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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 400 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 ng per 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 0.5-mg DW 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 22.5 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-1000 (Sanplatec, Osaka, Japan) filled with 99.9995% (G3 grade) dry nitrogen.<br /><br />
GC-TOF-MS conditions<br />
Using the splitless mode of a CTC CombiPALautosampler (CTC Analytics, Zwingen, Switzerland), 1 µl of each sample (equivalent to 5.6 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.
Supplementary Material 4 Alkane standard mixtures (C8 - C20 and C21 - C40) purchased from Sigma-Aldrich (Tokyo, Japan) were used for calculating the retention index (RI) 1
. 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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