Application of QbD based approach in development and validation of RP-HPLC method for simultaneous estimation of Methotrexate and Baicalin in Dual-Drug Loaded Liposomes.

The present study delineates the development of a novel rugged and sensitive stability indicating risk-based HPLC method for the concurrent estimation of methotrexate (MTX) and baicalin (BCL) 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, % orthophosphoric acid content and % acetonitrile content that influences critical quality attributes. Box-Behnken design (BBD) was utilized for the optimization of the tailing factor as response for methotrexate and baicalin 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 optimized zone within the confines of design space, were chosen as mobile phase water: acetonitrile adjusted to pH 3.0 with 0.05% orthophosphoric acid (60:40, %v/v), flow rate 1.0 ml/min using an analytical column C 18 at an isobestic wavelength of 282 nm. Furthermore, the validation of the optimized method was done in accordance with ICH guidelines and were reckoned to be in the prescribed limits. Thereby the developed RP-HPLC method has high degree of practical utility for synchronous detection of methotrexate and baicalin in pharmaceutical nano-dosage forms such as protein-based-nanoparticles, nanocrystals, polymeric-nanoparticles, metallic-nanoparticles, etc. in in vivo and invitro studies.