BaA and BaP analytical standards were purchased from Supelco Inc. (Bellefonte, PA, USA), BbF and BkF were from Aldrich Chemical Co. (Steinheim, Germany). Hexane, cyclohexane and N,N-dimethylformamide (HPLC grade) were purchased from Tedia Company Inc. (Fairfield, selleck chemical OH, USA). Acetonitrile (HPLC grade) and reagent grade anhydrous sodium sulphate were from J.T. Baker (Phillipsburg, NJ, USA). Water was obtained from a Millipore Milli-Q water purification system (Milford, MA, USA).

Millex HV 0.45 μm filters were purchased from Millipore and Bakerbond SPE silica columns (500 mg, 3 mL) were from J.T. Baker. Extraction and clean up procedures were based on the method described by Tfouni et al. (2009). In a separating funnel, 50 mL of N,N-dimethylformamide–water (9:1, mL:mL) and 60 mL of a sodium sulphate aqueous solution (1 g/100 g) were added to a 10 mL sample. PAHs were successively extracted with three aliquots (25 mL) of cyclohexane. The combined extract was dried find more with anhydrous sodium sulphate, concentrated on a rotary evaporator to approximately 2 mL at 40 °C and dried under a flow of nitrogen. Clean up was performed

by silica gel SPE. Cartridges were prepared by pre-washing with 12 mL of hexane followed by drying using a Vacuum Manifold from Supelco. The extracts were suspended with three aliquots (1 mL) of hexane, applied in the SPE cartridge and eluted with 7 mL hexane. Solvent was dried under a flow of nitrogen and the residue was dissolved in 1 mL acetonitrile, filtered through a 0.45 μm filter and analyzed by HPLC with fluorescence detection. The analyses were carried out using a Shimadzu (Kyoto, Japan) HPLC apparatus equipped with a LC-20AT pump, a SIL-20AT autosampler, a CTO-20A column oven and a RF-10A xl fluorescence detector. Data were acquired and processed with LCsolution software. A C18 column (Vydac 201 TP54, 250 × 4.6 mm, 5 μm particle size; Vydac, Hesperia, CA, mafosfamide USA) at 30 °C and isocratic mobile phase consisting

of 75% acetonitrile and 25% water at a flow rate of 1 mL/min were used. The detector was set at 290 nm (excitation wavelength) and 430 nm (emission wavelength); injection volume was 20 μL. The external standard plot method was used for quantification. Duplicate HPLC injections of six concentration levels (0.1–2.0 ng/mL) of PAHs standard solutions, in acetonitrile, were used to construct linear regressions lines (peak area ratios versus PAH concentration). The limit of detection (LOD) for each PAH was defined as the concentration of the analyte that produced a signal-to-noise ratio of three. Accuracy and precision data were obtained through recovery studies carried out by spiking a coffee brew sample with PAHs standard solutions at concentration levels of 0.3, 0.5 and 1.