GC-MS Analysis of Rat Fecal Metabolites

Feces from the 4-week-old rats were thawed on ice, and approximately 10 mg of feces were homogenized in 50 μl of 15% phosphoric acid. The suspensions were homogenized with a vortex and centrifuged at 12,000 rmp for 10min at 4°C. The supernatants were processed for gas chromatography-mass spectrometry on an Agilent 7890A/5975C mass spectrometer (Agilent Technologies, California, USA). The injection volume was 1 μl, and the split ratio was 10:1. Samples were separated with an Agilent HP-INNOWAX capillary GC column (30 m × 0.25 mm ID × 0.25 µm). The initial temperature was 90°C and was increased to 120°C at 10°C/min, after which the temperature was increased to 150°C at 5°C /min and then to 250°C at 25°C /min, where it remained for 2 min. The carrier gas was helium (1.0 ml/min). The temperatures of the injection port and ion source were 250°C and 230°C respectively under SIM model. Agilent MSD ChemStation software was used for quantitative analysis of chromatographic peak area and retention time (25 (link)).