| SE45 > SS01 | |
| ID | SE45_SS01 |
| Title | Sample processing and extraction |
| Description | Each sample was extracted at a concentration of 5 mg flesh weight (FW) of tissues per ml extraction medium (methanol / chloroform/water [3:1:1 v/v/v]) containing 10 stable isotope reference compounds: • [2H4]-succinic acid • [13C5,15N]-glutamic acid • [2H7]-cholesterol • [13C3]-myristic acid • [13C5]-proline • [13C12]-sucrose • [13C4]-hexadecanoic acid • [2H4]-1,4-butanediamine • [2H6]-2-hydoxybenzoic acid • [13C6]-glucose using a Retsch mixer mill MM310 at a frequency of 30 Hz for 3 min at 4°C. Each isotope compound was adjusted to a final concentration of 15 ng µl-1 for each 1-µl injection. After 5-min centrifugation at 15,100 × g, a 100 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°C with shaking. For methoximation, 30 µl of methoxyamine hydrochloride (20 mg ml-1 in pyridine) were added to the sample. After 24 h of derivatization at room temperature, the sample was trimethylsilylated for 1 h using 30 µl of MSTFA at 37°C with shaking. After silylation 30 µl of n-heptane were added. All derivatization steps were performed in the vacuum glove box VSC-100 (Sanplatec, Japan) filled with 99.9995% (G3 grade) of dry nitrogen. |
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