Trace 1310 gas

A Thermo Scientific TRACE 1310 gas chromatograph, equipped with a split/splitless-injector (SSL) and a programmable temperature vaporization (PTV) injector, a TriPlus 100 LS autosampler, and coupled to a TSQ 8000 EVO triple quadrupole mass spectrometer with electron ionization source was used. The GC was equipped with a TraceGold TG-5MS amine 15 m × 0.25 mm × 0.25 µm column (Thermo Fisher Scientific Inc., Waltham, MA, USA) and was connected to the respective injector. Splitless injections on the SSL injector were performed on quartz wool (Thermo Fisher Scientific Inc., Waltham, MA, USA) or CarboFrit (Restek Coorperation, Bellefonte, PA, USA) injection port liner (dimensions: 4 mm × 6.5 mm × 78.5 mm), while large-volume injections were carried out on the PTV-injector with packed quartz wool liners (2 mm × 2.75 mm × 120 mm, Thermo Fisher Scientific Inc., Waltham, MA, USA)). Helium served as a carrier gas for the GC, and Argon as collision gas for the mass spectrometer (both Alphagaz, purity 99.999%). Spectra were recorded and analyzed in TraceFinder 4.1 and Chromeleon 7.2 (Thermo Fisher Scientific Inc., Waltham, MA, USA). GC-MS/MS programs developed for this study are outlined in Table 1.

Analysis of short-chain fatty acids in stool samples was conducted using a Trace 1310 gas chromatograph (Thermo Scientific) equipped with an auto sampler, a flame ionization detector (FID), a split injector, and a Stabilwax DA column (Restek; 30 m, 0.25 mm ID, 0.25 µm), according to previously described procedures.^{76 (link)} Briefly, thawed fecal samples were dissolved in water and homogenized in Fastprep (MP Biomedicals). Supernatant was collected and mixed 1:1 (vol/vol) with internal standard (solution of 0.4% formic acid and 2000 µM 2-methylvaleric acid). Samples were centrifuged, and supernatant was transferred into spin columns (VWR; 0.2 µm filter) and centrifuged again. Eluates were transferred into GC vials and analyzed in the GC-FID instrument. The software Chromeleon (v. 7.2) was used for instrument control, quantification and data analysis. SCFA quantification was calculated based on a standard curve made from two-fold dilutions of SCFA standards.