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Journal of Lipid Research, Vol. 48, 458-464, February 2007
Copyright © 2007 by American Society for Biochemistry and Molecular Biology

Methods

Highly sensitive quantification of 7-hydroxy-4-cholesten-3-one in human serum by LC-ESI-MS/MS

Akira Honda^1,*, Kouwa Yamashita, Mitsuteru Numazawa, Tadashi Ikegami, Mikio Doy^*, Yasushi Matsuzaki and Hiroshi Miyazaki^**

^* Ibaraki Prefectural Institute of Public Health, Mito, Ibaraki 310-0852, Japan
Faculty of Pharmaceutical Science, Tohoku Pharmaceutical University, Sendai, Miyagi 981-8558, Japan
Department of Medicine, Tokyo Medical University, Kasumigaura Hospital, Ami, Ibaraki 300-0395, Japan
^** Pharmax Institute, Kawasaki, Kanagawa 213-0021, Japan

Published, JLR Papers in Press, November 8, 2006.

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

We describe a highly sensitive and specific method for the quantification of serum 7-hydroxy-4-cholesten-3-one (C4), which has been used as a biomarker for bile acid biosynthesis. This method is based upon a stable isotope dilution technique by liquid chromatography-tandem mass spectrometry (LC-MS/MS). C4 was extracted from human serum (2–50 µl) by a salting-out procedure, derivatized into the picolinoyl ester (C4-7-picolinate), and then purified using a disposable C₁₈ cartridge. The resulting picolinoyl ester derivative of C4 was quantified by LC-MS/MS using the electrospray ionization mode. The detection limit of the C4 picolinoyl ester was found to be 100 fg (signal-to-noise ratio = 10), which was 1,000 times more sensitive than the detection limit of C4 with a conventional HPLC-ultraviolet method. The relative standard deviations between sample preparations and between measurements by our method were calculated to be 5.7% and 3.9%, respectively, by one-way layout analysis. The recovery experiments were performed using serum spiked with 20.0–60.0 ng/ml C4 and were validated by a polynomial equation. The results showed that the estimated concentration with 95% confidence limit was 23.1 ± 2.8 ng/ml, which coincided completely with the observed ₀ ± SD = 23.3 ± 1.0 ng/ml with a mean recovery of 93.4%. This method provides highly reliable and reproducible results for the quantification of C4, especially in small volumes of blood samples.

Supplementary key words bile acid synthesis • cholesterol 7-hydroxylase • picolinic acid • liquid chromatography electrospray ionization-tandem mass spectrometry

Abbreviations: C4, 7-hydroxy-4-cholesten-3-one; CYP7A1, cholesterol 7-hydroxylase; ESI, electrospray ionization; GC-SIM, gas chromatography-mass spectrometry with selected ion monitoring; LC-MS/MS, liquid chromatography-tandem mass spectrometry; SRM, selected reaction monitoring; UV, ultraviolet

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