NOTE—In the following Assays,where more than one Assaymethod is given for an individual ingredient,the requirements may be met by following any one of the specified methods,the method used being stated in the labeling only if Method 1is not used.

Mobile phase— Use n-hexane.

Standard preparation— Dissolve an accurately weighed quantity of USP Vitamin A RSin n-hexane,and dilute quantitatively,and stepwise if necessary,to obtain a solution having a known concentration of about 15µg of retinyl acetate per mL.

System suitability preparation— Dissolve an accurately weighed quantity of retinyl palmitate in n-hexane to obtain a solution having a concentration of about 15µg of retinyl palmitate per mL.Mix equal volumes of this solution and the Standard preparationto obtain a solution having concentrations of 7.5µg each of retinyl acetate and retinyl palmitate per mL.

Assay preparation— Weigh and finely powder not fewer than 20Tablets.Transfer an accurately weighed portion of the powder,equivalent to 5Tablets,to a container having a polytef-lined screw cap,add 10mLof dimethyl sulfoxide and 15mLof n-hexane,and shake for 45minutes on a wrist-action shaker in a water bath maintained at 60.[NOTE—Set up the wrist-action shaker to ensure that the contents of the container are mixed vigorously and thoroughly.]Centrifuge at 3000rpm for 10minutes,and transfer the hexane layer by means of a pipet to a 100-mLvolumetric flask.Add 15mLof n-hexane to the dimethyl sulfoxide layer,shake thoroughly for 5minutes,and transfer the hexane layer by means of a pipet to the 100-mLvolumetric flask.Repeat this extraction with three additional 15-mLportions of n-hexane.Dilute the extracts in the volumetric flask with n-hexane to volume,and mix.Quantitatively dilute a 10-mLvolume of this solution with n-hexane to obtain a solution having a final concentration of about 15µg of vitamin Aper mL.Retain the remaining solution for use in the assays for vitamin D,vitamin E,and phytonadione.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 325-nm detector and a 4.6-mm ×15-cm column that contains 3-µm packing L8.The flow rate is about 1mLper minute.Chromatograph the System suitability preparation,and measure the peak responses as directed for Procedure:the resolution,R,between retinyl acetate and retinyl palmitate is not less than 10;and the relative standard deviation for replicate injections is not more than 3.0%.

Procedure— Separately inject equal volumes (about 40µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the peak area for retinyl acetate obtained from the Standard preparationand the peak area for retinyl acetate or retinyl palmitate in the chromatogram of the Assay preparation.For products containing vitamin Aacetate or vitamin Apalmitate,calculate the quantity,in mg,of vitamin Aas the retinol equivalent (C20H30O)in the portion of Tablets taken by the formula: in which 0.872is the factor used to convert retinyl acetate,obtained from USP Vitamin A RS,to its retinol equivalent;Cis the concentration,in mg per mL,of USP Vitamin A RSin the Standard preparation;Dis the dilution factor,in mL,for the Assay preparation;and rUand rSare the peak areas of the all-transretinyl ester obtained from the Assay preparationand the Standard preparation,respectively.[NOTE—The molar responses of retinyl acetate and retinyl palmitate are equivalent.]

3N Methanolic sulfuric acid solution— Cautiously add 9mLof sulfuric acid to 80mLof methanol in a 100-mLvolumetric flask.Cool,dilute with methanol to volume,and mix.

Sodium ascorbate–pyrogallol solution— Transfer 10g of sodium ascorbate and 5g of pyrogallol to a 100-mLvolumetric flask,and add sufficient water to dissolve.Add 1.7mLof sulfuric acid,dilute with water to volume,and mix.

Lecithin solution— Transfer 0.5g of lecithin to a 100-mLvolumetric flask,dissolve in and dilute with 2,2,4-trimethylpentane to volume,and mix.

Mobile phase— Prepare a mixture of n-hexane and ethyl acetate (99.7:0.3).

Standard preparation— Dissolve an accurately weighed quantity of USP Vitamin A RSin 2,2,4-trimethylpentane,and dilute quantitatively,and stepwise if necessary,with 2,2,4-trimethylpentane to obtain a solution having a known concentration of about 15µg of retinyl acetate per mL.

System suitability preparation— Dissolve an accurately weighed quantity of retinyl palmitate in 2,2,4-trimethylpentane to obtain a solution having a concentration of about 15µg per mL.Mix equal volumes of this solution and the Standard preparationto obtain a solution having concentrations of 7.5µg each of retinyl acetate and retinyl palmitate per mL.

Assay preparation— [NOTE—This preparation is suitable for the determination of vitamin A,vitamin D,and vitamin E,when present in the formulation.]Weigh and finely powder not fewer than 20Tablets.If vitamin Dis present in the formulation,transfer an accurately weighed portion of the powder,equivalent to about 30µg of cholecalciferol or ergocalciferol,to a container having a polytef-lined screw cap.If vitamin Dis not present in the formulation,use a portion of the powder equivalent to about 90mg of vitamin E(as alpha tocopherol,alpha tocopheryl acetate,or alpha tocopheryl hemisuccinate).If vitamin Eis not present in the formulation,use a portion of the powder equivalent to about 2.5mg of retinyl acetate or retinyl palmitate.Add about 0.5g of sodium bicarbonate,1.5mLof Lecithin solution,and 12.5mLof 2,2,4-trimethylpentane,and disperse on a vortex mixer.Add 6mLof Sodium ascorbate–pyrogallol solution,shake slowly,and allow the solution to degas.Continue shaking until the evolution of gas has ceased,and then shake for an additional 12minutes.Add 6mLof dimethyl sulfoxide,mix on a vortex mixer to form a suspension,and shake for 12minutes.Add 6mLof 3N Methanolic sulfuric acid solution,mix on a vortex mixer to form a suspension,and shake for 12minutes.Add 12.5mLof 2,2,4-trimethylpentane,mix on a vortex mixer to form a suspension,and shake for 10minutes.Centrifuge for about 10minutes to break up the emulsion and to clarify the supernatant.[NOTE—The supernatant is used for the determination of vitamin A,and also vitamin Dand vitamin E,if present in the formulation.]If necessary,quantitatively dilute a volume of the supernatant with 2,2,4-trimethylpentane to obtain a solution having a concentration close to that of the Standard preparation.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 325-nm detector and a 4.6-mm ×25-cm column that contains 5-µm packing L24.The flow rate is about 1.5mLper minute.Chromatograph the System suitability preparation,and measure the peak areas as directed for Procedure:the resolution,R,between retinyl acetate and retinyl palmitate is not less than 8.0;and the relative standard deviation for replicate injections is not more than 3.0%.

