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Lipophilic compound extraction<br /> … Lipophilic compound extraction<br />
The method for the extraction of lipophilic compounds was modified from Furuhashi and Weckwerth (2013). Approximately 5 mg of lyophilized Euglena sample was homogenized with quartz sands (Wako, Osaka, Japan) (approximately 300 mg) in 1 mL of MCA solvent (methanol/chloroform/2 % acetic acid, 5:2:1, v/v/v) using a Mixer Mill MM400 (Retsch, Haan, Germany) for 90 s at 30 Hz. For absolute quantification, 60 µg C20:0 FAME (Larodan, Sweden) was added into 1 mL MCA solvent before homogenizing. The sample was centrifuged at 21,000×g for 3 min. The supernatant was divided into two fractions, retaining 200 µL for wax ester analysis and the remaining 800 µL for total lipophilic compound analysis. Phase separation was conducted by adding 500 µL of water and 400 µL of chloroform to the supernatant. The sample was centrifuged at 21,000×g for 3 min, and an upper polar phase was removed. The remaining inter-phase and the bottom apolar phase were desiccated using a centrifugal evaporator (Thermo Fischer, Waltham, MA, USA).
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Derivatization for wax ester profiling<br />
For wax esters and other free form lipophilic compound profiling, the dried apolar (with inter-phase) pellet was incubated with 20 µL methoxamine-HCl (Sigma-Aldrich, St. Louis, MO, USA) solution (40 mg/mL pyridine) for 90 min at 30 °C. N-methyl-N-(TMS)trifuoroacetamide (80 µL; MSTFA: Sigma-Aldrich) for silylation was added and incubated for 30 min at 37 °C, and 1 µL of the sample was injected into the GC–MS.<br /><br />
Methyl esterification and derivatization for total lipophilic compound profiling<br />
Alkaline methyl esterification converted only the bounded form of fatty acids (e.g., glycerides and wax esters) into FAMEs. The resulting glycerol and fatty alcohols (FALs) with free fatty acids (FFAs) and steroids contained in the original sample were silylated during the subsequent MSTFA treatment (Furuhashi and Weckwerth, 2013). For the lipophilic compound analysis, the dried apolar (with inter-phase) pellet was incubated with 500 µL of 0.5 M sodium methoxide in methanol for 90 min at 60 °C. The sample was then cooled to room temperature and 1 mL of 1 % acetic acid was added to stop the reaction. Chloroform (400 µL) was added and phase separation was conducted by centrifugation at 21,000×g for 3 min. An upper polar phase was removed, 1 mL of ultrapure water was added and vortexed. The sample was then centrifuged at 21,000×g for 3 min and an upper polar phase was removed again, leaving an inter-phase and a bottom apolar phase that were desiccated using a centrifugal evaporator (Thermo Fischer). The dried pellet was incubated with 20 µL of methoxamine solution (40 mg/mL pyridine) for 90 min at 30 °C. MSTFA (80 µL) was added and incubated for 30 min at 37 °C. The sample (1 µL) was injected into the GC–MS. Retention time was compared to that of authentic alkane standard compounds according to a previous study (Furuhashi and Weckwerth, 2013). Measurements were performed in a batch.<br /><br />
GC–MS conditions<br />
GC–MS measurements were carried out on a gas chromatography-time of flight-mass spectrometer (GC–TOF-MS) (Auto sampler, JASCO PAL GC-xt; GC, Agilent 6890N; MS, Waters GTC premier) instrument (JASCO, Tokyo, Japan; Agilent Technologies, Santa Clara, CA; Waters, Milford, MA, USA). The injector temperature was 230 °C using a cold trap splitless mode. The flow rate of carrier He gas was 1 ml/min. GC separation was performed on an HP-5MS capillary column (30 m × 0.25 mm × 0.25 µm) (Agilent Technologies, Santa Clara, CA). After a 1-min isothermal period at 70 °C, the temperature was programmed to 76 °C at a heating rate of 1 °C min−1, then to 350 °C at a heating rate of 6 °C min−1 and maintained for 1 min. The temperatures of the GC–MS transfer line and the source of the mass spectrometer were 340 and 250 °C, respectively. The mass spectrometer was operated in an electron-impact mode (EI) at 70 eV in a scan range of m/z 40–600. I) at 70 eV in a scan range of m/z 40–600.
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