DoE-based validation of a HPLC-UV method for quantification of rotigotine nanocrystals: Application to in vitro dissolution and ex vivo nasal permeation studies.

The current work is focused on optimization, development, and validation of a sensitive and specific reversed-phase high-performance liquid chromatography (RP-HPLC) method for the estimation of rotigotine (RTG) in bulk and nanoformulations. The RP-HPLC method was effectively optimized using the concepts of design of experiments. Critical method variables (CMVs) were screened using Plackett-Burman design. Box-Behnken, a surface response methodology-based design, was further used for the optimization of CMVs with the number of theoretical plates and retention time (min) as responses. The optimized chromatographic conditions for the RP-HPLC method were: acetonitrile proportion: 54% v/v, pH of buffer: 5.0 (10 mM), and flow rate: 0.65 mL/min. The number of theoretical plates and retention time in the study were found to be 11206 and 7.65 min, respectively. The developed method exhibited good linearity (R = 0.9995) within a range of 25-600 ng/mL and LOD and LOQ were found to be 9 and 12 ng/mL, respectively. The developed RP-HPLC method was found sensitive, accurate, precise, specific, robust, and stability indicating according to the regulatory guidelines. The validated method was efficiently applied for in vitro dissolution study, ex vivo nasal permeation study, and estimation of drug content of RTG nanocrystals.