Development and validation of RP-UPLC method for the determination of darifenacin hydrobromide, its related compounds and its degradation products using design of experiments

Abstract

A selective stability-indicating ultra-performance liquid chromatographic (UPLC) method was developed for the quantitative determination of darifenacin hydrobromide (DFN) and its related compounds in API and pharmaceutical dosages. The chromatographic separation was achieved on an Acquity UPLC BEH C18 column (100, 2.1 mm and 1.7 μm) at a flow rate of 0.3 mL/min, and detection was performed at 210 nm. The typical retention behaviors of impurities at various pH values were depicted graphically. The LC conditions were optimized by design of experiments (DOE) to obtain optimal separation in the shortest possible run time. A central composite design (CCD) was employed to study the main effects and interactions of the independent variables. The drug and its thirteen impurities were eluted within 13 min. The method exhibited consistent, high-quality recoveries (93.8 ± 2.1 to 99.8 ± 1.5 (mean ± RSD)) with a high precision for the drug and impurities. Linear regression analysis revealed an excellent correlation between peak responses and concentrations (R(2) values of 0.9991-0.9999) for the drug and impurities. The stability-indicating capability of the method was verified by forced degradation experiments and mass balance study. LC-MS revealed protonated molecular ion peaks [M+H](+) at m/z 428.20, m/z 425.20 and m/z 281.30 for the acid (Imp-4), oxidized (Imp-6) and N-dealkylated (Imp-1) forms of DFN, respectively. Possible degradation pathways were established based on the known reactivity of the drug through hydrolysis, oxidation, N-dealkylation, phenyl hydroxylation, dihydrobenzofuran ring hydroxylation and ring opening. The m/z values of unknown degradation products were matched with the proposed structures and reported DFN metabolites.