Isolation and analysis of bacterial cobamides by high-performance liquid chromatography

Cyanocobamides were extracted from diverse bacterial species, purified by XAD-4 and neutral aluminum oxide column chromatography, and separated by isocratic reversed-phase high-performance liquid chromatography (HPLC). Retention times are given for seven cobamide types: dicyanocobinamide (factor B), Co alpha-(alpha-benzimidazolyl)-Co beta-cyanocobamide, Co alpha-(5-hydroxybenzimidazolyl)-Co beta-cyanocobamide (factor III), Co alpha-(5-methoxybenzimidazolyl)-Co beta-cyanocobamide (factor IIIm), Co alpha-(5-methylbenzimidazolyl)-Co beta-cyanocobamide, cyanocob(III)alamin (vitamin B-12) and Co alpha-(naphthimidazolyl)-Co beta-cyanocobamide. Other Co beta-ligandyl-cobamides such as hydroxycobamide and the light-sensitive methyl-, acetyl-, propyl-, and adenosylcobamides were separated by HPLC in a gradient mode. The recovery of total cell cobamide after extraction, purification, and separation was 75-80%. The method was useful in preparative and analytical work. Less than 10 ng cyanocobamide was detectable.