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.

Tomato juice preparation— Centrifuge commercially canned tomato juice so that most of the pulp is removed.Suspend about 5g per liter of 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 yeast extract,0.75g of dried peptone,1.0g of anhydrous dextrose,and 0.20g of monobasic potassium phosphate in 60to 70mLof water.Add 10mLof Tomato juice preparationand 1mLof Polysorbate 80solution.Adjust 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 for Culture medium.

Stock culture of Lactobacillus leichmannii— To 100mLof Culture medium,add 1.0to 1.5g of agar,and heat the mixture on a steam bath,with stirring,until the agar dissolves.Place 10-mLportions of the hot solution in test tubes,cover the tubes,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.3[NOTE—Before first using a fresh culture in this assay,make not fewer than 10successive transfers of the culture in a 2-week period.]Incubate for 16to 24hours at a temperature between 30and 40held constant to within ±0.5.Store in a refrigerator.

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 two sterile tubes containing 10mLof the Culture mediumeach.Incubate these cultures for 16to 24hours at a temperature between 30and 40held 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.The cell suspension so obtained is the Inoculum.[NOTE—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— Because of the high sensitivity of the test organism to minute amounts of vitamin B12activity and to traces of many cleansing agents,cleanse meticulously by suitable means,followed preferably by heating at 250for 2hours,using hard-glass 20-mm ×150-mm test tubes,and other necessary glassware.

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 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 Vitamin B12Activityunder 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.

Reagent 1— Use a 25%solution of tetrabutylammonium hydroxide in methanol.

Reagent 2— Transfer 5.0g of pentetic acid to a 50-mLvolumetric flask.Using sonication if necessary,dissolve in and dilute with 1Nsodium hydroxide to volume,and mix.

Mobile phase— Transfer 2g of monobasic potassium phosphate to a 1-Lvolumetric flask,and dissolve in 650mLof water.Add 12.0mLof Reagent 1,7.0mLof 3Nphosphoric acid,and 240mLof methanol.Cool to room temperature,adjust with phosphoric acid or ammonia TSto a pHof 7.0,dilute with water to volume,mix,and filter.Recheck the pHbefore use.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).[NOTE—The methanol and water content may be varied (between 1%and 3%)by adding water or methanol to the prepared Mobile phaseto obtain baseline separation of folic acid and the internal standard.The pHmay be increased up to 7.15to obtain better separation.]

Internal standard preparation— Transfer about 40mg of methylparaben,accurately weighed,to a 1000-mLvolumetric flask,and add 220mLof methanol to dissolve.Dissolve 2.0g of monobasic potassium phosphate in about 300mLof water in a separate beaker,quantitatively transfer this solution to the flask containing the methylparaben solution,and add an additional 300mLof water.Add 19mLof Reagent 1,7mLof 3Nphosphoric acid,and 30mLof Reagent 2.Adjust with ammonia TSto a pHof 9.8,bubble nitrogen through the solution for 30minutes,dilute with water to volume,and mix.

Standard preparation— Dissolve an accurately weighed quantity of USP Folic Acid RSin Internal standard preparationto obtain a solution having a known concentration of about 0.2mg per mL.Transfer 2.0mLof this solution to a 25-mLvolumetric flask,dilute with Internal standard preparationto volume,and mix.

Assay preparation— Weigh and finely powder not fewer than 30Tablets.Transfer an accurately weighed portion of the powder,equivalent to about 0.4mg of folic acid,to a 50-mLamber-colored centrifuge tube.Add 25.0mLof Internal standard preparation,insert a stopper,shake by mechanical means for 10minutes,and centrifuge.Filter a portion of the clear supernatant,and use the filtrate.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 280-nm detector and a 3.9-mm ×30-cm column that contains packing L1.The flow rate is about 1mLper minute.Chromatograph the Standard preparation,and record the peak areas as directed for Procedure:the relative retention times are about 0.8for folic acid and 1.0for methylparaben;and the relative standard deviation for replicate injections is not more than 3.0%.

