Acquity 1.7um beh amide hilic column

Plasma was separated immediately after sampling and stored at −140°C. Metabolites were analyzed with ACQUITY UPLC‐MS/MS system (Waters Corporation) as in Khan et al (2014). The column was 2.1 × 100 mm Acquity 1.7um BEH amide HILIC column and the detection system Xevo^® TQ‐S tandem triple‐quadrupole mass spectrometer (Waters Corporation). In order to analyze differences among groups, univariate analysis T‐test2 was performed on autoscaled data. In order to explain the separation among groups, unsupervised multivariate analysis, principal component analysis (PCA), was performed. Non‐transformed data were mean‐centered and autoscaled. Five components were used in all the PCA models. Dendrogram was plotted on normalized data using Ward's linkage clustering algorithm and Pearson's correlation similarity measure and visualized as heatmap.

Targeted metabolomics of 102 metabolites in serum samples were analyzed by mass spectrometry-based methods at the Metabolomics Unit, Technology Centre, Institute for Molecular Medicine Finland FIMM, University of Helsinki. Briefly, final analysis for all metabolites were performed on an ACQUITY UPLC-MS/MS system (Waters Corporation, Milford, MA, USA). Chromatographic separation was done using 2.1 × 100 mm Acquity 1.7um BEH amide HILIC column (Waters Corporation, Milford, MA, USA), and temperature was maintained at 45°C. The total run time was 17.5 minutes including 2.5 minutes of equilibration step at a flow rate of 600 µL/min. For the full list of the measured metabolites and specific data on the processing of samples see appendix, Table S2 and Supplementary Methods.