Cyanocobalamin
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C63H88CoN14O14P 1355.37

Vitamin B12.
Vitamin B12 [68-19-9].
» Cyanocobalamin contains not less than 96.0 percent and not more than 100.5 percent of C63H88CoN14O14P, calculated on the dried basis.
Packaging and storage— Preserve in tight, light-resistant containers, and store at controlled room temperature.
Identification—
A: The absorption spectrum of the solution employed for measurement of absorbance in the Assay exhibits maxima within ±1 nm at 278 nm and 361 nm and within ±2 nm at 550 nm. The ratio A361/A278 is between 1.70 and 1.90, and the ratio A361/A550 is between 3.15 and 3.40.
B: Fuse about 1 mg with 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 dissolve by boiling. Add 1 drop of phenolphthalein TS, and add sodium hydroxide solution (1 in 10), dropwise, until just pink. Add 500 mg of sodium acetate, 0.5 mL of 1 N acetic acid, and 0.5 mL of nitroso R salt solution (1 in 500): a red or orange-red color appears at once. Add 0.5 mL of hydrochloric acid, and boil for 1 minute: the red color persists.
C: Dissolve about 5 mg in 5 mL of water in a 50-mL distilling flask connected with a short, water-cooled condenser. To the flask add 2.5 mL of hypophosphorous acid, close, boil gently but short of distillation for 10 minutes, then distill 1 mL into a test tube containing 1 mL of sodium hydroxide solution (1 in 50). To the test tube add 4 drops of cold saturated ferrous ammonium sulfate solution, shake gently, then add about 30 mg of sodium fluoride, and bring the contents to a boil. Immediately add, dropwise, 5 N sulfuric acid until a clear solution results, then add 3 to 5 drops more of the acid: a blue or blue-green color develops within a few minutes.
Loss on drying 731 Heat about 25 mg, accurately weighed, in a suitable vacuum drying apparatus at 105 and at a pressure of not more than 5 mm of mercury for 2 hours, cool, and weigh: it loses not more than 12.0% of its weight.
Pseudo cyanocobalamin— Dissolve 1.0 mg in 20 mL of water contained in a small separator, add 5 mL of a mixture of equal volumes of carbon tetrachloride and m-cresol, and shake well for about 1 minute. Allow to separate, draw off the lower layer into a second small separator, add 5 mL of 5 N sulfuric acid, shake well, and allow to separate completely (the complete separation of the layer may be facilitated by centrifuging): the separated upper layer is colorless or has no more color than a mixture of 0.15 mL of 0.10 N potassium permanganate and 250 mL of water.
Assay— Transfer about 30 mg of Cyanocobalamin, accurately weighed, with the aid of water to a 1-L volumetric flask, dilute with water to volume, and mix. Dissolve an accurately weighed quantity of USP Cyanocobalamin RS in water, and dilute quantitatively and stepwise with water to obtain a Standard solution having a known concentration of about 30 µg per mL. Concomitantly determine the absorbances of both solutions in 1-cm cells at the wavelength of maximum absorbance at about 361 nm, with a suitable spectrophotometer, using water as the blank. Calculate the quantity, in mg, of C63H88CoN14O14P in the Cyanocobalamin taken by the formula:
C(AU / AS)
in which C is the concentration, in µg per mL, of USP Cyanocobalamin RS in the Standard solution; and AU and AS are the absorbances of the solution of Cyanocobalamin and the Standard solution, respectively.
Auxiliary Information— Please check for your question in the FAQs before contacting USP.
Topic/Question Contact Expert Committee
Monograph Curtis Phinney

1-301-816-8540
(DSN05) Dietary Supplements - Non-Botanicals
Reference Standards Lili Wang, Technical Services Scientist
1-301-816-8129
RSTech@usp.org
USP32–NF27 Page 2036
Pharmacopeial Forum: Volume No. 31(5) Page 1350