As previously described [16 (link)] for GC/TOF MS analysis, the dried metabolites were derivatized with 5 μl of 40 mg/ml of methoxyamine hydrochloride in pyridine (Pierce, Rockford, IL) at 30°C for 90 min and 45 μl of N-methyl-N-(trimethylsilyl) trifluoroacetamide (Fluka, Buchs, Switzerland) at 37°C for 30 min. A mixture of fatty acid methyl esters (from C8 to C30) was added to the samples as internal retention index markers. GC/TOF MS analysis was conducted using an Agilent 7890B GC (Agilent Technologies, Wilmington, DE) equipped with a Pegasus HT TOF MS (Leco, St. Joseph, MI). For the separation of compounds in metabolite samples, an RTX-5Sil MS capillary column (30 m × 25 mm, 0.25 μm film thickness; Restek, Bellefonte, PA) with an additional 10-m long integrated guard column was used. Then, 1 μl of sample was injected into the GC in a splitless mode with oven temperature held at 50°C for 1 min, which was increased to 330°C at 20°C/min and held for 5 min. Mass spectra of metabolites were acquired in a mass range of 85 to 500 m/z at an acquisition rate of 17 spectra/s. The ionization mode was subjected to electron impact at 70 eV and the temperature for the ion source was set to 250°C. The transfer line was set to 250°C and 280°C, respectively.
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