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One hundred milligrams of each material wa … One hundred milligrams of each material was extracted with extraction buffer [methanol/chloroform/water (3:1:1, v/v/v)] at a concentration of 100 mg/ml and containing 10 stable isotope reference compounds. Each isotope compound was adjusted to a final concentration of 15 ng/μl for each 1-μl injection. After centrifugation, a 200-μl aliquot of the supernatant was drawn and transferred into a glass insert vial. The extracts were evaporated to dryness in an SPD2010 SpeedVac® concentrator from ThermoSavant (Thermo electron corporation, Waltham, MA, USA).<br /><br />
One microliter of each sample was injected in the splitless mode by an CTC CombiPAL autosampler (CTC analytics, Zwingen, Switzerland) into an Agilent 6890N gas chromatograph (Agilent Technologies, Wilmingston, USA) equipped with a 30 m × 0.25 mm inner diameter fused-silica capillary column with a chemically bound 0.25-μl film Rtx-5 Sil MS stationary phase (RESTEK, Bellefonte, USA) for metabolome analysis. Four replicates were subjected to 1D-GC-TOF/MS analysis. Helium was used as the carrier gas at a constant flow rate of 1 ml/min. The temperature program for metabolome analysis started with a 2-min isothermal step at 80 °C and this was followed by temperature ramping at 30 °C to a final temperature of 320 °C, which was maintained for 3.5 min. Data acquisition was performed on a Pegasus III TOF mass spectrometer (LECO, St. Joseph, MI, USA) with an acquisition rate of 30 spectra/s in the mass range of a mass-to-charge ratio of m/z = 60–800. Alkane standard mixtures (C8–C20 and C21–C40) were purchased from Sigma–Aldrich (Tokyo, Japan) and were used for calculating the retention index (RI). The normalized response for the calculation of the signal intensity of each metabolite from the mass-detector response was obtained by each selected ion current that was unique in each metabolite MS spectrum to normalize the peak response, using the method of Kopka et al. The normalized responses are peak areas corrected using the sample weight and a constant amount of the representative internal standard compound ([13C4]-hexadecanoic acid). ndard compound ([13C4]-hexadecanoic acid).
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