## 3 Results and Discussion  
### Optimization of SPME Parameters  
Starting from the optimization of the HS-SPME conditions, preliminary experiments were performed in order to compare the extraction efficiency of different fiber coatings, namely, polyethylene glycol (PEG), polyacrylate (PA), carboxen/polydimethylsiloxane (CAR/PDMS), carboxen (CAR), carboxen/divinylbenzene/polydimethylsiloxane (CAR/ DVB/PDMS) and polydimethylsiloxane (PDMS), using the experimental conditions reported in Section 2.2 (exposure time 30 min, after 30 min of sample heating at 37 \({}^{\circ}\)C). A good compromise for the simultaneous extraction of all the selected targets compounds \begin{table}
\begin{tabular}{c c c c} \hline
**RT \({}^{*}\)** & **Compound** & **Segment Start–End (min)** & **Characteristic Ions \({}^{**}\)** \\ \hline
5.79 \(\pm\) 0.03 & toluene & 4.00–7.50 & 65, **91** \\ \hline
8.44 \(\pm\) 0.02 & ethylbenzene & 7.50–10.50 & **91**, 106 \\ \hline
11.01 \(\pm\) 0.02 & benzaldheyde & 10.50–11.50 & 77, **105** \\ \hline
11.66 \(\pm\) 0.01 & octanal & 10.50–13.50 & **43**, 56 \\ \hline
14.30 \(\pm\) 0.03 & benzoic acid & 13.50–15.50 & 91, **121** \\ \hline
16.59 \(\pm\) 0.03 & dodecane & 15.50–18.50 & **57**, 71, 85 \\ \hline
23.05 \(\pm\) 0.04 & tetradecane & 18.50–24.50 & **57**, 71, 85 \\ \hline \end{tabular} \({}^{*}\) Retention time (RT); \({}^{**}\) Quantification ion in bold.  
\end{table} 
Table 1: SIM operation mode parameters. was reached employing the bipolar fibers CAR/PDMS and CAR/DVB/PDMS, which were then selected for further experiments. Figure 1 compares the instrumental response obtained for each analyte using the two fibers. Then, desorption conditions in the GC injector (splitless mode) were optimized testing different combinations of temperature and time, evaluating the carry-over after each desorption. It was found that 2 min at 200 \({}^{\circ}\)C were sufficient to completely remove the volatiles from the fiber. The optimization of the extraction temperature, pH, ionic strength and stirring were not performed, since the method is intended to be applied to ex vivo experiments that involve surgical resected tissues of CRC patients and their subsequent cultivation in laboratory, under suitable conditions of sterility. Therefore, only extraction time profiles at 37 \({}^{\circ}\)C (ex vivo incubation temperature) were evaluated for both selected fibers and the relevant results reported in Figure 2. As apparent, 30 min of HS sampling were required to obtain the best response for the analytes with both fibers, with the only exception of benzaldehyde and tetradecane with CAR/DVB and CAR/DVB/PDMS fibers, respectively, that reached equilibrium after 15 min. DI-SPME extraction was also considered, since it could provide useful information for the evaluation of metabolites directly released from the tissues into the culture medium. Then, all the main variables (time, temperature, ionic strength and pH) that influence the extraction process were evaluated, under constant agitation (700 rpm). It is worth noting that, to preserve the polymer phase, especially due to the presence of salt, the fibers were left overnight in distilled water without exposing the coating. This practice permitted to perform more than 80 consecutive extractions per fiber. Generally speaking, salt addition often improves the recovery, due to the salting-out effect. Thus, experiments were performed by increasing progressively the ionic strength of the extraction solutions. Figure 3 reports the results obtained performing the extraction for 1 h at room temperature, without and after the addition (30%) of NaCl, respectively. As apparent, a positive effect of salt on the extraction was mostly observed. Figure 1: Instrumental response obtained extracting the selected compounds from the HS using the CAR/PDMS and CAR/DVB/PDMS fibers, respectively. Figure 3: Effect of the NaCl concentration on the DI extraction efficiency obtained with both selected fibers.