Development and Validation of a High-Performance Liquid Chromatography Method for Hesperidin in Pharmaceutical Formulations

Background: Hesperidin, a bioflavonoid with diverse pharmaceutical applications, necessitates a reliable analytical method for its quantification in formulations. This study developed a straightforward, sensitive, and economical High Performance Liquid Chromatography (HPLC) method to address this need. Aim: To develop an easy, rapid, accurate, precise and reliable analytical method for the hesperidin. Methods: The separation was performed using a C-18 column HSS (250 x 4.0 mm, 5μm particles). The mobile phase used was a mixture of Methanol:Water in ratio of 50:50 v/v isocratic at 40° C, with a flow rate of 0.9 mL/min and an injection volume of 10 μL. Detection was carried out at 280 nm using a PDA detector. Results: The new RP-HPLC technique yielded a correlation value of 0.99 and was demonstrated to be linear in the 10-30 μg/ml range. Hesperidin was shown to have retention period of 4.888 minute. The technique was validated using criteria such as specificity, accuracy, LOD, LOQ, precision, repeatability, robustness. Conclusion: The validated HPLC method shows excellent reliability and reproducibility, making it suitable for routine quantitative analysis of hesperidin in pharmaceutical formulations. This technique is effective for quality control, offering a cost-efficient and rapid approach for the determination of hesperidin. It met validation criteria for specificity, accuracy, and precision, proving to be a reliable and cost-effective tool for routine analysis in pharmaceutical formulations. Major Findings: The newly developed RP-HPLC method for quantifying hesperidin exhibited high linearity (correlation coefficient of 0.9907) within the 10-30 μg/mL range, with a retention time of 4.892 minutes. It met validation criteria proving to be a reliable and cost-effective tool for routine analysis in pharmaceutical formulations.