Investigation of Human Blood Plasma Sample Preparation for Performing Metabolomics Using Ultrahigh Performance Liquid Chromatography/Mass Spectrometry

Abstract

The following study investigates the preparation of human blood plasma for metabolomic profiling analysis by ultrahigh performance liquid chromatography coupled to time-of-flight mass spectrometry (UPLC/TOFMS) in a novel two-step design study. Four different organic solvents (acetonitrile, acetone, methanol, and ethanol) were used to assess human blood plasma preparation via protein precipitation. The optimal conditions for sample preparation were investigated, with consideration to the number of extracted markers, data quality/reproducibility, and column lifetime prolongation. Isotopically labeled internal standards were used to monitor data quality/reproducibility. Gel electrophoresis was also used to measure the protein content in the supernatant of the "first design step" allowing assessment of the amount of protein that would be injected/accumulate onto the column after many injections that would be apparent in a global metabolic profiling study. The second design step followed on from the results obtained in step one, with four of the best conditions selected and further investigated, looking at the effects of vortex time and temperature on precipitation/extraction. Two choices of solvent compositions were found to be "optimal" for preparation of plasma for global metabolic profiling analysis; these were "methanol/ethanol" (1:1, v/v) and "methanol/acetonitrile/acetone" (1:1:1, v/v/v) added to plasma (4:1 ratio, 400 microL total volume).