Bpx70

Isotopologue distribution analysis of intracellular and extracellular metabolites was performed by gas chromatography coupled to mass spectrometry (GC-MS). All GC-MS analysis was carried out using an Agilent 7890 A GC equipped with HP5 capillary column connected to an Agilent 5975 C MS. GC-MS analysis of fatty acids was carried out using a GCMS-QP 2012 Shimadzu coupled with bpx70 (SGE) column. For all measurements, 1 µL of sample was injected at 250 °C, helium as the carrier gas, at a flow rate of 1 mL per minute. Each metabolite or metabolite set had different isolation, derivatization and detection procedures as explained in^{49 (link)–51 (link)}. Raw mass spectra of metabolites were corrected for natural abundance of ¹³C, ²⁹Si, ³⁰Si, ³³S, ³⁴S to compute the fractions of ¹³C incorporated into the analyzed metabolic products from artificially labeled substrates. Data are available via Metabolights with identifier MTBLS183. (https://www.ebi.ac.uk/metabolights)

The content of the fatty acids was determined according to LST EN ISO 15304:2003/AC:20052 [29 ] with gas chromatograph Shimadzu GC—2010 PLUS (Shimadzu Corporation, Kyoto, Japan) using a flame ionization detector and column BPX—70, 120 m. The meat samples were prepared according to the procedures described in LST EN ISO 12966-2: 2011 [30 ]. The fatty acids were methylated with anhydrous KOH in methanol.
A total of 25 ± 0.01 g of meat in a plastic test tube was mixed with 25 mL of hexane, then mixed thoroughly by vortex for 1 h. From the test tube, 4 mL of extract was mixed with 200 µL 2 mol/L KOH solution then centrifuged at the speed of rpm and left to layer for 30 min. The amount of 1 µL of the extract was taken for HPLC analysis.
The oven temperature program was as follows: 60 °C for 2 min; 20 °C min⁻¹ ramp to 230 °C; and kept for 25 min. The carrier gas was nitrogen. The temperature of the evaporator was 250 °C and the detector was 270 °C. The fatty acid kit used for fatty acid identification was the “Supelco 37 Component FAME Mix”.

The fatty acid profile was determined according to the official method of the Regulation EEC/2568/91, Annex IV with amendments [23 ]. The fatty acid methyl esters (FAME) were obtained by cold alkaline transesterification with methanolic potassium hydroxide solution and extracted with n-heptane. FAME were analyzed on a model GC-2010 Shimadzu chromatograph, equipped with an BPX-70, (60 m × 0.25 mm × 0.25 μm), capillary column and a flame ionization detector (FID). The carrier gas was helium, with a flow of 1.5 mL/min. The temperatures of the injector and detector were set at 250 and 260 °C respectively and the oven temperature was increased gradually from 165 to 225 °C in 35 min. The injection volume was 1 μL. Quantification was achieved using a FAME standard mixture purchased from Sigma (St. Louis, MO, USA). The results were expressed as a percentage of individual fatty acids. Analytical-grade methanol, heptane, and potassium hydroxide were purchased from Sigma (St. Louis, MO, USA).