| MS Description
|
Extraction procedures<br />
All meta … Extraction procedures<br />
All metabolite extraction procedures were kept on ice, and the quantities for sample aliquots were 25 μL for blood plasma, 5 x 10^6 for cells, 5 mg for tissues, and 2 mL for algae cultures. Metabolites were extracted with 1,000 μL of degassed acetonitrile:isopropanol:water (3:3:2, v/v/v) and then homogenized, centrifuged, decanted, and evaporated. Extracts were cleaned with 500 μL of degassed acetonitrile:water (1:1, v/v) to remove triglycerides and membrane lipids, and evaporated again. For GC-MS analysis, internal standard C8–C30 fatty acid methyl esters were added to determine the retention index. The dried samples were derivatized with 10 μL of methoxyamine hydrochloride (or ethoxyamine hydrochloride) in pyridine and subsequently by 90 μL of MSTFA (or MSTFA-d9) for trimethylsilylation of acidic protons.<br />
<br />
Briefly, for GC-MS analysis, we used an Agilent 7890A GC system (Agilent Technologies) and an Agilent 7200 accurate mass Q-TOF mass spectrometer (Agilent Technologies) with the transfer line temperature maintained at 290 °C. Chromatography was done on an Rxi-5Sil MS column (30 × 0.25 mm, 0.25 μm; Restek) with helium (99.999%; Airgas) at a constant flow of 1 mL/min. The GC temperature program was set as follows: initial temperature of 60 °C with a hold time of 1 min, a temperature ramp of 10 °C/min to 325 °C, and a final hold time of 9.5 min at 325 °C. The injection volume was 1 μL in splitless mode at 250 °C. Mass spectra were acquired from m/z 50 to m/z 800 at a 5-Hz scan rate and 750-V detector voltage in both electron ionization (EI) mode and chemical ionization (CI) mode. Other data acquisition parameters were as follows: EI ion source temperature, 230 °C; EI electron energy, 70 eV; CI ion source temperature, 300 °C; CI electron energy, 135 eV; CI gas flow rate, 20%; CI gas, methane (99.999%; Airgas). e, 20%; CI gas, methane (99.999%; Airgas).
|
