Priyal Desai, Niyatee Thakor, Sonal Desai*
Background: HPLC-PDA method was developed using factorial design approach and validated for the estimation of hesperidin in orange peel powder. A 32 factorial design was applied to evaluate the effect of the two factors that are mobile phase composition and flow rate on the various chromatographic responses such as retention time, area, and tailing factor. Chromatographic separation was achieved using Sunniest ECO (250 mm × 4.6 mm, 5 μm) column as a stationary phase and optimized mobile phase consisted of Methanol: water in ratio of 51.5:48.5 v/v at a flow rate of 1.1 mL/min. Detection wavelength was 285 nm. The retention time of hesperidin was found to be 4.01 min.
Results: In validation studies, calibration curve was found to be linear over the range of 0.2-6 μg/mL with the regression coefficient of 0.996. The LOD and LOQ were found to be 0.066 μg/mL and 0.2 μg/mL; respectively. The % recovery of hesperidin was found to be between 96%-105%. The method was found to be precise and robust as the values of % RSD were found to be less than 2.0% for both intraday and interday precision study and in robustness study for hesperidin. The validated method was successfully applied for quantitative determination of hesperidin in dried orange peel powder.
Conclusion: Factorial design assisted HPLC-PDA method was developed and validated for the quantification of hesperidin in orange peel. This method can be used for routine quality control of orange peel powder using hesperidin as marker compound by the herbal manufacturer.