Agilent 7890a gc oven

Briefly, 20 mg of faeces were mixed with 500 µL internal standard (hexanoic acid-d3, 10 µg/mL). After homogenization and centrifugation (15,000 rpm, 5 min, 4 °C), the supernatant was transferred into an Eppendorf tube with 5% concentrated sulfuric acid (supernatant vs. 5% concentrated sulfuric acid, volume 10:1). Then, an equivalent volume of ethyl acetate was added to the mixture. After centrifugation and incubation at 4 °C for 30 min, the supernatant was transferred to chromatographic vials equipped with 150 µL inserts before the gas chromatography mass spectrometry (GC-MS) analysis. The analysis was performed on an Agilent 7890A GC oven coupled to an Agilent 5975C inert mass selective detector (Agilent Technologies, Santa Clara, CA, USA). SCFAs were identified by the times of retention in accordance with the standard solutions. Standard mixes of 0.01, 0.1, 1, 10, 100, 1000 µL/mL and blank were also prepared in the same way as the samples.

SCFAs were extracted by mixing 20 mg of feces with 500 µL internal standard (hexanoic acid-d3, 10 µg/mL). After homogenization and centrifugation (12,000 rpm, 5 min, 4 °C), the supernatant was transferred to an Eppendorf tube with 5% concentrated sulfuric acid at a ratio of 10:1. Subsequently, an equivalent volume of ethyl acetate was added to the mixture. After centrifugation and incubation at 4 °C for 30 min, the supernatant was removed and used in gas chromatography–mass spectrometry (GCMS) analysis. The analysis was performed in an Agilent 7890 A GC oven coupled to an Agilent 5975 C inert mass selective (MS) detector (Agilent Technologies, Santa Clara, CA, USA). SCFAs were identified by retention times of compounds when compared to standard solutions. Standard solutions of acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, and valeric acid (at 0.01, 0.1, 1, 10, 100, and 1000 µL/mL) and blank solutions were prepared following the samples preparation procedure.