A simple, sensitive and specific reverse-phase high performance liquid chromatographic (RP-HPLC) method with UV detection was developed and validated for simultaneous analysis of caffeine, paracetamol and sulfasalazine, markers of high, medium and low intestinal permeability, respectively. The method was used to determine their apparent permeability coefficient (Papp) using rat everted gut sac technique. Chromatography was carried out on C-18 column with mobile phase comprising of ammonium

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... te buffer (0.01 M, pH 5.0) and acetonitrile gradient pumped at a flow rate of 0.5 ml min −1. The retention times for caffeine, paracetamol and sulfasalazine were 8.85 0.02 min, 7.8 0.02 min and 12.2 0.05 min, respectively. Method validation parameters included specificity, accuracy, precision, sensitivity and stability studies. The applicability and reliability of the analytical method was evaluated by analyzing samples obtained from the rat everted gut sac experiment. Papp values obtained for the marker compounds, studied separately or in a cassette, correlated well with human fraction absorption, enabling their classification as high, medium and low permeable compounds. In conclusion, the developed RP-HPLC method for permeability markers caffeine, paracetamol and sulfasalazine can be used for high throughput cassette validation of rat everted gut sac model for intestinal permeability assessment.