Procedure— Separately inject equal volumes (about 40µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the peak area of retinyl acetate obtained from the Standard preparationand the peak area of retinyl acetate or retinyl palmitate obtained from the Assay preparation.Calculate the quantity,in mg,of vitamin A,as the retinol (C20H30O)equivalent,in the portion of Tablets taken by the formula: in which Eis the factor used to convert the retinyl acetate,obtained from USP Vitamin A RS,to its retinol equivalent,the factor being 0.872;Cis the concentration,in mg per mL,of USP Vitamin A RSin the Standard preparation;Dis the dilution factor,in mL,used to prepare the Assay preparationfrom the supernatant;and rUand rSare the peak responses of the all-transretinyl ester obtained from the Assay preparationand the Standard preparation,respectively.[NOTE—The initial extraction into 26.5mLof 2,2,4-trimethylpentane is already accounted for in this equation.The molar responses of retinyl acetate and retinyl palmitate are equivalent.]

Extraction solvent— Prepare a mixture of n-hexane and methylene chloride (3:1).

Potassium hydroxide solution— Cautiously add 80g of potassium hydroxide to 100mLof water,mix,and cool.

Diluting solution— Transfer 1.0g of pyrogallol to a 100-mLvolumetric flask,and add alcohol to dissolve.Dilute with alcohol to volume,and mix.

Mobile phase— Prepare a mixture of n-hexane and isopropyl alcohol (92:8).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard stock solution— Dissolve an accurately weighed quantity of USP Vitamin A RSin Diluting solution,and dilute quantitatively,and stepwise if necessary,with Diluting solutionto obtain a solution having a known concentration of about 30µg per mL.This solution may be stored in a refrigerator for 1week.

Standard preparation— Quantitatively dilute an accurately measured volume of Standard stock solutionwith Diluting solutionto obtain a solution having a known concentration of about 1µg of USP Vitamin A RSper mL.Transfer 10.0mLof this solution to a stoppered 125-mLflask,and add 5mLof water,5mLof Diluting solution,and 3mLof Potassium hydroxide solution.Insert the stopper tightly,shake for 15minutes over a water bath maintained at 60±5,and cool to room temperature.Add 7mLof water and 25.0mLof Extraction solvent.Insert the stopper tightly,and shake vigorously for 60seconds.Rinse the sides of the flask with about 60mLof water,and allow to stand for about 10minutes until the layers separate.Withdraw a portion of the organic layer for injection into the chromatograph.This Standard preparationcontains about 0.34µg of retinol per mL.

Assay preparation— Weigh and finely powder a counted number of Tablets.Transfer an accurately weighed portion of the powder,equivalent to about 1.5mg of retinyl acetate,to a stoppered 125-mLflask.Add 5mLof water,15mLof Diluting solution,and 3mLof Potassium hydroxide solution.Insert the stopper tightly,shake for 15minutes over a water bath maintained at 60±5,and cool to room temperature.Add 7mLof water and 25.0mLof Extraction solvent.Insert the stopper tightly,and shake vigorously for 60seconds or longer,if necessary,for complete extraction.Rinse the sides of the flask with about 60mLof water,and allow to stand for about 10minutes until the layers separate.[NOTE—Do not shake,as an emulsion may form.]Withdraw a portion of the organic layer,and dilute quantitatively,and stepwise if necessary,with Extraction solventto obtain a solution having a concentration of about 0.34µg of retinol per mL.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 335-nm detector and a 6.2-mm ×8-cm column that contains packing L3.The column temperature is maintained at 40,and the flow rate is about 4mLper minute.Chromatograph the Standard preparation,and measure the peak areas as directed for Procedure:the relative retention times are about 0.92for 13-cisretinol and 1.0for all-transretinol;and the relative standard deviation for replicate injections is not more than 5.0%.

Procedure— Separately inject equal volumes (about 50µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the peak areas for all-transretinol and 13-cisretinol.Calculate the quantity,in mg,of vitamin A,as the retinol (C20H30O)equivalent,in the portion of Tablets taken by the formula: in which 0.872is the factor used to convert retinyl acetate,obtained from USP Vitamin A RS,to its retinol equivalent;Cis the concentration,in mg per mL,of USP Vitamin A RSin the Standard preparation;Dis the dilution factor,in mL,used to prepare the Assay preparation;rUis the sum of the areas of the all-transretinol and 13-cisretinol peaks obtained from the Assay preparation;and rSis the peak area of all-transretinyl acetate obtained from the Standard preparation.

Mobile phase— Prepare a filtered and degassed mixture of n-hexane and isopropyl alcohol (99:1).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard preparation— Dissolve an accurately weighed quantity of USP Cholecalciferol RSor USP Ergocalciferol RSin n-hexane,and dilute quantitatively,and stepwise if necessary,to obtain a solution having a known concentration of about 2µg per mL.

System suitability preparation— Heat a volume of the Standard preparationat 60for 1hour to partially isomerize vitamin D(cholecalciferol or ergocalciferol)to its corresponding precursor.

Assay preparation— Transfer not less than 20mL,accurately measured,of the solution retained as specified in the directions for Assay preparationin the Assay for vitamin A,Method 1to a suitable container,and evaporate,if necessary,in vacuum at room temperature to obtain a solution having a concentration of about 2µg of cholecalciferol or ergocalciferol per mL.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 265-nm detector and a 4.6-mm ×15-cm column that contains 3-µm packing L8.The flow rate is about 1mLper minute.Chromatograph the System suitability preparation,and record the peak heights as directed for Procedure:the resolution,R,between the vitamin Dform present and its corresponding precursor is not less than 10.Chromatograph the Standard preparation,and record the peak heights as directed for Procedure:the relative standard deviation for replicate injections is not more than 3.0%.

