To separate the MCF derivatives from the reactive mixture, 400 μL of chloroform was added to the mixture and then mixed vigorously for 10 s followed by the addition of 400 μL of sodium bicarbonate solution

(50 mM) and vigorous mixing for an additional 10 s. The upper aqueous layer was discarded and the chloroform phase was subjected to GC-MS analysis. GC-MS directly analysis and compound identifications GC-MS analysis was performed Inhibitors,research,lifescience,medical with a Shimadzu GCMS-QP2010 system, equipped with a quadrupole mass selective detector on electron impact (EI) mode operated at 70 eV. The column used for all analyses was a ZB1701 (Zebron, Phenomenex), 30 m × 250 μm i.d. × 0.15 μm film thickness. The MS was operated in scan mode (start after 4.5 min,

mass range 40-650 a.m.u. at 0.15 s/scan). The parameters for separation and analysis of TMS and MCF derivatives are described in Villas-Bôas et al. [6] and Smart et al. [15], respectively. For compounds forming more Inhibitors,research,lifescience,medical than one major derivative, the most promotion intense peak was selected for quantitation. We have used the Automated Mass Spectral Deconvolution and Identification System (AMDIS) to identify compounds present in each sample based on mass spectra and retention times against our in-house MS library of spectra. AMDIS is a software Inhibitors,research,lifescience,medical freely distributed by the National Institute of Standards and Technology and has been largely applied Inhibitors,research,lifescience,medical to metabolomics. Repeatability of the GC-MS

equipment To assess the repeatability of the analytical instrument (GC-MS), we derivatized two different concentrations of standards known to produce stable derivatives by both derivatization methods and we analyzed the same sample 6 times in sequence. The repeatability was assessed by determining Inhibitors,research,lifescience,medical the relative standard deviation (RSD) of the GC-peak area, using Equation (1), of each metabolite derivative between the 6 analyses. RSD=SD/mean×100 (1) Stability The standard mixture containing all metabolites listed in Table 1 was derivatized in two different concentrations (n = 2) and immediately injected into the GC-MS. The Batimastat same samples were re-injected after 24, 48 and 72 hours. The stability of the metabolite derivatives was assessed by determining the relative standard deviation (RSD) of the GC-peak area, Equation (1), of each derivative within 72 hours. Repeatability of derivatization To assess the repeatability of the derivatization reactions we derivatized 6 replicates samples of the standard mixture listed in Table 1 in two different concentrations. Each sample was injected into the GC-MS immediately after derivatization. The repeatability was assessed by determining the relative standard deviation (RSD) of the GC-peak area, using Equation (1), of each metabolite derivative between the 6 replicate samples.