Agilent 6890 gas chromatograph

Lipids were extracted from ileum via the Folch Method [32 (link)] and phospholipid fractions were separated by thin layer chromatography (TLC) as previously described [29 (link)]. Briefly, samples were spotted on H-plates (EMD Chemicals, Gibbstown, NJ, USA) to separate phospholipid species. Bands corresponding to lyso-phosphatidylcholine (lyso-PC), sphingomyelin (SM), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PI), and phosphatidylethanolamine (PE) were collected and methylated with 14% boron trifluoride-methanol (Fisher Scientific, Mississauga, ON, Canada). Fatty acid methyl esters were separated on a DB-FFAP fused-silica capillary column (15 m, 0.1 m film thickness, 0.1 mm i.d.; Agilent, Mississauga, ON, Canada) and quantified on an Agilent 6890 gas chromatograph. Peaks were identified by retention times of fatty acid methyl ester standards (Nu-Chek-Prep, Elysian, MN, USA) using EZchrom Elite version 3.2.1 software. Fatty acid results were calculated as percent composition or ug/0.1 g tissue with 10 µg of heptadecanoic acid (17:0) added to each fraction for the internal standard.

GC-MS analysis was performed to determine the molecular composition of AME. The filtered AME was analysed using Agilent 6890 Gas Chromatograph with a 5975C Mass-selective Detector (Agilent, Palo Alto, CA, USA). The column used was ZB-5 MSI (30 m × 0.25 mm × 0.25 μm) and the mobile phase was 5% phenyl/95% dimethylpolysiloxane. Helium was used as the carrier gas at a constant flow rate of 1 mL/min. The initial oven temperature was 40°C and it was maintained at this temperature for 1.5 min; the temperature was gradually increased to 230°C at a rate of 4°C/min and was maintained for 8 min. The temperature of the injection port was 250°C and the flow rate of helium was 1 mL/min. The compounds discharged from the column were detected by a quadrupole mass detector. The ions were generated by electron ionization method. The temperatures of the MS quadrupole and source were 150 and 230°C, respectively, electron energy was 70 eV, temperature of the detector was 230°C, the emission current multiplier voltage was 1624 V, the interface temperature was 280°C, and the mass range was from 20 to 460 u. The relative mass fraction of each chemical component was determined by peak area normalization method. The National Institute Standard and Technology Library was used to analyse the spectrum and identify the compounds detected.

Untargeted metabolomics analysis was performed by the West Coast Metabolomics Center at the University of California (Davis, CA) on a fee-for-service basis. Urine aliquots were normalized by urine creatinine concentration measured by a SIRRUS Clinical Chemistry Analyzer and extracted by degassed acetonitrile. Internal standards C8-C30 fatty acid methyl ethers were added, and the samples were derivatized by methoxyamine hydrochloride in pyridine and subsequently by N-methyl-N-trimethylsilyltrifluoroacetamide for trimethylsilylation of acidic protons. Analytes were separated using an Agilent 6890 gas chromatograph (Santa Clara, CA) and mass spectrometry was performed on a Leco Pegasus IV time of flight mass spectrometer (St. Joseph, MI) following a published protocol [21 (link)]. Unnamed peaks were excluded from statistical analysis.

For the determination of the SCFA content, 2 g of the sample were collected in a 15 mL conical centrifuge tube with 5 mL of HPLC-grade acetone and 1 mL of HPLC-grade water. This was mixed by vortexing for 15 s, followed by the addition of 100 μL of 85% (w/v) phosphoric acid, and the solution was mixed again by vortexing for 10 s and centrifuged for 23 min at 21,000× g at 4 °C; the supernatant was separated by passing through a 0.20 μm filter. The filtrate was subsequently placed in amber vials with a lid to carry out the liquid chromatography analysis and stored at −80 °C until used. Liquid chromatography was performed using an Agilent 6890 gas chromatograph with a flame ionization detector and a DB-FFAP 30 m × 0.25 mm × 0.25 μm column (Agilent Technologies, Wilmington, NC, USA) for the analysis of the filtered supernatant with a 0.2 μm syringe filter [40 (link)]. The conditions followed a split mode (20:1) with an inlet temperature of 220 °C and pressure of 168 kPa. A volume of 1 μL was injected, with a constant flow of 1.4 mL/min, using helium as a carrier. The detector temperature was 250 °C. The column initially operated at 35 °C, with a hold period of 30 s, before increasing at 10 °C/min until 90 °C was reached. This temperature was held for 2 min and was followed by an increase of 12 °C/min until 230 °C was reached, and this was held for 6 min.

The essential oils were analyzed by an Agilent 6890 gas chromatograph (GC) equipped with an HP-5MS capillary column (60 m × 0.25 mm, 0.25 μm film thickness) and a flame ionization detector (FID) (Agilent Technologies Inc., CA, USA). The split ratio was 1 : 20 (injection volume: 1 μL) with helium as carrier gas (1 mL/min). The GC oven temperature was as follows: held at 70°C (2 min), 2°C/min to 180°C (55 min), 10°C/min to 310°C (13 min), and kept at 310°C (4 min). The GC-MS analysis was carried out using an Agilent 6890 gas chromatograph equipped with an Agilent 5975C mass selective detector. The Agilent 6890 gas chromatograph (GC) coupled to an Agilent 5975C mass selective detector (MS) was used to identify the chemical composition of the essential oils. The parameters of GC and capillary column were the same as in GC-FID. The MS was operated in the electron ionization mode at 70 eV and the mass range (m/z 29 to 500). The ion source temperature and interface temperature were 230°C and 280°C, respectively. The relative percentage of chemical constituents was determined by the peak area normalization method. A series of n-alkanes (C₈–C₃₀) were injected to calculate the retention index. The components of the essential oils were identified by comparison of their mass spectrum and calculated retention index and with those listed in Wiley 275 and NIST 17 databases.

GC–TOF MS analysis and data processing were performed as previously described^{30 (link)}, using a Leco Pegasus IV time-of-flight mass spectrometer (Leco Corporation) coupled to an Agilent 6890 gas chromatograph (Agilent Technologies) equipped with an Rtx5Sil-MS column (30 m × 0.25 mm; 0.25 µm phase; Restek) and a Gerstel MPS2 automatic liner exchange system (Gerstel GMBH & Co. KG). Raw data files were processed using the metabolomics BinBase database³¹ . All database entries in BinBase were matched against UC Davis metabolomics center’s mass spectral library.