Metabolite extraction and derivatization was conducted as previously described; briefly, a volume of 300μL of the extraction solvent (acetonitrile: water, 1:1 v/v) was added to the cell pellets (2 million cells per pellet), and the supernatants were collected and transferred to gas chromatography (GC) vials for drying using EZ-2 Plus (GeneVac-Ipswich, UK) at 37 ± 1°C (Bataineh MT et al., 2021 (link)). First, the dry samples were dissolved in 25μL of 20 mg/mL methoxyamine hydrochloride in pyridine, followed by the silylation step. Then, for complete derivatization, the samples were incubated at 50°C for 30 min, transferred to 200μL micro-inserts, and analyzed by GC–MS. GC–MS analysis was performed using a QP2010 gas chromatography-mass spectrometer (GC-2010 coupled with a GC–MS QP-2010 Ultra) equipped with an auto-sampler (AOC-20i+s) from Shimadzu (Tokyo, Japan). Chemstation software (Shimadzu). In addition, GC total ion chromatograms (TIC) and fragmentation patterns of the compound were identified using the NIST/EPA/NIH Mass Spectral Library (NIST 14).
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