Procedure— Separately inject equal volumes (about 15µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the peak areas for folic acid and methylparaben.Calculate the quantity,in mg,of folic acid (C19H19N7O6)in the portion of Tablets taken by the formula: in which Cis the concentration,in mg per mL,of USP Folic Acid RSin the Standard preparation;and RUand RSare the peak area ratios of folic acid to methylparaben obtained from the Assay preparationand the Standard preparation,respectively.

Reagent— Dissolve 7.5g of edetate disodium,with stirring,in 500mLof water containing 10mLof ammonium hydroxide.

Diluting solution— Prepare a solution of ammonium hydroxide containing 60µg per mL.

Mobile phase— Transfer 0.4mLof triethylamine,15.0mLof glacial acetic acid,and 350mLof methanol to a 2000-mLvolumetric flask,dilute with 0.008Msodium 1-hexanesulfonate to volume,and mix.Filter,and degas.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard stock solution— Dissolve an accurately weighed quantity of USP Folic Acid RSin Diluting solutionto obtain a solution having a known concentration of about 60µg per mL.Prepare this solution fresh daily.

Standard preparation— Transfer 5.0mLof Standard stock solutionto a stoppered 125-mLflask,and add 10.0mLof methanol and 35.0mLof Reagent.Insert the stopper,shake for 15minutes in a water bath maintained at 60,and cool.Filter,discarding the first few mLof the filtrate.

Assay preparation— Weigh and finely powder a counted number of Tablets.Transfer an accurately weighed portion of the powder,equivalent to about 0.3mg of folic acid,to a stoppered 125-mLflask.Add 10.0mLof methanol and 35.0mLof Reagent,insert the stopper,shake for 15minutes in a water bath maintained at 60,and cool.Filter,discarding the first few mLof the filtrate.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 270-nm detector and a 4.6-mm ×25-cm column that contains packing L7.The column temperature is maintained at 50,and the flow rate is about 2.0mLper 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 2.0%.

Procedure— Separately inject equal volumes (about 5µL)of the Assay preparationand the Standard preparationinto the chromatograph,record the chromatograms,and measure the areas of the major peaks.Calculate the quantity,in mg,of folic acid (C19H19N7O6)in the portion of Tablets taken by the formula: in which Cis the concentration,in mg per mL,of USP Folic Acid RSin the Standard preparation;and rUand rSare the peak areas of folic acid obtained from the Assay preparationand the Standard preparation,respectively.

Mobile phase— Prepare a filtered and degassed mixture of water and phosphoric acid (1000:1).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Internal standard preparation— Transfer about 80mg of p-hydroxybenzoic acid,accurately weighed,to a 1000-mLvolumetric flask,and dissolve in 3mLof alcohol.Add about 50mLof water and 7.1g of dibasic sodium phosphate,dilute with water to volume,and mix.Adjust with phosphoric acid to a pHof 6.7,and mix.

Standard preparation— Dissolve an accurately weighed quantity of USP Calcium Pantothenate RSin Internal standard preparationto obtain a solution having a known concentration of about 0.6mg per mL.

Assay preparation— Weigh and finely powder not fewer than 30Tablets.Transfer an accurately weighed portion of the powder,equivalent to about 15mg of calcium pantothenate,to a centrifuge tube.Add 25.0mLof the Internal standard preparation,and shake vigorously for 10minutes.Centrifuge,filter,and use the clear filtrate.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 210-nm detector and a 3.9-mm ×15-cm column that contains packing L1.The flow rate is about 1.5mLper minute.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the relative retention times are 0.5for calcium pantothenate and 1.0for p-hydroxybenzoic acid;and the relative standard deviation for replicate injections is not more than 3.0%.

