## 3 Results and Discussion
### Analyses of Ex Vivo Colon Tissues from CRC Patients  
The colon tissues (healthy tissue and tumor) from CRC patients were analyzed by HS- and DI-SPME-GC-MS, employing the optimized experimental conditions, and identities were attributed to the secreted VOCs. At first, peaks identification was carried out by comparing the mass spectra of the chromatographic peaks with those reported in the National Institute of Standards and Technology (NIST) library ([[http://webbook.nist.gov/chemistry/2018](http://webbook.nist.gov/chemistry/2018)]([http://webbook.nist.gov/chemistry/2018](http://webbook.nist.gov/chemistry/2018)) (accessed on 6 July 2021)). Furthermore, the relative retention index (RI) of each compound was calculated using the RT of the C7-C30 series of homologous \(n\)-alkanes (1000 ug/mL in hexane from Supelco) and compared with those reported for substances analyzed under similar conditions. The protocols allowed to individuate 31 different compounds, shown in Table 3, clearly demonstrating the potential of the present approach. \begin{table}
\begin{tabular}{c c c c} \hline
**N \#** & **RT (min)** & **RI *** & **Compound** \\ \hline
1 & 3.2 & 671 & 1-butanol \\ \hline
2 & 3.45 & 687 & 2-pentanone \\ \hline
3 & 4.75 & 723 & 1-butanol, 3-methyl \\ \hline
4 & 5.00 & 732 & disulfide, dimethyl \\ \hline
5 & 5.1 & 753 & pyridine \\ \hline
6 & 5.79 & 761 & toluene \\ \hline
7 & 8.44 & 851 & ethylbenzene \\ \hline
8 & 9.02 & 875 & 2-heptanone \\ \hline
9 & 9.14 & 885 & 2,4-dithiapentane \\ \hline
10 & 9.67 & 895 & 2,5-dimethyl pyrazine \\ \hline
11 & 9.91 & 917 & oxime, methoxy-phenyl \\ \hline
12 & 10.69 & 921 & pentanoic acid \\ \hline
13 & 11.01 & 961 & benzaldehyde \\ \hline
14 & 11.14 & 996 & furan, 2-pentyl \\ \hline
15 & 11.66 & 1001 & octanal \\ \hline
16 & 12.14 & 1023 & benzyl Alcohol \\ \hline
17 & 12.9 & 1027 & phenol, 2-methyl \\ \hline
18 & 13.02 & 1094 & 2-nonanone \\ \hline
19 & 13.28 & 1080 & nonanal \\ \hline
20 & 13.63 & 1083 & phenethyl alcohol \\ \hline
22 & 13.88 & 1143 & disulfide, methyl (methylthio)methyl \\ \hline
23 & 14.30 & 1171 & benzoic acid \\ \hline
24 & 13.90 & 1138 & phenol, 2-ethyl \\ \hline
25 & 13.95 & 1143 & phenol, 3,5-dimethyl \\ \hline \end{tabular}
\end{table} 
Table 3: List of the VOCs secreted by surgical resected colon mucosa tissues of CRC patients. The DI-SPME-GC-MS procedure was then used to monitor the progressive emission of seven selected VOCs from the healthy tissues and diseased mucosa, respectively, of three CRC patients, for 1 week in ex vivo conditions. The DI-SPME technique was preferred to carry out kinetic studies, because it permitted to be independent from the metabolic evolution of the ex vivo systems, which also makes impossible to perform replicate analyses on the same vials to evaluate intra- and inter-day variability. Figure 6 shows the kinetic profiles obtained for each analyte while Table 4 shows the concentration ranges of the analytes estimated in healthy tissues and diseased mucosa. Data reported in Figure 6 suggest that the secretion of most of the selected compounds, i.e., benzaldehyde, ethylbenzene, octanal and tetradecane, from both type of tissues was observable for 6 days, with a constant increase for 4 days, when a plateau was reached. On the contrary, dodecane and toluene showed a fast reduction that lead to their total disappearance after only 2 days of incubation. In the case of ethylbenzene, octanal (after 2 days) and benzaldehyde, quantitative differences were observed between sick and healthy tissues of the same patient. Moreover, compared to the normal colonic mucosa, cancer tissue is characterized by higher release of benzaldehyde, likely due to the increase in the rate of specific metabolic processes catalyzed by the disease which, on the contrary, slows down the production of ethylbenzene. Figure 6: Kinetic profiles of seven selected analytes secreted by surgical resected colon tissues (healthy tissue and tumor) from three CRC patients.