*4.2. Analysis of Metabolites Present in Shrimp Waste Samples by HPLC-ESI-MS/TOF*

The differential analysis of the metabolites contained in the four substances tested was carried out by HPLC-ESI-MS-TOF. Before the analysis, each sample was subjected to specific pretreatments. In particular, "EtOAc-extract" and "MetOH-extract" were dissolved in a methanol solution at 1% of acetic acid. Finally, "Water-extract" and "Nitric-extract" were mixed to acidified water. Each sample was finally filtered with 0.22 μm filter and then analyzed using an UPLC (1290 Infinity LC, Agilent Technologies, Santa Clara, CA, USA) coupled with a quadrupole time of flight mass spectrometer (Agilent 6546 LC/Q-TOF) operating in positive and negative ionization mode. Chromatographic separation was performed with an Agilent Zorbax RRHD SB-C18, 2.1 mm × 50 mm, 1.8 μm column. Mobile phase A was composed of Milli-Q water and acetonitrile was used for mobile phase B (both phases were acidified with 0.1% formic acid), with gradient elution, as follows: 0 min, 2% B; 22 min 95% B; 25 min, 5% B. The column was equilibrated for 3 min before every analysis. The flow rate was 0.4 mL/min, and 5 μL of sample was injected. Dual AJS ESI source conditions were as follows: gas temperature: 325 ◦C; gas flow: 10 L/min; nebulizer pressure: 40 psig; sheath gas temperature: 295 ◦C; sheath gas flow: 12 L/min; capillary voltage: 4000 V; nozzle voltage: 500 V; Fragmentor: 120 V; skimmer: 70 V; product ion scan range: 100–1500 Da; MS scan rate: 5 spectra/s; MS/MS scan rate: 3 spectra/s; maximum precursors per cycle: 2; and collision energy: 10, 20, 40 eV. The analysis of the metabolites was carried out in triplicate. Untargeted LC/Q-TOF based metabolomics approach was used to identify the metabolic profiling of shrimp waste extracts. Integration, data elaboration and identification of metabolites were managed using MassHunter Qualitative Analysis software B.08.00 and library PCDL Manager B.08.00.
