SE143:/MS2

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Sample Set Information

ID SE143
Title Application of a metabolomic method combining one-dimensional and two-dimensional gas chromatography-time-of-flight/mass spectrometry to metabolic phenotyping of natural variants in rice.
Description We have developed a comprehensive method combining analytical techniques of one-dimensional (1D) and two-dimensional (GC × GC) gas chromatography-time-of-flight (TOF)–mass spectrometry. This method was applied to the metabolic phenotyping of natural variants in rice for the 68 world rice core collection (WRC) and two other varieties. Ten metabolites were selected as metabolite representatives, and the selected ion current of each metabolite peak obtained from both techniques were statistically compared. Our method of combining 1D- and GC × GC-TOF/MS is useful for the metabolic phenotyping of natural variants in rice for further studies in breeding programs.
Authors Kusano M, Fukushima A, Kobayashi M, Hayashi N, Jonsson P, Moritz T, Ebana K, Saito K.
Reference J Chromatogr B Analyt Technol Biomed Life Sci. 2007 Aug;855(1):71-9. Epub 2007 May 16.
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Analytical Method Details Information

ID MS2
Title Two-dimensional gas chromatography-time-of-flight/mass spectrometry (GC × GC-TOF/MS)
Instrument GC:Agilent 6890N gas chromatograph (Agilent Technologies, Wilmingston, USA)
MS:Pegasus III TOF mass spectrometer (LECO, St. Joseph, MI, USA)
Instrument Type
Ionization EI
Ion Mode Positive
Description 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).

The GC × GC-TOF/MS system consisted of the Agilent 6890N gas chromatograph and Pegasus 4D TOF-MS (Leco, St. Joseph, MI, USA). An Rtx-5 Sil MS column containing 5% diphenyl–95% dimethylpolysiloxane as the stationary phase was used as the first column (I.D., 30 m × 0.25 mm and film thickness, 0.25 μl) and an Rtx-50 column containing 50% methyl–50% phenyl polysiloxane was used as the second column (I.D., 1.0 m × 0.18 mm and film thickness, 0.20 μl). Both columns were purchased from Restek (Bellefonte, USA). They were connected using a universal Press-Tight® connector (Restek). A thermal modulator and the first and second ovens were independently controlled by Leco ChromaTOF optimized for Pegasus 4D software version 2.32 (Leco, St. Joseph, MI, USA). The second column was housed within the second oven. A 1-μl portion of each extract was injected in the splitless mode; three replicate injections were made for each biological sample. The temperature of the GC inlet and transfer line was set at 250 °C. The first column was maintained at 70 °C for 2 min and then increased at the rate of 15 °C/min to 320 °C, and this temperature was maintained for 4 min. The second column was initially set at 80 °C and the temperature program followed was the same as that used for the first column. The modulator was operated at 30 °C for a 3.0-s period; this temperature was higher than that of the first GC oven. The carrier gas (helium) was maintained at a constant flow rate of 1 ml/min. Signals within the mass range from m/z 60 to 600 were collected at a rate of 100 spectra/s after 273 s of solvent delay. The linear retention time (RTcompound x) was calculated using the RT value of each compound on the first (1st RTcompound x) and second dimensions (2nd RTcompound x) as mentioned below:

The alkane mixtures used in GC × GC-TOF/MS analysis were analyzed to determine the RI. The algorithm was applied in the Leco ChromaTOF software to calculate the RI. The normalized response was then calculated as described by Kopka et al.

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