30 m db 35ms 5 m duraguard capillary column

Dried non-polar fatty acids were dissolved in 500 μl 2% (w/v) H₂SO₄ in methanol and incubated at 50°C for 90 min. Afterwards, 100 μl saturated NaCl solution and 200 μl hexane were added, followed by vigorously mixing and short centrifugation. The last steps were repeated three times and the upper phase was transferred to a GC-MS vial. The hexane was evaporated under vacuum and samples were re-dissolved in 50 μL hexane and vials were caped immediately. 1 μL of sample was injected into an SSL injector at 270 °C in splitless mode. GC/MS analysis was performed using an Agilent 7890A GC equipped with a 30-m DB-35MS 5-m Duraguard capillary column. Helium was used as carrier gas at a flow rate of 1.0 mL/min. The GC oven temperature was held at 55°C for 5 min and increased to 325 °C at 6 °C/min. The GC was connected to an Agilent 5975C inert XL MSD, operating under electron ionization at 70 eV. The MS source was held at 230 °C and the quadrupole at 150 °C. The MS was operated in scanning monitoring. The total run time of one sample was 60 min. All GC/MS chromatograms were processed by using Metabolite Detector software. Protein concentrations were determined using the DC Protein Assay (Bio-Rad Laboratories) and data were normalized to protein content of the respective sample.

Metabolite derivatization was performed using a Gerstel MPS. Dried polar metabolites were dissolved in 15 µL of 2% methoxyamine hydrochloride in pyridine and shaken for 60 min at 40°C. An equal volume of N-tert-butyldimethylslyl-N-methyltrifluoroacetamide (MTBSTFA) was added and held for 60 min at 40°C. 1 µL of sample was injected into an SSL injector at 270°C in splitless mode. GC/MS analysis was performed using an Agilent 7890A GC equipped with a 30-m DB-35MS 5-m Duraguard capillary column. As carrier gas helium was used at a flow rate of 1.0 mL/min. The GC oven temperature was held at 100 °C for 2 min and increased to 300 °C at 10 °C/min. After 3 min, the temperature was increased to 325 °C. The GC was connected to an Agilent 5975C inert XL MSD, operating under electron ionization at 70 eV. The MS source was held at 230 °C and the quadrupole at 150 °C. The MS was operated in selected ion monitoring. The total run time of one sample was 25.00 min. All GC/MS chromatograms were processed by using Metabolite Detector software 22 (link). Protein concentrations were determined using the DC Protein Assay (Bio-Rad Laboratories) and data were normalized to protein content of the respective sample.

Metabolite derivatization was performed using a Gerstel MPS. Dried polar metabolites were dissolved in 15 μl of 2 % methoxyamine hydrochloride in pyridine at 40 °C under shaking. After 60 min, an equal volume of MTBSTFA was added and held for 30 min at 40 °C under continuous shaking. One microliter sample was injected into an SSL injector at 270 °C in splitless mode. GC-MS analysis was performed using an Agilent 7890A GC equipped with a 30-m DB-35MS + 5-m Duraguard capillary column. Helium was used as carrier gas at a flow rate of 1 ml/min. The GC oven temperature was held at 100 °C for 2 min and increased to 300 °C at 10 °C/min. After 3 min, the temperature was increased to 325 °C. The GC was connected to an Agilent 5975C inert XL MSD, operating under electron ionization at 70 eV. The MS source was held at 230 °C and the quadrupole at 150 °C. The detector was operated in single ion mode (see Additional file 1: Table S1 for details). The total run time of one sample was 25.00 min.