Synthesis and Characterization of Cobinamide

Cobinamide was synthesized from hydroxocobalamin acetate (Wockhardt, LTD, Mumbai, India) by base hydrolysis using cerium (III) hydroxide19 , produced by adding sodium hydroxide to cerium nitrate; in this reaction, the Ce⁺³ ion serves a catalytic function20 . The cobinamide product was purified on a carboxymethyl cellulose cation exchange column (CM52, Whatman), and concentrated and de-salted on a reversed-phase C-18 column. Final concentration was performed by flash evaporation and lyophilization; the cobinamide product is highly stable and can be stored for months at 4°C. Purity of cobinamide preparations was evaluated by HPLC by converting all of the cobinamide to dicyanocobinamide, and analyzing the sample on a C-18 reversed-phase column eluted with a gradient from 20 mM potassium phosphate, pH 4.6 containing 0.2 mM KCN (solvent A) to 60% methanol/water (solvent B): one minute to 40% B, 11 min to 50% B, and 1 min to 100%B (flow rate 1 ml/min). The dicyanocobinamide eluted at 16 min and was detected by spectral absorption at 366 nm21 (link). The concentration of cobinamide solutions was determined spectrophotometrically in 0.1 M HCl (as diaquocobinamide) using an extinction coefficient of 2.8×10⁴ /M/cm at 348 nm 22 (link). At neutral pH, cobinamide exists as aquohydroxocobinamide, and at a pH of > 11, it exists as dihydroxocobinamide 23 .