GC-MS Analysis of Metabolites

GC-MS samples from the above extraction were re-dissolved and derivatized. Eight microliters of a retention time standard mixture (0.029% v/v n-dodecane, n-pentadecane, n-nonadecane, n-docosane, n-octacosane, n-dotracontane, and n-hexatriacontane dissolved in pyridine) was added. The sample set also included a reference quality control of authentic metabolite standards (1 mg ml⁻¹ each) (Additional file 2: Table S1A). Volumes of 1 μL were then injected onto 30-m VF-5 ms GC column with 0.25 mm i.d., film thickness of 0.25 μ m, and + 10 m EZ-Guard (Agilent) in splitless and split mode (32:1) allowing a more accurate comparison of highly abundant metabolites (e.g. tartarate, sugars, and inositol). The GC-MS system consisted of an AS 3000 autosampler, a TRACE GC ULTRA gas chromatograph, and a DSQII quadrupole mass spectrometer (Thermo-Fisher ltd). The parameters of the machine were exactly as described in [69 (link)]. Spectral searching was done by consulting the National Institute of Standards and Technology (NIST, Gaithersburg, USA) algorithm incorporated in the Xcalibur® data software (version 2.0.7) against RI libraries from the Max-Planck Institute for Plant Physiology in Golm, Germany (http://www.mpimp-golm.mpg.de/mms-library/) and finally normalized by the total metabolites and correcte d for the dilution factor.