Procedure— Separately inject equal volumes (about 10µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the peak responses for calcium pantothenate and the internal standard.Calculate the quantity,in mg,of calcium pantothenate (C18H32CaN2O10)in the portion of Tablets taken by the formula: in which Cis the concentration,in mg per mL,of USP Calcium Pantothenate RSin the Standard preparation;and RUand RSare the peak response ratios of calcium pantothenate to p-hydroxybenzoic acid obtained from the Assay preparationand the Standard preparation,respectively.

Standard stock solution— Dissolve about 50mg of USP Calcium Pantothenate RS,previously dried and stored in the dark over phosphorus pentoxide and accurately weighed while protected from absorption of moisture during the weighing,in about 500mLof water in a 1000-mLvolumetric flask.Add 10mLof 0.2Nacetic acid and 100mLof sodium acetate solution (1in 60),and dilute with water to volume to obtain a solution containing 50µg of USP Calcium Pantothenate RSper mL.Store under toluene in a refrigerator.

Standard preparation— On the day of the assay,dilute an accurately measured volume of Standard stock solutionwith water to obtain a solution having a known concentration of 0.01to 0.04µg of calcium pantothenate per mL,the exact concentration being such that the responses obtained as directed for Procedure,2.0and 4.0mLof the Standard preparationbeing used,are within the linear portion of the log-concentration response curve.

Assay preparation— Weigh and powder not fewer than 30Tablets.Transfer an accurately weighed portion of the powder,equivalent to about 50mg of calcium pantothenate,to a 1000-mLvolumetric flask containing 500mLof water.Add 10mLof 0.2Nacetic acid and 100mLof sodium acetate solution (1in 60),dilute with water to volume,and filter.Dilute an accurately measured volume of this solution quantitatively,and stepwise if necessary,to obtain a solution having approximately the same concentration as that of the Standard preparation.

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 and 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.

Riboflavin–thiamine hydrochloride–biotin solution— Prepare a solution of riboflavin,thiamine hydrochloride,and biotin in 0.02Nacetic acid containing 20µg of riboflavin,10µg of thiamine hydrochloride,and 0.04µg of biotin 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 the 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 plantarum4incubating 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 0.2µg of calcium pantothenate.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.

Buffer solution— Dissolve 10.0g of monobasic potassium phosphate in 2000mLof water,and adjust with phosphoric acid to a pHof 3.5.

Mobile phase— Prepare a filtered and degassed mixture of Buffer solutionand methanol (90:10).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard stock solution— Dissolve an accurately weighed quantity of USP Calcium Pantothenate RSin water to obtain a solution having a known concentration of about 0.25mg per mL.Prepare fresh every 4weeks.Store in a refrigerator.

Standard preparation— Quantitatively dilute an accurately measured volume of Standard stock solutionwith water to obtain a solution having a known concentration of about 40µg of USP Calcium Pantothenate RSper mL.

Assay preparation— Weigh and finely powder not fewer than 20Tablets.Transfer an accurately weighed portion of the powder,equivalent to about 10mg of calcium pantothenate,to a 250-mLvolumetric flask.Add 10mLof methanol,and swirl to disperse the test specimen.Dilute with water to volume,mix,and filter.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 205-nm detector and a 3.9-mm ×30-cm column that contains 5-µm packing L1.The column temperature is maintained at 50,and the flow rate is about 2mLper 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 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 for calcium pantothenate.Calculate the quantity,in mg,of calcium pantothenate (C18H32CaN2O10)in the portion of Tablets taken by the formula: in which Cis the concentration,in mg per mL,of USP Calcium Pantothenate RSin the Standard preparation;and rUand rSare the peak areas obtained from the Assay preparationand the Standard preparation,respectively.

Diluting solution— Prepare a mixture of water,acetonitrile,and glacial acetic acid (94:5:1).

