7890b cg system

Tissues were disrupted in 2:1 chloroform:methanol using a mechanical homogenizer submerged in a cooling bath of acetone and dry ice to maintain a temperature of approximately −80 °C. The lipid extract, after solvent evaporation to dryness, was then treated with 0.5 M KOH/MeOH for 10 min at room temperature under stirring for the derivatization of fatty acid residues of the glycerol esters-containing lipids into their corresponding fatty acid methyl esters (FAME) [40 (link)]. After this transesterification step, FAME were extracted with n-hexane; n-hexane phase was dehydrated with anhydrous Na₂SO₄, evaporated and analyzed via an Agilent 7890B CG system equipped with a 60 m × 0.25 mm × 0.25 μm (50%-cyanopropyl)-methylpolysiloxane column (DB23, Agilent, USA) and a flame ionization detector (FID), with an injector temperature at 230 °C and split injection of 50:1. Oven temperature started at 165 °C, was held for 3 min, then increased by 1 °C/min up to 195 °C, held again for 40 min, then increased by 10 °C/min up to 240 °C and held for 10 min. A constant pressure mode (29 psi), with helium as the carrier gas, was used. Methyl esters were identified by comparison with the retention times of commercially available standards or trans fatty acid references, which were obtained as described elsewhere [41 (link),42 (link)].

A total of 200 mg stool samples were taken from −80 °C, thawed at 4 °C, and vortexed with 1000 µL distilled water for 10 min, and centrifuged at 5000 rpm at 4 °C for 10 min. Subsequently, 500 µL of the supernatant was filtrated using 0.22 µm Millipore filter, thoroughly mixed with 50 µL of 50% sulfuric acid solution (v/v). Then, 800 µL diethyl ether was used for SCFAs extraction for 20 min and centrifuged at 10,000 rpm at 4 °C. The supernatant was taken for SCFAs determination by gas chromatograph (GC, 7890B CG System, Agilent Technologies Corporation, CA, USA) equipped with DB-FFAP (0.25 µm × 0.32 mm × 30 m). Moreover, 2-ethyl butyric acid was used as the internal standard.
The main parameters were set as follows: (a) the carrier gas was nitrogen gas at a rate of 2.0 mL/min; (b) the injection volume was 1.0 µL; (c) the injection and ionization temperatures were 230 and 250 °C, respectively; (d) the gradient conditions were set as the initial temperature of 100 °C for 0.5 min, rose to 170 °C at a rate of 8 °C/min and maintained for 0.5 min, and then rose to 220 °C at a rate of 20 °C/min and maintained for 2 min.