*2.5. Determination of PAHs in Soil, Forage, and Milk by Gas Chromatography–Tandem Mass Spectrometry (GC-MS/MS)*

Soil samples (10 g) were extracted with dichloromethane:acetone [1:1 (*v*/*v*)] in a Soxtherm system (Gerhardt, Bonn, Germany). The extracts were cleaned with silica gel, then concentrated by rotary evaporation (Heidolph, Schwabach, Germany). PAH concentrations were determined after injection into a 7890A GC System coupled to a 5975C Inert XL MSD with a Triple-Axis Detector (Agilent Tech., Santa Clara, CA, USA), following EPA Method 8272, with modifications. The samples were run on a capillary column DB-5ms with a length of 30 m, an inner diameter of 0.25 mm, and a film thickness of 0.25 μm (Agilent Tech. Santa Clara, CA, USA), with He as the carrier gas at 1 mL min−1. The initial oven temperature of 70 ◦C was held for 2 min; ramped up to 220 ◦C at 20 ◦C min−1, then to 270 ◦C at 10 ◦C min<sup>−</sup>1, where it was held for 1 min; ramped up to 290 ◦C at 10 ◦C min<sup>−</sup>1, where it was held for 1 min; and finally ramped up to 300 ◦C at 10 ◦C min<sup>−</sup>1, where it was held for 7 min. The total run time of GC separation was 30 min The gas chromatography injector was operated in splitless mode for 2 min, and its temperature was maintained at 260 ◦C. The mass spectrometer was operated in electron ionization mode (EI) at 70 eV and calibrated daily by auto-tuning with perfluorotributylamine (PFTBA). PAH calibration standards (AccuStandard, New Haven, CT, USA) were used. Blanks (one for every five samples), duplicate samples, and cross correlation were used for quality assurance and quality control (QA/QC) purposes. RSD for individual PAHs was below 10% in all cases. The following species (*m*/*z*) were quantified: 128 (naphthalene), 152 (acenaphthylene), 153 and 154 (acenapthene), 165 and 166 (fluorene), 178 (anthracene/phenanthrene), 202 (fluoranthene/pyrene), 228 (benzo(a)anthracene/chrysene), 252 (benzo(b)fluoranthene/benzo(k)fluoranthene), 276 (indene(1,2,3-c,d)pyrene/benzo(g,h,i)perylene), and 278 (benzo(a,h)anthracene). In Appendix A, the most representative chromatogram of soil samples has been included.

Forage and milk samples were treated according to the "QuEChERS" extraction method, with some modifications [26]. Briefly, samples (10 g) were extracted with 30 mL acetonitrile and vortexed at 3000 rpm for 1 min. A total of 4 g anhydrous MgSO4 and 1 g NaCl were added and immediately vortexed for 1 min, then 50 μL of an internal standard solution were added and the mixture was vortexed for another 30 s. The mixture was centrifuged at 2800× *g* for 5 min at room temperature and the supernatant was purified by a dispersive solid-phase extraction method [26]. An aliquot of supernatant (5 mL) was transferred to a flat-bottomed flask, concentrated in a 40 ◦C water bath until neardrying, and dissolved in 5 mL of cyclohexane [26]. An aliquot of 1 μL was injected into a 7890B gas chromatograph (Agilent Tech.) equipped with a Select PAH CP7462 capillary column with a length of 30 m, an inner diameter of 0.25 mm, and a film thickness of 0.15 μm. He was used as carrier gas at 2 mL min−1. The initial oven temperature of 70 ◦C was held for 0.7 min, ramped up to 180 ◦C at 85 ◦C min−<sup>1</sup> and then to 230 ◦C at 3 ◦C min<sup>−</sup>1, where it was held for 7 min; ramped up to 280 ◦C at 28 ◦C min<sup>−</sup>1, where it was held for 10 min; and, finally, ramped up to 350 ◦C at 14 ◦C min−1, where it was held for 3 min. The total run time of GC separation was 60 min. The GC injector was operated in splitless mode for 1 min and its temperature was maintained at 300 ◦C. The compounds were detected using a 7000D mass spectrometer (Agilent Tech.), which was operated in electron ionization mode (EI) at 70 eV. The following *m*/*z* ratios were monitored: 178 (anthracene/phenanthrene), 202 (fluoranthene/pyrene), 228 (benzo(a)anthracene/chrysene), 252 (benzo(b)fluoranthene/benzo(k)fluoranthene/benzo(a)pyrene), 276 (benzo(g,h,i) perylene/Indene(1,2,3-c,d)pyrene), and 278 (benzo(a,h)anthracene). Each sample was analyzed per duplicate. The method was validated using five internal standards (AccuStandard, New Haven, CT, USA), prepared by adding isotopically labelled PAHs to sample extracts. Concentrations of PAHs in the samples were determined by comparing their peak areas to those of the internal standards.
