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Utilization of a deuterated derivatization agent to synthesize internal standards for gas chromatography-tandem mass spectrometry quantification of silylated metabolites.

Abstract

GC-MS analysis of silylated metabolites is a sensitive method that covers important metabolite groups such as sugars, amino acids and non-amino organic acids, and it has become one of the most important analytical methods for exploring the metabolome. Absolute quantitative GC-MS analysis of silylated metabolites poses a challenge as different metabolites have different derivatization kinetics and as their silyl-derivates have varying stability. This report describes the development of a targeted GC-MS/MS method for quantification of metabolites. Internal standards for each individual metabolite were obtained by derivatization of a mixture of standards with deuterated N-methyl-N-trimethylsilyltrifluoroacetamide (d9-MSTFA), and spiking this solution into MSTFA derivatized samples prior to GC-MS/MS analysis. The derivatization and spiking protocol needed optimization to ensure that the behaviour of labelled compound responses in the spiked sample correctly reflected the behaviour of unlabelled compound responses. Using labelled and unlabelled MSTFA in this way enabled normalization of metabolite responses by the response of their deuterated counterpart (i.e. individual correction). Such individual correction of metabolite responses reproducibly resulted in significantly higher precision than traditional data correction strategies when tested on samples both with and without serum and urine matrices. The developed method is thus a valuable contribution to the field of absolute quantitative metabolomics.

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  • Publisher Full Text

    • Authors

    Lien SK, Kvitvang HF, Bruheim P

    Institution

    Department of Biotechnology, Norwegian University of Science and Technology, Sem Sælands vei 6/8, N-7491 Trondheim, Norway.

    Source

    Journal of chromatography. A 1247: 2012 Jul 20 pg 118-24

    MeSH

    Amino Acids
    Carbohydrates
    Carboxylic Acids
    Deuterium
    Gas Chromatography-Mass Spectrometry
    Metabolomics
    Reference Standards
    Tandem Mass Spectrometry
    Trifluoroacetic Acid
    Trimethylsilyl Compounds

    Pub Type(s)

    Journal Article
    Research Support, Non-U.S. Gov't

    Language

    eng

    PubMed ID

    22695696