Abstract
A comprehensive UPLC-MS/MS method was developed and validated for the simultaneous separation and quantification of atenolol, propranolol, quinidine, and verapamil, using established intestinal permeability standards in the Caco-2 cell monolayer model. This in vitro model is widely accepted for predicting intestinal drug permeability and is formally recognized by global regulatory agencies, including the FDA, EMA, and WHO, as a surrogate for assessing drug permeability in biowaiver applications under the Biopharmaceutics Classification System (BCS) framework. Despite its regulatory importance, standardized methods for the simultaneous quantification of key permeability markers remain scarce. The selected compounds represent distinct transport pathways: paracellular (atenolol), passive transcellular (propranolol, verapamil), and P-glycoprotein-mediated efflux (quinidine). Method validation followed FDA guidelines and demonstrated high selectivity, linearity (r(2) > 0.998), precision, and accuracy. Solid-phase extraction enhanced recovery and reduced matrix effects. Application to Caco-2 permeability assays confirmed expected transport profiles, including P-gp inhibition effects with verapamil. By integrating multiple analytes in a single workflow, the method improves analytical throughput, supports mechanistic interpretation, and ensures consistency across assays. This advanced separation strategy, combined with sensitive mass spectrometric detection, supports regulatory and BCS-based classification studies, contributing to the standardization of permeability assessments in drug development.