Protocol for the analysis of phenolic compounds using nano-liquid chromatography-mass spectrometry and Caco-2 assays: from the evaluation of the uptake to the enterocyte metabolism

Abstract

When using the Caco-2 model, the analysis of phenolic compounds may be a challenge for those presenting low uptake rates, as well as the determination of cellular accumulation or metabolism is not an easier task. In this sense, the combination of nano-liquid chromatography (nano-LC) with high resolution mass spectrometry (MS) is a potential analytical alternative. Here we provide a nano-LC-MS protocol for analysing phenolic compounds in samples from Caco-2 assays that could be applied routinely. On the one hand, the samples from transport experiments are simply acidified. On the other hand, trace metabolites at cellular levels can be detected after a simple step of preconcentration. As an example, the absorption and enterocyte metabolism of N-feruloyltyramine is determined. Our outcomes show that the mass accuracy using lock-mass calibration is satisfactory in order to measure accurate m/z values and obtain the molecular formula of N-feruloyltyramine metabolites. For quantitative purposes, the method is adequate in terms of linearity, repeatability, and sensitivity. Consequently, the application of this global methodology may provide clues to understand the physiological distribution and mechanism of action of phenolic compounds.