Rapid analysis of cobalamin coenzymes and related corrinoid analogs by high-performance liquid chromatography

Cobalamin coenzymes and a series of related corrinoid analogs have been analyzed by high-performance liquid chromatography on reverse-phase C₈ and C₁₈ columns using both isocratic and gradient elution systems and 254-nm absorbance detection. In the isocratic mode, retention times for sulfitocobalamin, cyanocobalamin, methylcobalamin, adenosylcobalamin, and aquacobalamin on a LiChrosorb C₈ column were 1.1, 1.6, 2.2, 2.9, and 4.7 min, respectively. In the gradient mode, corresponding retention times were 9.7, 10.2, 12.8, 11.4, and 9.0 min. Closely related structural analogs of adenosylcobalamin such as 1,N⁶-ethenoadenosylcobalamin, formycinylcobalamin, and 2,6-diaminonebularinylcobalamin were clearly resolved from naturally occurring cobalamins by isocratic elution. In the gradient system, the order of elution of cobalamins was related to the hydrophobicity of the upper-axial ligand. This was demonstrated by determining the retention times of aminoalkylcobalamin homologs (C₂, 9.8; C₃, 10.5; C₅, 11.2; C₈, 12.8; and C₁₁, 14.9 min). The usefulness of this method was demonstrated by analyzing ⁵⁷Co-labeled cobalamins present in extracts of Lactobacillus leichmannii and murine leukemia L1210 cells.