyclododecanes (α-, β-, and γ-HBCDs), tris-2-chloroethyl phosphate (TCEP), tris(1chloro-2-propyl) phosphate (TCIPP), and tris(1,3-dichloro-2propyl) phosphate (TDCIPP), were separated in a single run for the first time using alternating positive and negative heated ESI source. Further metabolite separation and identification

was achieved using full scan (70,000 full width at half maximum (FWHM)), accurate mass (up to 1 ppm) spectrometry. Structural confirmation was performed via all ion fragmentation (AIF) spectra using the optional higher collisional dissociation (HCD) cell and MS/MS analysis. First insights into human metabolism of HBCDs revealed several hydroxylated and debrominated phase I metabolites, in addition to conjugated phase II glucuronides. Furthermore, various hydroxylated, oxidized, and conjugated metabolites of chlorinated phosphorous FRs were identified, leading to the suggestion of α-oxidation as a significant metabolic pathway for these compounds. Keywords High-resolution mass spectrometry . Orbitrap . Metabolism . Indoor dust . HBCD . Organophosphate flame retardants

Electronic supplementary material The online version of this article (doi:10.1007/s00216-015-8466-z) contains supplementary material, which is available to authorized users. M. A.66 %) and tris(1chloropropyl) phosphate (∼30 %). The formation of two BCIPP isomers was attributed to exposure of the microsomes to two TCIPP isomers [13]. Therefore, the detection of one

M.A.-E. Abdallah et al. Table 3

Metabolic profile of TCIPP by human HepG2 cell lines

BCIPP isomer following exposure of human HepG2 cells to D2 can be attributed to the presence of pure tris(1chloro-2-propyl) phosphate in the Wellington® standard for TCIPP, which was used to prepare the dosing mixture D2 in this study. Furthermore, a hydroxylated metabolite, bis(1-chloro-2propyl) hydroxy 2-propyl phosphate (TCEP-M1), and a

carboxylic acid, bis(1-chloro-2-propyl) carboxy 2-ethyl phosphate (TCEP-M2), were also identified (Table 3). This suggests that α-oxidation (Fig. 5) may constitute a major metabolic pathway for TCIPP in human hepatocytes. As a result of phase II metabolism, the glutathione conjugate of TCIPP was identified (Table 3). However, no glucuronide or sulfate conjugates could be confirmed.

Orbitrap™-MS provides novel insights into pollutants metabolism Table 4

Metabolic profile of TDCIPP by human HepG2 cell lines

TDCIPP metabolic profile Biotransformation of TDCIPP in indoor dust by human hepatocytes followed a similar profile to that observed for TCEP

and TCIPP (Table 4). The diester, bis(1,3-dichloro-2-propyl) hydrogen phosphate (BDCIPP), was the major metabolite formed after 24 h of exposure (based on the relative peak area to that of the parent TDCIPP). This is in agreement with a

M.A.-E. Abdallah et al.

previous in vivo study, which reported BDCIPP as the major urinary, fecal and biliary metabolite in rats following intravenous administration of radiolabeled TDCIPP [37]. Another in vitro study also reported BDCIPP as the major metabolite of TDCIPP by human liver microsomes [13]. In addition to the hydroxylated metabolite (TDCIPP-M1) and the carboxylic acid metabolite (TDCIPP-M2), the monoester, 1,3-dichloro-2propyl dihydrogen phosphate (DCIPP), was also identified (Table 4). Further hydrolysis of BDCIPP by esterases was previously suggested following in vitro incubation of TDCIPP with human liver microsomes [38]. The glutathione conjugate of TDCIPP was identified as a result of phase II metabolism (Table 4). This is in agreement with the results of previous in vitro studies using human liver microsomes [13, 38].

Conclusion A novel multi-residue analytical method was developed and applied to study the metabolic products formed when human HepG2 cell lines were challenged simultaneously—for the first time—with several widely used organic flame retardants present in indoor dust. To mimic real-life exposure scenarios, human hepatocytes were concomitantly exposed for 24 h to αHBCD, β-HBCD, γ-HBCD, TCEP, TCIPP, and TDCIPP extracted from indoor dust. To identify the large number of metabolites formed, an ExactiveTM Plus OrbitrapTM highresolution mass spectrometer was applied following chromatographic separation via UPLC. For the first time, target parent compounds were separated and monitored in a single run using an alternating positive and negative heated ESI source. Further metabolite separation and identification was performed using the high-resolution (70,000 FWHM) accurate mass (up to 1 ppm) features of the OrbitrapTM-MS. Structural confirmation of the detected metabolites was achieved via all ion fragmentation (AIF) spectra using the optional higher collisional dissociation (HCD) cell of the MS. Hepatic metabolism of HBCDs in human was investigated for the first time. Several hydroxylated and debrominated phase I metabolites were identified, while conjugated phase II glucuronides of HBCDs were also confirmed. Hydroxylated, oxidized, and conjugated metabolites of chlorinated phosphorous flame retardants were also observed, and αoxidation was proposed as a metabolic pathway for target PFRs in human hepatocytes. Acknowledgments The research leading to these results has received funding from the European Union Seventh Framework Programme FP7/ 2007-2013 under grant agreements PIIF-GA-2012-327232 (ADAPT project), 316665 (A-TEAM project), and 264600 (INFLAME project).

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