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).

Preanalytical Aspects and Sample Quality Assessment in Metabolomics Studies of Human Blood

“…For this reason, the preanalytical process is a critical factor for the success of metabolomics research studies. There are 2 major preanalytical phases: first, the period comprising patient preparation, blood sample collection, and storage in a freezer; and second, the pretreatment of stored samples for subsequent metabolomics analysis, e.g., by LC-MS (2)(3)(4)(5)(6). Typically, the sample pretreatment process follows a standard operating procedure (SOP) 7 based on evaluated protocols (7 ).…”

Method for liver tissue metabolic profiling study and its application in type 2 diabetic rats based on ultra performance liquid chromatography–mass spectrometry

“…Principal component analysis (PCA) is a typical unsupervised pattern recognition technique in metabonomics study [24]. The score plot and loading plot of PCA are often used to classify the different classes of samples and discover biomarkers [25]. The differential metabolites could be found out according to loading plot of PCA and p value of t-test [6].…”

Postgenomics Diagnostics: Metabolomics Approaches to Human Blood Profiling

“…More selective procedures, such as liquid-liquid extraction, are usually avoided in metabolomics studies (Tulipani et al, 2013;Vuckovic, 2012). Deproteinization is the minimum sample treatment required prior to MS analysis of blood metabolites and is commonly achieved using organic solvent precipitation (Bruce SJ et al, 2009), protein denaturation, or ultrafiltration (Psychogios et al, 2011). Protein precipitation with an organic solvent is the most commonly used method in the global metabolomics of blood (Vuckovic, 2012).…”