J. Lipid Res.
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A more recent version of this article appeared on February 1, 2007

Papers In Press, published online ahead of print November 8, 2006
J. Lipid Res., doi:10.1194/jlr.D600032-JLR200
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Submitted on August 28, 2006
Revised on October 31, 2006
Accepted on November 7, 2006

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

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

Ibaraki Prefectural Institute of Public Health, Mito, Ibaraki 310-0852

Corresponding Author: AkiraHonda{at}aol.com

We describe a highly sensitive and specific method for the quantification of serum 7alpha -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 mu l) by a salting-out procedure, derivatized into the picolinoyl ester (C4-7alpha -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 (ESI) mode. The detection limit of the C4 picolinoyl ester was found to be 100 fg (S/N = 10), which was approximately 1,000 times more sensitive than the detection limit of C4 with a conventional HPLC-UV method. The relative standard deviations between sample preparations and between measurements by the present 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 of C4 and were validated by polynomial equation. The results showed that the estimated concentration with 95% confidence limit was 23.1 ± 2.8 ng/ml that coincided completely with the observed X0 = 23.3 ± 0.1 ng/ml with a mean recovery of 93.4%. The present method provides highly reliable and reproducible results for the quantification of C4, especially in small volumes of blood samples.


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