The metabolomic profile was assessed with a validated targeted metabolomics approach, implementing liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), and using the AbsoluteIDQ p180 kit (Biocrates Life Sciences AG AbsoluteIDQ® p180 Kit, Innsbruck, Austria), which benefits of an established good interlaboratory reproducibility (27 (link)). Briefly, the serum samples were placed on a 96-well plate pre-loaded with the isotopic labeled internal standards, along with a phosphate buffer solution as blank sample, a calibration curve (7 levels), and three levels of quality control samples. Two different plates were implemented for this study. The sample preparation consisted in the derivatization of amino acids and biogenic amines with phenyl isothiocyanate, evaporation, extraction with 5 mM ammonium acetate in methanol, centrifugation, and dilution. Amino acids and biogenic amines were separated and analyzed through an analytical column before the mass spectrometry (LC-MS/MS), while lipids and the hexose were analyzed with a simple flow injection analysis (FIA-MS/MS). A total of 188 metabolites were measured, including 21 amino acids, 21 biogenic amines, the sum of hexoses, 40 acylcarnitine, 15 sphingolipids (SM), and 90 glycerophospholipids among which 14 lysophosphatidylcholines (LysoPC), 38 diacylphosphatidylcholine (PC aa), and 38 acylalkylphosphatidylcholine (PC ae). Further instrumental and analytical details have been previously reported (28 (link)).
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