á171ñVITAMIN B12ACTIVITY ASSAY

USP Reference Standards á11ñ— USP Cyanocobalamin RS.

Assay Preparation— Place a suitable quantity of the material to be assayed,previously reduced to a fine powder if necessary and accurately measured or weighed,in an appropriate vessel containing,for each g or mLof material taken,25mLof an aqueous extracting solution prepared just prior to use to contain,in each 100mL,1.29g of disodium phosphate,1.1g of anhydrous citric acid,and 1.0g of sodium metabisulfite.Autoclave the mixture at 121for 10minutes.Allow any undissolved particles of the extract to settle,and filter or centrifuge,if necessary.Dilute an aliquot of the clear solution with water so that the final test solution contains vitamin B12activity approximately equivalent to that of the Standard Cyanocobalamin Solutionwhich is added to the assay tubes.

Standard Cyanocobalamin Stock Solution— To a suitable quantity of USP Cyanocobalamin RS,accurately weighed,add sufficient 25percent alcohol to make a solution having a known concentration of 1.0µg of cyanocobalamin per mL.Store in a refrigerator.

Standard Cyanocobalamin Solution— Dilute a suitable volume of Standard Cyanocobalamin Stock Solutionwith water to a measured volume such that after the incubation period as described for Procedure,the difference in transmittance between the inoculated blank and the 5.0-mLlevel of the Standard Cyanocobalamin Solutionis not less than that which corresponds to a difference of 1.25mg in dried cell weight.This concentration usually falls between 0.01ng and 0.04ng per mLof Standard Cyanocobalamin Solution.Prepare a fresh standard solution for each assay.

Basal Medium Stock Solution— Prepare the medium according to the following formula and directions.Adehydrated mixture containing the same ingredients may be used provided that,when constituted as directed in the labeling,it yields a medium comparable to that obtained from the formula given herein.
Add the ingredients in the order listed,carefully dissolving the cystine and tryptophane in the hydrochloric acid before adding the next eight solutions in the resulting solution.Add 100mLof water,mix,and dissolve the dextrose,sodium acetate,and ascorbic acid.Filter,if necessary,add the polysorbate 80solution,adjust the solution to a pHbetween 5.5and 6.0with 1Nsodium hydroxide,and add purified water to make 250mL.
L-Cystine 0.1g
L-Tryptophane 0.05g
1NHydrochloric Acid 10mL
Adenine-Guanine-Uracil Solution 5mL
Xanthine Solution 5mL
Vitamin Solution I 10mL
Vitamin Solution II 10mL
Salt Solution A 5mL
Salt Solution B 5mL
Asparagine Solution 5mL
Acid-hydrolyzed Casein Solution 25mL
Dextrose,Anhydrous 10g
Sodium Acetate,Anhydrous 5g
Ascorbic Acid 1g
Polysorbate 80Solution 5mL
Acid-Hydrolyzed Casein Solution— Prepare as directed under Calcium Pantothenate Assay á91ñ.
Asparagine Solution— Dissolve 2.0of L-asparagine in water to make 200mL.Store under toluene in a refrigerator.
Adenine–Guanine–Uracil Solution— Prepare as directed under Calcium Pantothenate Assay á91ñ.
Xanthine Solution— Suspend 0.20g of xanthine in 30mLto 40mLof water,heat to about 70,add 6.0mLof 6Nammonium hydroxide,and stir until the solid is dissolved.Cool,and add water to make 200mL.Store under toluene in a refrigerator.
Salt Solution A— Dissolve 10g of monobasic potassium phosphate and 10g of dibasic potassium phosphate in water to make 200mL.Add 2drops of hydrochloric acid,and store under toluene.
Salt Solution B— Dissolve 4.0g of magnesium sulfate,0.20g of sodium chloride,0.20g of ferrous sulfate,and 0.20g of manganese sulfate in water to make 200mL.Add 2drops of hydrochloric acid,and store under toluene.
Polysorbate 80Solution— Dissolve 20g of polysorbate 80in alcohol to make 200mL.Store in a refrigerator.
Vitamin Solution I— Dissolve 10mg of riboflavin,10mg of thiamine hydrochloride,100µg of biotin,and 20mg of niacin in 0.02Nglacial acetic acid to make 400mL.Store,protected from light,under toluene in a refrigerator.
Vitamin Solution II— Dissolve 20mg of para-aminobenzoic acid,10mg of calcium pantothenate,40mg of pyridoxine hydrochloride,40mg of pyridoxal hydrochloride,8mg of pyridoxamine dihydrochloride,and 2mg of folic acid in dilute neutralized alcohol (1in 4)to make 400mL.Store,protected from light,in a refrigerator.