Procedure— Separately inject equal volumes (about 100µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the peak heights for vitamin D.Calculate the quantity,in µg,of cholecalciferol (C27H44O)or ergocalciferol (C28H44O)in the portion of Tablets taken by the formula: in which 1.09is a correction factor to account for the average amount of previtamin Dpresent in the formulation;Cis the concentration,in µg per mL,of USP Cholecalciferol RSor USP Ergocalciferol RSin the Standard preparation;Dis the dilution factor,in mL,for the Assay preparation;and rUand rSare the peak heights for cholecalciferol or ergocalciferol obtained from the Assay preparationand the Standard preparation,respectively.

3N Methanolic sulfuric acid solution,Sodium ascorbate–pyrogallol solution,Lecithin solution,and Assay preparation— Proceed as directed in the Assay for vitamin A,Method 2.

Mobile phase— Prepare a filtered and degassed mixture of n-hexane and tertiary butyl alcohol (98.75:1.25).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard preparation— Dissolve an accurately weighed quantity of USP Cholecalciferol RSor USP Ergocalciferol RSin 2,2,4-trimethylpentane,and dilute quantitatively,and stepwise if necessary,with 2,2,4-trimethylpentane to obtain a solution having a known concentration of about 1µg per mL.

System suitability preparation— Heat a volume of the Standard preparationat 60for 1hour to partially isomerize vitamin D(cholecalciferol or ergocalciferol)to its corresponding precursor.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 265-nm detector and a 4.6-mm ×25-cm column that contains 5-µm packing L24.The flow rate is about 1.0mLper minute.Chromatograph the System suitability preparation,and record the peak areas as directed for Procedure:the resolution,R,between the vitamin Dform present and its corresponding precursor is not less than 4.0.Chromatograph the Standard preparation,and record the peak areas as directed for Procedure:the relative standard deviation for replicate injections is not more than 3.0%.

Procedure— Separately inject equal volumes (about 40µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the peak areas for vitamin D.Calculate the quantity,in µg,of cholecalciferol (C27H44O)or ergocalciferol (C28H44O)in the portion of Tablets taken by the formula: in which Cis the concentration,in µg per mL,of USP Cholecalciferol RSor USP Ergocalciferol RSin the Standard preparation;and rUand rSare the peak responses of cholecalciferol or ergocalciferol in the Assay preparationand the Standard preparation,respectively.

Dilute acetic acid— Transfer 10mLof glacial acetic acid to a 100-mLvolumetric flask,dilute with water to volume,and mix.

Phenolphthalein solution— Prepare a solution containing 1g of phenolphthalein in 100mLof alcohol.

Potassium hydroxide solution— Slowly dissolve 14g of potassium hydroxide in a mixture of 31mLof dehydrated alcohol and 5mLof water.Prepare fresh daily.

Extraction solvent— Prepare a mixture of methylene chloride and isopropyl alcohol (99.8:0.2).

Mobile phase— Prepare a mixture of acetonitrile and methanol (91:9).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard stock solution— Dissolve an accurately weighed quantity of USP Cholecalciferol RSor USP Ergocalciferol RSin dehydrated alcohol to obtain a solution having a known concentration of about 0.2mg per mL.Prepare fresh every 4weeks.Store in a freezer.

Standard preparation— [NOTE—Condition the solid-phase extraction column specified for use in the Standard preparationand the Assay preparationby initially washing the column with 4.0mLof a mixture of methylene chloride and isopropyl alcohol (80:20),followed by 5.0mLof Extraction solvent.Do not allow the column to dry.]Dilute an accurately measured volume of Standard stock solutionwith dehydrated alcohol to obtain a solution having a known concentration of about 5µg of USP Cholecalciferol RSor USP Ergocalciferol RSper mL.Prepare this solution fresh daily.Transfer 2.0mLof this solution to a stoppered 125-mLflask.Add 15.0mLof water and 15.0mLof Potassium hydroxide solution,insert the stopper,and shake for 30minutes in a water bath maintained at 60.Allow to cool to room temperature,and transfer the contents of the flask to a 250-mLseparatory funnel.Add 15.0mLof water to the flask,insert the stopper,shake vigorously,and transfer this solution to the separatory funnel.Rinse the flask with 60mLof n-hexane,and transfer the rinsing to the separatory funnel.Insert the stopper,shake vigorously for 90seconds,and allow to stand for about 15minutes until the layers separate.Drain and discard the aqueous layer.Add 15.0mLof water to the hexane layer in the separatory funnel,insert the stopper,and shake vigorously.Allow to stand for about 10minutes until the layers separate,and discard the aqueous layer.Add 1drop of Phenolphthalein solutionand 15.0mLof water to the separatory funnel.Add Dilute acetic aciddropwise,with shaking,until the washing is neutral.Allow to stand for about 10minutes until the layers separate.Drain and discard the aqueous layer.Filter the hexane layer through anhydrous sodium sulfate supported by a small pledget of cotton into a 100-mLround-bottom flask.Rinse the funnel and sodium sulfate with a few mLof n-hexane,and collect the rinsings in the same flask.Evaporate the hexane in the flask on a rotary evaporator at 50to dryness.Immediately add 2.0mLof Extraction solventto dissolve the residue.Transfer this solution to a freshly conditioned solid-phase extraction column containing silica packing with a sorbent mass to column volume ratio of 500mg to 2.8mLor equivalent,rinse the round-bottom flask with 1.0mLof Extraction solvent,and transfer to the column.Elute the column with 2.0mLof Extraction solvent,and discard this fraction.Elute the column with 7.0mLof Extraction solvent,and collect the eluate in a suitable flask.Place the flask in a warm water bath maintained at about 42,and evaporate the solvent with the aid of a stream of nitrogen.Immediately add 2.0mLof acetonitrile to the residue,and use the solution for injection into the chromatograph.

Assay preparation— Weigh and finely powder not fewer than 20Tablets.Transfer an accurately weighed portion of the powder,equivalent to about 10µg of cholecalciferol or ergocalciferol,to the stoppered 125-mLflask,and proceed as directed for the Standard preparation,beginning with “Add 15.0mLof water and 15.0mLof Potassium hydroxide solution”.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 265-nm detector and a 4.6-mm ×25-cm column that contains 5-µm packing L1.The column temperature is maintained at 27,and the flow rate is about 0.7mLper minute.Chromatograph the Standard preparation,and record the peak heights as directed for Procedure:the relative standard deviation for replicate injections is not more than 4.0%.

