Development of an HPLC-FLD Method for Determination of Rhodamine-123 in Caco-2 Cell-Based Permeability Studies

Abstract

P-glycoprotein (P-gp) overexpression and its efflux pump function have been associated with chemotherapy failure in many cancers. Many cytotoxic agents have been P-gp substrates. Thus, it was essential to develop a simple and validated method to determine the P-gp substrate in the presence of P-gp inhibitors. In this study, the HPLC-FLD method was developed combined with Caco-2 cell-based permeability studies. First, we developed the HPLC method to quantify Rhodamine-123 (Rho-123) in transport media. It was validated to be sensitive, precise, accurate, linear and specific. Then, the method was employed to determine the Rho-123 concentration in the presence or absence of two known P-gp inhibitors, verapamil and tetrandrine, in a Caco-2 transepithelial transport experiment. The results showed that the two P-gp inhibitors decreased the secretory and increased the absorptive transport of Rho-123. It illustrated that the HPLC-FLD method for determination of Rho-123 in the presence of two P-gp inhibitors in Caco-2 cell-based permeability studies was accurate and reliable.