Quality by design assisted a stability indicating RP-HPLC method for the estimation of hesperidin in transfersome and marketed product

Abstract

The present study aimed to develop and validate a Quality by Design (QbD)-assisted simple stability-indicating reverse-phase high-performance liquid chromatography (RP-HPLC) method for the estimation of hesperidin in transfersomes and marketed formulations. This QbD strategic approach is innovative as it allows for optimization with minimal trial and error, making the HPLC method more efficient while enhancing robustness, reliability, and regulatory compliance. Hesperidin is a naturally occurring polyphenolic flavonoid widely distributed in citrus species, including oranges and lemons. Due to its extensive and attractive therapeutic potential, an efficient, accurate, and specialized HPLC technique was developed and validated according to ICH guidelines. A central composite design was employed for the analysis, offering the advantage of using a reduced number of design points and experimental runs. Using a C18 column (4.6 mm × 250 mm, 5 μm) and acetonitrile (0.1 % orthophosphoric acid) (70:30) as the mobile phase, chromatographic separation was performed at a wavelength of 284 nm and a flow rate of 1 ml/min. Hesperidin peaked around 4.6 min using the innovative analytical method. The method was found to be linear over a concentration range of 0.5–16 μg/ml. The developed method was robust, precise, and accurate, with a 98 % recovery. The limit of detection (LOD) was 0.453 μg/ml, and the limit of quantification (LOQ) was 1.375 μg/ml, with a relative standard deviation (RSD) of less than 2 %. Moreover, forced degradation studies of hesperidin were conducted, and the relevance of the developed method was evaluated on transfersome nanoformulations and marketed formulations, demonstrating its versatility and broad applicability. The hesperidin peak was well separated in the presence of degradation products. It was found that the developed method was adequate, sensitive, and specific. This research provides a comprehensive and innovative approach to hesperidin estimation, which can significantly impact quality control and formulation development.