Procedure— Separately inject equal volumes (about 15µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the peak heights for vitamin D.Calculate the quantity,in µg,of cholecalciferol (C27H44O)or ergocalciferol (C28H44O)in the portion of Tablets taken by the formula: in which 1.09is a correction factor to account for the average amount of previtamin Dpresent in the formulation;Cis the concentration,in µg per mL,of USP Cholecalciferol RSor USP Ergocalciferol RSin the Standard preparation;and rUand rSare the peak heights for cholecalciferol or ergocalciferol obtained from the Assay preparationand the Standard preparation,respectively.

Mobile phase— Dilute 10mLof phosphoric acid with water to 1000mLto obtain Solution A.Prepare a filtered and degassed mixture of methanol and Solution A(95:5).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard preparation— Dissolve an accurately weighed quantity of USP Alpha Tocopherol RS,USP Alpha Tocopheryl Acetate RS,or USP Alpha Tocopheryl Acid Succinate RSin methanol,and dilute quantitatively with methanol to obtain a solution having a known concentration of about 2mg per mL.

System suitability preparation— Dissolve an accurately weighed quantity of USP Ergocalciferol RSin methanol,and dilute quantitatively,and stepwise if necessary,with methanol to obtain a solution having a concentration of 0.65mg per mL.Transfer 1.0mLof this solution to a 100-mLvolumetric flask containing about 100mg of USP Alpha Tocopheryl Acetate RS,accurately weighed.Dissolve in 30mLof methanol,with the aid of sonication if necessary,dilute with methanol to volume,and mix.Store this solution in a refrigerator.

Assay preparation— Transfer not less than 20mL,accurately measured,of the solution retained as specified in the directions for Assay preparationin the Assay for vitamin A,Method 1to a suitable container,and evaporate in vacuum at room temperature to dryness.Transfer the residue with the aid of methanol to a suitable volumetric flask,and dilute with methanol to volume to obtain a solution having a concentration of about 2mg of alpha tocopherol,alpha tocopheryl acetate,or alpha tocopheryl acid succinate per mL.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 254-nm detector and an 8-mm ×10-cm column that contains 5-µm packing L1.The flow rate is about 2mLper minute.Chromatograph the System suitability preparation,and record the peak areas as directed for Procedure:the relative retention times are about 0.5for ergocalciferol and 1.0for alpha tocopheryl acetate;the resolution,R,between ergocalciferol and alpha tocopheryl acetate is not less than 12;and the tailing factor is between 0.8and 1.2.Chromatograph the Standard preparation,and record the peak areas as directed for Procedure:the relative standard deviation for replicate injections is not more than 3.0%.

Procedure— Separately inject equal volumes (about 100µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the peak areas.Calculate the quantity,in mg,of alpha tocopherol (C29H50O2),alpha tocopheryl acetate (C31H52O3),or alpha tocopheryl acid succinate (C33H54O5)in the portion of Tablets taken by the formula: in which Cis the concentration,in mg per mL,of the corrresponding USP Reference Standard in the Standard preparation;Dis the dilution factor,in mL,for the Assay preparation;and rUand rSare the peak responses for the relevant vitamin Eform obtained from the Assay preparationand the Standard preparation,respectively.Calculate the alpha tocopherol equivalent of alpha tocopheryl acetate or alpha tocopheryl acid succinate by multiplying the content,in mg,by the factor 0.91or 0.81,respectively.

Mobile phase— Transfer 240mLof methanol to a 1000-mLvolumetric flask,add 10mLof water followed by 0.5mLof 50%phosphoric acid,dilute with acetonitrile to volume,mix,filter,and degas.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

System suitability preparation— Dissolve accurately weighed quantities of USP Alpha Tocopherol RS,USP Alpha Tocopheryl Acetate RS,and USP Alpha Tocopheryl Acid Succinate RSin methanol,and dilute quantitatively with methanol to obtain a solution having known concentrations of about 2mg of each USP Reference Standard per mL.

Standard preparation— Dissolve an accurately weighed quantity of USP Alpha Tocopherol RS,USP Alpha Tocopheryl Acetate RS,or USP Alpha Tocopheryl Acid Succinate RSin methanol,and dilute quantitatively with methanol to obtain a solution having a known concentration of about 2mg per mL.

Assay preparation— Proceed as directed for Assay preparationin the Assay for vitamin A,Method 2.Transfer an accurately measured volume of the supernatant 2,2,4-trimethylpentane to a suitable volumetric flask,the volume of the specimen withdrawn from the 2,2,4-trimethylpentane and the size of the volumetric flask being such that the final concentration of the Assay preparationis equivalent to that of the Standard preparation.Evaporate nearly to dryness,add several mLof methanol,and evaporate the remaining 2,2,4-trimethylpentane.Dilute with methanol to volume,and mix.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 280-nm detector and a 4.6-mm ×25-cm column that contains 5-µm packing L1.The flow rate is about 1.5mLper minute.Chromatograph theSystem suitability preparation,and record the peak areas as directed for Procedure:the relative retention times for alpha tocopheryl acid succinate,alpha tocopherol,and alpha tocopheryl acetate are about 0.6,0.8,and 1.0,respectively;the resolution between alpha tocopheryl acid succinate and alpha tocopherol is not less than 4.0;and the resolution between alpha tocopherol and alpha tocopheryl acetate is not less than 3.0.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the relative standard deviation for replicate injections is not more than 3.0%.

Procedure— Separately inject equal volumes (about 25µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the peak areas.Calculate the quantity,in mg,of alpha tocopherol (C29H50O2),alpha tocopheryl acetate (C31H52O3),or alpha tocopheryl acid succinate (C33H54O5)in the portion of Tablets taken by the formula: in which Cis the concentration,in mg per mL,of the corresponding USP Reference Standard in the Standard preparation;Dis the dilution factor used in exchanging the solvent from 2,2,4-trimethylpentane to methanol;and rUand rSare the peak areas of the relevant vitamin Eform obtained from the Assay preparationand the Standard preparation,respectively.[NOTE—The initial extraction to 26.5mLof 2,2,4-trimethylpentane has been accounted for in the calculation formula.]Calculate the alpha tocopherol equivalent of alpha tocopheryl acetate or alpha tocopheryl acid succinate by multiplying the content,in mg,by the factor 0.91or 0.81,respectively.

