| MS Description
|
Leaf samples were crushed, extracted, and … Leaf samples were crushed, extracted, and their metabolite profiles were analyzed according to (Gullberg et al., 2004). Stable isotope reference compounds (15 ng μl−1 each of [13C3]-myristic acid, [13C4]-hexadecanoic acid, [2H4]-succinic acid, [13C5, 15N]-glutamic acid, [2H7]-cholesterol, [13C5]-proline, [13C4]-disodium α-ketoglutarate, [13C12]-sucrose, [2H4]-putrescine, [2H6]-salicylic acid, and [13C6]-glucose) were added to an extraction mixture of chloroform:MeOH:H2O (3:1:1). The samples (10 mg fresh weight each) were then extracted in 1 ml of the extraction mixture using a MM 301 Vibration Mill (Retsch GmbH & Co. KG, Haan, Germany) at a frequency of 30 Hz s−1 for 3 min using a 3-mm of tungsten carbide bead (Retsch GmbH & Co. KG, Haan, Germany) per tube to increase the extraction efficiency. After extraction samples were placed in an Eppendorf centrifuge (Model 5417C) for 10 min at 14,000 rpm. Following this, 200 μl of the supernatant was transferred to a GC-vial and evaporated to dryness. The samples were then derivatized by shaking them with 30 μl of methoxyamine hydrochloride (15 mg ml−1) in pyridine for 10 min at 5°C. Samples were then incubated overnight at room temperature. The samples were then trimethylsilylated by adding 30 μl of MSTFA with 1% TMCS and incubating for 1 h at room temperature. After silylation, 30 μl of heptane was added.<br />
<br />
The samples were analyzed according to Gullberg et al. (2004) using gas chromatography–time-of-flight mass spectrometry (GC–MS). We used blank control samples and a series of n-alkanes (C12–C40) to allow us to calculate retention indices (Schauer et al., 2005). One microliter of each derivatized sample was injected using a split/splitless injector in splitless mode of an Agilent 7683 autosampler (Agilent, Atlanta, GA, USA) into an Agilent 6890 gas chromatograph equipped with a 10-m × 0.18-mm i.d. fused silica capillary column with a chemically bonded 0.18 μm DB 5-MS stationary phase (J&W Scientific, Folsom, CA, USA). The injector temperature was 270°C, the septum purge flow rate was 20 ml min−1 and the purge was turned on after 60 s. The gas flow rate through the column was 1 ml min−1, the column temperature was held at 70°C for 2 min, then increased by 40°C min−1 to 320°C, and held for 2 min. The column effluent was introduced into the ion source of a Pegasus III time-of-flight mass spectrometer, GC–MS (LECO Corp., St Joseph, MI, USA). 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, and 30 spectra s−1 were recorded in the mass range 50–800 m/z. The acceleration voltage was turned on after a solvent delay. ltage was turned on after a solvent delay.
|
