Zebron zb wax capillary column

The RBC ghost moiety (i.e., porous, lysed cells) not used for enzyme assay was directly lipid-extracted according to the method of Folch et al. (1957) [48 (link)] and the gained complex lipids’ (FA) composition was determined with gas chromatography (Shimadzu Nexis 2030, Kyoto, Japan), in the form of fatty acid methyl esters [36 (link)], after a separation on a Phenomenex Zebron ZB-Wax capillary column (30 m × 0.25 mm × 0.25 micrometer film, Phenomenex Inc., Torrance, CA, USA). The chromatographic evaluation was performed with the LabSolutions 5.93 software, using the PostRun module (Shimadzu, Kyoto, Japan) with manual peak integration. Fatty acid composition was expressed as weight % of total FA methyl esters. The identification of the FAs was performed based on the retention time of a CRM external standard (Supelco 37 Component FAME Mix, Merck-Sigma Aldrich, CRM47885).

Measurement of the of SCFA production was measured using the Laurentin and Edwards [29 (link)] method. Briefly, immediately after sampling, aliquots of faecal sample fermentation fluid were added in a ratio of 2:1 to bijoux bottles containing 1ml NaOH and stored at −20 °C for SCFA measurement. The SCFAs were analysed on a TRACE 2000 gas chromatograph equipped with a flame ionisation detector (GC-FID;). The oven was programmed at an initial temperature of 80 °C for an initial 1 min, increasing 15 °C/min, thereafter increasing to 210 °C; using a Zebron ZB-Wax capillary column (15 m × 0.53 mm id × 1 μm-film thickness, Phenomenex, Cheshire, UK). The carrier gas (nitrogen) was set at a flow rate of 30mL/min. An internal standard (2-ethylbutyrate, 73.8 mmol/L) and orthophosphoric acid (0.1 mL) were added to 0.8 mL of faecal slurry aliquots. Extraction was carried out 3 times with diethyl ether (3 mL). Samples were centrifuged, and fractions pooled. Extractions were done in duplicate for each sample.

FAMEs were quantified by a Gas Chromatograph (GC) equipped with a Flame Ionization Detector (FID, SCION 436, Bruker, UK) and an Agilent CP-Wax 52CB column (Agilent Technologies, USA). Supelco 37 component standards (Sigma–Aldrich, MA, USA) were used for identification and quantification of the FAMEs, with additional standards for unsaturated C16 and C18 fatty acids. To identify unusual fatty acids, the FAMEs structures of ARK-S01-19 and ARK-S13-19 were verified by Helsinki University Lipidomics Unit (HiLIPID). The analyses were performed based on their electron-impact mass spectra recorded by GC-MS-QP2010 Ultra (Shimadzu Scientific Instruments, Kyoto, Japan), compared to the spectra of analytical standard mixtures of FAMEs (Supelco® Analytical Products, Merck) and published reference mass spectra (Christie 2021 ). The GC-mass spectrometer (GC-MS) was equipped with a Zebron ZB-wax capillary column (30 m, 0.25 mm ID and film thickness 0.25 μm; Phenomenex, Torrence CA, USA), which gave the FAMEs similar retention time patterns as the column used for the quantitative GC-FID analyses.