Diluting solution— Prepare a mixture of acetonitrile and ethyl acetate (1:1).

Mobile phase— Prepare a filtered and degassed mixture of methanol,acetonitrile,and n-hexane (46.5:46.5:7.0).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard preparation— Dissolve an accurately weighed quantity of USP Alpha Tocopherol RS,USP Alpha Tocopheryl Acetate RS,or USP Alpha Tocopheryl Acid Succinate RSin methanol,and quantitatively dilute with methanol to obtain a solution having a known concentration of about 0.3mg per mL.

Assay preparation— Weigh and finely powder not fewer than 20Tablets.Transfer an accurately weighed portion of the powder,equivalent to about 8mg of alpha tocopherol,to a 125-mLflask fitted with a ground-glass joint.Add 25.0mLof water,25.0mLof dehydrated alcohol,and 3.5g of potassium hydroxide pellets.Shake for 1hour in a water bath maintained at 55,cool,and transfer with the aid of a minimum volume of water to a 125-mLseparatory funnel.Rinse the flask with 50mLof n-hexane,and add the rinsing to the separatory funnel.Insert the stopper,shake vigorously for 60seconds,and allow the layers to separate.Drain the aqueous layer into a second 250-mLseparatory funnel,and repeat the extraction with 50mLof n-hexane.Discard the aqueous layer,and combine the hexane extracts.Wash the combined extracts with 25mLof water,allow the layers to separate,and discard the aqueous layer.Add 3drops of glacial acetic acid,and repeat the washing procedure two more times.Filter the washed hexane layer through anhydrous sodium sulfate into a 250-mLround-bottom flask.Rinse the funnel and sodium sulfate with a few mLof n-hexane,and add the rinsing to the hexane solution in the flask.Place the flask in a water bath maintained at 50,and evaporate the hexane solution with the aid of a rotary evaporator to dryness.Immediately add 25.0mLof Diluting solution,and swirl to dissolve the residue.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 291-nm detector and a 4.6-mm ×25-cm column that contains packing L1.The column temperature is maintained at about 40,and the flow rate is about 3mLper minute.Chromatograph the Standard preparation,and record the peak areas as directed for Procedure:the relative standard deviation for replicate injections is not more than 5.0%.

Procedure— Separately inject equal volumes (about 20µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the peak areas.Calculate the quantity,in mg,of alpha tocopherol (C29H50O2),alpha tocopheryl acetate (C31H52O3),or alpha tocopheryl acid succinate (C33H54O5)in the portion of Tablets taken by the formula: in which Cis the concentration,in mg per mL,of the corresponding USP Reference Standard in the Standard preparation;and rUand rSare the peak areas for the relevant vitamin Eform obtained from the Assay preparationand the Standard preparation,respectively.Calculate the alpha tocopherol equivalent of alpha tocopheryl acetate or alpha tocopheryl acid succinate by multiplying the content,in mg,by the factor 0.91or 0.81,respectively.

Mobile phase— Prepare a filtered and degassed mixture of methanol and water (95:5).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard stock solution— Dissolve an accurately weighed quantity of USP Phytonadione RSin methanol,with the aid of sonication if necessary,and quantitatively dilute with methanol to obtain a solution having a known concentration of about 200µg per mL.

Standard preparation— Pipet 10mLof Standard stock solutioninto a 100-mLvolumetric flask,dilute with methanol to volume,and mix.

System suitability preparation— Transfer 65mg of USP Alpha Tocopheryl Acetate RSto a 100-mLvolumetric flask,and dissolve in about 75mLof methanol.Add 10mLof Standard stock solution,dilute with methanol to volume,and mix.

Assay preparation— Transfer not less than 20mL,accurately measured,of the solution retained as specified in the directions for Assay preparationin the Assay for vitamin A,Method 1to a suitable container,and evaporate in vacuum at room temperature to dryness.Transfer the residue with the aid of methanol to a suitable volumetric flask,and dilute with methanol to volume to obtain a solution having a concentration of about 20µg of phytonadione per mL.

Chromatographic system(see Chromatography á621ñ)— The liquid chromatograph is equipped with a 254-nm detector and an 8-mm ×10-cm column that contains 5-µm packing L1.Chromatograph the System suitability preparation,and record the peak areas as directed for Procedure:the resolution,R,between alpha tocopheryl acetate and phytonadione is not less than 5.Chromatograph the Standard preparation,and record the peak areas as directed for Procedure:the relative retention times are about 0.68for alpha tocopheryl acetate and 1.0for phytonadione;and the relative standard deviation for replicate injections is not more than 3.0%.

Procedure— Separately inject equal volumes (about 100µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the peak areas.Calculate the quantity,in µg,of phytonadione (C31H46O2)in the Tablets taken by the formula: in which Cis the concentration,in µg per mL,of USP Phytonadione RSin the Standard preparation;Lis the labeled amount,in µg,of phytonadione in each Tablet;Dis the concentration,in µg per mL,of phytonadione in the Assay preparation,based on the labeled quantity per Tablet and the extent of dilution;and rUand rSare the peak areas for phytonadione obtained from the Assay preparationand the Standard preparation,respectively.

Solvent— Prepare a mixture of methanol and isopropanol (95:5).

Mobile phase— Prepare a filtered and degassed mixture of 800mLof methanol,200mLof methylene chloride,0.1mLof glacial acetic acid,1.36g of zinc chloride,and 0.41g of sodium acetate.

Internal standard solution— Prepare a solution of menaquinone 4(vitamin K2)in Solventhaving a concentration of about 5µg per mL.[NOTE—Aconcentrated stock solution of menaquinone 4(100µg per mL)can be stored for 2months in a refrigerator.]

Standard stock solution— Dissolve an accurately weighed quantity of USP Phytonadione RSin methylene chloride with the aid of sonication.Dilute with Solventquantitatively,and stepwise if necessary,to obtain a solution having a known concentration of about 5µg per mL.

Standard preparation— Pipet 1.0mLof Standard stock solution and 1.0mLof Internal standard solutioninto a 5.0-mLvolumetric flask,dilute with Solventto volume,and mix.Filter through a membrane having a 0.45-µm or finer porosity.

