Tian Qingguo, Failla Mark L, Bohn Torsten, Schwartz Steven J
Department of Food Science and Technology, The Ohio State University, Columbus, OH 43210, USA.
Rapid Commun Mass Spectrom. 2006;20(20):3056-60. doi: 10.1002/rcm.2700.
A simple, sensitive and selective liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometric method (LC/APCI-MS/MS) was developed and applied to quantitative determination of uptake of cholesterol by Caco-2 human intestine cells. Caco-2 cells were cultured in medium containing cholesterol-3,4-13C2 and phytosterols from nutritional supplements after in vitro digestion. Cellular cholesterol (cholesterol-3,4-13C2) and endogenous cholesterol were extracted using methanol/chloroform (1:2, v/v) and directly analyzed using LC/APCI-MS/MS with selected reaction monitoring (SRM), using cholesterol-2,2,3,4,4,6-d6 as an internal standard. Detection and quantification limits were 2.2 and 7.2 pmol, respectively. This method provides an effective tool for rapid determination of cholesterol uptake by cells with increased selectivity and sensitivity in comparison to previously reported LC/APCI-MS analysis using selected ion monitoring (SIM).
建立了一种简单、灵敏且具选择性的液相色谱/大气压化学电离串联质谱法（LC/APCI-MS/MS），并将其应用于定量测定Caco-2人肠细胞对胆固醇的摄取。Caco-2细胞在体外消化后，培养于含有胆固醇-3,4-13C2和来自营养补充剂的植物甾醇的培养基中。使用甲醇/氯仿（1:2，v/v）提取细胞胆固醇（胆固醇-3,4-13C2）和内源性胆固醇，并使用胆固醇-2,2,3,4,4,6-d6作为内标，通过选择反应监测（SRM）模式的LC/APCI-MS/MS直接进行分析。检测限和定量限分别为2.2和7.2 pmol。与先前报道的使用选择离子监测（SIM）的LC/APCI-MS分析相比，该方法为快速测定细胞对胆固醇的摄取提供了一种有效的工具，具有更高的选择性和灵敏度。
