Dphp d 10

Tris(hydroxmethyl) aminomethane (TRIS), CaCl 2 , paraoxon, 4-nitrophenol, DPHP, and DPHP-d 10 were purchased from Sigma Aldrich (Bornem, Belgium). TPHP was purchased from Chiron AS (>98% purity, Trondheim, Norway). 2-ethylhexyl phenyl phosphate (EHPHP) was synthesized by dr. Vladimir Belov (Max Planck Institute, Göttingen, Germany) as an ammonium salt (98% purity). EHDPHP was bought from Sigma Aldrich at a purity of 91%. Serum was collected in the frame of an ongoing study (registered at http://clinicaltrials.gov/ with number NCT01778868). This study was approved by the Ethical Committee of the Antwerp University Hospital (Belgian Registry number B30020097009) and all participants provided their written informed consent. Serum from 10 lean volunteers was pooled and stored at -20°C until analysis. Acetonitrile (analytical grade) was obtained from Merck KgA Chemicals (Darmstadt, Germany) and ultrapure water (18.2 MΩ) from an Elga LabWater water purification instrument (Saint Maurice, France).

The abbreviations and structures of OPE metabolites and their parent compounds are shown in Table S1 and Figure S1 in supporting information (SI). Standards of 4-HO-TPHP, 4-HO-DPHP, 3-HO-TBOEP, BBOEP, BBOEHEP, BCIPP, BCIPHIPP, BCEP, BDCIPP, EHPHP, and 5-HO-EHDPHP, and mass-labeled standards of TCEP-D12, BBOEHEP-D4, BBOEP-D4, BCEP-D8, BDCIPP-D10, and TBOEP-D6 were synthesized by Dr. Vladimir Belov (Max Planck Institute, Göttingen, Germany). TCEP was obtained from Chiron AS (Trondheim, Norway). DNBP, DPHP, DPHP-D10, and TPHP-D15 were supplied by Sigma-Aldrich (Bornem, Belgium).
The purities of all target analytes were >98%. Stock solutions of both native and internal standards (IS) were prepared in acetonitrile (ACN) at 2 and 10 ng/μL, respectively. Liquid chromatography-grade (LC-grade) ACN and methanol (MeOH) were obtained from Merck (LiChrosolv, Merk, Darmstadt, Germany). Formic acid (99-100%) and ammonium acetate (NH4AC) were of LC-grade and purchased from Sigma-Aldrich (Bornem, Belgium). Milli-Q water was generated by a PURELAB Flex system (ρ = 18.2 MΩ/cm, Elga Veolia, Tienen, Belgium). β-Glucuronidase enzyme solution was purchased from Sigma-Aldrich (lyophilized powder from E. coli, >10,000,000 unit/g). Solid-phase extraction (SPE) was performed using a Visiprep SPE vacuum manifold with 24 ports (Sigma-Aldrich).

HO-TBOEP, EHDPHP, HO-TPHP, HO-DPHP, BBOEHEP, BCIPP, BCIPHIPP, EHPHP, HO-EHDPHP, BCEP, TCEP-D12, BBOEHEP-D 4 , BDCIPP-D 10 , and TBOEP-D 6 were custom synthesized by Dr. Vladimir Belov (Max Planck Institute, Göttingen, Germany). Purity was more than 98% as measured by MS and NMR techniques. TCEP was purchased from Chiron AS (Trondheim, Norway). TBOEP, DPHP and DPHP-D 10 were purchased from Sigma-Aldrich (Bornem, Belgium). TPHP standard was purchased from Chiron AS (Trondheim, Norway). TCIPP standard was acquired from Pfalz & Bauer (Waterbury, USA). Methanol was LC-grade and was purchased from Merck (LiChrosolv®, Merk, Darmstadt, Germany), whilst hydrochloric acid (37%), formic acid (99-100%) and ammonium acetate were of analytical-grade and were purchased from Sigma Aldrich (Bornem, Belgium). Ultrapure water (UPW) was obtained from a PURELAB Flex system (ρ = 18.2 MΩ/cm, Elga Veolia, Tienen, Belgium). β-glucuronidase enzyme solution was purchased from Sigma Aldrich (lypophilized powder from E. coli, >10 000 000 unit/g). Solid-phase extraction (SPE) was performed using a Visiprep SPE vacuum manifold with 24 ports (Sigma Aldrich). Filtration of wastewater samples was carried out using glass microfiber filters (GF/A, 1.6 µm, Whatman, Sigma-Aldrich).