Development and Validation of a Cost-Effective and Sensitive UHPLC-ESI-MS/MS Method for Vitamin B12 Determination in Yogurt

The accurate determination of vitamin B12 in fortified food products presents considerable analytical challenges due to its low concentrations (μg kg –1) and its binding to proteins in dairy products. This study aimed to develop a reliable and accurate method for determining vitamin B12 in yogurts that offers a robust, sensitive, and accurate tool for the routine quantification of vitamin B12 in complex dairy matrices, contributing to nutritional assessment and quality control in the food industry. Therefore, sample preparation including hydrolysis at high temperatures in an excess of cyanide, and utilization of SPE cartridges for analyte purification and preconcentration is essential. UHPLC-ESI-MS/MS analysis was conducted using a C18 column in positive ionization mode (ESI+). The experimental procedure was meticulously optimized, examining extraction conditions, such as the solvent used, the extraction time, and the effectiveness of SPE cartridges. The optimized method demonstrated excellent linearity (R2 > 0.990), high recoveries (e.g., 82.7–96.6%), good precision (RSD < 20%), and adequate sensitivity (LOD: 0.38 μg kg −1 and LOQ: 1.1 μg kg −1). The method was successfully validated according to the Eurachem Guide, evaluated for uncertainty and corroborated through successful interlaboratory testing in cereal and milk matrices.