Liver and brain samples (2 g) were homogenized with dry Na2SO4, packed into columns, and extracted with acetone:cyclohexane (1:3,v/v). The extract was evaporated, and the lipid content (%, w/w) was determined. The extract was further cleaned up using gel permeation chromatography for lipid removal. All plasma, liver, and brain extracts were fractionated using Florisil. The collected Florisil fractions included fraction 1––dichloromethane:n-hexane (1:9,v/v) containing neutral compounds (PCBs, pesticides, polybrominated diphenyl ethers); fraction 2––acetone:n-hexane (1:9,v/v) containing MeSO2 compounds; and fraction 3––methanol:dichloromethane (1:5,v/v) containing OH compounds. The third fraction was gently evaporated to dryness and derivatized using diazomethane. A final clean-up of fractions 2 and 3 was performed on a solid-phase extraction column of 25% sulfuric acid silica with neutral silica on top, and extraction was performed with dichloromethane. All plasma, liver, and brain extracts were evaporated and transferred to gas chromatography (GC) vials with octachloronaphthalene added as volume corrector. The analysis of the compounds was performed on an Agilent 7890AGC with a 5975C mass spectrometer (MS) operated in single-ion monitoring mode. The MS was run in negative ion chemical ionization mode for the metabolites and pesticides and in electron impact mode for PCBs and DDTs. Polybrominated diphenyl ethers (PBDEs) were analyzed in the electron impact mode on an Autospec GC/high-resolution MS system (Waters). All sample fractions were analyzed separately. Fraction 1 (containing PCBs, pesticides, PBDEs, and MeO-PBDEs) was injected three times to the GC/MS or GC/high-resolution MS analysis instrument grouped into subgroups: PCBs/DDTs, pesticides/MeOBDEs, and PBDEs. Fraction 2 was analyzed for MeSO2-PCBs/dichlorodiphenyldichloroethylene (DDE), and fraction 3 was analyzed for the derivatized OH-PCBs, pentachlorophenol (PCP), 4-OH-heptachlorostyrene (4-OH-HpCS), tetrabromobisphenol-A (TBBP-A), and OH-PBDEs.
Polychlorinated biphenyls analyzed included the following congeners and metabolites: CB18, 28, 31, 33, 37, 47/49, 52, 99, 101, 105, 118, 123, 128, 138, 141, 149, 153, 156, 157, 167, 170, 180, 183, 187, 189, 194, 4-OH-CB120, 4-OH-CB107, 3-OH-CB153, 4-OH-CB146, 3-OH-CB138, 4-OH-CB130, 4-OH-CB163, 4-OH-CB187, 4-OH-CB172, and 4′-OH-CB193, as well as 3-MeSO2-CB52, 4-MeSO2-CB52, 3-MeSO2-CB49, 4-MeSO2-CB49, 3-MeSO2-CB91, 4-MeSO2-CB91, 3-MeSO2-CB101, 4-MeSO2-CB101, 3-MeSO2-CB87, 3-MeSO2-CB110, 4-MeSO2-CB110, 3-MeSO2-CB149, 4-MeSO2-CB149, 3-MeSO2-CB132, 4-MeSO2-CB132, 3-MeSO2-CB141, 4-MeSO2-CB141, 3-MeSO2-CB174, and 4-MeSO2-CB174. Quantified organochlorine pesticides and their metabolites included o,p′-DDT, o,p′-DDE, and o,p′-dichlorodiphenyldichlorethane (DDD), p,p′-DDT, p,p′-DDE, and p,p′-DDD, heptachlor, oxychlordane, trans-chlordane, cis-chlordane, trans-nonachlor, cis-nonachlor, PCP, 4-OH-HpCS, and MeSO2-DDE. The native [12C] PCBs and organochlorines (OCs) were purchased from Ultra Scientific Europe, and the isotopically labeled [13C] PCBs and OCs used as internal standards were purchased from Cambridge Isotope Laboratory. The native OH-PCBs and MeSO2-PCBs as well as PCP, 4-OH-HpCS, and MeSO2-DDE and the isotopically labeled OH-PCBs and PCP were kindly donated by the U.S. Centers for Disease Control and Prevention. In addition, a surrogate internal standard for the analysis of MeSO2-PCBs was used, 3-MeSO2-4-Me-2′,3′,4′,5,5′-PentaCB, which was also purchased from Cambridge Isotope Laboratory. The following brominated compounds were analyzed: BDE 28, 47, 66, 71/49, 77, 85, 99, 100, 119, 138, 153, 154, 183, 206, 207, and 209, 2,4,6,-tribromoanisole (TBA), TBBP-A, 2-OH-BDE68, 6-OH-BDE47/75, 5-OH-BDE47, 4-OH-BDE49, 5-OH-BDE100, 4-OH-BDE103, 5-OH-BDE99 4-OH-BDE101, 2-methoxy (MeO)-BDE68, 6-MeO-BDE47/2-MeO-BDE75, 5-MeO-BDE47, 4-MeO-BDE49, 5-MeO-BDE100, 4-MeO-BDE103, 5-MeO-BDE99, and 4-MeO-BDE101. Polychlorinated biphenyls 18, 33, and 37, o,p′-DDD, and TBA were not quantified in the brain samples, whereas 4-OH-HpCS was not quantified in the plasma samples. The native [12C] PBDEs, TBA, and TBBPA, MeO-BDEs, and isotopically labeled [13C] standard PBDEs were purchased from Wellington Laboratories.
All chemical analyses followed international requirements for quality assurance and control, for example, recommendations of the Arctic Monitoring and Assessment Program. In addition, the Norwegian Institute for Air Research participates in the Arctic Monitoring and Assessment Ring Test Program for persistent organic pollutants in human serum. Quality assurance and control of the sample preparation and analysis were ensured through the use of mass labeled internal standards for the PCBs, organochlorine pesticides, brominated compounds, and OH-PCBs, whereas a surrogate standard was used for the MeSO2 compounds in addition to sample preparation and analysis of certified reference materials and laboratory blanks. For each batch of 20 plasma samples, one standard reference (U.S. National Institute of Standards and Technology [NIST] human serum 1589a) and three blank samples were prepared, whereas for each batch of 10 brain and liver samples one standard reference (NIST cod oil 1588b) and one blank were prepared. The average recovery, determined from the added mass labeled internal standards ([13C]PCBs, pesticides, PBDEs, OH-PCBs) or surrogate standard (3-MeSO2-4-Me-2′,3′,4′,5,5′-PentaCB) for the MeSO2-PCBs was 76 ± 10% (standard deviation) for PCBs, 93 ± 13% for DDTs, 55 ± 15% for chlordanes (CHLs), 58 ± 18% for PBDEs, 60 ± 18% for OH compounds, 55 ± 18% for MeSO2 compounds, and 60 ± 16% for MeO-BDEs.