A Cyanide-Free UHPLC-MS/MS Workflow for the Analysis of Major Cobalamin Vitamers in Foods

Accurate determination of cobalamin vitamers in foods remains analytically challenging because conventional cyanidation-based methods convert native cobalamins into cyanocobalamin (CNCbl) and may distort their original distribution. In this study, a cyanide-free UHPLC-MS/MS workflow was developed for the analysis of major cobalamin vitamers in foods, with particular emphasis on preserving native forms during sample preparation. Light, temperature, and cleanup procedures were systematically evaluated. Methylcobalamin (MeCbl) and adenosylcobalamin (AdoCbl) showed pronounced light sensitivity, whereas red-light handling better preserved vitamer integrity during pre-analytical operations. A tandem cleanup procedure combining immunoaffinity and Oasis HLB solid-phase extraction improved extract cleanliness in complex food matrices. The workflow showed good chromatographic separation and excellent linearity (R² > 0.999). The validated limits of detection were 0.5 μg/kg for CNCbl, 1.0 μg/kg for AdoCbl, and 0.75 μg/kg for MeCbl. Application to food samples showed no detectable target cobalamins in the tested plant-derived foods, whereas animal liver and oyster samples showed comparatively high levels of the target cobalamin vitamers, with AdoCbl predominating in liver. The proposed workflow may serve as a practical cyanide-free option for exploratory or comparative native-vitamer analysis of CNCbl, AdoCbl, and MeCbl in foods within the current validation scope, particularly when full sets of matched isotope-labeled standards are not readily available.