4.3. UPLC-ESI-QToF-MS/MS Untargeted Metabolomic Profiling

Reconstituted extracts were analyzed by ultra-high-performance liquid chromatography–tandem mass spectrometry (UPLC-ESI-QToF-MS/MS). An untargeted metabolomics approach was used for an unbiased analysis of the endo-metabolomes (Figure 1b). Five µL of each sample were subjected to chromatographic separation using a UltiMate 3000 RS (Thermo Scientific Dionex, Waltham, MA, USA) UPLC system, equipped with a Kinetex C18 reversed phase column (1.7 µm, 150 × 2.1 mm from Phenomenex, Aschaffenburg, Germany) kept at 40 °C, with a flow rate of 300 µL/min. A gradient elution with water (+0.1% v/v formic acid) as eluent A and acetonitrile (+0.1% v/v formic acid) as eluent B, was run as follows: 1% B for 0 to 2 min, linear gradient from 1% B to 100% B from 2 to 20 min, hold 100% B until 25 min and linear gradient from 100% B to 1% B from 25 to 30 min.

Column eluates were analyzed in positive electrospray ionization mode on a Bruker maXis HD QToF mass spectrometer equipped with an Apollo II electrospray source (Bruker, Bremen, Germany). Raw data were acquired in full scan mode (50–1500 Da) in a data dependent MS/MS mode, performing collision-induced fragmentation of the five most abundant ions in each MS scan and using of Bruker’s “smart exclusion” functionality to minimize multiple fragmentation of the same ion. The collision energy was ramped from 80% to 200% of the default auto-MS/MS collision energy in order to get more information rich spectra.

The samples preparation and chromatographic separation carried out and described above are not optimal for the detection of lipids, which, containing polar heads and nonpolar fatty acyl chains, have distinct physicochemical properties and thus require specific methods of analysis; however, the present method was put in place to the aim of detecting the largest number of compounds possible.