Mobile phase— Prepare a mixture of water,methanol,and glacial acetic acid (73:27:1)containing 140mg of sodium 1-hexanesulfonate per 100mL.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard preparation— [NOTE—Use USP Niacin RSin place of USP Niacinamide RSfor formulations containing Niacin.]Transfer about 80mg of USP Niacinamide RS,20mg of USP Pyridoxine Hydrochloride RS,20mg of USP Riboflavin RS,and 20mg of USP Thiamine Hydrochloride RS,each accurately weighed,to a 200-mLvolumetric flask,and add about 180mLof Diluting solution.Immerse the flask in a hot water bath maintained at 65to 70for about 10minutes with regular shaking or using a vortex mixer,until all the solid materials are dissolved.Chill rapidly in a cold water bath for about 10minutes to room temperature,dilute with Diluting solutionto volume,and mix.

Assay preparation— Weigh and finely powder not fewer than 30Tablets,and transfer an accurately weighed portion of the powder,equivalent to about 10mg of niacinamide and 2.5mg each of pyridoxine hydrochloride,riboflavin,and thiamine hydrochloride,to a 50-mLcentrifuge tube.Add 25.0mLof Diluting solution,and mix,using a vortex mixer,for 30seconds to completely suspend the powder.Immerse the centrifuge tube in a hot water bath maintained at 65to 70,heat for 5minutes,and mix on a vortex mixer for 30seconds.Return the tube to the hot water bath,heat for another 5minutes,and mix on a vortex mixer for 30seconds.Filter a portion of the solution,cool to room temperature,and use the clear filtrate.[NOTE—Use the filtrate within 3hours of filtration.]

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 280-nm detector and a 3.9-mm ×30-cm column that contains packing L1.The flow rate is about 1mLper minute.Chromatograph the Standard preparation,and record the peak areas as directed for Procedure:the relative retention times of niacinamide,pyridoxine,riboflavin,and thiamine are about 0.3,0.5,0.8,and 1.0,respectively;and the relative standard deviation for replicate injections is not more than 3.0%.

Procedure— Separately inject equal volumes (about 10µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the peak areas for niacin or niacinamide,pyridoxine,riboflavin,and thiamine.Calculate the quantity,in mg,of niacinamide (C6H6N2O)in the portion of Tablets taken by the formula: in which Cis the concentration,in mg per mL,of USP Niacinamide RSin the Standard preparation;and rUand rSare the peak responses of niacinamide obtained from the Assay preparationand the Standard preparation,respectively.For formulations containing niacin,Cis the concentration,in mg per mL,of USP Niacin RSin the Standard preparation;and rUand rSare the peak areas for niacin obtained from the Assay preparationand the Standard preparation,respectively.Separately calculate the quantities,in mg,of pyridoxine hydrochloride (C8H11NO3·HCl),riboflavin (C17H20N4O6),and thiamine hydrochloride (C12H17ClN4OS·HCl)in the portion of Tablets taken by the same formula,in which Cis the concentration,in mg per mL,of the relevant USP Reference Standard in the Standard preparation;and rUand rSare the peak areas for the corresponding vitamin obtained from the Assay preparationand the Standard preparation,respectively.For products containing thiamine mononitrate,calculate the quantity,in mg,of thiamine mononitrate (C12H17N5O4S),in the portion of Tablets taken by the formula: in which 327.36and 337.27are the molecular weights of thiamine mononitrate and thiamine hydrochloride,respectively;and the other terms are as previously defined.

Extraction solvent— Transfer 1mLof glacial acetic acid and 2.5g of edetate disodium to a 100-mLvolumetric flask,dissolve in and dilute with water to volume,and mix.Prepare a mixture of this solution and methanol (75:25).

Mobile phase— Prepare a 0.1Msodium acetate solution by dissolving 13.6g of sodium acetate in 1000mLof water.Adjust with acetic acid to a pHof 5.4,and mix.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).[NOTE—Asmall amount of methanol (up to 1%)may be added to the Mobile phaseto improve resolution.]

Standard stock solution— Dissolve an accurately weighed quantity of USP Niacin RSin Extraction solvent,and dilute quantitatively,and stepwise if necessary,with Extraction solventto obtain a solution having a known concentration of about 1mg per mL.

