Methods
A validated LC-MS/MS assay for quantification of 24(S)-hydroxycholesterol in plasma and cerebrospinal fluid

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24(S)-hydroxycholesterol [24(S)-HC] is a cholesterol metabolite that is formed almost exclusively in the brain. The concentrations of 24(S)-HC in cerebrospinal fluid (CSF) and/or plasma might be a sensitive marker of altered cholesterol metabolism in the CNS. A highly sensitive 2D-LC-MS/MS assay was developed for the quantification of 24(S)-HC in human plasma and CSF. In the development of an assay for 24(S)-HC in CSF, significant nonspecific binding of 24(S)-HC was observed and resolved with the addition of 2.5% 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) into CSF samples. The sample preparation consists of liquid-liquid extraction with methyl-tert-butyl ether and derivatization with nicotinic acid. Good linearity was observed in a range from 1 to 200 ng/ml and from 0.025 to 5 ng/ml, for plasma and CSF, respectively. Acceptable precision and accuracy were obtained for concentrations over the calibration curve ranges. Stability of 24(S)-HC was reported under a variety of storage conditions. This method has been successfully applied to support a National Institutes of Health-sponsored clinical trial of HP-β-CD in Niemann-Pick type C1 patients, in which 24(S)-HC is used as a pharmacodynamic biomarker.

two-dimensional liquid chromatography-tandem mass spectrometry
validation
Niemann-Pick C1 disease
2-hydroxypropyl-β-cyclodextrin

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This work was supported by the Bench-to-Bedside Program, the Office of Rare Diseases Research, the Therapies for Rare and Neglected Diseases (TRND) Program in the National Center for Advancing Translational Sciences of the National Institutes of Health (NIH), and National Institutes of Health Grant 1ZIAHD008824-07 (F.D.P.). Additional support was provided by a grant from Dana's Angels Research Trust (D.S.O). Quantification of 24(S)-HC was performed in the Metabolomics Facility at Washington University (P30 DK020579).

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

    Abbreviations

    APCI

    atmospheric pressure chemical ionization

    AUC

    area under the curve

    CSF

    cerebrospinal fluid

    CV

    coefficient of variance

    D7-24-HC

    25,26,26,26,27,27,27-[2H7]-24(R/S)-hydroxycholesterol

    DQC

    dilution quality control

    HP-β-CD

    2-hydroxypropyl-β-cyclodextrin

    HQC

    high quality control

    LLOQ

    lower limit of quantification

    LQC

    low quality control

    MQC

    middle quality control

    MRM

    multiple reaction monitoring

    NIH

    National Institutes of Health

    NPC1

    Niemann-Pick type C1

    QC

    quality control

    RE

    relative error

    24(S)-HC

    24(S)-hydroxycholesterol

    ULOQ

    upper limit of quantification