Sample Collection
Three-month-old female C57BL/6J conventional and germ-free mice were exposed by oral gavage to corn oil (vehicle control, n=7), low-dose PCB mixture S4 (6 mg/kg, n=8) or high-dose PCB mixture (30 mg/kg, n=8). Fecal samples were collected 24 h after PCB administration and pooled by exposure group [Ref. 1].
Sample Extraction
A total of 200 mg of pooled feces from each of the 6 exposure groups was homogenized in 2 mL of water (TissueRuptor, Qiagen) for 30 seconds. Two solvent blanks without feces were extracted in parallel to control for any background contamination. The 3-F,4’PCB 3 sulfate (5 ng in acetonitrile) was spiked to each homogenate as internal standard, 6 mL of acetonitrile was added and the samples were mixed and centrifuged at 1,800 g for 5 min. Supernatants were transferred to new glass vials. Four mL of an acetonitrile/water mixture (3:1, v/v) was added and the extraction steps described above were repeated. The combined supernatants were subjected to the same pretreatment and SBSE extraction process as reported previously for polar bear serum, and 10-30 uL of the final extract was injected to a HPLC-Orbitrap, with instrumental blanks (i.e. 50% H2O/50% MeOH) used to monitor for carryover [Ref. 2].
Instrument Analysis
An Orbitrap EliteTM hybrid mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) coupled with an Accela HPLC was used for the analyses. An Xselect CSH C18 XP column (130 Å pore size, 2.5 um particle size, 3 mm i.d., 150 mm length, Waters, Milford, MA) was used with a mobile flow rate and pressure of 0.5 mL/min and 400-900 bar, respectively. Water (mobile phase A) and methanol (mobile phase B) were used. Both mobile phases contained 2 mM ammonium acetate and 2 mM 1-methyl piperidine (1-MP, pH of mobile phase A = 9.8). The HPLC gradient program was as follows: starting at 38% B, increased linearly to 50% B between 0.17 and 2.67 min, then to 99% B at 41 min, held for 6 min, and returning to 38% B with a hold for 6 min before the next injection. The sample injection volume was 20 to 50 uL [Ref. 2].
Method for Processing the Raw Data
Raw data are provided in “raw” format and were processed with Xcalibur software (version 1.0.51.0, Thermo Scientific). The chromatograms were extracted with a mass tolerance of 5 ppm, Boxcar smoothing point of 7, and mass precision decimals of 5. Only peaks that matched the theoretical isotope pattern were integrated. A representative peak from the extracted chromatogram was chosen for each homolog group to determine the molecular formulas of the respective metabolite class, with a charge value of -1, mass tolerance of 20 ppm, and a ring double bound (RDB) equivalent of 8.0-10.0.