Standard preparation— Transfer 5.0mLof Standard stock solutionto a 25-mLvolumetric flask,dilute with Extraction solventto volume,and mix.

Assay preparation— [NOTE—This preparation is suitable for the determination of niacin or niacinamide,pyridoxine,and riboflavin,when present in the formulation.]Weigh and finely powder not fewer than 20Tablets,and transfer an accurately weighed portion of the powder,equivalent to about 2mg of riboflavin,to a 200-mLvolumetric flask.If riboflavin is not present in the formulation,transfer a portion equivalent to about 2mg of pyridoxine.If pyridoxine is not present in the formulation,transfer a portion equivalent to about 20mg of niacin or niacinamide.Add 100.0mLof Extraction solvent,and mix for 20minutes,using a wrist-action shaker.Immerse the flask in a water bath maintained at 70to 75,and heat for 20minutes.Mix on a vortex mixer for 30seconds,cool to room temperature,and filter.Use the clear filtrate.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 254-nm detector and a 4.6-mm ×25-cm column that contains packing L1.The flow rate is about 1mLper 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 3.0%.If necessary,flush the column with methanol between injections.

Procedure— Separately inject equal volumes (about 20µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the peak areas for niacin.Calculate the quantity,in mg,of niacin (C6H5NO2)in the portion of Tablets taken by the formula: in which Cis the concentration,in mg per mL,of USP Niacin RSin the Standard preparation;and rUand rSare the peak responses of niacin obtained from the Assay preparationand the Standard preparation,respectively.

Extraction solvent,Mobile phase,Standard stock solution,Standard preparation,Assay preparation,and Chromatographic system— Using USP Niacinamide RSin place of USP Niacin RS,proceed as directed in the Assay for niacin,Method 2.

Procedure— Separately inject equal volumes (about 20µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the peak areas for niacinamide.Calculate the quantity,in mg,of niacinamide (C6H6N2O)in the portion of Tablets taken by the formula: in which Cis the concentration,in mg per mL,of USP Niacinamide RSin the Standard preparation;and rUand rSare the peak responses of niacinamide obtained from the Assay preparationand the Standard preparation,respectively.

Extraction solvent and Mobile phase— Prepare as directed in the Assay for niacin,Method 2.

Standard stock solution— Dissolve an accurately weighed quantity of USP Pyridoxine Hydrochloride RSin Extraction solvent,and dilute quantitatively,and stepwise if necessary,with Extraction solventto obtain a solution having a known concentration of about 0.1mg per mL.

Standard preparation— Transfer 5.0mLof Standard stock solutionto a 25-mLvolumetric flask,dilute with Extraction solventto volume,and mix.

Assay preparation— Prepare as directed for Assay preparationin the Assay for niacin,Method 2.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 254-nm detector and a 4.6-mm ×25-cm column that contains packing L1.The flow rate is about 1mLper 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 3.0%.

Procedure— Separately inject equal volumes (about 20µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the peak areas for pyridoxine.Calculate the quantity,in mg,of pyridoxine hydrochloride (C8H11NO3·HCl)in the portion of Tablets taken by the formula: in which Cis the concentration,in mg per mL,of USP Pyridoxine Hydrochloride RSin the Standard preparation;and rUand rSare the peak areas obtained from the Assay preparationand the Standard preparation,respectively.

Extraction solvent— Prepare as directed in the Assay for niacin,Method 2.

Mobile phase— Dissolve 6.8g of sodium acetate in 1000mLof water,and mix.Prepare a mixture of this solution and methanol (65:35).Add 2mLof triethylamine for every 1000mLof the mixture,adjust with glacial acetic acid to a pHof 5.2,and mix.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard stock solution— Transfer about 20mg of USP Riboflavin RS,accurately weighed,to a 200-mLvolumetric flask.Add about 180mLof Extraction solvent,and immerse the flask for about 5minutes in a water bath maintained at 65to 75.Mix well,and repeat if necessary until dissolved.Chill rapidly in a cold water bath to room temperature,dilute with Extraction solventto volume,and mix.

