Methoxyamine chloride in pyridine

Prior to GC/MS analysis, the samples were derivatized by methoxyamination and trimethylsilylation. For methoxyamination, 10 µL of 40 mg/mL methoxyamine chloride in pyridine (Sigma-Aldrich) was added to the samples and incubated for 90 min at 30 °C and 200 rpm. The samples were then trimethylsilylated by adding 45 µL of N-methyl-N-trimethylsilyltrifluoroacetamide (Sigma-Aldrich) with incubation for 30 min at 37 °C and 200 rpm.
For GC/MS, the derivatized metabolite samples were applied to an Agilent 7890A GC/5975C MSD system (Agilent Technologies) equipped with an RTX-5Sil MS capillary column (30 m × 0.25 mm, 0.25 µm film thickness; Restek, Bellefonte, PA) and an additional 10-m-long integrated guard column. One microliter of the derivatized sample was injected into the GC inlet in splitless mode. The oven temperature was initially set to 150 °C for 1 min, after which the temperature was increased to 330 °C at 20 °C/min, where it was held for 5 min. The mass spectra were recorded in a scan range 85–500 m/z at electron impact of 70 eV, and the temperatures of the ion source and transfer line were 230 and 280 °C, respectively.

To analyze AB and ethyl-AB using gas chromatography/mass spectrometry (GC/MS), 5 μL each of the samples containing 1 g/L of AB or ethyl-AB was dried using a centrifugal vacuum evaporator (Labconco, Kansas, MO, USA). The dried samples were derivatized by adding 10 μL of 40 mg/mL methoxyamine chloride in pyridine (Sigma-Aldrich, St. Louis, MO, USA) and then incubating at 30 °C for 90 min. The samples were then subjected to trimethylsilylation by adding 45 μL of N-methyl-N-(tri-methylsilyl)-trifluoroacetamide (MSTFA; Fluka, St. Louis, MO, USA) and then incubating at 37 °C for 30 min.
The derivatized samples were analyzed using an Agilent 7890A GC/5975C MSD system (Agilent Technologies) with a RTX-5Sil MS column (30 m length, 0.25 mm inner diameter, and 0.25 µm film thickness; Restek, Bellefonte, PA, USA) with an additional 10-m guard column. The GC/MS operating conditions were as follows. The derivatized sample of 1 μL was injected into the GC column in splitless mode. The GC oven temperature was initially programmed at 50 °C for 1 min, and then it rapidly increased by 20 °C/min to 280 °C, and then it was held constant for 5 min. Electron ionization was performed at 70 eV. The temperatures of the ion source and transfer line were 250 °C and 280 °C, respectively. Mass spectra were recorded in the scan range of 80–700 m/z.