Agilent 6890n gc

Maternal cecal SCFA levels were quantified as described previously^{20 (link)} with minor modifications, by the McMaster Regional Centre of Mass Spectrometry (MRCMS). Thirty mg of frozen cecal contents was acidified in a weight equivalent volume of 3.7% HCl. Ten μl of internal standards were added to each sample and SCFA extraction was performed by adding 500 μl of diethyl ether to each sample, and the resultant solution was vortexed for 15 minutes. After vortexing, 800 μl of diethyl ether cecal extract was transferred to a clean 1.5 ml centrifuge tube. A 60 μl aliquot of each diethyl ether cecal extract was derivatized with 20 μl of N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA; Sigma-Aldrich) in a chromatographic vial containing an insert. The resultant organic extract-MTBSTFA mixture was incubated at ambient temperature for 1 hour and SCFA levels were quantified using a gas chromatograph (Agilent 6890 N GC), coupled to a mass spectrometer detector (Agilent 5973 N Mass Selective Detector).

Fatty acids were determined through derivatization to fatty acid methyl esters (FAMEs). To prepare FAMEs of the extracts, samples (2 mL) were mixed with 400 μL of internal standard (undecanoic acid 1000 mg/L in hexane/acetone 80:20 v/v) and 2 mL of methanol/sulfuric acid (93:7 v/v). These mixtures were maintained for 60 min at 80 °C. Then, the samples were allowed to cool down to room temperature. After addition of 5 mL of hexane, the samples were mixed in a vortex for 1 min and two phases were formed. The organic phase was transferred to a volumetric flask (10 mL), and the re-extraction of the polar phase was carried out three to four more times until a volume of 10 mL was obtained.
The FAMEs were analyzed with an Agilent 6890N GC (Agilent Technologies, Santa Clara, CA, USA), equipped with a SP2380 capillary column (30 m, 0.25 mm i.d., 0.20 μm film thickness) and flame ionization 19244–80560 as a detector. Nitrogen gas was used as carrier at a flow rate of 0.8 mL/min. The column temperature was kept initially at 50 °C for 2 min, then gradually increased to 240 °C at 4 °C/min, and finally, maintained at 240 °C for 1 min. An aliquot of the extracts (1 μL) was injected automatically with 20:1 in split mode. Injector and detector temperatures were set at 260 and 280 °C, respectively.

Quantitative and qualitative data were determined by GC and GC-MS, respectively. The fraction was injected onto a Shimadzu GC-17 A system, equipped with an AOC-20i autosampler and a split/splitless injector. The column used was an DB-5 (Optima-5), 30 m, 0.25 mm i.d., 0.25 μm df, coated with 5% diphenyl-95% polydimethylsiloxane, operated with the following oven temperature program: 50 °C, held for 1 min, rising at 3 °C/min to 250 °C, held for 5 min, rising at 2 °C/min to 280 °C, held for 3 min; injection temperature and volume, 250 °C and 1.0 μL, respectively; injection mode, split; split ratio, 30:1; carrier gas, nitrogen at 30 cm/s linear velocity and inlet pressure 99.8 KPa; detector temperature, 280 °C; hydrogen, flow rate, 50 mL/min; air flow rate, 400 mL/min; make-up (H₂/air), flow rate, 50 mL/min; sampling rate, 40 ms. Data were acquired by means of GC solution software (Shimadzu). Agilent 6890 N GC was interfaced with a VG Analytical 70–250 s double-focusing mass spectrometer. Helium was used as the carrier gas. The MS operating conditions were: ionization voltage 70 eV, ion source 250 °C. The GC was fitted with a 30 m × 0.32 mm fused capillary silica column coated with DB-5. The GC operating parameters were identical with those of GC analysis described above.