Development and Validation of Normal Phase HPLC Method for Determination of Vitamin A (Retinyl Palmitate) in Fortified Rice Kernels (FRKs)

Fortifying rice with vitamin A represents a crucial step toward addressing nutritional deficiencies in Bangladesh. The increasing interest in food enrichment necessitates straightforward and precise methods for analyzing fortified products. This study introduces a simple and reliable approach based on normal-phase high-performance liquid chromatography (HPLC) analysis to quantify retinyl palmitate in fortified rice kernels. The method was validated in accordance with the International Conference on Harmonization guidelines. Analysis was conducted using a DB silica column (250 cm × 4.6 mm; 5 μm), with a mobile phase consisting of n-hexane and isopropyl alcohol in a 96:04 v/v ratio and a flow rate of 1.0 mL/min. Detection occurred at a wavelength of 326 nm, with a retention time for vitamin A of approximately 3 min and a total run time of 5 min. The calibration plot exhibited a linear relationship (R² = 1.00) across the concentration range of 0.48–30.80 μg/mL. The limit of detection (LOD) and limit of quantification (LOQ) were 0.015 and 0.051 μg/mL for standard and 0.35 and 1.07 μg/mL for spiked samples, respectively. Accuracy, determined through recovery studies, showed a mean recovery rate of over 90%. The relative standard deviation (RSD%) for intraday and interday precision was found to be less than 2%. Robustness and solution stability results were within acceptable limits. The proposed method, meeting all validation criteria including linearity, accuracy, precision, and robustness, offers a rapid and cost-effective solution for routine quality control of vitamin A-fortified rice, with potential to support food fortification monitoring in resource-limited settings.