J. Lipid Res.
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Originally published In Press as doi:10.1194/jlr.D800017-JLR200 on May 23, 2008

Papers In Press, published online ahead of print September 1, 2008
J. Lipid Res., doi:10.1194/jlr.D800017-JLR200
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Journal of Lipid Research, Vol. 49, 2063-2073, September 2008
Copyright © 2008 by American Society for Biochemistry and Molecular Biology

Methods

Highly sensitive analysis of sterol profiles in human serum by LC-ESI-MS/MSboxs

Akira Honda*, Kouwa Yamashita{dagger}, Hiroshi Miyazaki§, Mutsumi Shirai**, Tadashi Ikegami{dagger}{dagger}, Guorong Xu§§, Mitsuteru Numazawa{dagger}, Takashi Hara** and Yasushi Matsuzaki1,{dagger}{dagger}

* Center for Collaborative Research, Tokyo Medical University, Kasumigaura Hospital, Ami, Ibaraki 300-0395, Japan
{dagger}{dagger} Department of Gastroenterology, Tokyo Medical University, Kasumigaura Hospital, Ami, Ibaraki 300-0395, Japan
{dagger} Faculty of Pharmaceutical Science, Tohoku Pharmaceutical University, Sendai, Miyagi 981-8558, Japan
§ Pharmax Institute, Kawasaki, Kanagawa 213-0021, Japan
** Ibaraki Prefectural Institute of Public Health, Mito, Ibaraki 310-0852, Japan
§§ Department of Medicine, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Newark, NJ 07103

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

Published, JLR Papers in Press, May 23, 2008.

This work was supported in part by a Grant-in-Aid for Scientific Research (C20591309) from Japan Society for the Promotion of Science and a grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

1 To whom correspondence should be addressed. e-mail: ymatsuzaki-gi{at}umin.ac.jp

We have developed a highly sensitive and specific method for the analysis of serum sterol profiles. Sterols in 1 µl of dried serum were derivatized into picolinyl esters (3β-picolinate) and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using the electrospray ionization (ESI) mode. In addition to cholesterol, 19 cholesterol precursors, cholestanol, campesterol, sitosterol, and sitostanol were identified simultaneously. Quantitative analyses for the picolinyl esters of 11 available sterols were performed, and detection limits were found to be less than 1 pg on-column. Reproducibilities and recoveries of 8 noncholesterol sterols were validated according to one-way layout and polynomial equation, respectively. The variances between sample preparations and between measurements by this method were calculated to be 1.6% to 8.2% and 2.5% to 16.5%, respectively. The recovery experiments were performed using 1 µl aliquots of normal human serum spiked with 1 ng to 6 ng of sterols, and recoveries of the sterols ranged from 88.1% to 102.5% with a mean recovery of 98.1%. The present method provides reliable and reproducible results for the identification and quantification of neutral sterols, especially in small volumes of blood samples, which is useful for serological diagnosis of inherited disorders in cholesterol metabolism and for noninvasive evaluation of cholesterol biosynthesis and absorption in humans.

Supplementary key words cholestanol • cholesterol precursors • congenital birth defect • liquid chromatography-electrospray ionization-tandem mass spectrometry • picolinic acid • plant sterols

Abbreviations: CDPX2, X-linked dominant chondrodysplasia punctata type 2; CTX, cerebrotendinous xanthomathosis; ESI, electrospray ionization; GC, gas chromatography; LC-APCI-MS, liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry; LC-MS/MS, liquid chromatography-tandem mass spectrometry; SLOS, Smith-Lemli-Opitz syndrome; SRM, selected reaction monitoring


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