Derivatization-independent cholesterol analysis in crude lipid extracts by liquid chromatography/mass spectrometry: Applications to a rabbit model for atherosclerosis
Derivatization-independent cholesterol analysis in crude lipid extracts by liquid chromatography/mass spectrometry: Applications to a rabbit model for atherosclerosis
复制标题
DOI：
10.1016/j.chroma.2011.05.011
复制
发表时间：
2011-07-15
影响因子：
4.1
通讯作者：
Wenk, Markus R.
中科院分区：
文献类型：
--
作者：
Shui, Guanghou;Cheong, Wei Fun;Wenk, Markus R.
从有限的生物样本中直接测量粗脂提取物中的各种甾醇在一次实验中是具有挑战性的。目前基于质谱学(MS)的方法通常需要在进行MS分析之前进行化学衍生化。在这里，我们提出了一种不依赖于衍生化的方法来分析各种甾醇，包括胆固醇及其同系物，使用液相色谱和大气压化学电离质谱仪。基于胆固醇的特定串联质谱图，多反应监测(MRM)跃迁被用来定量游离胆固醇及其脂肪酸酯。几种胆固醇氧化产物也可以使用前向液相色谱分离和特定的MRM转变来测量。在测量总胆固醇方面，该方法与已建立的酶分析方法一起得到了验证。作为概念的验证，我们分析了给予高胆固醇饮食(HCD)的兔的血浆类固醇，这是一种经典的动脉粥样硬化模型。HCD组兔血浆中游离胆固醇、胆固醇酯、7-羟基胆固醇和7-酮胆固醇升高。该方法也可以作为定量分析哺乳动物以外其他生物中的其他甾醇，如麦角固醇和谷甾醇的一种很好的工具。在酿酒酵母中，我们的结果表明，在yeh2 Delta和tgl1 Delta细胞中，麦角甾醇酯与游离麦角甾醇的比率都显著增加，这与各自酶的功能一致。(C)2011爱思唯尔B.V.保留所有权利。
Direct measurement of various sterols in crude lipid extracts in a single experiment from limited biological samples is challenging. Current mass spectrometry (MS) based approaches usually require chemical derivatization before subjecting to MS analysis. Here, we present a derivatization-independent method for analyzing various sterols, including cholesterol and its congeners, using liquid chromatography and atmospheric pressure chemical ionization mass spectrometry. Based on the specific tandem mass spectrometry pattern of cholesterol, multiple reaction monitoring (MRM) transitions were used to quantify free cholesterol and its fatty acyl esters. Several cholesterol oxidation products could also be measured using the upfront liquid chromatography separation and specific MRM transitions. The method was validated alongside established enzymatic assays in measuring total cholesterol. As a proof of concept, we analyzed plasma sterols in rabbits administrated with a high cholesterol diet (HCD) which is a classical atherosclerotic model. Free cholesterol, cholesterol esters, 7-hydroxycholesterol, and 7-ketocholesterol were elevated in plasma of rabbits on HCD. This method could also serve as an excellent tool for quantitative analysis of other sterols such as ergosterol and sitosterol in other organisms beside mammalian. In Saccharomyces cerevisiae, our results indicated dramatic increases of the ratio of ergosterol esters to free ergosterol in both yeh2 Delta, and tgl1 Delta, cells, which are consistent with the function of the respective enzymes. (C) 2011 Elsevier B.V. All rights reserved.