5890 gas chromatograph

The qualitative and quantitative evaluation of fecal SCFAs and the preparation of standard curves was performed by an Agilent GC-MS system composed of a 5971 single quadrupole mass spectrometer, 5890 gas chromatograph and 7673 autosampler, through our previously described GC-MS method [29 (link)]. Briefly, the SCFAs were extracted as follows: an aliquot of 100 µL of fecal extract solution (corresponding to 0.1 mg of stool sample) was added to 50 μL of an ISTDs mixture, 1 mL of tert-butyl methyl ether and 50 µL of 1.0 M HCl 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 finally the solvent layer was transferred to an autosampler vial and processed three times.

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)}.