4.2.4. HPLC–MS Plasma Analysis

To obtain a metabolic profiling of plasma, an HPLC–MS full scan method was used, according to [44]. A Varian MS 500 equipped with a Prostar 430 autosampler and binary chromatograph 212 series (Varian, Palo Alto, CA, USA), was used as the HPLC–MS system. An Agilent (Milan, Italy) Eclipse XDB C−8 column (2.1 × 150 mm 3.5 µm) was used as a stationary phase. The mobile phase was composed of solvent A (acetonitrile with 0.5% acetic acid) and solvent B (water with 2% formic acid). Linear gradients of A and B were used as follows: 0 min, 10% A; 20 min, 85% A; 21 min, 100% A, 21.30 min, 10% A; 27 min, 10% A. The flow rate was 200 µL/min and the injection volume was 10 µL. The mass range explored was 50–1000 *m/z*. The mass spectra were recorded both in positive standard mode and in turbo data depending scanning (tdds) mode that allows the elucidation of the fragmentation patterns of the detected ions. Collected plasma samples were centrifuged (13,000× *g* for 10 min) and directly injected in the HPLC. Each HPLC–MS data set was processed with the MZmine 2.9 software; from the raw data files, a data set composed of 102 variables was obtained. The Median Fold Change normalization was applied to take into account the effects of sample dilution. Data were log-transformed and mean-centered.
