5971 single quadrupole mass spectrometer

Faeces were freshly collected and stored at − 80 °C. At use, each sample was thawed and weighted (weight range 500–800 mg); then added sodium bicarbonate 10 mM (1:1 w/v) in a 1.5 mL centrifuge tube. The obtained suspension was then mixed with the aid of a sterile wooden stick and briefly shaken in a vortex apparatus, extracted in ultrasonic bath (15 min) and then centrifuged at 4 °C at 13.000 rpm for 90 min. The supernatant was collected, transferred in 1.5 mL centrifuge tube and stored at − 20 °C until use.
For the analysis, these supernatant samples were thawed, briefly centrifuged at 5000 rpm and resuspended for 5 min in ultrasonic bath. The SCFAs were then extracted as follows: an aliquot of 100 μL of sample solution (corresponding to 0.1 mg of stool sample) was added with 10 μL of internal standard (ISTD) mixture, 1 mL of tert-butyl-methyl ether and 50 μL of 1.0 M HCl solution in 1.5 mL centrifuge tube. Afterwards, each tube was shaken in a vortex apparatus for 2 min, centrifuged at 10,000 rpm for 5 min; the solvent layer was finally transferred in auto-sampler vial and analysed by Gas-Chromatography–Mass Spectrometry (GC–MS) method, using an Agilent GC–MS system composed with 5971 single quadrupole mass spectrometer, 5890 gas-chromatograph and 7673 autosampler. Details of the method are described in Supplementary Materials.

Each collected sample was thawed and weighted (weight range 500–800 mg), then added sodium bicarbonate 10 mM (1:1 w/v) in a 1.5 mL centrifuge tube. The obtained suspension was then mixed with the aid of a sterile wooden stick and briefly shaken in a vortex apparatus, extracted in ultrasonic bath (15 min) and then centrifuged at 4 °C at 13.000 rpm for 90 min. The supernatant was collected, transferred in 1.5 mL centrifuge tube and stored at − 20 °C until use. For the analysis, these supernatant samples were thawed, briefly centrifuged at 5000 rpm and resuspended for 5 min in an ultrasonic bath. The SCFAs and MCFAs were then extracted as follows: an aliquot of 100 μL of sample solution (corresponding to 0.1 mg of stool sample) was added with 10 μL of internal standard (ISTD) mixture, 1 mL of tert-butyl-methyl ether and 50 μL of 1.0 M HCl solution in 1.5 mL centrifuge tube. Afterwards, each tube was shaken in a vortex apparatus for 2 min, centrifuged at 10,000 rpm for 5 min; the solvent layer was finally transferred in auto-sampler vial and analysed by Gas-Chromatography–Mass Spectrometry (GC–MS) method, using an Agilent GC–MS system composed with 5971 single quadrupole mass spectrometer, 5890 gas-chromatograph and 7673 autosampler^{72 (link)}.

The analysis of FFAs was performed by Agilent gas chromatography-mass spectrometry (GC-MS) system composed of 5971 single quadrupole mass spectrometer, 5890 gas-chromatograph, and 7673 autosampler, with a dedicated previously described protocol (Baldi et al., 2021 (link)). The chemicals, GC-MS conditions, and calibration parameters were reported in supporting information SX. Just before the analysis, each sample was thawed. The FFAs were extracted as follows: an aliquot of 300 μl of serum sample was added to 10 μl of ISTD mixture, 100 μl of tert-butyl methyl ether, and 20 μl of 6 M HCl + 0.5 M NaCl solution in 0.5 ml centrifuge tube. Afterward, each tube was stirred in a vortex for 2 min, centrifuged at 10,000 rpm for 5 min, and finally, the solvent layer was transferred to a vial with a microvolume insert and analyzed.

The qualitative and quantitative evaluation of fecal SCFAs and MCFAs was performed by Agilent gas chromatography-mass spectrometry (GC-MS) system composed with 5971 single quadrupole mass spectrometer, 5890 gas chromatograph and 7673 autosampler, through our previously described GC-MS method (18 (link)).
Briefly, just before the analysis, stool samples were thawed and added with sodium bicarbonate 0.25 mM solution (1:1 w/v) in a 1.5 mL centrifuge tube. Then, the obtained suspensions were sonicated for 5 minutes, centrifuged at 5000 rpm for 10 minutes and then the supernatants were collected. The SCFAs were finally extracted as follow: an aliquot of 100μL of sample solution (corresponding to 0.1 mg of stool sample) was added of 50 μL of internal standards mixture, 1 mL of tert-butyl methyl ether and 50 μL of HCl 6 M + 0.5 M NaCl solution in a 1.5 mL centrifuge tube. Subsequently, each tube was shaken in a vortex apparatus for 2 min, centrifuged at 10,000 rpm for 5 min, and lastly the solvent layer was transferred to an autosampler vial and processed three times.

The FFA analysis was performed using an Agilent GC–MS system composed of a 5971 single quadrupole mass spectrometer, a 5890 gas-chromatograph, and a 7673 autosampler. The chemicals, GC–MS conditions, and calibration parameters were reported in Section 1 of the Supplementary Material.

The plasma levels of short, medium and long chain fatty acids were measured using Gas Chromatography coupled with Mass Spectrometry. The preparation of standard curves was carried out using an Agilent GC-MS 114 system, which consisted of a 5971 single quadrupole mass spectrometer, 5890 gas chromato-115 graph and 7673 autosampler. The details of the methodology can be found in a previous publication [45 (link)].