Highly sensitive quantification of key regulatory oxysterols in biological samples by LC-ESI-MS/MS

We describe a highly sensitive and specific method for the quantification of key regulatory oxysterols in biological samples. This method is based upon a stable isotope dilution technique by liquid chromatography-tandem mass spectrometry (LC-MS/MS). After alkaline hydrolysis of human serum (5 μl) or...

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Bibliographic Details
Published in:Journal of lipid research 2009-02, Vol.50 (2), p.350-357
Main Authors: Honda, Akira, Yamashita, Kouwa, Hara, Takashi, Ikegami, Tadashi, Miyazaki, Teruo, Shirai, Mutsumi, Xu, Guorong, Numazawa, Mitsuteru, Matsuzaki, Yasushi
Format: Article
Language:English
Subjects:
Animals
Calibration
Chromatography, Liquid - methods
Humans
Hydroxycholesterols - analysis
Microsomes, Liver - chemistry
Rats
Reproducibility of Results
Sensitivity and Specificity
Spectrometry, Mass, Electrospray Ionization - methods
Tandem Mass Spectrometry - methods
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Summary:We describe a highly sensitive and specific method for the quantification of key regulatory oxysterols in biological samples. This method is based upon a stable isotope dilution technique by liquid chromatography-tandem mass spectrometry (LC-MS/MS). After alkaline hydrolysis of human serum (5 μl) or rat liver microsomes (1 mg protein), oxysterols were extracted, derivatized into picolinyl esters, and analyzed by LC-MS/MS using the electrospray ionization mode. The detection limits of the picolinyl esters of 4β-hydroxycholesterol, 7α-hydroxycholesterol, 22R-hydroxycholesterol, 24S-hydroxycholesterol, 25-hydroxycholesterol, 27-hydroxycholesterol, and 24S,25-epoxycholesterol were 2-10 fg (5-25 amol) on-column (signal-to-noise ratio = 3). Reproducibilities and recoveries of these oxysterols 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 1.8% to 12.7% and 2.9% to 11.9%, respectively. The recovery experiments were performed using rat liver microsomes spiked with 0.05 ng to 12 ng of oxysterols, and recoveries of the oxysterols ranged from 86.7% to 107.3%, with a mean recovery of 100.6%. This method provides reproducible and reliable results for the quantification of oxysterols in small amounts of biological samples.
ISSN:0022-2275
1539-7262
DOI:10.1194/jlr.d800040-jlr200