Centrifugal vacuum evaporator

Plasma samples were prepared following a procedure described previously^{7 (link)}. Please refer to Table S1 for patient characteristics. The samples were thawed at 4 °C. In all, 400 μl of ice-cold methanol/ethanol 50:50 [v/v] was added to 100 μl of plasma and mixed using a vortex mixer for 15 s. For the samples used for metabolic profiling the ice-cold methanol/ethanol mixture contained five internal standards (caffeine-d3 0.88 μmol/L, hippuric-d5 acid 0.22 μmol/L, nicotinic-d4 acid 0.88 μmol/L, octanoyl-l-carnitine-d3 0.22 μmol/L, l-phenyl-d5-alanine 0.44 μmol/L (all from C/D/N Isotopes, Pointe-Claire, Canada)). Next, these dilutions were incubated for 20 min at 4 °C and subsequently centrifuged at 18,600 × g for 15 min at 4 °C. In all, 350 μl of the supernatant was transferred into new tubes and dried in a centrifugal vacuum evaporator (Eppendorf). The sample was reconstituted in 100 ml 0.1% formic acid in deionised water/methanol 90:10 [v/v], mixed using a vortex mixer for 15 s and centrifuged at 18,600 × g for 15 min at room temperature. In all, 90 μl of the supernatant was transferred to an autosampler vial and used for LC-MS analysis.

Frozen human heparin-anticoagulated plasma was thawed at 4 °C and mixed by vortexing. An aliquot of 100 μl of plasma was transferred into a 1.5-ml polypropylene microcentrifuge tube. Then, 400 μl ice-cold methanol/ethanol (50:50 vol/vol) containing five internal standards (IS) [caffeine-d3 0.88 μmol/L, hippuric-d5 acid 0.22 μmol/L, nicotinic-d4 acid 0.88 μmol/L, octanoyl-L-carnitine-d3 0.22 μmol/L, L-phenyl-d5-alanine 0.44 μmol/L (all from C/D/N Isotopes, Pointe-Claire, Canada)] was added to each plasma aliquot. Samples were thoroughly mixed on a vortex mixer for 30 s, incubated at 4 °C for 20 min, and centrifuged at 18,600 g for 15 min at 4 °C. An aliquot of 350 μl of the supernatant was transferred into a 1.5-ml polypropylene microcentrifuge tube. Samples were dried in a centrifugal vacuum evaporator (Eppendorf, Hamburg, Germany) at room temperature, reconstituted in 100 μl of water containing 0.1% (vol/vol) formic acid, vortexed for 15 s, and centrifuged at 18,600 g for 15 min at room temperature. An aliquot of 90 μl was transferred into 250 -μl polypropylene autosampler vials. These samples were either placed in an autosampler at 4 °C for direct analysis or stored at −80 °C. Stored samples were thawed at room temperature and centrifuged at 18,600 g for 15 min at room temperature before analysis.