Development and validation of stability indicating UPLC method for the simultaneous determinationof beta-blockers and diuretic drugs in pharmaceutical dosage forms

With the objective of reducing analysis time and maintaining good efficiency, there has been substantial focus on high-speed chromatographic separations. In this work, rapid, precise and specific stability indicating ultra performance liquid chromatography (UPLC) method has been developed and validated for the simultaneous determination of beta-blockers and diuretic drugs in pharmaceutical formulations. The chromatographic separations of all the drugs were achieved on a Waters Acquity BEH C18, 50×2.1 mm, 1.7 µm UPLC column within a short runtime of 3.3 min. The newly developed method was validated according to the ICH guidelines with respect to specificity, linearity, accuracy, precision and robustness. Forced degradation studies were also performed for all the drug samples to demonstrate the stability indicating power of the developed UPLC method. Two unknown degradants were detected in the alkaline degradation of Amiloride hydrochloride with sodium hydroxide having not been reported previously. The structure of the degradants was determined using a combination of UPLC/MS and mechanistic chemistry. This degradant result from destruction of the guanidine moiety of amiloride hydrochloride. The two impurities were characterized as 3,5-diamino-6-chloropyrazine-2-carboxylic acid (impurity A) and 3-chloropyrazine-2,6-diamine (impurity B), respectively. Comparison of system performance with conventional HPLC was made with respect to analysis time, efficiency and sensitivity.