A: The visible absorption spectrum of the solution, prepared for measurement of the absorption as directed under pH-dependent cobalamins, exhibits maxima at 426 ± 2 nm, 516 ± 2 nm, and 550 ± 2 nm.

B: Fuse a mixture of about 1 mg of Hydroxocobalamin and about 50 mg of potassium pyrosulfate in a porcelain crucible. Cool, break up the mass with a glass rod, add 3 mL of water, and boil until dissolved. Add 1 drop of phenolphthalein TS, and add 2 N sodium hydroxide dropwise until a pink color appears. Add 0.5 g of sodium acetate, 0.5 mL of 1 N acetic acid, and 0.5 mL of nitroso R salt solution (1 in 100): a red or orange-red color appears immediately. Add 0.5 mL of hydrochloric acid, and boil for 1 minute: the red or orange-red color persists.

pH 4.0 Buffer— Dissolve 2.61 g of sodium acetate and 20.5 g of sodium chloride in 5.25 mL of glacial acetic acid and sufficient water to make 1500 mL of solution, and mix.

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.

Procedure— [note—Perform the following test in subdued light.] Transfer about 40 mg of Hydroxocobalamin, accurately weighed, to a 25-mL volumetric flask, dissolve in carbon dioxide-free water, dilute with carbon dioxide-free water to volume, and mix. Transfer 1.0-mL portions of this solution to each of two glass-stoppered test tubes. To one of the tubes, designated B, add 3.0 mL of pH 4.0 Buffer, and mix. To the other tube, designated U, add 3.0 mL of pH 9.3 Buffer, and mix. Determine the absorbance of solution U, in a 1-cm cell, at the wavelength of maximum absorbance at about 550 nm, with a suitable spectrophotometer, using solution B as the blank. Calculate the percentage of pH-dependent cobalamins, as hydroxocobalamin, by the formula: in which A is the absorbance of solution U, and W is the weight, in mg, of Hydroxocobalamin taken: the content, calculated on the dried basis, is between 95.0% and 102.0%.

Cyanocobalamin tracer reagent, Cresol-carbon tetrachloride solution, Butanol-benzalkonium chloride solution, and Alumina-resin column— Prepare as directed under Cobalamin Radiotracer Assay 371.

Procedure— Transfer about 50 mg of Hydroxocobalamin, accurately weighed, to a 25-mL volumetric flask, dissolve in water, dilute with water to volume, and mix. Transfer 5.0 mL of this solution to a glass-stoppered, 50-mL centrifuge tube, and add 5.0 mL of Cyanocobalamin tracer reagent and 15 mL of Cresol-carbon tetrachloride solution. Insert the stopper, shake gently, centrifuge, carefully remove the upper, aqueous layer by aspiration, and discard the aspirated liquid. Add 25 mL of 5 N sulfuric acid, insert the stopper, shake gently, centrifuge, and remove and discard the upper, aqueous layer. Repeat the washing with additional 25-mL portions of the 5 N sulfuric acid until the acid wash is colorless (six to eight washings), and discard the acid washings. Add Cresol-carbon tetrachloride solution as necessary during the acid washings to maintain the volume of this phase at not less than 10 mL. Wash this solution successively with two 10-mL portions of saturated dibasic sodium phosphate solution and one 10-mL portion of water, and discard all of the aqueous washings. Proceed as directed for Procedure under Cobalamin Radiotracer Assay 371, beginning with “To the washed extract add 30 mL of a mixture of 2 volumes of Butanol-Benzalkonium Chloride Solution and 1 volume of carbon tetrachloride.”

Calculation— Calculate the cyanocobalamin content, in µg, of the Hydroxocobalamin taken by the formula: in which R is the quantity, in µg, of cyanocobalamin in the portion of the Standard solution taken; CS and CU are the corrected average radioactivity values, expressed in counts per minute per mL, of the Standard solution and test solution, respectively; and AU and AS are the absorbances, determined at 361 nm, of the test solution and the Standard solution, respectively: the limit, calculated on the dried basis, is 5.0%.

Cyanocobalamin tracer reagent, Cresol-carbon tetrachloride solution, Phosphate-cyanide solution, Butanol-benzalkonium chloride solution, and Alumina-resin column— Prepare as directed under Cobalamin Radiotracer Assay 371.

Assay preparation— Transfer about 40 mg of Hydroxocobalamin, accurately weighed, to a 2000-mL volumetric flask, dissolve in and dilute with water to volume, and mix. Transfer 25.0 mL of this solution to a beaker, add 5.0 mL of Cyanocobalamin tracer reagent, and proceed as directed for Assay Preparation under Cobalamin Radiotracer Assay 371, beginning with “Add, while working under a hood, 5 mg of sodium nitrite.”

Procedure— Proceed as directed for Procedure under Cobalamin Radiotracer Assay 371. Calculate the quantity, in µg, of C62H89CoN13O15P in the Hydroxocobalamin taken by the formula: in which 1346.36 and 1355.37 are the molecular weights of hydroxocobalamin and cyanocobalamin, respectively; R is the quantity, in µg, of cyanocobalamin in the portion of the Standard solution taken; CS and CU are the corrected average radioactivity values, expressed in counts per minute per mL, of the Standard solution and test solution, respectively; and AU and AS are the absorbances, determined at 361 nm, of the test solution and the Standard solution, respectively.