Tomato Juice Preparation— Centrifuge commercially canned tomato juice so that most of the pulp is removed.Suspend about 5g per Lof analytical filter-aid in the supernatant,and filter,with the aid of reduced pressure,through a layer of the filter-aid.Repeat,if necessary,until a clear,straw-colored filtrate is obtained.Store under toluene in a refrigerator.

Culture Medium— [NOTE—Adehydrated mixture containing the same ingredients may be used provided that,when constituted as directed in the labeling,it yields a medium equivalent to that obtained from the formula given herein.]Dissolve 0.75g of water-soluble yeast extract,0.75g of dried peptone,1.0g of anhydrous dextrose,and 0.20g of potassium biphosphate in 60mLto 70mLof water.Add 10mLof Tomato Juice Preparationand 1mLof Polysorbate 80Solution.Adjust the solution with 1Nsodium hydroxide to a pHof 6.8,and add water to make 100mL.Place 10-mLportions of the solution in test tubes,and plug with cotton.Sterilize the tubes and contents in an autoclave at 121for 15minutes.Cool as rapidly as possible to avoid color formation resulting from overheating the medium.

Suspension Medium— Dilute a measured volume of Basal Medium Stock Solutionwith an equal volume of water.Place 10-mLportions of the diluted medium in test tubes.Sterilize,and cool as directed above for the Culture Medium.

Stock Culture of Lactobacillus leichmannii— To 100mLof Culture Mediumadd 1.0g to 1.5g of agar,and heat the mixture,with stirring,on a steam bath,until the agar dissolves.Place approximately 10-mLportions of the hot solution in test tubes,cover the tubes suitably,sterilize at 121for 15minutes in an autoclave (exhaust line temperature),and allow the tubes to cool in an upright position.Inoculate three or more of the tubes,by stab transfer of a pure culture of Lactobacillus leichmannii.*(Before first using a fresh culture in this assay,make not fewer than 10successive transfers of the culture in a 2-week period.)Incubate 16to 24hours at any selected temperature between 30and 40but held constant to within ±0.5,and finally store in a refrigerator.
Prepare fresh stab cultures at least three times each week,and do not use them for preparing the inoculum if more than 4days old.The activity of the microorganism can be increased by daily or twice-daily transfer of the stab culture,to the point where definite turbidity in the liquid inoculum can be observed 2to 4hours after inoculation.Aslow-growing culture seldom gives a suitable response curve,and may lead to erratic results.

Inoculum— [NOTE—Afrozen suspension of Lactobacillus leichmanniimay be used as the stock culture,provided it yields an inoculum comparable to a fresh culture.]Make a transfer of cells from the Stock Culture of Lactobacillus leichmanniito 2sterile tubes containing 10mLof the Culture Mediumeach.Incubate these cultures for 16to 24hours at any selected temperature between 30and 40but held constant to within ±0.5.Under aseptic conditions,centrifuge the cultures,and decant the supernatant.Suspend the cells from the culture in 5mLof sterile Suspension Medium,and combine.Using sterile Suspension Medium,adjust the volume so that a 1in 20dilution in saline TSproduces 70%transmittance when read on a suitable spectrophotometer that has been set at a wavelength of 530nm,equipped with a 10-mm cell,and read against saline TSset at 100%transmittance.Prepare a 1in 400dilution of the adjusted suspension using Basal Medium Stock Solution,and use it for the test inoculum.(This dilution may be altered,when necessary,to obtain the desired test response.)

Calibration of Spectrophotometer— Check the wavelength of the spectrophotometer periodically,using a standard wavelength cell or other suitable device.Before reading any tests,calibrate the spectrophotometer for 0%and 100%transmittance,using water and with the wavelength set at 530nm.

