Application of an HPLC Method for Selective Determination of Phenazopyridine Hydrochloride: Theoretical and Practical Investigations

Abstract

Abstract HPLC method was developed for the selective determination of phenazopyridine hydrochloride (PAP) in the presence of its computationally selected metabolite. Density functional theory was applied as a computational model to study the energy of PAP metabolites, and the results revealed that 2,3,6-triaminopyridine (TAP) is the most stable metabolite. Good resolution and separation of PAP from TAP was achieved using a reversed-phase BDS Hypersil C18 column with a mobile phase consisting of acetonitrile–water (75 + 25, v/v) at flow rate of 1 mL/min and with UV detection at 280 nm. The linear regression analysis data for the calibration plot of PAP showed a good linear relationship over the concentrationrange of 5–45 μg/mL, with an LOD of 0.773 μg/mL. Moreover, a theoretical investigation of the relationship between the stationary phaseand the studied molecules was performed to confirm the experimental results. The proposed method was successfully applied for the selective determination ofPAP in pharmaceutical formulation. In addition, the obtained results were statistically compared to a reported method, with no significant differences foundbetween the investigated method and the reported onewith respect to accuracy and precision.