Development of Stable Isotope Dilution Assays for the Analysis of Natural Forms of Vitamin B12 in Meat

The first multiple stable isotope dilution assay method was developed for the simultaneous determination of four cobalamins, namely, hydroxocobalamin (OHCbl), adenosylcobalamin (AdoCbl), methylcobalamin (MeCbl), and cyanocobalamin (CNCbl), in their native forms. The sample preparation was optimized with enzyme treatment and immunoaffinity purification. The analysis was performed by LC-MS/MS using respective¹⁵N-labeled cobalamins as internal standards. Method validation resulted in limits of detection ranging from 0.19 to 0.58 ng/g and limits of quantification ranging from 0.68 to 1.73 ng/g. Recoveries at three levels were between 82 and 121%. Intra-day and inter-day precisions were below 6% and 11% RSD, respectively. The analysis of a reference material resulted in a variance of <1% from the certified value. The newly developed method demonstrated excellent sensitivity, recovery, accuracy, and reproducibility and was further applied to quantitate the four cobalamins in various meats.