Standard preparation— Quantitatively dilute 5.0mLof Standard stock solutionwith Extraction solventto 25.0mL,and mix.

Assay preparation— Prepare as directed for Assay preparationin the Assay for niacin,Method 2.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 254-nm detector and a 4.6-mm ×25-cm column that contains packing L1.The flow rate is about 1mLper 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 3.0%.

Procedure— Separately inject equal volumes (about 20µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the peak areas for riboflavin.Calculate the quantity,in mg,of riboflavin (C17H20N4O6)in the portion of Tablets taken by the formula: in which Cis the concentration,in mg per mL,of USP Riboflavin RSin the Standard preparation;and rUand rSare the peak areas obtained from the Assay preparationand the Standard preparation,respectively.

Mobile phase— Dissolve 1.88g of sodium 1-hexanesulfonate in 1Lof 0.1%phosphoric acid.Prepare a mixture of this solution and acetonitrile (92:18).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard stock solution— Dissolve an accurately weighed quantity of USP Thiamine Hydrochloride RSin 0.2Nhydrochloric acid,and dilute quantitatively,and stepwise if necessary,with 0.2Nhydrochloric acid to obtain a solution having a known concentration of about 0.1mg per mL.

Standard preparation— Transfer 5.0mLof Standard stock solutionto a 25-mLvolumetric flask,dilute with 0.2Nhydrochloric acid to volume,and mix.

Assay preparation— Weigh and finely powder not fewer than 20Tablets.Transfer an accurately weighed portion of the powder,equivalent to about 2mg of thiamine,to a 200-mLvolumetric flask.Add 100.0mLof 0.2Nhydrochloric acid,shake for 15minutes with a wrist-action shaker,and heat to boiling for 30minutes.Cool to room temperature,and filter.Use the clear filtrate.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 254-nm detector and a 4.6-mm ×25-cm column that contains packing L1.The flow rate is about 2mLper 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 3.0%.

Procedure— Separately inject equal volumes (about 20µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the areas for the major peaks.For products containing thiamine hydrochloride,calculate the quantity,in mg,of thiamine hydrochloride (C12H17ClN4OS·HCl)in the portion of Tablets taken by the formula: in which Cis the concentration,in mg per mL,of USP Thiamine Hydrochloride RSin the Standard preparation;and rUand rSare the peak areas for thiamine obtained from the Assay preparationand the Standard preparation,respectively.For products containing thiamine mononitrate,calculate the quantity,in mg,of thiamine mononitrate (C12H17N5O4S)in the portion of Tablets taken by the formula: in which 327.36and 337.27are the molecular weights of thiamine mononitrate and thiamine hydrochloride,respectively;and the other terms are as previously defined.

Reagent— Prepare a solution containing 25g of edetate disodium in 1000mLof water.

Mobile phase— Transfer 0.4mLof triethylamine,15.0mLof glacial acetic acid,and 350mLof methanol to a 2000-mLvolumetric flask,dilute with 0.008Msodium 1-hexanesulfonate to volume,and mix.Filter,and degas.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard stock solution— Dissolve accurately weighed quantities of USP Niacin RSor USP Niacinamide RS,USP Pyridoxine Hydrochloride RS,USP Riboflavin RS,and USP Thiamine Hydrochloride RSin Reagent,with heating if necessary,to obtain a solution having known concentrations of about 1.5mg of USP Niacin RSor USP Niacinamide RSper mL,0.24mg of USP Pyridoxine Hydrochloride RSper mL,0.08mg of USP Riboflavin RSper mL,and 0.24mg of USP Thiamine Hydrochloride RSper mL.

