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Extraction and derivatization for GC-TOF-M … Extraction and derivatization for GC-TOF-MS<br />
Each frozen sample with a 5-mm zirconia bead was extracted with 40 fold amount of solvent (methanol/chloroform/water [3:1:1 v/v/v]) containing 10 stable isotope reference compounds at 4°C in a mixer mill (MM301; Retsch, Haan, Germany) at a frequency of 15 Hz. Each isotope compound was adjusted to a final concentration of 15 ngper 1-μl injection volume. After 5-min centrifugation at 15,100 × g, a 200-μl aliquot of the supernatant was transferred to a glass insert vial. The extracts were evaporated to dryness in an SPD2010 SpeedVac® concentrator (Thermo Fisher, Scientific, Waltham, MA, USA). We used extracts from 5-mg FW samples for derivatization, i.e., methoxymation and silylation. For methoxymation, 30 μl of methoxyamine hydrochloride (20 mg/ml in pyridine) were added to the sample. After 17 h of derivatization at room temperature the sample was trimethylsilylated for 1 h using 30 µl of MSTFA at 37°C with shaking. All derivatization steps were performed in a vacuum glove box VSC-100 (Sanplatec, Osaka, Japan) filled with 99.9995% (G3 grade) dry nitrogen.<br />
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GC-TOF-MS conditions<br />
Using the splitless mode of a CTC CombiPALautosampler (CTC Analytics, Zwingen, Switzerland), 1 μl of each sample (equivalent to 1.4 µg DW) was injected into an Agilent 6890N gas chromatograph (Agilent Technologies, Wilmingston, DE, USA) featuring a 30 m × 0.25 mm inner diameter fused-silica capillary column and a chemically bound 0.25-μl film Rxi-5 Sil MS stationary phase (RESTEK, Bellefonte, PA, USA) with a tandem connection to a fused silica tube (1 m, 0.15 mm). An MS column change interface (msNoVent-J; SGE, Yokohama, Japan) was used to prevent air and water from entering the MS during column change-over. Helium was the carrier gas at a constant flow rate of 1 ml min-1. The temperature program for GC-MS analysis started with a 2-min isothermal step at 80°C followed by 30°C temperature-ramping to a final temperature of 320°C that 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 sec. Data acquisition was on a Pegasus IV TOF mass spectrometer (LECO, St. Joseph, MI, USA); the acquisition rate was 30 spectras-1 in the mass range of a mass-to-charge ratio of m/z = 60–800.
Alkane standard mixtures (C8 - C20 and C21 - C40) purchased from Sigma-Aldrich (Tokyo, Japan) were used for calculating the retention index (RI) (Schauer et al., 2005). For quality control we injected methylstearate into every 6th sample. The sample run order was randomized in single-sequence analyses. We analyzed the standard compound mixtures using the same sequence analysis procedures. ing the same sequence analysis procedures.
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