John Clara, Werner Philipp, Worthmann Anna, Wegner Katrin, Tödter Klaus, Scheja Ludger, Rohn Sascha, Heeren Joerg, Fischer Markus
Department of Biochemistry and Molecular Cell Biology, University Medical Center Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, Germany.
Hamburg School of Food Science, Institute of Food Chemistry, University of Hamburg, Grindelallee 117, 20146 Hamburg, Germany.
J Chromatogr A. 2014 Dec 5;1371:184-95. doi: 10.1016/j.chroma.2014.10.064. Epub 2014 Oct 29.
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 (R2) 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.
最近，羟基甾醇和胆汁酸作为能量稳态和炎症的重要调节因子，越来越受到关注。由于存在大量不同的羟基甾醇和胆汁酸种类，因此需要强大的分析工具来定量这些结构和化学性质相似的分析物。在此，我们介绍一种基于液相色谱 - 串联质谱（LC-MS/MS）的方法，用于快速定量34种甾醇（羟基甾醇、初级、次级胆汁酸及其牛磺酸和甘氨酸共轭物）。使用坚固的酰胺嵌入C18（极性嵌入）固定相可实现异构体羟基甾醇和胆汁酸的色谱基线分离。当前方法具有简单的提取方案，该方案已在血浆、尿液、胆囊、肝脏、粪便和脂肪组织中得到验证，避免了固相萃取以及衍生化程序。代表性分析物在血浆中的总提取回收率在58 - 86%之间，尿液中为85%，肝脏中为79 - 92%，脂肪组织中为76 - 98%，粪便中为93 - 104%，胆囊中为62 - 79%。验证程序表明，97%的分析物在选定浓度范围内校准曲线呈线性，计算得到的决定系数（R2）大于0.99。一项对野生型小鼠进行的标准饲料和富含胆固醇的西式饮食喂养研究表明，本文所述方案提供了一种强大的工具，可同时定量代谢活跃组织中的胆固醇衍生物和胆汁酸，并追踪肠肝循环。