Procedure— Cleanse meticulously by suitable means,followed preferably by heating at 250for 2hours,hard-glass test tubes,about 20mm ×150mm in size,and other necessary glassware because of the high sensitivity of the test organism to minute amounts of vitamin B12activity and to traces of many cleansing agents.
To test tubes add,in duplicate,1.0mL,1.5mL,2.0mL,3.0mL,4.0mL,and 5.0mL,respectively,of the Standard Cyanocobalamin Solution.To each of these tubes and to four similar empty tubes add 5.0mLof Basal Medium Stock Solutionand water to make 10mL.
To similar test tubes add,in duplicate,respectively,1.0mL,1.5mL,2.0mL,3.0mL,and 4.0mLof the Assay Preparation.To each tube add 5.0mLof Basal Medium Stock Solutionand water to make 10mL.Place one complete set of standard and assay tubes together in one tube rack and the duplicate set in a second rack or section of a rack,preferably in random order.
Cover the tubes suitably to prevent bacterial contamination,and sterilize the tubes and contents in an autoclave at 121for 5minutes,arranging to reach this temperature in not more than 10minutes by preheating the autoclave,if necessary.Cool as rapidly as practicable to avoid color formation resulting from overheating the medium.Take precautions to maintain uniformity of sterilizing and cooling conditions throughout the assay,since packing tubes too closely in the autoclave,or overloading it,may cause variation in the heating rate.
Aseptically add 0.5mLof Inoculumto each tube so prepared,except two of the four containing no Standard Cyanocobalamin Solution(the uninoculated blanks).Incubate the tubes at a temperature between 30and 40held constant to within ±0.5,for 16to 24hours.
Terminate growth by heating to a temperature not lower than 80for 5minutes.Cool to room temperature.After agitating its contents,place the container in a spectrophotometer that has been set at a wavelength of 530nm,and read the transmittance when a steady state is reached.This steady state is observed a few seconds after agitation when the reading remains constant for 30seconds or more.Allow approximately the same time interval for the reading on each tube.
With the transmittance set at 100%for the uninoculated blank,read the transmittance of the inoculated blank.If the difference is greater than 5%or if there is evidence of contamination with a foreign microorganism,disregard the results of the assay.
With the transmittance set at 100%for the uninoculated blank,read the transmittance of each of the remaining tubes.Disregard the results of the assay if the slope of the standard curve indicates a problem with sensitivity.

Calculation— Prepare a standard concentration-response curve by the following procedure.Test for and replace any aberrant individual transmittances.For each level of the standard,calculate the response from the sum of the duplicate values of the transmittances (S)as the difference,y=2.00–S.Plot this response on the ordinate of cross-section paper against the logarithm of the mLof Standard Cyanocobalamin Solutionper tube on the abscissa,using for the ordinate either an arithmetic or a logarithmic scale,whichever gives the better approximation to a straight line.Draw the straight line or smooth curve that best fits the plotted points.
Calculate the response,y,adding together the two transmittances for each level of the Assay Preparation.Read from the standard curve the logarithm of the volume of the Standard Preparationcorresponding to each of those values of ythat falls within the range of the lowest and highest points plotted for the standard.Subtract from each logarithm so obtained the logarithm of the volume,in mL,of the Assay Preparationto obtain the difference,x,for each dosage level.Average the values of xfor each of three or more dosage levels to obtain bar(x)=M¢,the log-relative potency of the Assay Preparation.Determine the quantity,in µg,of USP Cyanocobalamin RScorresponding to the cyanocobalamin in the portion of material taken for assay by the equation antilogM=antilog (M¢+log R),in which Ris the number of µg of cyanocobalamin that was assumed to be present in each mg (or capsule or tablet)of the material taken for assay.

Replication— Repeat the entire determination at least once,using separately prepared Assay Preparations.If the difference between the two log potenciesMis not greater than 0.08,their mean,bar(M),is the assayed log-potency of the test material (see Vitamin B12Activity Assayunder Design and Analysis of Biological Assays á111ñ).If the two determinations differ by more than 0.08,conduct one or more additional determinations.From the mean of two or more values ofMthat do not differ by more than 0.15,compute the mean potency of the preparation under assay.
*  Pure cultures of Lactobacillus leichmanniimay be obtained as No.7830from the American Type Culture Collection,10801University Blvd.,Manassas,VA20110.