Pegasus two time of flight mass spectrometer

To detect phenolics and vanillin production from enzymatic reactions, we used a quantitative GC–MS analysis. The resulting phenolics from enzymatic reactions were extracted by adjusting the pH of the samples to below 2 with 6 mol l⁻¹ HCl and addition of butyl acetate (1:1, v:v) and then derivatized [59 (link)]. The derivatized samples (1 µl) were analyzed on an Combi-PAL autosampler (Agilent Technologies GmbH, Waldbronn, Germany) coupled to an Agilent 7890 gas chromatograph in split less mode coupled to a Leco Pegasus two time-of-flight mass spectrometer (LECO, St. Joseph, MI, USA) as described by Weckwerth et al. [60 (link)]. The chromatograms were exported from the Leco ChromaTOF software (version 3.25) to the R software. Peak detection, retention time alignment, and library matching were performed using the Target Search R-package [61 ]. Metabolites were quantified by the peak intensity of a selective mass. The intensity normalization procedure was performed by dividing the fresh weight, followed by sum of the total ion count and global outlier replacement [62 (link), 63 (link)].

Metabolites were extracted using the MTBE method as described elsewhere^{78 (link)}. One hundred-fifty μl vacuum-dried polar phases samples were derivatized and subjected to GC‒MS analysis as described previously^{79 (link)}. The GC‒MS data were obtained using an Agilent 7683 series auto-sample (Agilent Technologies, http://www.home.agilent.com), coupled to an Agilent 6890 gas-chromatograph‒Leco Pegasus two time-of-flight mass spectrometer (Leco: http://www.leco.com/). Identical chromatogram acquisition parameters were applied to those previously used^{80 (link)}. Chromatograms were exported from the LECO CHROMATOF software (version 3.34) to the R software. Ion extraction, peak detection, retention time alignment and library searching were obtained using the TargetSearch package from Bioconductor⁸¹ . The resulting data matrix was used for further analysis.