A Validated Stability-indicating RP-HPLC Method for Piperine Estimation in Black Pepper, Marketed Formulation and Nanoparticles

Aim: The aim of the study was to develop and validate a simple stability indicating reversephase High Performance Liquid Chromatography (RP-HPLC) method for quantitative analysis of piperine in Ayurvedic marketed formulation, black pepper and cubosome nanoformulation. Methods: The method was established by using Luna C18 HPLC column using a mobile phase consisting of acetonitrile: 0.01% ortho phosphoric acid (60:40, v/v; pH 3), delivered isocratically with flow rate of 1 mL/min and detected at 340 nm. The validation of chromatographic parameters and stress testing were performed in accordance with International Conference on Harmonization (ICH) guidelines. Results: The developed method was observed to be specific, linear (r2 > 0.999) over the selected range of concentration 0.5 to 20 μg/mL, precise (percentage relative standard deviation < 2%), with the detection and quantification limit as 0.015 and 0.044 μg/mL respectively. The relevancy of the developed method was analyzed on the piperine entrapped cubosome nanoformulation, which was formulated by fragmentation technique. The entrapment efficiency of piperine for prepared cubosome was observed to be 87.01 %. The method was implemented for the estimation of piperine in black pepper. The concentration of piperine in marketed formulation was found to be similar with the labeled concentration. The analyte peak was found to be complete resolved without any interference of additives and degrading products. Conclusion: The validated method was observed to be specific, sensitive and sufficient for the routine analysis of food products, marketed formulations and nanoparticles containing piperine.