Optimization of deproteinization. (a) In the step of monophasic deproteinization, two samples containing either plasma:solvent ratios of 1:3 or 1:9 were examined. In the condition of a 1:3 ratio, 300 μL of either methanol or ethanol was added to 100 μL of plasma; whereas in the setting of a 1:9 ratio, 900 μL of methanol or ethanol was added to 100 μL of plasma. In the aspect of biphasic deproteinization, a two-step solvent addition was introduced, starting with an addition of 100 μL chloroform with 200 μL of either methanol or ethanol to 200 μL of plasma, followed by an addition of 400 μL 50% chloroform/H2O. Metabolites extracted via both mono- and biphasic methods were subjected to a 1-minute vortexing, and a 20-minute incubation on ice. After centrifugation, the metabolites in the supernatants were subsequently lyophilized at 45°C for 2 hours. (b) To further optimize the step of monophasic deproteinization, various combinations of methanol/ethanol were prepared, and added to the plasma at a ratio of either 1:3 or 1:9 (plasma:solvent).