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Extraction and derivatization for GC-M … Extraction and derivatization for GC-MS<br />
One hundred milligrams of each sample was extracted with extraction buffer [methanol/chloroform/water(3:1:1,v/v/v)]
at a concentration of 100 mg/ml containing 10 stable isotope reference compounds as follows:
・[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 />
Each isotope compound was adjusted to a final concentration of 15ng/μl for each 1-μl injection.
After centrifugation, a 200-μl aliquot of the supernatant (ca. 25 mg of each sample) was drawn
and transferred into a glass insert vial. The extracts were evaporated to dryness in an SPD2010
Speed-Vac® concentrator from ThermoSavant (Thermo electron corporation, Waltham, MA, USA).
For methoximation, 30μl of methoxyamine hydrochloride (20mg/ml in pyridine) was added to the sample.
After 24 h of derivatization at room temperature, the sample was trimethylsilylated
for 1h using 30μl of MSTFA with 1% TMCS at 37°C 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.
For methoximation, 30μl 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
30μl of MSTFA with 1% TMCS at 37°C 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 extracts (ca. 277.8μg 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. 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. The transfer line and the ion source temperatures were 250 and 200°C, 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 222 and 237 s. Data acquisition was performed on a Pegasus III and Pegasus IV TOF mass spectrometers (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.<br /> Alkane standard mixtures (C8–C20 and C21–C40) were purchased from Sigma–Aldrich (Tokyo,Japan) and were used for calculating the retention index (RI) [10, 11]. 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. a was normalized using the CCMN algorithm.
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