Application of a Quality by Design-based approach in development and validation of an RP-HPLC method for simultaneous estimation of methotrexate and mangiferin in dual drug-loaded liposomes.

Abstract

The present study delineates the development of a novel rugged and sensitive stability-indicating risk-based HPLC method for the concurrent estimation of methotrexate and mangiferin in dual drug-loaded nanopharmaceuticals based on an analytical QbD approach. Preliminary screening trials along with systemic risk analysis were performed, endeavouring to explicate the critical method attributes, namely pH, percentage orthophosphoric acid content and percentage methanol content, that influence critical quality attributes. Box-Behnken design was utilized for the optimization of the tailing factor as response for methotrexate and mangiferin in short run time. The chromatographic conditions were optimized by performing 17 experimental runs acquired from Design-Expert software. The chromatographic conditions after the analysis of an optimized zone within the confines of the design space were chosen as mobile phase water-methanol adjusted to pH3.0 with 0.05% orthophosphoric acid (65:35, v/v) and flow rate 1.0ml/min using a C18 analytical column at an isosbestic wavelength of 265 nm. Furthermore, the validation of the optimized method was done in accordance with International Conference on Harmonization guidelines and were reckoned to be in the prescribed limits. The developed RP-HPLC method has a high degree of practical utility for synchronous detection of methotrexate and mangiferin in pharmaceutical nano-dosage forms such as protein-based-nanoparticles, nanocrystals, polymeric nanoparticles and metallic nanoparticles in in vivo and in vitro studies.