13c4 pfos

Liver PFOS was extracted using a slightly modified previously published method (Chang et al., 2017 (link)). Frozen liver tissues (~50 mg) was homogenized in 2 mL Omni Hard Tissue Homogenizing tubes containing 1.4 mm ceramic beads, with 400 μL cold, deionized water spiked with a fixed amount of a stable isotope-labeled internal standard (13C4-PFOS, Wellington Laboratories, Ontario, Canada, Product code: MPFOS). Using an Omni Bead Ruptor Elite (Omni International, Kennesaw, GA), the mixture was homogenized for 30 seconds at 4 m/s. 250 μL of homogenate was digested overnight at room temperature in 10% 1N KOH. 100 μL of digested homogenate was further treated with 100 μL of 2N HC1, 500 μL 1N formic acid, 500 μL of saturated ammonium sulfate, and 5 mL methyl tert-butyl ether (MTBE). The solution was mixed on a shaker (20-30 min at room temperature). The organic and aqueous layers were separated by centrifugation (2500 × g, 5 min), and an exact volume of MTBE (4.5 mL) was removed from the solution. The top organic layer was subsequently transferred to a new tube and evaporated. The resulting sample was reconstituted with 20 mL of acetonitrile and water (1:1) prior to LC-MS/MS analysis.

Serum collected at necropsy was prepared according to methods described in Hansen et al. (2001) (link). Briefly, 20 μL of sera, 10 μL of isotope-labeled internal standard (¹³C4-PFOS, Wellington Laboratories, Ontario, Canada, Product code: MPFOS), 200 μL of 0.5 M tetrabutylammonium bisulfate (TBA; adjusted to pH 10), and 400 μL of 0.25 M sodium carbonate were added to a 15-mL polypropylene tube, and thoroughly mixed. 3 mL of MTBE was added to the solution, and the mixture was placed on a shaker for 20–30 min at room temperature. The organic and aqueous layers were separated by centrifugation (2500 x g, 5 min), and an exact volume of MTBE (2.5 mL) was removed from the solution. The top organic layer was subsequently transferred to a new tube and evaporated overnight. The resulting sample was reconstituted with 1.5 mL of acetonitrile and water (1:1) prior to LC-MS/MS analysis

Serum collected at necropsy was prepared according to methods described by Hansen (Hansen et al., 2001 (link)). Briefly, 20 μL of sera, 2 μL of isotope-labeled internal standard (13C4-PFOS, Wellington Laboratories, Ontario, Canada, Product code: MPFOS), 200 μL of 0.5M tetrabutylammonium bisulfate (TBA; adjusted to pH 10), and 400 μL of 0.25M sodium carbonate were added to a 15-mL polypropylene tube, and thoroughly mixed. 5 mL of MTBE was added to the solution, and the mixture was placed on a shaker for 20-30 min at room temperature. The organic and aqueous layers were separated by centrifugation (2500 × g, 5 min), and an exact volume of MTBE (4.5 mL) was removed from the solution. The top organic layer was subsequently transferred to a new tube and evaporated overnight. The resulting sample was reconstituted with 20 mL of acetonitrile and water (1:1) prior to LC-MS/MS analysis

Liver PFOS extraction method was modified from Chang et al. (2017) (link). Briefly, frozen liver tissues (~100 mg) were homogenized in 2 mL Omni Hard Tissue Homogenizing tubes containing 1.4 mm ceramic beads, with 400 μL cold, deionized water spiked with a fixed amount of a stable isotope-labeled internal standard (¹³C4-PFOS, Wellington Laboratories, Ontario, Canada, Product code: MPFOS). Using an Omni Bead Ruptor Elite (Omni International, Kennesaw, GA), the mixture was homogenized for 30 sec at 4 m/s. 250 μL of homogenate was then digested overnight at room temperature in 10% 1N KOH. 100 μL of digested homogenate was further treated with 100 μL of 2N HCl, 500 μL 1N formic acid, 500 μL of saturated ammonium sulfate, and 5 mL methyl tert-butyl ether (MTBE). The solution was mixed on a shaker (20–30 min at room temperature). The organic and aqueous layers were separated by centrifugation (2500 x g, 5 min), and an exact volume of MTBE (4.5 mL) was removed from the solution. The top organic layer was subsequently transferred to a new tube and evaporated. The resulting sample was reconstituted with 10 mL of acetonitrile and water (1:1) prior to LC-MS/MS analysis.

Calibration solutions were created by combining two solutions produced by the NIST Reference Materials (RMs) 8446 Perfluorinated Carboxylic Acids and Perfluorooctane Sulfonamide in Methanol and RM 8447 Perfluorinated Sulfonic Acids in Methanol. Together, the solution contained 15 PFAAs as follows: perfluorobutyric acid (PFBA), perfluoropentanoic acid (PFPeA), perfluorohexanoic acid (PFHxA), perfluoroheptanoic acid (PFHpA), PFOA, PFNA, perfluorodecanoic acid (PFDA), perfluoroundecanoic acid (PFUnA), perfluorododecanoic acid (PFDoA), perfluorotridecanoic acid (PFTriA), perfluorotet-radecanoic acid (PFTA), perfluorobutanesulfonic acid (PFBS), perfluorohexanesulfonic acid (PFHxS), PFOS, and perfluorooc-tanesulfonamide (PFOSA).
Internal standards (ISs) were purchased from Cambridge Isotope Laboratories (Andover, MA), RTI International (Research Triangle Park, NC), and Wellington Laboratories (Guelph, Ontario) to create an IS mixture comprised of eleven isotopically labeled PFAAs, and they were as follows: ¹³C₄-PFBA, ¹³C₂-PFHxA, ¹³C₈-PFOA, ¹³C₉-PFNA, ¹³C₉-PFDA, ¹³C₂-PFUnA, ¹³C₂-PFDoA, ¹⁸O₂-PFBS, ¹⁸O₂-PFHxS, ¹³C₄-PFOS, and ¹⁸O₂-PFOSA.