Simultaneous quantification of 4-hydroxytamoxifen and hesperidin in liposomal formulations: Development and validation of a RP-HPLC method

Abstract

Breast cancer treatment options are diverse, with tamoxifen commonly used as a selective estrogen receptor modulator (SERM) for hormone receptor-positive breast cancer. However, tamoxifen can have adverse systemic effects. Local transdermal therapy offers a potential solution by delivering the drug directly to the breast and minimizing systemic exposure. Hesperidin, a flavonoid, exerts synergistic effects when combined with anticancer agents. This combination therapy may be a more effective approach to breast cancer management. Analytical methods have been developed to quantify 4-Hydroxytamoxifen (4-HT) and hesperidin separately; however, no method currently exists for their simultaneous quantification in pharmaceutical formulations. This study aimed to develop and validate a reverse-phase high-performance liquid chromatography (RP-HPLC) method for the simultaneous quantification of 4-HT and hesperidin in liposomal formulations. A Design of Experiments (DoE) approach was employed using a Box-Behnken design (BBD) to optimize the RP-HPLC method. BBD allowed for a reduction in the number of required tests by creating a statistical model to estimate the significance of various factors and interactions. The methanol concentration, flow rate, and injection volume were considered as independent variables for optimization. A mobile phase (90:10 ratio of methanol: 0.1% v/v orthophosphoric acid) with a flow rate of 0.4 mL/min, and an injection volume of 10 μL was selected as optimized chromatographic condition. 4-HT showed a retention time (Rt) of 5.05 min and hesperidin showed an Rt of 7.11 min using an optimized analytical method and was detected at 275 nm. The developed RP-HPLC method was validated according to the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines, confirming its accuracy, precision, linearity, selectivity, and robustness. The validated method was then successfully applied to determine the entrapment efficiency and permeation of 4-HT and hesperidin into loaded liposomes. This study fills a gap in the literature by providing a simple and reliable RP-HPLC method for the simultaneous quantification of 4-HT and hesperidin in liposomal formulations.