á371ñCOBALAMIN RADIOTRACER ASSAY
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 500and 5000counts per minute per mL.Add 1drop of cresol per Lof solution prepared,and store in a refrigerator.
Standardization— Prepare a solution of a weighed quantity of USP Cyanocobalamin RSin water to contain 20to 50µg per mL.Perform the entire assay on a 10.0-mLportion 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 100mg of potassium cyanide in 1000mLof a saturated solution of dibasic sodium phosphate,and mix.

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

Alumina-Resin Column— Place a pledget of glass wool in the bottom of a constricted glass tube such as a 50-mLburet.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 7cm in height.When the solid has settled somewhat,allow the water to drain so that there is only 1cm 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 10cm,and allow the water to drain to about 1cm from the top of the alumina.Add a pledget of glass wool,and wash the column,using a total of 50mLof water,and again drain to within 1cm 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 B12activity to that of 200to 500µg of cyanocobalamin.Add water to make a measured volume of not less than 25mL,then add 5.0mLof Cyanocobalamin Tracer Reagent.Add,while working under a hood,5mg of sodium nitrite and 2mg of potassium cyanide for each mLof the resulting solution.Adjust the solution with diluted hydrochloric acid to a pHof approximately 4,and heat on a steam bath for 15minutes.Cool,and adjust the solution with 1Nsodium hydroxide to a pHbetween 7.6and 8.0.Centrifuge or filter to remove any undissolved solids.

Procedure— Transfer the Assay Preparationto a 250-mLcentrifuge bottle,add 10mLof Cresol–Carbon Tetrachloride Solution,suitably close the bottle with a glass,polyethylene,or foil-wrapped rubber stopper,shake vigorously for 2to 5minutes,and centrifuge.Remove and save the lower,solvent layer.Repeat the extraction using a 5-mLportion of Cresol–Carbon Tetrachloride Solution,and combine the lower,solvent-layer extracts in a centrifuge bottle or separator of 50-to 100-mLcapacity.
Wash the combined extracts with successive 10-mLportions of 5Nsulfuric acid until the last washing is practically colorless (two washings usually suffice).During each washing,shake for 2to 5minutes,allow the layers to separate,centrifuge,if necessary,and discard the acid layer.Wash further with two successive 10-mLportions of Phosphate–Cyanide Solution.Finally,wash with 10mLof water.Discard all of the washings.
To the washed extract add 30mLof a mixture of Butanol-Benzalkonium Chloride Solutionand carbon tetrachloride (2:1).Extract with two 5-mLportions of water,each time shaking vigorously for 1minute,centrifuging,and removing and saving the upper,aqueous layer.
Pass the combined aqueous extracts through the Alumina-Resin Columnat a rate of about 1mLper 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 5mL),and collect the colored eluate (usually about 10mL)in a 50-mLcentrifuge tube or separator containing 500µLof diluted acetic acid.Extract the eluate by shaking for 2to 5minutes with 5mLof Cresol–Carbon Tetrachloride Solution,and discard the upper,aqueous layer.To the extract add 5.0mLof water,5mLof carbon tetrachloride,and 10mLof 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 361nm and 550nm,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 3.10and 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.

Calculation— 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 Ris the quantity,in µg,of cyanocobalamin in the portion of the standard solution taken;CSand CUare the corrected average radioactivity values,expressed in counts per minute per mL,of the standard and assay solutions,respectively;and AUand ASare the absorbances determined at 361nm of the assay and standard solutions,respectively.
*  Asolution of cyanocobalamin made radioactive by the incorporation of 60Co is available from Merck and Co.,Inc.,Rahway,NJ07065.