Assay preparation— Weigh and finely powder not fewer than 20Tablets.To a centrifuge tube fitted with a cap,transfer an accurately weighed amount of powder,not exceeding 800mg,and equivalent to an amount of phytonadione not exceeding 50µg.Add 4mLof water.Insert the stopper,and mix using a vortex mixer until the sample is dispersed.Place the tube in a water bath at 60for 5minutes.Remove from the bath,and again shake or mix using a vortex mixer for 1minute while the preparation is still hot.Add 8mLof alcohol,and swirl the contents to mix.Place the tube in a water bath at 60for 5minutes.Remove from the bath,and again shake or mix using a vortex mixer for 2minutes while the preparation is still hot.Cool to room temperature.Add an accurately measured volume of Internal standard solution,equivalent to 1.0mLper each 5µg of the expected amount of phytonadione in the aliquot taken.Add 20.0mLof petroleum ether,and cap the tube tightly.Shake or mix using a vortex mixer for 15minutes to thoroughly mix the contents.Centrifuge to separate the two layers.Transfer a volume of the top layer of petroleum ether,equivalent to 5to 50µg of the expected amount of phytonadione,to an appropriate flask.Place the flask in a water bath at 35to 45,and evaporate the solvent under a stream of nitrogen until an oily residue is left.Dissolve the residue in a volume of Solventto obtain a solution having a concentration of about 1µg per mLof phytonadione.

Chromatographic system(see Chromatography á621ñ)— The liquid chromatograph is equipped with a fluorometric detector set at 320nm for excitation and 420nm for emission,a 4.6-mm ×25-cm column that contains 5-µm,end-capped packing L1,and a postcolumn reactor constituted with a 4.6-mm ×3-cm PEEKcolumn tightly packed with zinc powder.[NOTE—Prepare the postcolumn reactor daily,or as necessary,to meet the system suitability requirements.]The flow rate is about 1.0mLper minute.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the relative retention times are 1.0for the internal standard and 1.4for phytonadione;the column efficiency for the phytonadione peak is not less than 2500theoretical plates;the tailing factor for the phytonadione peak is not more than 1.5;and the relative standard deviation for replicate injections is not more than 3.0%.

Procedure— Separately inject equal volumes (about 25µL)of the Standard preparation and the Assay preparationinto the chromatograph,record the chromatograms,and measure the peak responses.Calculate the quantity,in µg,of phytonadione (C31H46O2)in the portion of Tablets taken by the formula: in which Cis the concentration,in µg per mL,of USP Phytonadione RSin the Standard preparation;Dis the volume,in mL,of Internal standard solutionused to prepare the Assay preparation;and RUand RSare the peak response ratios of phytonadione to that of the internal standard obtained from the Assay preparationand the Standard preparation,respectively.

Potassium hydroxide solution— Dissolve 58.8g of potassium hydroxide in 50mLof water.

Iodine solution— Transfer about 10mg of iodine to a 100-mLvolumetric flask.Dissolve in cyclohexane,dilute with cyclohexane to volume,and mix.Dilute 10mLof this solution with cyclohexane to 100mL,and mix.[NOTE—Prepare this solution fresh daily.]

Assay preparation— Weigh accurately not fewer than 20Tablets.Grind the Tablets to a fine powder,and transfer an accurately weighed quantity of the powder,equivalent to about 2mg of beta carotene,to a 500-mLsaponification flask.Add 100mLof alcohol,6mLof Potassium hydroxide solution,and a magnetic stirring bar.Attach an air condenser to the flask,and heat under reflux for 45minutes with constant stirring.Cool to room temperature,add 170mLof solvent hexane,and stir for 30minutes.Quantitatively transfer the contents of the flask to a 500-mLseparatory funnel with portions of solvent hexane.Allow the layers to separate for 5to 10minutes,and transfer the upper organic layer to a 500-mLvolumetric flask.Transfer the lower aqueous layer into the saponification flask,add 170mLof solvent hexane,and stir for an additional 20minutes.Quantitatively transfer the contents of the saponification flask to the separatory funnel with the aid of portions of solvent hexane.Allow the layers to separate for 10minutes.Drain the lower aqueous layer,and discard.Transfer the organic layer to the volumetric flask containing the previously collected organic layer.Rinse the separatory funnel with small portions of solvent hexane,and transfer the washings to the volumetric flask.Dilute the hexane extracts with solvent hexane to volume,add 3g of anhydrous sodium sulfate,shake,and allow to settle.Quantitatively transfer a volume of this solution,equivalent to about 100µg of beta carotene,to a 50-mLvolumetric flask.Evaporate under a stream of nitrogen to dryness,and immediately add cyclohexane.Add 2mLof Iodine solution,and heat for 15minutes in a water bath maintained at 65.Cool rapidly,dilute with cyclohexane to volume,and mix.

Procedure— Determine the absorbance of the Assay preparationat the wavelength of maximum absorbance at about 452nm,using cyclohexane as the blank.Calculate the quantity,in mg,of beta carotene (C40H56)in the Tablets taken by the formula: in which Lis the labeled amount,in mg,of beta carotene in each Tablet;Dis the concentration,in mg per mL,of beta carotene in the Assay preparation,based on the labeled quantity per Tablet and the extent of dilution;AUis the absorbance of the Assay preparation;and 223is the absorptivity of beta carotene at 452nm.

Mobile phase— Transfer 85mLof acetonitrile,1g of sodium perchlorate,and 1mLof phosphoric acid to a 1000-mLvolumetric flask,dilute with water to volume,mix,filter,and degas.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard preparation— Transfer about 67mg of USP Biotin RS,accurately weighed,to a 200-mLvolumetric flask,dissolve in and dilute with dimethyl sulfoxide to volume,and mix.Transfer 3.0mLof this solution to a 200-mLvolumetric flask,dilute with water to volume,and mix to obtain a solution having a known concentration of about 5µg of USP Biotin RSper mL.

Assay preparation— Weigh and finely powder not fewer than 20Tablets.Transfer an accurately weighed portion of the powder,equivalent to about 1mg of biotin,to a 200-mLvolumetric flask.Add 3mLof dimethyl sulfoxide,and swirl to wet the contents.Place the flask in a water bath at 60to 70for 5minutes.Sonicate for 5minutes,dilute with water to volume,mix,and filter.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 200-nm detector and a 4.6-mm ×15-cm column containing 3-µm packing L7.The flow rate is about 1.2mLper minute.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the relative standard deviation for replicate injections is not more than 3%.

