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All radioactive determinations required by this method should be made with a suitable counting assembly over a period of time optimal for the particular counting assembly used. All procedures should be performed in replicate to obtain the greatest accuracy.

Cyanocobalamin Tracer Reagent—
Dilute an accurately measured volume of a solution of radioactive cyanocobalamin* with water to yield a solution having a radioactivity between 500 and 5000 counts per minute per mL. Add 1 drop of cresol per L of solution prepared, and store in a refrigerator.
Standardization— Prepare a solution of a weighed quantity of USP Cyanocobalamin RS in water to contain 20 to 50 µg per mL. Perform the entire assay on a 10.0-mL portion of this solution, proceeding as directed under Assay Preparation, beginning with “Add water to make a measured volume.”

Cresol–Carbon Tetrachloride Solution—
Mix equal volumes of carbon tetrachloride and freshly distilled cresol.

Phosphate–Cyanide Solution—
Dissolve 100 mg of potassium cyanide in 1000 mL of a saturated solution of dibasic sodium phosphate, and mix.

Butanol-Benzalkonium Chloride Solution—
Dilute benzalkonium chloride solution (17 in 100) with water (3:1), and mix with 36 volumes of butyl alcohol.

Alumina-Resin Column—
Place a pledget of glass wool in the bottom of a constricted glass tube such as a 50-mL buret. With the tube held in an upright position, add a volume of a slurry of ion-exchange resin (see in the section Reagents, Indicators, and Solutions), in water, sufficient to give a column of settled resin 7 cm in height. When the solid has settled somewhat, allow the water to drain so that there is only 1 cm of liquid above the resin column, and tamp the resin lightly. Then add a volume of a slurry of anhydrous alumina (not acid-washed) in water sufficient to increase the height of the settled column to 10 cm, and allow the water to drain to about 1 cm from the top of the alumina. Add a pledget of glass wool, and wash the column, using a total of 50 mL of water, and again drain to within 1 cm of the top of the column. Prepare a fresh column for each determination.

Assay Preparation—
Transfer to a beaker a weighed quantity or measured volume of the preparation to be assayed, equivalent in vitamin B12 activity to that of 200 to 500 µg of cyanocobalamin. Add water to make a measured volume of not less than 25 mL, then add 5.0 mL of Cyanocobalamin Tracer Reagent. Add, while working under a hood, 5 mg of sodium nitrite and 2 mg of potassium cyanide for each mL of the resulting solution. Adjust the solution with diluted hydrochloric acid to a pH of approximately 4, and heat on a steam bath for 15 minutes. Cool, and adjust the solution with 1 N sodium hydroxide to a pH between 7.6 and 8.0. Centrifuge or filter to remove any undissolved solids.

Transfer the Assay Preparation to a 250-mL centrifuge bottle, add 10 mL of Cresol–Carbon Tetrachloride Solution, suitably close the bottle with a glass, polyethylene, or foil-wrapped rubber stopper, shake vigorously for 2 to 5 minutes, and centrifuge. Remove and save the lower, solvent layer. Repeat the extraction using a 5-mL portion of Cresol–Carbon Tetrachloride Solution, and combine the lower, solvent-layer extracts in a centrifuge bottle or separator of 50- to 100-mL capacity.
Wash the combined extracts with successive 10-mL portions of 5 N sulfuric acid until the last washing is practically colorless (two washings usually suffice). During each washing, shake for 2 to 5 minutes, allow the layers to separate, centrifuge, if necessary, and discard the acid layer. Wash further with two successive 10-mL portions of Phosphate–Cyanide Solution. Finally, wash with 10 mL of water. Discard all of the washings.
To the washed extract add 30 mL of a mixture of Butanol-Benzalkonium Chloride Solution and carbon tetrachloride (2:1). Extract with two 5-mL portions of water, each time shaking vigorously for 1 minute, centrifuging, and removing and saving the upper, aqueous layer.
Pass the combined aqueous extracts through the Alumina-Resin Column at a rate of about 1 mL per minute, maintaining a 1-cm layer of liquid on the head of the column by adding water as needed. Discard as much of the forerun as is colorless (usually about 5 mL), and collect the colored eluate (usually about 10 mL) in a 50-mL centrifuge tube or separator containing 500 µL of diluted acetic acid. Extract the eluate by shaking for 2 to 5 minutes with 5 mL of Cresol–Carbon Tetrachloride Solution, and discard the upper, aqueous layer. To the extract add 5.0 mL of water, 5 mL of carbon tetrachloride, and 10 mL of butyl alcohol. Shake, allow to separate until the upper layer is clear, and remove the upper, aqueous layer.
Determine the absorbances of the aqueous extract, in a 1-cm cell, at 361 nm and 550 nm, with a suitable spectrophotometer, using a tungsten light source. Make the 361-nm reading using a filter capable of reducing stray light. Calculate the ratio A361/A550: the purity of the aqueous extract is acceptable if the ratio is between 3.10 and 3.40. If a ratio outside this range is observed, purify the aqueous extract by repeating the extraction cycle, proceeding as directed in the foregoing paragraph.
If an acceptable absorbance ratio is observed in the aqueous extract, determine the radioactivity, in counts per minute, using a suitable counter over a period optimal for the particular counting assembly used. Average the results, and correct the average for the observed background radioactivity determined over two or more 30-minute periods.

Calculate the cobalamin content, expressed in µg of cyanocobalamin, of the portion taken for assay by the formula:
R(CS / CU)(AU / AS),
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 and assay solutions, respectively; and AU and AS are the absorbances determined at 361 nm of the assay and standard solutions, respectively.

*  A solution of cyanocobalamin made radioactive by the incorporation of 60Co is available from Merck and Co., Inc., Rahway, NJ 07065.

Auxiliary Information—
Staff Liaison : Andrzej Wilk, Ph.D., Senior Scientific Associate
Expert Committee : (RMI05) Radiopharmaceuticals and Medical Imaging Agents 05
USP29–NF24 Page 2566
Phone Number : 1-301-816-8305