2.6. Short Chain Fatty Acids Detection

Short chain fatty acids were detected in fecal media using gas chromatography-mass spectrometry (GC-MS) according to Dixon et al. [30]. Determination was performed using the same samples indicated in Section 2.5. After preconditioning (according to the manufacturer’s instructions) a polydimethylsiloxane/divinylbenzene (PDMS/DVB) fiber (65 μm), a manual solid-phase micro-extraction (SPME) holder (Supelco Inc., Bellefonte, PA, USA) was used. Before headspace sampling, the fiber was exposed to the gas chromatography (GC) inlet for 1 h for thermal desorption at 250 °C. Three grams of sample were placed into 10 mL glass vials, and 10 μL of 4-methyl-2-pentanol (final concentration 33 mg/L) was added as the internal standard. Samples were then equilibrated for 10 min at 40 °C. The SPME fiber was exposed to each sample for 40 min. Equilibration and absorption phases were conducted under stirring. The fiber was then inserted into the injection port of the gas chromatograph for 10 min of sample desorption. GC-mass spectrometry (MS) analyses were performed with an Agilent 7890A gas chromatograph (Agilent Technologies, Palo Alto, CA, USA) coupled to an Agilent 5975C mass selective detector, operating in electron impact mode (ionization voltage, 70 eV). A Supelcowax 10 capillary column (length, 60 m; inner diameter, 0.32 mm; Supelco, Bellefonte, PA, USA) was used. The temperature program was 50 °C for 1 min, followed by an increase at a rate of 4.5 °C/min to 65 °C, an increase at a rate of 10 °C/min to 230 °C, and then holding at 230 °C for 25 min [31]. The injector, interface, and ion source temperatures were kept at 250, 250, and 230 °C, respectively. The mass-to-charge ratio interval was 30 to 350 Da and spectra were acquired at a rate of 2.9 scans per second. Injection was carried out in splitless mode, and helium (flow rate = 1 mL/min) was used as the carrier gas. Molecules were identified based on the comparison of their retention times with those of pure compounds (Sigma-Aldrich, Milan, Italy). Identities were confirmed by searching mass spectra in the available databases (NIST, version 2005; Wiley, version 1996). Quantitative data for the identified compounds were obtained by interpolation of the relative area versus the internal standard area. All the GC-MS raw files were converted into netCDF format via Chemstation (Agilent Technologies, Santa Clara, CA, USA) and subsequently processed through the XCMS toolbox (http://metlin.scripps.edu/download/; accessed date: 1 December 2020). XCMS software allows for automatic and simultaneous retention time alignment, matched filtration, peak detection, and peak matching. GC-MS/SPME data were organized in matrices for subsequent statistical analysis.