Polar metabolites were derivatized and analysed by GC-MS (Agilent 7890B-5977A) and identification and abundance of individual metabolites was estimated as previously described 113 (link)
. In brief, dried metabolite samples were washed with methanol (twice), and derivatized overnight at RT with methoxyamine (20 mg/mL in pyridine, Sigma) followed by addition of BSTFA + 1% TMCS (Sigma) for > 1 hr at RT. GC-MS was performed using splitless injection (injection temperature 270°C) onto a 30 m + 10 m × 0.25 mm DB-5MS+DG column (Agilent J&W), with helium carrier gas, in electron impact ionization mode. The oven temperature was initially 70°C (2 min), followed by a temperature increase to 295°C at 12.5°C/min and subsequently to 320°C at 25°C/min (held for 3 min). GAVIN software 114 (link)
was used for metabolite identification and quantification by comparison to the retention times, mass spectra, and responses of known amounts of authentic standards.
. In brief, dried metabolite samples were washed with methanol (twice), and derivatized overnight at RT with methoxyamine (20 mg/mL in pyridine, Sigma) followed by addition of BSTFA + 1% TMCS (Sigma) for > 1 hr at RT. GC-MS was performed using splitless injection (injection temperature 270°C) onto a 30 m + 10 m × 0.25 mm DB-5MS+DG column (Agilent J&W), with helium carrier gas, in electron impact ionization mode. The oven temperature was initially 70°C (2 min), followed by a temperature increase to 295°C at 12.5°C/min and subsequently to 320°C at 25°C/min (held for 3 min). GAVIN software 114 (link)
was used for metabolite identification and quantification by comparison to the retention times, mass spectra, and responses of known amounts of authentic standards.
