|SE53 > MS01|
|Instrument||Agilent 6890N gas chromatograph (Agilent Technologies) and Pegasus IV TOF mass spectrometer (LECO)|
|Description||<Extraction and derivatization for GC-MS>
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:
・ [13C3]-myristic acid,
・[2H6]-2-hydoxybenzoic acid and
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.
For methoximation, 30 µl of methoxyamine hydrochloride (20 mg ml-1 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.
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.
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) [7, 8]. 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 .