Simultaneous LC−MS/MS Determination of Reference Pharmaceuticals as a Method for the Characterization of the Caco-2 Cell Monolayer Absorption Properties

Since its introduction a decade ago, the Caco-2 in vitro model for testing intestinal permeability has found wide application, in particular for screening new molecules in the pharmaceutical industry. An important issue for the validation of the model is to verify integrity and proper functionality of the Caco-2 cells monolayer, to ensure reproducibility and consistency of results. Several of these methods are based on measuring the apparent permeability coefficients (Papp) of well-characterized reference compounds, having known absorption characteristics, and comparing the observed values to those expected. Most of them use fluorescent or radioactive chemicals to test different parameters. The main limitation of such approaches is that each parameter to be tested is associated with a single specific method, so that multiple procedures are needed to adequately characterize the cell monolayer. This work describes the use of a unique LC-MS/MS method to simultaneously determine the Papp of a set of reference substances having well-characterized absorption behavior, i.e., phenylalanine, atenolol, and propranolol. The method is routinely used in our laboratory to check on model reproducibility and proved useful in verifying the consistency of the results derived from the experiments.