Samples analysis and data processing

The derivatized samples were analyzed by using a global unbiased mass spectrometry-based platform with GC-MS incorporating an Agilent 5975C interfaced to the GC 7820 (Agilent Technologies, Palo Alto, CA, USA). The system was equipped with a DB-5ms column (Agilent J&W Scientific, Folsom, CA, USA); the injection temperature was set at 230 °C and the detector temperature at 280 °C. Carrier gas (helium) flow rate was equal to 1 mL/min. GC oven starting temperature program was 90 °C with 1 min hold time and ramping at a rate of 10 °C per minute, reaching a final temperature of 270 °C with 7 min hold time. 1 µL of the derivatized sample was injected in split (1:20) mode. After a solvent delay of 3 min, mass spectra were acquired in full scan mode using 2.28 scans per second, with a mass range of 50–700 Amu. Each acquired chromatogram was analyzed by means of the free software Automated Mass Spectral Deconvolution and Identification System (AMDIS): http://chemdata.nist.gov/mass-spc/amdis. Each peak was identified by comparing the corresponding mass spectra and retention times with those stored in an in-house made library including 255 metabolites. Other metabolites were identified by using the National Institute of Standards and Technology’s mass spectral database (NIST08) (31) and the Golm Metabolome Database (GMD) (32). As a result, 122 compounds were accurately identified, whereas 5 additional metabolites were tentatively identified on the basis of GMD data. AMDIS analysis produced a matrix spreadsheet (Microsoft Excel^®, Microsoft Co, Redmond, WA, USA) as a preliminary tool for the subsequent chemometric analysis.