Standard preparation— Transfer 5.0mLof Standard stock solutionto a stoppered 125-mLflask.Add 10.0mLof a mixture of methanol and glacial acetic acid (9:1)and 30.0mLof a mixture of methanol and ethylene glycol (1:1).Insert the stopper,shake for 15minutes in a water bath maintained at 60,and cool.Filter,discarding the first few mLof the filtrate.

Assay preparation— Weigh and finely powder not fewer than 20Tablets.Transfer an accurately weighed portion of the powder,equivalent to about 7.5mg of niacin or niacinamide,1.2mg of pyridoxine hydrochloride,0.4mg of riboflavin,and 1.2mg of thiamine hydrochloride,to a stoppered 125-mLflask.Add 10.0mLof a mixture of methanol and glacial acetic acid (9:1)and 30.0mLof a mixture of methanol and ethylene glycol (1:1).Insert the stopper,shake for 15minutes in a water bath maintained at 60,and cool.Filter,discarding the first few mLof the filtrate.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 270-nm detector and a 4.6-mm ×25-cm column that contains packing L7.The column is maintained at 50,and the flow rate is about 2.0mLper 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 2.0%.

Procedure— Separately inject equal volumes (about 5µL)of the Assay preparationand the Standard preparationinto the chromatograph,record the chromatograms,and measure the areas of the peak responses.Calculate the quantity,in mg,of niacin (C6H5NO2)or niacinamide (C6H6N2O)in the portion of Tablets taken by the formula: in which Cis the concentration,in mg per mL,of USP Niacin RSor USP Niacinamide RSin the Standard preparation;and rUand rSare the peak areas for niacin or niacinamide obtained from the Assay preparationand the Standard preparation,respectively.Separately calculate the quantities,in mg,of pyridoxine hydrochloride (C8H11NO3·HCl)and riboflavin (C17H20N4O6)in the portion of Tablets taken by the same formula,in which the terms are as defined therein.For products containing thiamine hydrochloride,calculate the quantity,in mg,of thiamine (C12H17ClN4OS)in the portion of Tablets taken by the same formula.For products containing thiamine mononitrate,calculate the quantity,in mg,of thiamine mononitrate (C12H17N5O4S)in the portion of Tablets taken by the formula: in which 327.36and 337.27are the molecular weights of thiamine mononitrate and thiamine hydrochloride,respectively;and the other terms are as previously defined.

Lanthanum chloride solution— Dissolve 26.7g of lanthanum chloride heptahydrate in 0.125Nhydrochloric acid to make 100.0mL.

Calcium standard stock solution— Weigh accurately about 1.001g of calcium carbonate,previously dried at 300for 3hours and cooled in a desiccator for 2hours,and dissolve in 25mLof 1Nhydrochloric acid.Boil to expel carbon dioxide,and dilute with water to 1000mLto obtain a solution having a known concentration of about 400µg of calcium per mL.

Standard preparations— Quantitatively dilute a volume of Calcium standard stock solutionwith 0.125Nhydrochloric acid to obtain a standard solution having a known concentration of about 100µg of calcium per mL.Into separate 100-mLvolumetric flasks,pipet 1.0,1.5,2.0,2.5,and 3.0mLof the standard solution.To each flask add 1.0mLof Lanthanum chloride solution,dilute with water to volume,and mix to obtain solutions having known concentrations of about 1.0,1.5,2.0,2.5,and 3.0µg of calcium per mL.

Assay preparation— Weigh and finely powder not fewer than 20Tablets.Transfer an accurately weighed portion of the powder,equivalent to about 5Tablets,to a porcelain crucible.Heat the crucible in a muffle furnace maintained at about 550for 6to 12hours,and cool.Add about 60mLof hydrochloric acid,and boil gently on a hot plate or steam bath for 30minutes,intermittently rinsing the inner surface of the crucible with 6Nhydrochloric acid.Cool,and quantitatively transfer the contents of the crucible to a 100-mLvolumetric flask.Rinse the crucible with small portions of 6Nhydrochloric acid,and add the rinsings to the flask.Dilute with water to volume,mix,and filter,discarding the first 5mLof the filtrate.Dilute this solution quantitatively,and stepwise if necessary,with 0.125Nhydrochloric acid to obtain a solution having a concentration of about 2µg of calcium per mL,adding 1mLof Lanthanum chloride solutionper 100mLof the final volume.

