We incubated the lyophilized polar extracts with 50 µL of methoxyamine in pyridine (40 µg/µL) for 45 min at 60 °C. To increase volatility of the compounds, we silylated the samples by using 25 µL of N-methyl-N-trimethylsilyltrifluoroacetamide with 1% trimethylchlorosilane (Thermo Fisher Scientific) for 30 min at 60 °C.
A 7890A GC system coupled to a 7200 QTOF or 7000 QqQ MS (Agilent Technologies, Palo Alto, CA) was used for isotopolog determination. Derivatized samples were injected (1 µL) in the gas chromatograph system with a split inlet equipped with a J&W Scientific DB5-MS + DG stationary-phase column (30 mm × 0.25 mm i.d., 0.1-µm film, Agilent Technologies). Helium was used as a carrier gas at a flow rate of 1 mL/min in constant flow mode. The injector split ratio was adjusted to 1:5, and oven temperature was programmed at 70 °C for 1 min and increased at 10 °C/min to 325 °C. The ionization performed was positive chemical ionization with isobutene as reagent gas. Mass spectral data on the 7200 QTOF were acquired in full-scan mode from m/z 35 to 700 with an acquisition rate of 5 spectra per second. Mass spectral data on the 7000 QqQ were acquired in scan mode monitoring selected ion clusters of the different metabolites.
A 7890A GC system coupled to a 7200 QTOF or 7000 QqQ MS (Agilent Technologies, Palo Alto, CA) was used for isotopolog determination. Derivatized samples were injected (1 µL) in the gas chromatograph system with a split inlet equipped with a J&W Scientific DB5-MS + DG stationary-phase column (30 mm × 0.25 mm i.d., 0.1-µm film, Agilent Technologies). Helium was used as a carrier gas at a flow rate of 1 mL/min in constant flow mode. The injector split ratio was adjusted to 1:5, and oven temperature was programmed at 70 °C for 1 min and increased at 10 °C/min to 325 °C. The ionization performed was positive chemical ionization with isobutene as reagent gas. Mass spectral data on the 7200 QTOF were acquired in full-scan mode from m/z 35 to 700 with an acquisition rate of 5 spectra per second. Mass spectral data on the 7000 QqQ were acquired in scan mode monitoring selected ion clusters of the different metabolites.