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

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 concentration range of 5-45 μg/mL, with an LOD of 0.773 μg/mL. Moreover, a theoretical investigation of the relationship between the stationary phase and the studied molecules was performed to confirm the experimental results. The proposed method was successfully applied for the selective determination of PAP in pharmaceutical formulation. In addition, the obtained results were statistically compared to a reported method, with no significant differences found between the investigated method and the reported one with respect to accuracy and precision.