An ultra performance liquid chromatographic method for determining phytosterol uptake by Caco-2 cells

A simple method for the determination of cellular uptake of phytosterols by Caco-2 cells has been developed by ultra performance liquid chromatography with ultraviolet detection (UPLC–UV). UPLC–UV was established using an ODS column, acetonitrile/H2O (9:1, v/v) as a mobile phase, and a detection wavelength at 210 nm. As analytes, β-sitosterol, campesterol, stigmasterol, and brassicasterol were selected based on the abundance in foods and the similarity of their structures. A linear relation was observed between the peak area and the amount of sterol injected from 50 to 2000 pmol (r > 0.999) with a relative standard deviation (RSD) of less than 2.5% (n = 6). This method was applied to the determination of cellular uptake of phytosterols by Caco-2 cells. Recovery tests showed that phytosterols were extracted from the cell lysates by chloroform and determined by UPLC–UV with a recovery rate of more than 80.2% and an RSD of less than 11.3% (n = 3). When Caco-2 cells were incubated with phytosterols at 37 °C, their uptake was increased with time in a concentration-dependent manner. This method will be useful for the simultaneous determination of cellular phytosterols in an in vitro intestine model.