Hp innowax capillary gc column

With appropriate dose of acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid(Sigma, Germany) and caproic acid(Aladdin, The United States), diethyl ether (Sinopagic Chemical Reagent Company, China) was added to make 10 mixed-standard concentration gradients(0.02 μg/mL, 0.1 μg/mL,0.5 μg/mL, 2 μg/mL,10 μg/mL,25 μg/mL,50 μg/mL,100 μg/mL,250 μg/mL,500 μg/mL). Feces were thawed on ice, and approximately 50 mg of feces were homogenized in 50 μl 15% phosphoric acid. The suspensions were homogenized with a vortex and centrifuged at 15285g for 10min at 4°C. The supernatants were processed for gas chromatography-mass spectrometry on a Thermo TRACE 1310-ISQ gas mass spectrometer (Thermo, Massachusetts, 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 (27 (link)).

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)).

The serum samples were thawed on ice, and 100 µL aliquots were added into a 2-mL glass centrifuge tube mixing with 50μL of water with 15% phosphoric acid and 150 µL of 5 µg/mL 4-methyl valeric acid (IS). The suspensions were homogenized for about 1 min and centrifuged for 10 min at 12000×g. 1μl supernatant was taken for GC-MS analysis using an Agilent Model 7890A/5975C GC-MS system. To quantify short-chain fatty acid, a calibration curve for the concentration range of 0.1-100 ug/ml was constructed. The IS was used to correct for injection variability between samples and minor changes in the instrument response.
The 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 transmission line were 250 °C and 230 °C respectively. The electron bombardment ionization source, SIM (Selected ion Monitor) scanning mode, and electron energy were 70 eV.