2.2.4. HS-SPME-GC-MS Method Conditions and Performance Characteristics

The collection of BTEX in the volatile fraction of both calibration and sample solutions was carried out in 20-ml HS vials with magnetic screw caps provided with polytetrafluoroethylene (PTFE)/silicone septa (Agilent Technologies). BTEX were collected through adsorption onto the polydimethylsiloxane (PDMS) stationary phase-coated fused silica fiber (thickness 100 μm, length 1 cm) introduced into the sample vial. The PDMS fiber was left in the vial for 30 s at 50 ◦C. Mechanical stirring was performed for 5 s with a stirring speed of 500 rpm. Analyses were performed using a gas chromatograph (7890B Agilent Tecnologies, Santa Clara CA, USA) equipped with an automated sampler (Pal System, CTC Analytics AG, Zwingen, Switzerland), a split/splitless injector and a single-quad mass spectrometer (5977A Agilent Technologies, Santa Clara CA, USA). Once incubation was completed, the heated gas-tight syringe containing the fiber was automatically transferred into the GC injector via the automated sampler and BTEX were thermally desorbed at 250 ◦C for 300 s and injected into the GC column in split injection mode (split ratio 1:10). Separation was performed on capillary column semivolatiles, 30 m × 0.25 mm, i.d. 0.25 μm film thickness (Phenomenex). Helium (purity ≥ 99.999%) was applied as carrier gas at a constant flow rate of 1 ml/min. The GC oven temperature program used for optimal separation was: 40 ◦C for 2 min, ramped 8 ◦C/min up to 80 ◦C, then ramped 60 ◦C/min up to 250 ◦C. Transfer line and ion source temperatures were kept at 260 ◦C and 270 ◦C, respectively. The mass spectrometer was operated in electron impact (EI) ionization mode (70 eV). Identification of BTEX was based on comparison of the obtained mass spectra with those included in the National Institute of Standards and Technology (NIST) library (MassHunter software) and considered positive by library search match >800 for both forward and reverse matching. Further criteria for compounds identification were: (a) the matching of relative retention times (tR) with those of the authentic standards within the allowed deviation of ± 0.05 min; and (b) the matching of ion ratios collected with those of the authentic standards within a tolerance of ± 20%. Quantification was performed in a selected ion monitoring mode (SIM). One quantifier ion and two qualifier ions were selected for each compound on the basis of their selectivity and abundance: 79 m/z as quantifier ion and 51 and 39 m/z as qualifier ions for benzene; 91 m/z as quantifier ion and 65 and 39 m/z as qualifier ions for toluene; and 91 m/z as quantifier ion and 106 and 51 m/z as qualifier ions for ethylbenzene and xylenes. Five point matrix-matched calibration curves were constructed for quantification (r<sup>2</sup> > 0.995) reporting compound/benzene-d6 quantifier ion peak areas ratio vs amount ratio. Calibration curves were in the range 2.6–41.6 μg/L for benzene, 2.7–43.2 μg/L for toluene and xylenes isomers and 2.8–44.8 μg/L for ethylbenzene. The xylenes isomers were quantified on the basis of p-xylene response factor (e.g., p-xylene calibration curve) and reported as sum in Table 2. Chromatograms of a blank sample and a sample spiked with the BTEX standard solution (calibration level 3) were

compared in Figure S1 (Supplementary Material, Figure S1). The main performance characteristics of the HS-SPME-GC-MS method were also evaluated. Linearity was calculated on the basis of three sets of replicates for each calibration level on three different days. As for the results, all matrix-matched calibration curves were linear over the set concentration ranges: relative accuracy (%) for each point was within the <sup>±</sup> 5% of the expected concentrations, and all coefficients of determination (r2) were >0.995. Selectivity/specificity was assessed directly onto the chromatograms obtained from the blank and from spiked matrices. The occurrence of possible extra peaks was tested by monitoring in SIM mode qualifier and quantifier ions characteristic for each investigated compound onto the blank matrix chromatograms, within the retention time window expected for the analyte elution. Limit of detection (LOD) and LOQ values were assessed in the spiked matrix by determining the lowest concentration of the analytes that resulted in a signal-to-noise (S/N) ratio of ≥ 3 and ≥ 10, respectively. LOD values were 1.4 μg/L for benzene and toluene, 1.5 μg/L for xylenes, and 1.6 μg/L for ethylbenzene. LOQ values were 2.6 μg/L for benzene, 2.7 μg/L for toluene and xylenes and 2.8 μg/L for ethylbenzene. Repeatability expressed as intra-day coefficients of variation (CV%) was evaluated on a set of results (n = 6 replicates) obtained for each analyte at three validation levels (i.e., LOQ values; 10.4 μg/L for benzene, 10.8 μg/L for toluene and xylenes, 11.2 μg/L for ethylbenzene; 41.6 μg/L for benzene, 43.2 μg/L for toluene and xylenes and 44.8 μg/L for ethylbenzene). Intra-day CV% values were 1.2–4.5% for benzene, 1.2–9.9% for toluene, 3.2–10.9% for ethylbenzene and 2.8–11.4% for xylenes. Intermediate precision (expressed as inter-day CV%) and recovery were calculated by analyzing the series within the three different days (n = 18 replicates). Inter-day CV% values were 5.1–15.3% for benzene, 6.6–10.0% for toluene, 8.8–14.6% for ethylbenzene and 9.4–15.4% for xylenes. Finally, recoveries were in the range of 96.6–113.0%.
