High-performance liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry determination of cholesterol uptake by Caco-2 cells

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).