University of Pardubice Faculty of Chemical Technology

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Metabolomic analysis of human plasma by bio-inert UHPLC system coupled with high-resolution mass spectrometry

Authors: Langová Alena | Peterka Ondřej | Jirásko Robert | Holčapek Michal | Vaňková Zuzana

Year: 2023

Type of publication: ostatní - přednáška nebo poster

Page from-to: nestránkováno

Titles:

Language	Name	Abstract	Keywords
eng	Metabolomic analysis of human plasma by bio-inert UHPLC system coupled with high-resolution mass spectrometry	Metabolomics is a comprehensive study, which is concerned with both nontargeted and targeted analysis of endogenous metabolites and attempts to systematically identify and quantify metabolites from a biological sample. Metabolites are small molecules typically up to 1,500 Da with diverse structures, including lipids, amino acids, peptides, nucleic acids, organic acids, vitamins, thiols, or carbohydrates. The metabolomic analysis is mainly based on mass spectrometry (MS) coupled with ultrahigh-performance liquid chromatography in HILIC or RP modes. The metabolites often contain problematic functional groups for the separation, like phosphate, which may interact with the surface of the instrument or chromatographic column, leading to bad peak shapes. The new instrumentation and columns with bio-inert materials offer excellent separation efficiency for these problematic analytes. The essential step of the metabolomic analysis is a sample preparation. Protein precipitation by organic solvents is the most commonly used method for sample preparation, but abundant lipid classes could be present in a large excess, which could cause the suppression of low abundant metabolites. The bio-inert Acquity Premier UHPLC system with Acquity Premier BEH Amide column (150×2.1 mm; 1.7 µm) coupled to mass spectrometry with high-resolution (Xevo G2-XS QTOF, Waters) was used. The new separation method was developed with the following conditions: gradient elution, where the mobile phase A was acetonitrile with 0,005% acetic acid (AA), and phase B 15mM ammonium acetate with 0,005% AA, flow rate 0.4 mL/min, and column temperature 45°C. The total run time is 20 min, including the equilibration time. The MS conditions were optimized for both positive and negative ion modes. The method enables the separation of isomers, such as leucine/isoleucine/norleucine or determination of phosphate metabolites, such as ATP, ADP, and AMP. Double Folch extraction method was applied for the sample preparation