4.4. Targeted Metabolomics
Reagents, standards and controls were prepared according to manufacturer’s instructions (Biocrates UM-P180-SCIEX-13) for mass spectrometry. Analytical columns (Acquity UPLC BEH C18 1.7 µm 2.1 mm × 75 mm, Waters) and pre-column (Acquity BEH C18 1.7 VANGUARD) from Waters (Eschborn, Germany) were used. Targeted metabolomics was performed using the AbsoluteIDQ p180 Kit (BIOCRATES Life Sciences AG, Innsbruck, Austria) [Serum from each sample was processed according to the manufacturer’s recommendations. In brief, 10 μL of the internal standard solution was added to each well on a filter spot of the 96-well extraction plate. After drying under a gentle stream of nitrogen, 10 μL of each serum sample, quality control (QC) sample, blank, zero sample or calibration standard, was added to the appropriate wells. The plate was then dried under a gentle stream of nitrogen. Derivatization was performed using 5% phenyl isothiocyanate (33% ethanol, 33% water, 33% pyridine). Samples were incubated for 20 min followed by extraction with methanol. Electrospray ionization (ESI)—LC-MS/MS and flow injection (FIA) MS/MS was performed on Sciex 4500QTRAP MD (SCIEX, Framingham, MA, USA). MS-system was coupled to an Agilent 1290 UHPLC-system (G4226A autosampler, infinity BinPump, G1316C column-oven, G1330B thermostat (Agilent, Santa Clara, CA, USA)).Data were processed with Analyst Software version 1.6.2 MD (SCIEX, Framingham, MA, USA). LC- and MS/MS settings were set according to the manufacturer’s manual. MetIDQ software (5.5.4-DB 100 Boron-2623 (Biocrates, Innsbruck, Austria)) was used for validation and processing of MS data. Amino acids and biogenic amines were separated by UHPLC before injection into the mass spectrometer, while flow injection analysis was used for PCs, hexose, Cs and SMs. In this dataset, metabolites with more than 60% measurements at the limit of detection (LOD) or below lowest limit of quantification (LLOQ), and those samples with missing values for more than 60% of the metabolites were excluded from the analyses as described previously [