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The sample preparation process was as follows. Prepared samples were placed in 2 mL microtubes, and 1 mL of methanol:water:chloroform (2.5:1:1, v/v/v) containing 0.060 mL of 200 ppm ribitol as an internal standard was added for the extraction of primary metabolites. The mixture was centrifuged at 16,000 × g for 5 min at 4 °C, and 0.8 mL of the supernatant was transferred to a new tube. Then, 0.4 mL of distilled water was added, mixed thoroughly, and centrifuged again under the same conditions. Subsequently, 0.9 mL of the upper phase was transferred to a new tube and concentrated using a vacuum concentrator and a freeze dryer. The completely dried extracts were derivatized for GC–TOF–MS analysis. For derivatization, 0.08 mL of 2% methoxyamine hydrochloride in pyridine was added, and the mixture was incubated at 30 °C with shaking at 1200 rpm for 90 min. Afterward, 0.08 mL of MSTFA (N-methyl-N-trimethylsilyl-trifluoroacetamide) was added and reacted at 37 °C with shaking at 1200 rpm for 30 min. The derivatized samples were transferred to 2 mL screw-cap GC vials containing 0.25 mL insert vials, and 1 μL of each sample was injected into the GC–TOF–MS system for analysis.

Instrumental analysis was performed using gas chromatography and mass spectrometry. For chromatographic separation, a GC–TOF–MS system equipped with a CP-Sil 8 CB Low Bleed/MS column (30 m × 0.25 mm × 0.25 μm film thickness; CP 5860, Agilent) was used. The column oven temperature was initially set at 80 °C and then increased to 320 °C at a rate of 15 °C min⁻¹. Helium was used as the carrier gas at a flow rate of 1 mL min⁻¹, and samples were analyzed in split mode with a split ratio of 1:25. For mass spectrometric detection, the injector temperature was set to 230 °C, while the ion source and transfer line temperatures were maintained at 250 °C and 280 °C, respectively.

The mass scan range was set to 85–600 m/z, and data were acquired at a rate of 10 spectra per second.