Vf wax column

Gas chromatography–mass spectrometry (GC-MS) analyses followed the protocol described in Fiers et al. [30 (link)] and were performed on a gas chromatograph (Agilent Technologies 7890A, Santa Clara, CA, USA) coupled to a mass selective detector (Agilent Technologies 5975C) with enhanced Chemstation version E.02.00.493 software. The GC, fitted with a Gerstel MPS autosampler (Maestro 1, version 1.4.8.14/3.5; Müllheim/Ruhr, Germany), was used for SPME injection. After extraction, the volatile compounds were desorbed in the pulsed splitless mode for 5 min at 250 °C. Separation was performed on a VF-WAX column (Agilent Technologies, USA, 30 m × 0.250 mm I.D, 0.25 µm film thickness). Helium was used as a carrier gas at a constant flow rate of 1.5 mL.min⁻¹. The inlet temperature was 250 °C. Pulsed splitless injection mode in a 1.5 mm HS-liner was used (injection pulse pressure of 30 psi for 1 min). The following temperature programs were used: 35 °C for 2 min, 5 °C.min⁻¹ up to 155 °C, 20 °C.min⁻¹ up to 250 °C, and a final hold at 250 °C for 10 min. The mass spectrometer was operated in electron ionization (EI) mode at 70 eV, source temperature 230 °C, quadrupole temperature 150 °C, scanned mass range from 20 to 350 amu, threshold of 150 amu, and a scan speed of 4.27 scans.s⁻¹.

The details regarding GC-MS analysis have been previously described [17 (link)]. Briefly, GC-MS analysis of metabolites in plasma was carried out using an Agilent Technologies 7890 N gas chromatograph coupled to an Agilent Technologies 5977A quadrupole mass selective spectrometer with a triple-axis detector (Agilent, Palo Alto, CA) operated in electron ionization mode at 70 eV with a mass scan range of m/z 50–800. Derivatized samples were separated on a VF-WAX column (Agilent Technologies, Middelburg, The Netherlands) with an oven temperature ramp from 50 °C to 230 °C. The carrier gas was helium set at constant flow mode (1.0 mL/min). The identification of each metabolite in the samples was confirmed by comparing their relative retention times and mass spectra with those of authentic standard compounds. The relative levels of metabolites were calculated by comparing their peak areas to that of the internal standard compound.
Fatty acid desaturase activities and elongase activities were obtained indirectly by calculating fatty acid ratios of products to precursors. The equations are as follows: C16 Δ9-desaturase = Palmitoleic acid/Palmitic acid; C18 Δ9-desaturase = Oleic acid/Stearic acid; Δ6-desaturase = γ-Linolenic acid/Linoleic acid; Elongase activity = Stearic acid/Palmitic acid.

The details of GC-MS have been previously published [16 (link)]. Briefly, all analyses were performed on an Agilent Technologies 7890 N gas chromatograph coupled to an Agilent Technologies 5977A quadrupole mass selective spectrometer with a triple-axis detector (Agilent, Palo Alto, CA) in the electron ionization mode (70 eV) and full scan monitoring mode (m/z 50–800). Derivatized samples were separated on a VF-WAX column (Agilent Technologies, Middelburg, Netherlands) with helium as the carrier gas and a temperature ramp from 50 °C to 230 °C. Metabolites in the samples were identified by comparing their relative retention times and mass spectra with those of authentic reference standards. The relative metabolite levels were calculated by comparing their peak areas to that of the internal standard compound.