Journal of Lipid Research
Volume 53, Issue 10, October 2012, Pages 2231-2241
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Methods
Targeted profiling of circulating and hepatic bile acids in human, mouse, and rat using a UPLC-MRM-MS-validated method

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Bile acids (BAs) are a group of chemically related steroids recognized as regulatory molecules whose profiles can change in different physio-pathological situations. We have developed a sensitive, fast, and reproducible ultraperformance liquid chromatography/multiple reaction monitoring/mass spectrometry method to determine the tissue and sera BA profiles in different species (human, rat, and mouse) by quantifying 31 major and minor BA species in a single 21-min run. The method has been validated according to FDA guidelines, and it generally provides good results in terms of intra- and interday precision (less than 8.6% and 16.0%, respectively), accuracy (relative error measurement between –11.9% and 8.6%), and linearity (R2 > 0.996 and dynamic ranges between two and four orders of magnitude), with limits of quantification between 2.5 and 20 nM. The new analytical approach was applied to determine BA concentrations in human, rat, and mouse serum and in liver tissue. Our comparative study confirmed and extended previous reports, showing marked interspecies differences in circulating and hepatic BA composition. The targeted analysis revealed the presence of unexpected minoritary BAs, such as tauro-alpha-Muricholic acid in human serum, thus allowing us to obtain a thorough profiling of human samples. Its great sensitivity, low sample requirements (25 µl of serum, 5 mg of tissue), and comprehensive capacity to profile a considerable number of BAs make the present method a good choice to study BA metabolism in physiological and pathological situations, particularly in toxicological studies.

lipidomics
metabolomics
targeted analysis

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This work was supported by Instituto de Salud Carlos III of the Spanish Ministry of Science and Innovation (PI10/00923 and PI11/02942), by a Miguel Server contract (CP08/00125) from the Spanish Ministry of Science and Innovation/Instituto de Salud Carlos III. (A.L.), and by a predoctoral contract from the Vali+d program of the Conselleria d'Educació (Regional Valencian Ministry of Education) (J.C.G.-C.).

    Abbreviations:

    αMCA

    alpha-muricholic acid

    BA

    bile acid

    βMCA

    beta-muricholic acid

    CA

    cholic acid

    CDCA

    chenodeoxycholic acid

    CV

    coefficient of variation

    DCA

    deoxycholic acid

    DHCA

    dehydrocholic acid

    GCA

    glycocholic acid

    GCDCA

    glycochenodeoxycholic acid

    GDCA

    glycodeoxycholic acid

    GDHCA

    glycodehydrocholic acid

    GHCA

    glycohyocholic acid

    GHDCA

    glycohyodeoxycholic acid

    GLCA

    glycolithocholic acid

    GUDCA

    glycoursodeoxycholic acid

    HCA

    hyocholic acid

    HDCA

    hyodeoxycholic acid

    IS

    internal standard

    LCA

    lithocholic acid

    LLOQ

    lower limit of quantification

    LOD

    limit of detection

    MRM

    multiple reaction monitoring

    MuroCA

    murocholic acid

    QC

    quality control

    RME

    relative measurement error

    SPE

    solid-phase extraction

    TαMCA

    tauro-alpha-Muricholic acid

    TβMCA

    tauro-beta-Muricholic acid

    TCA

    taurocholic acid

    TCDCA

    taurochenodeoxycholic acid

    TDCA

    taurodeoxycholic acid

    TDHCA

    taurodehydrocholic acid

    THCA

    taurohyocholic acid

    THDCA

    taurohyodeoxycholic acid

    TLCA

    taurolithocholic acid

    TUDCA

    tauroursodeoxycholic acid

    TωMCA

    tauro-omega-muricholic acid

    UDCA

    ursodeoxycholic acid

    UPLC

    ultraperformance liquid chromatography

    ωMCA

    omega-Muricholic acid

The online version of this article (available at http://www.jlr.org) contains supplementary data in the form of seven figures and three tables.