Pretreatment of PBDEs in biological samples 2.3 Lipid extraction Internal standards (tri- through hexa-13C12-BDE) were spiked in samples of blood (30 g), bile (30 g), liver (1 g), and adipose tissue (1 g), respectively, and then extracted by liquid–liquid extraction following the method of Hirai et al. (2002). Briefly, in blood spiked with internal standard, 12 mL of ammonium sulfate, 6 mL of ethanol, and 18 mL of n-hexane were mixed and extracted 3 times. Internal standard spiked in bile was extracted with 50 mL of acetone/n-hexane (2:1 v/v) followed by 2 extractions with n-hexane. Liver and adipose tissues were homogenized in the presence of a 5-fold volume of sodium sulfate with internal standard, and extracted with 50 mL of acetone/n-hexane (2:1 v/v) followed by two extractions with n-hexane. The extract was washed with ultra-pure water. The organic layer was dried over sodium sulfate and then evaporated to dried by evaporation. The amount of lipid remaining was determined using the gravimetric method. 2.4 Alkaline degradation Extracted lipid was saponified with 10 mL of 2M potassium hydroxide/ethanol containing 10% water for 2 h with shaking. The PBDEs were extracted with 15 mL of n-hexane (2 times, 30 min each). The extract was concentrated to about 2 mL at 40ºC on a rotary evaporator and then passed through a multi-layer column. 2.5 Multi-layer column chromatography Extracted lipid was dissolved in 3 mL of n-hexane and passed through a glass column (250 mm 10 mm ID) equipped with a glass-wool plug and packed from bottom to top with 1.5 g of anhydrous sodium sulfate, 0.9 g of silica gel, 3 g (2% w/w) of potassium hydroxide-silica gel, 0.9 g of silica gel, 4.5 g (44% w/w) of sulfuric acid-silica gel, 6 g (22% w/w) of sulfuric acid-silica gel, 0.9 g of silica gel, 3 g of silver nitrate-silica gel, 0.9 g of silica gel, and 1.5 g of anhydrous sodium sulfate. Before loading the sample, the column was washed with 100 mL of n-hexane. Samples were loaded onto the column and dioxins and PCBs were eluted in the first fraction with 100 mL of n-hexane. PBDEs were eluted in the second fraction with 200 mL of methylene chloride:n-hexane (1:9 v/v) (Hirai et al. 2002). The flow rate was 2.5 mL/min. After addition of approximately 20 μL of n-nonane, the PBDE eluates were concentrated to a volume of about 0.5 mL at 40C on a rotary evaporator. 2.6 Preparation of samples for analysis Concentrated fractions containing PBDE were transferred to a gas chromatograph (GC) vial. The remaining solvent was evaporated under a stream of nitrogen. A total of 20 pg of C12-PBDE (BDE-77, BDE-126) was added to this solution and 1.5 μL was injected for 13 HRGC/HRMS analysis.