2.4. Gas Chromatography-Flame Ionization Detector (GC-FID)

Plasma acetate, butyrate, and propionate levels were measured using gas chromatography-mass spectrometry (GCMS-QP2010; Shimadzu, Kyoto, Japan) with flame ionization detector (FID), as previously reported [28]. Separation was performed on the SGE BP GC column (21 × 0.5 µm, 30 m × 0.53 mm; Shimadzu GLC Ltd., Tokyo, Japan). The working solutions of acetate, butyrate, and propionate used as internal and external standards were at the concentration of 10 mM and kept at −20 °C in the freezer. Dry air, nitrogen, and hydrogen were supplied to the FID at 300, 20, and 30 mL/min, respectively. An aliquot of 2 µl sample was injected into the column. The inlet and FID temperature were set at 200 °C and 240 °C, respectively. The total running time was 17.5 min. Analytical standard grades used as internal standards for acetate and propionate were obtained from Sigma-Aldrich (St. Louis, MO, USA) and for butyrate was from Chem Service (West Chester, PA, USA).