Analytical Validation Study with Uncertainty Evaluation of the Method for Quantitative Determination of Hesperidin in the Extracted Product and Mandarin Processing Waste

The aim of this study was to develop and validate an alternative, selective, reproducible, and high-yield extraction-based method by ultrasound-assisted technique combined with an effective, specific, sensitive, and rapid analytical HPLC procedure for obtaining hesperidin from mandarin (Citrus unshiu) peel as waste material in citrus processing industries and its quantitative determination in the obtained extracted product and citrus waste materials as well. The evaluation of the uncertainty of the developed method is also described in the present work. The proposed method is focused not only on obtaining hesperidin but also provides the possibility for obtaining other valuable bioactive compounds within the single stepwise extraction process. The proposed method was validated concerning the following validation parameters: robustness, system suitability test, specificity, linearity range, precision, accuracy, sensitivity, limits of quantification (LOQ), and detection (LOD). A design of experiments using the Placket-Burman approach was used for the robustness study of the combined method. The measurement uncertainty of the proposed method was evaluated based on the four-step process using a combination of two appropriate - bottom-up and top-down approaches using the method validation data. The LOD and LOQ of the analytical procedure were 0.00001 mg/mL and 0.000025 mg/mL, respectively. The calibration curve (0.000025-0.5 mg/mL) is linear and the square of the correlation coefficient is equal to 0.99992. The determined average amount of hesperidin in the dry citrus peel samples is equal to 35.36 ± 3.14 mg/g (k=2, 95 % level of confidence). The mean recovery of the combined method is 91.48 %. The purity of the dry-extracted product of hesperidin is not less than 90 %.