00 ng) was added as PCB surrogate common. The medium and homogenate samples had been acidified with six M hydrogen chloride (1 mL) and extracted with 2-propanol (3 mL) and hexane-MTBE (1:1, v/v; five mL). The organic phase was washed with 1 KCl (two mL). Just after derivatization with diazomethane, the samples were spiked with the internal standard (2,2, three,4,four,five,6,6-octachlorobiphenyl, 200 ng). The samples had been additional cleaned by shaking the sample with 2-propanol (two mL) and tetrabutylammonium sulfite (two mL), and reshaking by adding nanopure water (5 mL). The organic extract was mixed with concentrated sulfuric acid (two mL), kept overnight and transferred to vials. Analysis of PCB 136 and its metabolites The levels of PCB 136 and its hydroxylated metabolites (as methoxylated PCB derivatives) in liver and brain samples were determined simultaneously utilizing an Agilent 6980N gas chromatograph (GC) equipped using a 63Ni micro-electron capture detector (-ECD) in addition to a DB1-MS capillary column (60 m ?0.25 mm inner diameter (ID) ?0.25 m film thickness; Agilent, Santa Clara, CA, USA) (Kania-Korwel et al., 2012). The injector and detector temperatures have been 280 and 300 , respectively. The temperature system was as follows: one hundred for 1 min, five /min to 250 , hold for 20 min, five /min to 280 , hold for 3 min. The flow price was 1 mL/min. The concentrations of PCB 136 and its hydroxylated metabolites had been inside the linear array of calibration curves (1 1000 ng/mL) in all samples. Detailed information regarding detection limits are shown in Table A3. The recoveries of PCB 65, PCB 166 and 4-OH-PCB 159 were 92 ?8 , 86 ?12 and 87 ?15 , respectively. The levels of PCB 136 and its hydroxylated metabolites in liver slices had been adjusted for recoveries. Protein levels have been determined using the process of Lowry employing bovine serum albumin as normal (Lowry et al., 1951). Enantioselective analysis of PCB 136 and OH-PCB 136 atropisomers Atropisomers of PCB 136 and 5-OH-PCB 136 were separated around the very same instrument described above working with a Chirasil-Dex capillary column (25 m ?0.1394041-21-4 Price 25 mm inner diameter (ID), 0.25M film thickness; Agilent, Santa Clara, CA, USA) (Wu et al., 2011). The injector and detector temperatures had been kept at 250 . The flow rate was three mL/min. The temperature plan was as follows: 15 /min from 50 to 135 , hold for 800 min, 15 / min to 200 , hold for ten min. 4-OH-PCB 136 atropisomers have been separated with aXenobiotica. Author manuscript; readily available in PMC 2014 November 01.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptWu et al.PageCyclosil-B capillary column (30 m ?0.25 mm ID ?0.25 m film thickness; Agilent, Santa Clara, CA, US) (Kania-Korwel et al.m-PEG12-acid Chemscene , 2011; Wu et al.PMID:24377291 , 2011). The temperature program was as follows: 15 /min from 50 to 160 , hold for 360 min, 15 /min to 200 , hold for 10 min. The enantiomeric fraction (EF) values for PCB 136, 4-OH and 5-OH-PCB 136 have been determined as EF = Region E(two)/(Location E(1) + Region E(2)). Added facts about detection limits and resolution of PCB and OH-PCB atropisomers are offered in Table A4. Statistics GraphPad Prism 4 (version 4, GraphPad Application, La Jolla, CA, USA) or SAS software (version 9.three, SAS Institute, Cary, NC, USA) have been employed for the statistical evaluation. All data are presented because the imply ?regular deviation/ regular error of imply (as indicated inside the table or figure legends). One-way or two-way evaluation of variance (ANOVA) were used to recognize important differences in animal.