Abstract
This chapter describes methods for rapid analysis of naturally occurring corrinoids and corrinoid analogs based on high-performance liquid chromatography (HPLC). Since corrinoids have dissimilar amphipathic solubility properties, complex mixtures can be resolved on reverse-phase HPLC columns using either isocratic or gradient elution techniques. Body fluid and tissue corrinoids, which are usually associated with specific binding proteins, must be extracted and desalted prior to HPLC. Resolved corrinoids are detected directly by UV-VIS flow-cell absorbance spectrophotometry if chemical levels exceed 1 pmol per compound. Lower levels of radiolabeled corrinoids can be resolved, collected, and counted. The chapter also describes the synthesis of cobalamin coenzymes and other corrinoids, extraction of corrinoids from prokaryotic and eukaryotic cells, and resolution of corrinoids by HPLC (isocratic method and gradient method). Extracts obtained from L. leichmannii and L1210 cells grown in the presence of [57Co]CN-Cbl are analyzed by the gradient method. The column is developed at 0.5 ml/min (5 to 30% in 40 min) which allowed for more efficient collection of the column effluent. Columns are regenerated by increasing the gradient to 50% and then decreasing it to starting conditions.
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