5 m guard column

The dried pellets were resuspended in 200 µl of sodium hydroxide (1 mol/l) and then transferred into silanized glass tubes. The MCF derivatization was performed in accordance with the protocol published in Smart et al (37 (link)). The derivatives of the MCF metabolites were analyzed using an Agilent GC7890B system (Agilent Technologies, Inc.) coupled to a MSD5977A mass spectrometer (Agilent Technologies, Inc.) with the electron impact ionization set at 70 eV. The gas chromatograph used to separate the metabolites was a Zebron ZB-1701 Capillary GC column (30 mx250 µm id ×0.15 µm with a 5 m guard column; Phenomenex); 1 µl of derivatized sample was injected into the GC inlet that operated at 290°C in a split-less mode under 180 kPa for 1 min. Details of GC and MS parameters were also set up according to the methodology reported in Smart et al (37 (link)). Helium gas flow rate was constantly controlled at 1 ml/min. The GC oven temperature was initiated at 45°C for 2 min and raised to 180°C at 9°C/min for 5 min. Subsequently, the oven temperature was raised at 40°C/min to 220°C and held for 5 min. The temperature was then increased again at 40°C/min to 240°C and held for 11.5 min. The last temperature increase was at 40°C/min to 280°C and held for 2 min. The transfer interface between GC and MS was setup at 250°C, the MS ion source at 250°C and the quadrupole at 130°C.

Dried intracellular and extracellular metabolite extracts were resuspended in 200 μl of sodium hydroxide (1 M). The MCF derivatization step was performed according to the method published by Han et al. [20 (link)]. All MCF-derivatized samples were analyzed using an Agilent GC7890B chromatography system coupled to a MSD5975 mass spectrometer (Agilent, California, USA) operating at 70 eV. The GC column was a ZB-1701 GC capillary column (30 m × 250 μm id × 0.15 μm with a 5 m guard column, Phenomenex, USA). GC-MS parameters and temperature programs were set according to the protocol reported in Han et al. [20 (link)].

Polar metabolites were derivatised for 90 min at 55°C with 20 μL of methoxyamine (c = 20 mg/mL) in pyridine under continuous shaking and subsequently for 60 min at 55°C with 20 μL of MTBSTFA w/1% TBDMCS. GC-MS analysis was performed using an Agilent 7890B GC coupled to an Agilent 5977A Mass Selective Detector (Agilent Technologies). A sample volume of 1 μL was injected into a Split/Splitless inlet, operating in splitless mode at 270°C. Gas chromatograph was equipped with a 30 m (I.D. 250 μm, film 0.25 μm) ZB-35MS capillary column with 5 m guard column (Phenomenex). Helium was used as carrier gas with a constant flow rate of 1.2 mL/min. GC oven temperature was held at 100°C for 2 min and increased to 300 °C at 10 °C/min and held for 4 min. Total run time was 26 min. Transfer line temperature was set to 280°C. Mass selective detector (MSD) was operating under electron ionisation at 70 eV. MS source was held at 230°C and the quadrupole at 150°C. For precise quantification of the MID, measurements were performed in selected ion monitoring mode. Target ions (m/z) and Dwell times are shown in Table S1.
The MetaboliteDetector software package (v. 3.220180913) was used for mass spectrometric data post processing, quantification, MID calculations, correction of natural isotope abundance, and determinations of fractional carbon contributions.⁹⁶ (link)