Extraction and cleanup of nitro-PAH samples

Aliquots (10 g) of the homogenized samples were placed in flat-bottomed flasks (300 mL) and then spiked with 1 mL of 20 μg/kg Ace-d10, Phen-d10, Chrys-d12 and Pery-d12, respectively. The samples were extracted with 100 mL n-hexane. The extraction efficiency of the suspension was enforced by ultrasonication in an ultrasonic bath at 50–60 Hz (Bransonic 2200, Branson Ultrasonic Corp., Danbury, CT, USA) at 28 °C (room temperature) for 60 min. The obtained extracts were filtered to remove particulate matter (110 mm filter paper; Advantec, Toyo Roshi Kaisha, Ltd., Japan). Next, the solution was sequentially extracted with 50, 25, and 25 mL N,N-DMFA: water (9:1, v/v). The extract was then diluted with 100 mL of 1% sodium sulfate solution and sequentially re-extracted with 50, 35, and 35 mL n-hexane, respectively. The combined hexane solution was washed twice with 50 mL distilled water, dried with anhydrous sodium sulfate (15 g) and concentrated on an N–N Series SB-100 rotary vacuum evaporator (Eyela, Tokyo, Japan) to 2 mL at 30 °C. The concentrated extracts were cleaned by adsorption chromatography on silica gel cartridges. The cartridges were conditioned with 6 mL cyclohexane. The extract was transferred to the cartridge quantitatively. The column was first eluted with 4 mL cyclohexane and then with 6 mL cyclohexane/DCM (1:1); the whole eluent was collected. The fraction was again evaporated and the resulting residues were re-dissolved in 1 mL. The solution was filtered through a 0.45-μm PTFE membrane filter (25 mm, 0.45 μm) from Agilent Technologies (Wilmington, DE, USA), and transferred to 2-mL amber screw-cap vials. An aliquot of this solution was analyzed by gas chromatography/mass spectrometry (GC/MS).