Db5 ms stationary phase

A 1 μl aliquot of derivatised sample was injected in splitless mode by an Agilent 7683 Series autosampler (Aligent, Atlanta, GA) into an Aligent 6980 GC equipped with a 10 m × 0.18 mm id fused-silica capillary column chemically bonded with 0.18 μm DB5-MS stationary phase (J&W Scientific, Folsom, CA). The injector temperature was 270 °C. The carrier gas was helium, set at a constant flow of 1 mL/min through the purge flow rate of 20 ml/min and an equilibrant time of 1 min. The column temperature was initially kept at 70 °C for 2 min and then increased from 70 °C to 320 °C at 30 °C/min, where it was kept for 2 min. The effluent from the column was introduced to the ion source in the Pegasus III TOF-MS (Leco Corp., St Joseph, MI). The transfer temperature was set at 250 °C and the ion source temperature at 200 °C. Ions were generated by a 70 eV electron beam at a current of 2.0 mA. Masses were acquired from m/z 50 to 800 at a rate of 30 spectra per second, and the acceleration voltage was turned on after a solvent delay of 165 s. Retention indexes were calculated from the retention times obtained from the injection of a homologous series of n-alkanes (C₁₂–C₃₂) for each batch. All samples were run in randomised order58 (link).

Culture broth of B. methylotrophicum in medium supplemented with corn steep liquor (CSL) or yeast extract (YE) was sampled at 0 h, 36 h (middle exponential phase), 60 h (later exponential phase), and 84 h (stationary phase), respectively. After the centrifugation at 13,000 g for 10 min, the supernatant was taken and filtered. 200 μL of supernatant was lyophilized under low temperature under low temperature (− 60 °C). The dried samples were derivatized at 40 °C for 160 min with 50 μL methoxamine hydrochloride (20 mg/mL in pyridine) and 80 μL N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA). Derivatized samples were subsequently analyzed using a TRACE 1310 GC system (Thermo Fisher Scientific, USA) combined with ISQ 7000 MS system (Thermo Fisher Scientific, USA), which was equipped with a fused-silica capillary column (30 m × 0.25 mm i.d., 0.25 μm DB-5MS stationary phase, J&W Scientific, Folsom, CA). Four replicates were performed for each sample.

The derivatized samples were analyzed using a LECO Pegasus IV GC-TOF/MS system. An Agilent 6890 gas chromatograph (Agilent, San Jose, CA, USA) was used with a 30 m long, 0.25 mm i.d. (inside diameter) fused silica capillary column with 0.25 μm DB-5 MS stationary phase (J & W Scientific, Folsom, CA, USA). One μL was injected in splitless mode and 10:1 split ratio for lipophilic and polar extracts, respectively. Chromatography was performed at a flow rate of 1.5 mL/min. For the lipophilic metabolites, the initial column temperature was set as 100 °C and maintained for 2 min, 100 to 200 °C at 8 °C/min and held for 1 min, 200 to 260 °C at 3 °C/min and held for 1 min, and then 260 to 320 °C at 8 °C/min and held for 4 min. For the polar metabolites, the initial column temperature was set as 100 °C and maintained for 2 min, 100 to 260 °C at 4 °C/min and held for 1 min, 260 to 320 °C at 20 °C/min and held for 3 min. The column’s effluent was then directed to the ion source of a Leco Pegasus IV time off light mass (TOF) spectrometer with an ion source temperature of 200 °C, transferline temperature of 250 °C, and electron ionization at −70 eV. Mass spectra were obtained from m/z 80–500 at 20 scans per second. The detector voltage was set to 1800 V.