High Resolution Mass Spectrometric Analysis of Vitamin B₁₂ Opens the Door to Structural Identification of Cobalamin Impurities

Vitamin B₁₂ is one of nature's most structurally complex small molecules and an essential coenzyme that is only produced by prokaryotes. Due to its pivotal role in nerve function, metabolism, and the immune system in humans, vitamin B₁₂ deficiency is considered a major public health concern. Numerous mass spectrometric analytical methods exist to identify and quantify vitamin B₁₂ from a wide array of matrices, including food, diet supplements, tissues, and plasma. Nevertheless, structural elucidation of vitamin B₁₂ through tandem mass spectrometry (MS/MS) fragmentation remains a challenge due to its intricate structure, versatility of derivatives, and isobaric impurities. This work combined native and force-degraded samples with synthetic impurity standards to elucidate the structures of collision-induced dissociation (CID) fragment ions of vitamin B₁₂ over the full <i>m</i>/<i>z</i> range with a high mass accuracy. Our findings show that the complexity of vitamin B₁₂ MS/MS fragments can be deduced through a "simple" combination of four (4) major CID cleavage sites. These results enabled us to propose a general vitamin B₁₂ CID fragmentation pathway that allows for the calculation of the theoretical <i>m</i>/<i>z</i> for fragment ions of unknown cobalamin species and impurities. This work aims to provide a novel, universal analytical platform to identify and quantify vitamin B₁₂ and its derivatives through mass spectrometry with high mass accuracy.