GC-TOF MS Analysis of Derivatized Metabolites

An Agilent 7890A GC (Hewlett-Packard, Atlanta, GA) coupled to a Pegasus HT TOF MS (Leco, St. Joseph, MI) was used for the analysis of derivatized metabolite samples. The derivatized extract (1 µL) was injected into the GC in splitless mode. An RTX-5Sil MS capillary column (30 m length, 25 mm inner diameter, and 0.25 mm film thickness; Restek, Bellefonte, PA) and an additional 10-m long integrated guard column were used for GC separation. The sample was initially held at a constant temperature of 50°C for 1 min, after which it was ramped to 330°C at 20°C/min and then finally held for 5 min. The transfer line temperature was set at 280°C. Mass spectra were acquired in a scanning range of 85–500 m/z at an acquisition rate of 10 spectra/sec. The ionization mode was subjected to electron impact at 70 eV with an ion source temperature set at 250°C. GC/TOF MS data were preprocessed by Leco ChromaTOF software (version 3.34; Leco) by using automated peak detection and mass spectral deconvolution. Preprocessed MS data were processed using BinBase, an in-house programmed database for the identification of metabolites, as described previously [19] (link), [20] (link). The abundance of each identified metabolite was obtained by normalizing the peak intensity of each metabolite using the median of sums of peak intensities of all the identified metabolites in each sample [21] , [22] (link).