Targeted Metabolomic Profiling of Plasma

Freshly-thawed plasma samples were analyzed at Baylor College of Medicine Metabolomics Core facility. Metabolites were extracted from plasma using previously described standard procedures for targeted metabolomic profiling using ultrahigh-performance liquid chromatography (UPLC)-tandem mass spectrometry [4–8 (link)]. Pooled plasma samples were used as quality controls. For extraction of the metabolome, 100 μl of plasma was mixed with a methanol mixture containing equimolar amounts of eight internal standard compounds, and metabolic extraction was performed using consecutive application of ice-cold organic and aqueous solvents (water: methanol: chloroform: water, with a ratio of 1:4:3:1) followed by deproteinization and drying of the extract. The dried extract was resuspended in injection solvent and analyzed using UPLC-tandem mass spectrometry (Agilent 1290 series UPLC system equipped with a degasser, binary pump, thermostatted autosampler and column oven, Agilent Technologies, CA, USA). The multiple reaction monitoring-based measurement of relative metabolite levels was performed using normal phase chromatographic separation. All samples were kept at 4°C, and analysis was performed on aliquots of 5 and 10 μl.