Bis 2 ethylhexyl phthalate

Apoe^−/− mice, which were on the C57BL/6 genetic background, were generated from breeding pairs purchased from the Jackson Laboratory, Bar Harbor, ME. At 6 weeks of age, female mice were switched onto a Western diet containing 21% fat, 0.15% cholesterol, 34.1% sucrose, 19.5% casein, and 15% starch (TD88137, Harlan Laboratories) and maintained on the diet for 12 weeks. Female mice were chosen because they are more susceptible to atherosclerosis than male counterparts [21 (link)] and also they were used in our previous studies [22 (link)-25 (link)]. One group of mice received bis-(2-ethylhexyl) phthalate (Sigma) during Western diet consumption, and the control group received no phthalate. The phthalates were administered in drinking water at a daily dosage of 100 mg/kg of body weight for each mouse, as reported [26 (link)]. This dose has been shown to be effective in inducing estrogenic effects in mice [26 (link)]. All procedures were carried out in accordance with current National Institutes of Health guidelines and approved by the Institutional Animal Care and Use Committee.

We obtained dimethyl phthalate (DMP, ≥99%), dibutyl phthalate (DBP, 99%), di-isobutyl phthalate (DIBP, 99%), diethyl phthalate (DEP, 99.5%), and bis (2-ethylhexyl) phthalate (DEHP, ≥99%) from Sigma-Aldrich (Darmstadt, Germany). We purchased HPLC-grade petroleum ether and n-hexane from Merck Chemical Company (Darmstadt, Germany). We obtained analytical-grade H₂SO₄ (98.3%) from Shengqinghe Chemical Company (Nanjing, China). In addition, dealing with concentrated sulfuric acid and use special acid-resistant gloves and glasses
We used ultrapure water purified using a Millipore Purification System (Burlington, NJ, USA) in all experiments. To minimize the risk of contamination, we used glassware in all experiments, which we strictly washed and checked to ensure the lack of contamination with PAEs before use.
We prepared individual standard stock solutions of each phthalic acid ester (100 mmol L⁻¹) and stored them at 4 °C. We prepared serial dilutions of individual standard stocks and multicomponent solutions containing equal concentrations (100 µmol L⁻¹) of each compound on site by diluting the stock solution with methanol. We constructed calibration curves by plotting the means of triplicate measurements of peak highs against the concentrations of each compound.

Bis (2-ethylhexyl) phthalate (DEHP), dibutyl phthalate (DBP), benzyl butyl phthalate (BBP), 4-nonylphenol (NP), 4-tert-octylphenol (OP) and DMSO (dimethyl sulfoxide) were purchased from Sigma-Aldrich Co, (USA). Ethanol was acquired from Winkler (Chile).

Acetonitrile and acetone were HPLC grade and purchased from Fisher Scientific (Fair Lawn, NJ, USA). Water was purified by an Easy pure system (Thermo Scientific, Waltham, MA, USA). Standard PAEs containing butyl benzyl phthalate, bis(2-ethylhexyl) phthalate, dibutyl phthalate, diethyl phthalate, dimethyl phthalate, and di-n-octyl phthalate (EPA 606-M, mix, 200 μg/mL in methanol) were purchased from Sigma Aldrich (Laramie, WY, USA). Ceramic homogenizer, dispersive SPE adsorbent (4000 mg MgSO₄, 1000 mg NaCl, 500 mg Na₂H citrate·1.5 H₂O, and 1000 mg Na₃ citrate·2 H₂O), clean-up adsorbent (150 mg PSA, 150 mg C18, 900 mg MgSO₄), and sodium chloride were purchased from Agilent Technologies (Lake Forest, CA, USA). For heavy metals analysis, HNO₃ (69%, hiperpur) and H₂O₂ (30% w/v 100 vol.) were purchased from PanReac AppliChem (AppliChem GmbH, Darmstadt, Germany). CRM-141R—trace elements in calcareous loam soil, CRM-142R—light sandy soil (trace elements), and CRM-143R—sewage sludge amended soil (trace elements) were prepared by Joint Research Centre, Ispra, and purchased by Sigma-Aldrich Chemie GmbH (Buchs, Switzerland). Standard solutions of Cd, Cr, Co, Cu, Ni, Pb, and Zn (1000 mg/dm³) were purchased by J. T. Baker (SAD, Instra).

Phthalimide (PM), 3-Ethoxy-4-methoxyphenol (EMP), o-cyanobenzoic acid (CBA), dibutylamine (DBA), bis-2 (ethylhexyl) phthalate (BEP), indole (IND), hexahydro-3-(2-methylpropyl)-pyrrolopyrazine-1,4-dione (HMPP), and salicylic acid (SA) were purchased from Sigma-Aldrich Korea (Seoul, Korea) and dissolved in dimethylsulfoxide (DMSO). Phosphate-buffered saline (PBS, pH 7.4) was prepared with 100 mM phosphoric acid and 0.7% sodium chloride. Anticoagulant buffer (ACB, pH 4.5) was prepared to contain 186 mM NaCl, 17 mM Na₂EDTA, and 41 mM citric acid. A transfection reagent (Metafectene Pro) was purchased from Biontex (Plannegg, Germany).

Analytical standards of Benzophenone (CAS 119-61-9), Butyl stearate (CAS 123-95-5), Diisobutyl phthalate (CAS 84-69-5), and Tris (2,4-di-tert-butylphenyl) phosphite (Irgafos 168, CAS 31570-04-4), from LGC Standards (Bury, United Kingdom), and Bis (2-ethylhexyl) adipate (CAS 103-23-1), Bis (2-ethylhexyl) phthalate (CAS 117-81-7), Bis (2-ethylhexyl) terephthalate (CAS 6422-86-2), Butylated hydroxytoluene (CAS 128-37-0), Diphenyl sulphone (CAS 127-63-9), and Tri-n-butyl acetyl citrate (CAS 77-90-7), from Merck KGaA (Darmstadt, Germany), were used as standards for confirmation and quantification.
Analytical standards of Phenol-13C6 (CAS 89059-34-7), Benzophenone-D10 (CAS 22583-75-1), and Bis (2-ethylhexyl) phthalate-D4 (CAS 93951-87-2), all from LGC Standards (Bury, UK), were used as internal standards.
Standard n-alkane mixtures, C8-C20 and C10-C40 (all even), both from Merck KGaA (Darmstadt, Germany), were used for retention index calculation.
Acetone ≥ 99.8%, residue-analysis grade, from VWR International (Radnor, PA, USA), was used as solvent.
The analyzed sample was a post-consumer recycled LDPE film provided by Cadel Deinking, S.L. (Sant Vicent del Raspeig, Spain). The recycled LDPE film was obtained through a patented process that uses water-based chemicals to remove contaminants and includes grinding, washing, drying, and extrusion [37 ].