Agilent 5977a mass selective detector

First, polar metabolites were derivatized 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 additional 20 μl of MTBSTFA w/ 1% TBDMCS. GC-MS analysis was performed using an Agilent 7890B GC coupled to an Agilent 5977 A 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 ionization 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 S2.
The MetaboliteDetector software package (Version 3.220180913) was used for mass spectrometric data post processing, quantification, MID calculations, correction of natural isotope abundance, and determinations of fractional carbon contributions^{63 (link)}.

Dried samples were derivatized using Tri-Sil Z reagent (Sigma-Aldrich) and diluted in an equivalent volume of ethyl acetate prior to GC-MS analysis. GC was performed using an Agilent 7890B fitted with a Zebron ZB5-HT Inferno column (Phenomenex). Injections were performed in pulsed splitless mode (30 psi pulse pressure) with the inlet temperature set to 250 °C. The GC oven temperature program was 170 °C and held for 2 mins with subsequent increase to 300 °C (20 °C/min) and held at 300 °C for an additional 11.5 min (total run time 20 min). The GC oven was coupled to an Agilent 5977 A Mass Selective Detector set to scan mode from 60 to 800 mass units (solvent delay 8 min). For quantification, the detector was set to 7.2 scans/sec.