Highly sensitive quantification of key regulatory oxysterols in biological samples by LC-ESI-MS/MS
Highly sensitive quantification of key regulatory oxysterols in biological samples by LC-ESI-MS/MS
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DOI：
10.1194/jlr.d800040-jlr200
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发表时间：
2009-02-01
影响因子：
6.5
通讯作者：
Matsuzaki, Yasushi
中科院分区：
文献类型：
--
作者：
Honda, Akira;Yamashita, Kouwa;Matsuzaki, Yasushi
我们描述了一种高度灵敏且特异的方法，用于定量生物样品中关键调节氧甾醇。该方法基于液相色谱-串联质谱 (LC-MS/MS) 的稳定同位素稀释技术。将人血清（5μl）或大鼠肝微粒体（1mg蛋白质）碱解后，提取氧甾醇，衍生化为吡啶甲酰酯，并使用电喷雾电离模式通过LC-MS/MS进行分析。 4β-羟基胆固醇、7α-羟基胆固醇、22R-羟基胆固醇、24S-羟基胆固醇、25-羟基胆固醇、27-羟基胆固醇和 24S、25-环氧胆固醇的吡啶甲酰酯的柱上检测限为 2-10 fg (5-25 amol)（信噪比 = 3）。这些氧甾醇的重现性和回收率分别根据单向布局和多项式方程进行了验证。计算出样品制备之间和通过该方法测量之间的差异分别为1.8%至12.7%和2.9%至11.9%。使用掺有0.05 ng至12 ng氧甾醇的大鼠肝微粒体进行回收实验，氧甾醇的回收率范围为86.7%至107.3%，平均回收率为100.6%。该方法为少量生物样品中氧甾醇的定量提供了可重复且可靠的结果。 Honda, A.、K. Yamashita、T. Hara、T. Ikegami、T. Miyazaki、M. Shirai、G. Xu、M. Numazawa 和 Y. Matsuzaki。通过 LC-ESI-MS/MS 对生物样品中的关键调节氧甾醇进行高灵敏度定量。 J.脂质研究。 2009。50：350-357。
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 mu l) 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 4 beta-hydroxycholesterol, 7 alpha-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. Honda, A., K. Yamashita, T. Hara, T. Ikegami, T. Miyazaki, M. Shirai, G. Xu, M. Numazawa, and Y. Matsuzaki. Highly sensitive quantification of key regulatory oxysterols in biological samples by LC-ESI-MS/MS. J. Lipid Res. 2009. 50: 350-357.