Development of Pretreatment Method for Analysis of Vitamin B12 in Cereal Infant Formula using Immunoaffinity Chromatography and High-Performance Liquid Chromatography

Abstract Vitamin B12 deficiency may lead to serious health issues in both infants and adults. A simple analytical method involving sample pretreatment with enzyme, followed by cyanide addition under acidic conditions; separation on an immunoaffinity column; and high-performance liquid chromatography (HPLC) was developed for the rapid detection and quantitation of vitamin B12 in powdered milk. Detection limit and powdered milk recovery were determined by quantitative analysis. The limits of detection and quantitation were 2.71 and 8.21 μg/L, respectively. Relative standard deviations of the intra-day and inter-day precisions varied in the ranges of 0.98%–5.31% and 2.16%–3.90%, respectively. Recovery of the analysis varied in the range of 83.41%–106.57%, suggesting that the values were acceptable. Additionally, vitamin B12 content and recovery in SRM 1849a were 54.10 μg/kg and 112.24%, respectively. Our results suggested that the analytical method, including the sample pretreatment step, was valid. This analytical method can be implemented in many laboratory-scale experiments that seek to save time and labor. Therefore, this study shows that immunoaffinity–HPLC/ultraviolet is an acceptable technique for constructing a reliable database on vitamin B12 in powdered milk containing starch as well as protein and/or fat in high amounts.