Development and greenness assessment of a stability-indicating HPLC-DAD method for simultaneous determination of allopurinol and benzbromarone

The growing interest in Green Analytical Chemistry (GAC) principles through the replacement of polluting analytical procedures with greener ones, has encouraged us to develop an eco-friendly stability-indicating HPLC with diode array detection method (HPLC-DAD) for simultaneous determination of allopurinol (ALP) and benzbromarone (BNZ). Effective separation was accomplished using Durashell C18 column (4.6 x 250 mm, 5 mu m particle size) with gradient elution of the mobile phase composed of 0.02 M ammonium acetate (pH 5.0) and methanol. Quantification of ALP and BNZ was based on measuring their peak areas at 251 nm. ALP and BNZ peaks eluted at retention times 4.85 and 10.30 min respectively. The proposed HPLC procedure was carefully validated in terms of system suitability, linearity, ranges, precision, accuracy, specificity, robustness, detection, and quantification limits. The linearity range for both ALP and BNZ was 5-100 mu g/mL with correlation coefficients >0.9999. Forced degradation conditions of neutral, acidic, and alkaline hydrolysis, oxidation, and thermal degradation were applied on both drugs. Good resolution of the drugs from their forced degradation products proved that the proposed method is stability-indicating. In addition, the resolution of both drugs from about 10 pharmacologically or chemically related pharmaceutical compounds of different medicinal categories showed the high specificity of the proposed method. The validated HPLC method was successfully applied to the simultaneous determination of both drugs in their tablet dosage forms. Furthermore, greenness assessment and comparison with previously published methods were carried out using two different GAC metrics, namely, the national environmental method index (NEMI) and the analytical Eco-Scale.