Procedure— Separately inject equal volumes (about 100µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the responses for the biotin peaks.Calculate the quantity,in µg,of biotin (C10H16N2O3S)in the portion of Tablets taken by the formula: in which Cis the concentration,in µg per mL,of USP Biotin RSin the Standard preparation;and rUand rSare the biotin peak responses obtained from the Assay preparationand the Standard preparation,respectively.

Standard stock solution— Dissolve an accurately weighed quantity of USP Biotin RSin 50%alcohol,dilute with 50%alcohol to obtain a solution having a known concentration of about 50µg per mL,and mix.Store this solution in a refrigerator.

Standard preparation— On the day of the assay,dilute a volume of Standard stock solutionwith water to obtain a solution having a known concentration of about 0.1ng per mL.

Assay preparation— Weigh and finely powder not fewer than 30Tablets.Transfer an accurately weighed quantity of the powder,equivalent to about 100µg of biotin,to a 200-mLvolumetric flask,add 3mLof 50%alcohol,and swirl to wet the contents.Heat the flask in a water bath at 60to 70for 5minutes.Sonicate for 5minutes,dilute with 50%alcohol to volume,mix,and filter.Dilute an accurately measured volume of the filtrate,quantitatively,and stepwise if necessary,with water to obtain a solution having a concentration of about 0.1ng per mL.

Acid-hydrolyzed casein solution— Mix 100g of vitamin-free casein with 500mLof 6Nhydrochloric acid,and reflux the mixture for 8to 12hours.Remove the hydrochloric acid from the mixture by distillation under reduced pressure until a thick paste remains.Redissolve the resulting paste in water,adjust the solution with 1Nsodium hydroxide to a pHof 3.5±0.1,and add water to make 1000mL.Add 20g of activated charcoal,stir for 1hour,and filter.Repeat the treatment with activated charcoal.Store under toluene in a refrigerator at a temperature not below 10.Filter the solution if a precipitate forms during storage.

Cystine–tryptophan solution— Suspend 4.0g of L-cystine in 1.0g of L-tryptophan (or 2.0g of D,L-tryptophan)in 700to 800mLof water,heat to 70to 80,and add dilute hydrochloric acid (1in 2)dropwise,with stirring,until the solids are dissolved.Cool,and add water to make 1000mL.Store under toluene in a refrigerator at a temperature not below 10.

Adenine–guanine–uracil solution— Dissolve 200mg each of adenine sulfate,guanine hydrochloride,and uracil,with the aid of heat,in 10mLof 4Nhydrochloric acid,cool,and add water to make 200mL.Store under toluene in a refrigerator.

Polysorbate 80solution— Dissolve 25g of polysorbate 80in alcohol to make 250mL.

Calcium pantothenate solution— Prepare a solution of calcium pantothenate in 50%alcohol containing 10µg per mL.Store in a refrigerator.

Riboflavin–thiamine hydrochloride solution— Prepare a solution of riboflavin and thiamine hydrochloride in 0.02Nacetic acid containing 20µg of riboflavin and 10µg of thiamine hydrochloride per mL.Store under toluene,protected from light,in a refrigerator.

p-Aminobenzoic acid–niacin–pyridoxine hydrochloride solution— Prepare a solution in a mixture of water and neutralized alcohol (3:1)containing 10µg of p-aminobenzoic acid,50µg of niacin,and 40µg of pyridoxine hydrochloride per mL.Store in a refrigerator.

Salt solution 1— Dissolve 25g of monobasic potassium phosphate and 25g of dibasic potassium phosphate in water to make 500mL.Add 5drops of hydrochloric acid,and mix.Store under toluene.

Salt solution 2— Dissolve 10g of magnesium sulfate,0.5g of sodium chloride,0.5g of ferrous sulfate,and 0.5g of manganese sulfate in water to make 500mL.Add 5drops of hydrochloric acid,and mix.Store under toluene.

Basal medium stock solution—

Stock culture of Lactobacillus plantarum— Dissolve 2.0g of yeast extract in 100mLof water,add 500mg of anhydrous dextrose,500mg of anhydrous sodium acetate,and 1.5g of agar,and heat the mixture on a steam bath,with stirring,until the agar dissolves.Add 10-mLportions of the hot solution to test tubes,close or cover the tubes,sterilize in an autoclave at 121,and allow the tubes to cool in an upright position.Prepare stab cultures in three or more of the tubes,using a pure culture of Lactobacillus plantarum,2incubating for 16to 24hours at a temperature between 30and 37held constant to within ±0.5.Store in a refrigerator.Prepare a fresh stab of the stock culture every week,and do not use for inoculum if the culture is more than 1week old.

Culture medium— To each of a series of test tubes containing 5.0mLof Basal medium stock solution,add 5.0mLof water containing 0.5ng of biotin.Plug the tubes with cotton,sterilize in an autoclave at 121,and cool.

Inoculum— [NOTE—Afrozen suspension of Lactobacillus plantarummay 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 plantarumto a sterile tube containing 10mLof culture medium.Incubate this culture for 16to 24hours at a temperature between 30and 37held constant to within ±0.5.The cell suspension so obtained is the Inoculum.

Procedure— To similar separate test tubes add,in duplicate,1.0and/or 1.5,2.0,3.0,4.0,and 5.0mLof the Standard preparation.To each tube and to four similar empty tubes,add 5.0mLof Basal medium stock solutionand sufficient water to make 10mL.

Calculation— Prepare a standard concentration-response curve as follows.For each level of the Standard,calculate the response from the sum of the duplicate values of the transmittance (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 preparationper 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.

Replication— Repeat the entire determination at least once,using separately prepared Assay preparations.If the difference between the two log-potencies Mis not greater than 0.08,their mean,bar(M),is the assayed log-potency of the test material (see The Confidence Interval and Limits of Potencyunder 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 of Mthat do not differ by more than 0.15,compute the mean potency of the preparation under assay.

Add the following:

Buffer— Transfer 800mLof water and 100mLof triethylamine to a 1000-mLvolumetric flask.Add 80mLof 85%phosphoric acid,dilute with water to volume,and mix.

