A liquid chromatography-tandem mass spectrometry-based method for the simultaneous determination of hydroxy sterols and bile acids
A liquid chromatography-tandem mass spectrometry-based method for the simultaneous determination of hydroxy sterols and bile acids
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DOI：
10.1016/j.chroma.2014.10.064
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发表时间：
2014-12-05
影响因子：
4.1
通讯作者：
Fischer, Markus
中科院分区：
文献类型：
--
作者：
John, Clara;Werner, Philipp;Fischer, Markus
近年来，羟基甾醇和胆汁酸受到越来越多的关注，因为它们是能量平衡和炎症的重要调节者。大量不同的羟基甾醇和胆汁酸需要强大的分析工具来量化这些结构和化学上相似的分析物。在这里，我们介绍了一种基于LC-MS/MS的快速定量34种甾醇(羟基甾醇、初级、次级胆汁酸及其牛磺酸和甘氨酸结合物)的方法。使用坚固的酰胺包埋C18(极性包埋)固定相，实现了羟基甾醇和胆汁酸同分异构体的基线分离。目前的方法具有简单的提取方案，适用于血浆、尿液、胆汁、肝脏、粪便和脂肪组织，避免了固相萃取和衍生化过程。血浆、尿液、肝脏、脂肪组织、粪便和胆汁的总提取回收率分别为58-86%、85%、79-92%、76-98%、93-104%和62-79%。验证程序表明，对于97%的分析物，校准曲线在选定的浓度范围内是线性的，计算的决定系数(R-2)大于0.99。用标准饲料和高胆固醇西式饮食喂养野生型小鼠的研究表明，这里描述的方案提供了一个强大的工具，可以同时量化代谢活跃组织中的胆固醇衍生物和胆汁酸，并跟踪肠道-肝脏循环。(C)2014爱思唯尔B.V.保留所有权利。
Recently, hydroxy sterols and bile acids have gained growing interest as they are important regulators of energy homoeostasis and inflammation. The high number of different hydroxy sterols and bile acid species requires powerful analytical tools to quantify these structurally and chemically similar analytes. Here, we introduce a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method for rapid quantification of 34 sterols (hydroxy sterols, primary, secondary bile acids as well as their taurine and glycine conjugates). Chromatographic baseline separation of isomeric hydroxy sterols and bile acids is obtained using a rugged amide embedded C18 (polar embedded) stationary phase. The current method features a simple extraction protocol validated for blood plasma, urine, gall bladder, liver, feces, and adipose tissue avoiding solid phase extraction as well as derivatization procedures. The total extraction recovery for representative analytes ranged between 58-86% in plasma, 85% in urine, 79-92% in liver, 76-98% in adipose tissue, 93-104% in feces and 62-79% in gall bladder. The validation procedure demonstrated that the calibration curves were linear over the selected concentration ranges for 97% of the analytes, with calculated coefficients of determination (R-2) of greater than 0.99. A feeding study in wild type mice with a standard chow and a cholesterol-enriched Western type diet illustrated that the protocol described here provides a powerful tool to simultaneously quantify cholesterol derivatives and bile acids in metabolically active tissues and to follow the enterohepatic circulation. (C) 2014 Elsevier B.V. All rights reserved.