Comprehensive Metabolome Profiling of Plasma Samples

We used ultra-high-pressure liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) to acquire data in both positive and negative ionization modes that allowed for identification and quantification of 630 metabolites. Plasma samples were processed using the MxP Quant 500 Kit (Biocrates) according to the manufacturer’s instructions. In brief, 10 µl of plasma sample, calibration standard and control sample were transferred onto a filter containing internal standards for internal standard calibration. Filters were dried under a stream of nitrogen using a pressure manifold (Waters). Samples were incubated with derivatization reagent phenyl isocyanate for 60 min. After drying under nitrogen, analytes were extracted with 5 mmol l⁻¹ ammonium acetate in methanol and the eluate was further diluted for the UPLC–MS/MS analysis. The targeted analysis covered 630 metabolites (https://biocrates.com/mxp-quant-500-kit/) detected by MS/MS after UPLC separation and flow injection analysis (FIA). Each measurement required two UPLC runs and three FIA runs to cover all metabolites. All analyses were performed on an ACQUITY UPLC I-Class system (Waters) coupled to a Xevo TQ-S mass spectrometer (Waters). Reversed-phase chromatographic separation was accomplished using a C18 LC-column (Biocrates) with 0.2% formic acid in water with 0.2% formic acid in acetonitrile as the eluent system. The FIA solvent was methanol, with a modifier provided by the kit manufacturer. Data analysis of the UPLC–MS/MS results was based on a seven-point curve or one-point calibration and internal standard normalization. Values below the lower threshold were set to zero. Concentration data were analysed using MetaboAnalyst v.5. Concentrations were log-transformed before analysis and raw P values and log₂-transformed fold change values are shown in the graphics of Fig. 1.