Standard preparation— Transfer about 25mg of USP Biotin RS,accurately weighed,to a 250-mLvolumetric flask,dissolve in and dilute with water to volume,and mix.Transfer 1.5mLof this solution to a 250-mLvolumetric flask,dilute with water to volume,and mix to obtain a solution having a known concentration of about 0.6µg per mLof USP Biotin RS.[NOTE—Aportion of the Standard preparationwill be used to determine the percent recovery of biotin from the Solid-phase extraction procedure.]

Assay preparation— Weigh and finely powder not fewer than 20Tablets.Transfer an accurately weighed portion of the powder,equivalent to about 60µg of biotin,to a 100-mLvolumetric flask.Add about 80mLof water,and sonicate for 30to 40minutes with occasional mixing.Cool to room temperature,dilute with water to volume,mix,and filter.Adjust the pHof the solution with either dilute acetic acid or 0.1Nsodium hydroxide to between 6.0and 7.0.

Solid-phase extraction— [NOTE—Condition the extraction column specified in this procedure in the following manner.Wash the column with a 2-mLportion of methanol.Equilibrate with a 2-mLportion of water.]Separately pipet 5.0mLof the Assay preparationand Standard preparationinto freshly conditioned solid-phase extraction columns consisting of a mixed-mode packing with a sorbent-mass of 60mg.[NOTE—The mixed-mode packing consists of anion-exchange and reversed-phase sorbents.The reverse-phase component is a polymer of copolymer N-vinylpyrrolidone and divinylbenzene.The anion exchange moiety is a trialkyamino group.*]Wash the column with 10mLof 30%(v/v)methanol in water.Apply an appropriate volume (about 4.9mL)of 30%(v/v)methanol in 0.1Nhydrochloric acid to the column.Collect the eluate in a 5-mLvolumetric flask,containing 100µLof 40%(w/v)sodium acetate in water,and dilute with 30%(v/v)methanol in 0.1Nhydrochloric acid to volume.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 200-nm detector and a 4.6-mm ×25-cm column containing packing L1.The flow rate is about 2.0mLper minute.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the tailing factor is not more than 1.5;and the relative standard deviation for replicate injections is not more than 2%.Chromatograph the portion of Standard preparationthat has undergone solid-phase extraction,and record the peak responses as directed forProcedure:the relative standard deviation for replicate injections is not more than 2%;and the recovery is between 95%and 100%.

Procedure— Separately inject equal volumes (about 100µL)of the Standard preparationand the Assay preparationthat have undergone solid-phase extraction,into the chromatograph,record the chromatograms,and measure the responses for the biotin peak.Calculate the quantity,in µg,of biotin (C10H16N2O3S)in the portion of Tablets taken by the formula: in which Cis the concentration,in µg per mL,of USP Biotin RSin the Standard preparation;and rUand rSare the biotin peak responses obtained from the Assay preparationand the Standard preparation,respectively.USP28

Mobile phase— Prepare a filtered and degassed mixture of water and methanol (65:35).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard preparation— Dissolve an accurately weighed quantity of USP Cyanocobalamin RSin water to obtain a stock solution having a known concentration of about 10µg per mL.[NOTE—Store this stock solution in a dark place,and discard after 1week.]Dilute a portion of this stock solution quantitatively with water to obtain a solution having a known concentration of about 1µg per mL.

Assay preparation— Weigh and finely powder not fewer than 30Tablets.Transfer an accurately weighed portion of the powder,equivalent to about 100µg of cyanocobalamin,to a 250-mLflask.Quantitatively add 100.0mLof water,and carefully extract for 2minutes.Filter about 10mLof the extract,and use the filtrate.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 550-nm detector and a 4.6-mm ×15-cm column containing 5-µm packing L1.The flow rate is about 0.5mLper minute.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the relative standard deviation for replicate injections is not more than 3.0%.

Procedure— Separately inject equal volumes (about 200µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the peak responses for cyanocobalamin.Calculate the quantity,in µg,of cyanocobalamin (C63H88CoN14O14P)in the portion of Tablets taken by the formula: in which Cis the concentration,in µg per mL,of USP Cyanocobalamin RSin the Standard preparation;and rUand rSare the peak responses for cyanocobalamin obtained from the Assay preparationand the Standard preparation,respectively.

Standard cyanocobalamin stock solution— Dissolve an accurately weighed quantity of USP Cyanocobalamin RSin 25%alcohol to obtain a solution having a known concentration of about 1.0µg of USP Cyanocobalamin RSper mL.Store in a refrigerator.

Standard preparation— 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 preparationis not less than that which corresponds to a difference of 1.25mg in dried cell weight.This concentration usually falls between 0.01and 0.04ng per mLof Standard preparation.Prepare this solution fresh for each assay.

Assay preparation— Weigh and finely powder not fewer than 20Tablets.Transfer an accurately weighed portion of the powder,equivalent to about 1.0µg of cyanocobalamin,to an appropriate vessel containing,for each g of powdered Tablets taken,25mLof an aqueous extracting solution prepared just prior to use to contain,in each 100mL,1.29g of dibasic sodium 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 to obtain a final solution containing vitamin B12activity approximately equivalent to that of the Standard preparation.

Acid-hydrolyzed casein solution— Prepare as directed in the Assay for calcium pantothenate,Method 2.

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 in the Assay for calcium pantothenate,Method 2.

Xanthine solution— Suspend 0.20g of xanthine in 30to 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 1— Dissolve 10g of monobasic potassium phosphate and 10g of dibasic potassium phosphate in water to make 200mL,and add 2drops of hydrochloric acid.Store this solution under toluene.

Salt solution 2— 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,and add 2drops of hydrochloric acid.Store this solution under toluene.

Polysorbate 80solution— Dissolve 20g of polysorbate 80in alcohol to make 200mL.Store in a refrigerator.

Vitamin solution 1— Dissolve 10mg of riboflavin,10mg of thiamine hydrochloride,100µg of biotin,and 20mg of niacin in 0.02Nglacial acetic acid to make 400mL.Store under toluene,protected from light,in a refrigerator.

Vitamin solution 2— Dissolve 20mg of p-aminobenzoic acid,10mg of calcium pantothenate,40mg of pyridoxine hydrochloride,40mg of pyridoxal hydrochloride,8mg of pyridoxamine dihydrochloride,and 2mg of folic acid in a mixture of water and neutralized alcohol (3:1)to make 400mL.Store,protected from light,in a refrigerator.

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.