Direct species-selective determination of cobalamins by ionspray mass spectrometry and ionspray tandem mass spectrometry

Abstract

A direct method for the simultaneous compound-specific determination of vitamin B₁₂ (cyanocobalamin), its analogues methylcobalamin, hydroxocobalamin, adenosylcobalamin (coenzyme B₁₂) and cobinamides by ionspray MS was developed. Single and tandem MS modes were compared. For all the compounds except hydroxocobalamin, the most intense ion formation was observed as a result of protonation of the original molecular cation leading to an analytically useful doubly charged ion. For hydroxocobalamin a similar process leads to the loss of the functional group leading to the [Cbl + H]²⁺ ion, which is analytically useful only in the absence of other cobalamins. Hydroxocobalamin can be determined in the presence of other compounds using the minor [OH-Cbl]⁺ ion. The calibration curves are linear in the range 0.01–1 µg ml^–1, which can be extended up to 10 µg ml^–1 if alternative ions are chosen. Detection limits of 5–40 ng ml^–1 are obtained. Collision-induced dissociation leads first to the loss of the Co substituent (Me, CN, OH, adenosyl) and thus to a decrease in selectivity. The method was applied to the analysis of a synthetic mixture of the cobalamins and to the determination of hydroxocobalamin in a commercial pharmaceutical preparation.