Innovative High-Performance Liquid Chromatography with a Diode Array Detector Method for the Simultaneous Analysis of Thiamine, Folic Acid, and Niacin in Fortified Rice for Monitoring in Fortification Programs: Development and Validation

Abstract

Water-soluble vitamins such as thiamine, niacin, and folic acid are added to rice in Costa Rica as part of fortification programs to combat nutrient deficiencies. To assess the effectiveness of fortification, two technologies─fortified-coated and fortified-extruded kernels─were analyzed using Scanning electron microscopy (SEM). SEM revealed significant differences in starch structure between the two technologies, with gel and flour properties influenced by amylose, amylopectin, and protein concentrations. A phosphate buffer extraction method was used, followed by RP-HPLC-DAD analysis to evaluate vitamin content. The method showed reliability, with favorable system suitability test results, accurate linearity, and detection limits for folic acid, thiamine, nicotinamide, and nicotinic acid. Trueness assessments confirmed no significant differences from reference material analysis, and precision metrics met AOAC criteria, with acceptable HorRat values for repeatability and reproducibility. Recovery rates and homogeneity tests were within the 80–110% range. The RP-HPLC-DAD method was validated as effective for determining folic acid, thiamine, and niacin levels in fortified rice. From February 2021 to July 2023, 63% of samples met the national standards for folic acid, and 66% met thiamine standards. Continued monitoring is essential to ensure that fortified rice meets public health objectives and addresses micronutrient deficiencies.