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Cobinamide, dihydroxide, dihydrogen phosphate (ester), mono(inner salt), 3 ¢ -ester with 5,6-dimethyl-1- - D -ribofuranosyl-1 H -benzimidazole. Cobinamide dihydroxide dihydrogen phosphate (ester), mono(inner salt), 3 ¢ -ester with 5,6-dimethyl-1- - D -ribofuranosylbenzimidazole [ 13422-51-0 ].

» Hydroxocobalamin contains not less than 95.0 percent and not more than 102.0 percent of C 62 H 89 CoN 13 O 15 P, calculated on the dried basis.

Packaging and storage Preserve in tight, light-resistant containers, and store in a cool place. Identification

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 791 : between 8.0 and 10.0, in a solution (2 in 100).

Loss on drying 731 Dry it at a pressure below 5 mm of mercury at 100 for 2 hours: it loses between 14.0% and 18.0% of its weight.

pH-dependent cobalamins

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:

(100,000 A ) / (19.66 W ),

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%.

Limit of cyanocobalamin

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:

R ( C S / C U )( A U / A S ),

in which R is the quantity, in µg, of cyanocobalamin in the portion of the Standard solution taken; C S and C U are the corrected average radioactivity values, expressed in counts per minute per mL, of the Standard solution and test solution, respectively; and A U and A S 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%.

Residual solvents 467 : meets the requirements. (Official January 1, 2007)

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 C 62 H 89 CoN 13 O 15 P in the Hydroxocobalamin taken by the formula:

(1346.36 / 1355.37)( R )( C S / C U )( A U / A S ),

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; C S and C U are the corrected average radioactivity values, expressed in counts per minute per mL, of the Standard solution and test solution, respectively; and A U and A S are the absorbances, determined at 361 nm, of the test solution and the Standard solution, respectively.

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