pH 9.3 Buffer— Dissolve 23.8 g of sodium borate and 402 mg of boric acid in sufficient water to make 1500 mL of solution, and mix.

Standard preparation— Dissolve a suitable quantity of USP Cyanocobalamin RS, accurately weighed, in pH 9.3 Buffer and dilute quantitatively, and stepwise if necessary, to obtain a solution having a known concentration of about 30 µg per mL.

Assay preparation— Transfer an accurately measured volume of Injection, equivalent to about 5 mg of hydroxocobalamin, to a 50-mL volumetric flask containing about 25 mL of pH 9.3 Buffer. Add 5.0 mL of potassium cyanide solution (1 in 10,000), allow to stand at room temperature for 30 minutes, dilute with pH 9.3 Buffer to volume, and mix. Transfer 15.0 mL of this solution to a second 50-mL volumetric flask, dilute with pH 9.3 Buffer to volume, and mix.

Procedure— Concomitantly determine the absorbances of the solutions in 1-cm cells at the wavelength of maximum absorbance at about 361 nm, with a suitable spectrophotometer, using pH 9.3 Buffer as the blank. Calculate the quantity, in mg, of hydroxocobalamin (C62H89CoN13O15P) in each mL of the Injection taken by the formula: in which 1346.36 and 1355.37 are the molecular weights of hydroxocobalamin and cyanocobalamin, respectively; C is the concentration, in µg per mL, of USP Cyanocobalamin RS in the Standard preparation; V is the volume, in mL, of Injection taken; and AU and AS are the absorbances of the Assay preparation and the Standard preparation, respectively.