DUAL-DRUG QUANTIFICATION: HPLC METHOD VALIDATION FOR HESPERIDIN AND PIPERINE IN ETHOSOMAL DELIVERY SYSTEMS

Objective: The current study aims to develop and validate a simple, accurate, precise, and robust Reverse Phase-High Performance Liquid Chromatography (RP-HPLC) method to estimate hesperidin and piperine in an ethosomal formulation simultaneously. This method is intended to support the characterization of a novel ethosomal formulation designed to enhance the transdermal delivery of hesperidin and piperine. Methods: Chromatographic separation was done using a C18 column (250×4.6 mm) featuring a 5 μm particle size, maintained at 25 °C) with a Photodiode Array (PDA) detector. The mobile phase consisted of a mixture of methanol, Acetonitrile (ACN), and water (70:20:10 v/v, pH 3.2). Separation was performed at a flow rate of 0.8 ml/min with a 10 µl injection volume. All the detection was done at an isosbestic wavelength of 291 nm. Results: Hesperidin and piperine were eluted with retention time of 3.15 and 5.40 min, respectively. The developed RP-HPLC method demonstrated excellent accuracy, precision, and linearity for simultaneous quantification of hesperidin and piperine. According to the guidelines, the optimized ethosomal formulation exhibited a particle size of 208.4±0.3 nm, a Polydispersity Index (PDI) of 0.1294, and high entrapment efficiencies of hesperidin: 94.14±0.13%, and piperine: 79.57±0.11%. In vitro drug release of the ethosomal formulation was found to be 87.27±2.56% for hesperidin and 83.95±5.32% for piperine. Conclusion: This HPLC method could be used to measure both hesperidin and piperine simultaneously in ethosomes and in other nanoparticle-based formulations. The optimized ethosomal formulation demonstrated desirable characteristics for transdermal drug delivery, including sustained release property and good entrapment efficiency.