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MS Description <Sample processing and extraction>&l <Sample processing and extraction><br /> The lyophilized sample in a 2 ml tube was frozen and then homogenized with a 5 mm of zirconia bead by a Mixer Mill (Retsch, Haan, Germany) at 20 Hz for 1 min. Five mg dry weight (DW) of the lyophilized samples were weighed for GC-MS and LC-MS analyses, while 25 mg DW of the samples for CE-MS analysis.<br /><br /> <Extraction and derivatization for GC-MS><br /> Each sample was extracted with a concentration of 2.5 mg DW of tissues per ml extraction medium (methanol / chloroform/water [3:1:1 v/v/v]) containing 10 stable isotope reference compounds:<br /> [2H4]-succinic acid,<br /> [13C5,15N]-glutamic acid,<br /> [2H7]-cholesterol,<br /> [13C3]-myristic acid,<br /> [13C5]-proline,<br /> [13C12]-sucrose,<br /> [13C4]-hexadecanoic acid,<br /> [2H4]-1,4-butanediamine,<br /> [2H6]-2-hydoxybenzoic acid and<br /> [13C6]-glucose<br /> using a Retsch mixer mill MM310 at a frequency of 30 Hz for 3 min at 4℃. Each isotope compound was adjusted to a final concentration of 15 ng µl-1 for each 1-µl injection. After centrifugation for 5 min at 15,100 × g, 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). For methoximation, 30 µl of methoxyamine hydrochloride (20 mg/ml in pyridine) was added to the sample. After 24 h of derivatization at room temperature, the sample was trimethylsilylated for 1 h using 30 µl of MSTFA with 1% TMCS at 37℃ with shaking. Thirty µl of n-heptane was added following silylation. All the derivatization steps were performed in the vacuum glove box VSC-100 (Sanplatec, Japan) filled with 99.9995% (G3 grade) of dry nitrogen.<br /><br /> <GC-TOF/MS conditions><br /> One microliter of each sample was injected in the splitless mode by an CTC CombiPAL autosampler (CTC analytics, Zwin-gen, 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.<br /> Helium was used as the carrier gas at a constant flow rate of 1 ml min-1. The temperature program for metabolome analysis started with a 2-min isothermal step at 80 ℃ and this was followed by temperature ramping at 30 ℃ to a final temperature of 320 ℃, which was maintained for 3.5 min. The transfer line and the ion source temperatures were 250 and 200 ℃, respectively. Ions were generated by a 70-eV electron beam at an ionization current of 2.0 mA. The acceleration voltage was turned on after a solvent delay of 273 s. Data acquisition was performed on a Pegasus IV TOF mass spectrometer (LECO, St. Joseph, MI, USA) with an acquisition rate of 30 spectra s-1 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) (Wagner et al. 2003; Schauer et al. 2005). 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. For quality control, we injected methylstearate in every 6 samples. Data was normalized using the CCMN algorithm (Redestig et al. 2009). the CCMN algorithm (Redestig et al. 2009).
MS ID MS01  +
MS Instrument Agilent 6890N gas chromatograph (Agilent Technologies) and Pegasus IV TOF mass spectrometer (LECO)  +
MS Ion Mode Positive  +
MS Ionization EI  +
MS Title GC-TOF/MS  +
Modification dateThis property is a special property in this wiki. 7 September 2018 06:44:45  +
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