Procedure— Concomitantly determine the absorbances of the Standard preparationsand the Assay preparationat the calcium emission line at 422.7nm with an atomic absorption spectrophotometer (see Spectrophotometry and Light-Scattering á851ñ)equipped with a calcium hollow-cathode lamp and a nitrous oxide–acetylene flame,using 0.125Nhydrochloric acid containing 0.1%Lanthanum chloride solutionas the blank.Plot the absorbances of the Standard preparationsversus concentration,in µg per mL,of calcium,and draw the straight line best fitting the five plotted points.From the graph so obtained,determine the concentration,C,in µg per mL,of calcium in the Assay preparation.Calculate the weight,in mg,of calcium (Ca)in the portion of Tablets taken by the formula: in which Dis the dilution factor used to prepare the Assay preparation.

Chromium standard stock solution— Transfer about 2.829g of potassium dichromate,previously dried at 120for 4hours and accurately weighed,to a 1000-mLvolumetric flask,dissolve in and dilute with water to volume,and mix to obtain a solution having a known concentration of about 1000µg of chromium per mL.Store in a polyethylene bottle.

Standard preparations— Transfer 10.0mLof the Chromium standard stock solutionto a 1000-mLvolumetric flask,add 50.0mLof 6Nhydrochloric acid,dilute with water to volume,and mix to obtain a standard solution having a known concentration of about 10µg of chromium per mL.Transfer 10.0mLand 20.0mLof the standard solution to separate 100-mLvolumetric flasks,and transfer 15.0mLand 20.0mLof the standard solution to separate 50-mLvolumetric flasks.Dilute the contents of each of the four flasks with 0.125Nhydrochloric acid to volume,and mix to obtain solutions having known concentrations of about 1.0,2.0,3.0,and 4.0µg of chromium per mL.

Assay preparation— Proceed as directed for Assay preparationin the Assay for calcium,except to prepare the Assay preparationto contain about 1µg of chromium per mLand to omit the use of the Lanthanum chloride solution.

Procedure— Concomitantly determine the absorbances of the Standard preparationsand the Assay preparationat the chromium emission line at 357.9nm with an atomic absorption spectrophotometer (see Spectrophotometry and Light-Scattering á851ñ)equipped with a chromium hollow-cathode lamp and an air–acetylene flame,using 0.125Nhydrochloric acid as the blank.Plot the absorbances of the Standard preparationsversus concentration,in µg per mL,of chromium,and draw the straight line best fitting the four plotted points.From the graph so obtained,determine the concentration,C,in µg per mL,of chromium in the Assay preparation.Calculate the quantity,in µg,of chromium (Cr)in the portion of Tablets taken by the formula: in which Dis the dilution factor used to prepare the Assay preparation.

Copper standard stock solution— Dissolve about 1.00g of copper foil in a minimum volume of a 50%(v/v)solution of nitric acid,and dilute with a 1%(v/v)solution of nitric acid to 1000mL.This solution contains 1000µg of copper per mL.

Standard preparations— Transfer 10.0mLof Copper standard stock solutionto a 100-mLvolumetric flask,and dilute quantitatively with 0.125Nhydrochloric acid to volume to obtain a standard solution having a concentration of 100µg of copper per mL.To separate 200-mLvolumetric flasks,transfer 1.0,2.0,4.0,6.0,and 8.0mLof the standard solution,dilute with water to volume,and mix to obtain solutions having known concentrations of about 0.5,1.0,2.0,3.0,and 4.0µg of copper per mL.

Assay preparation— Proceed as directed for Assay preparationin the Assay for calcium,except to prepare the Assay preparationto contain about 2µg of copper per mLand to omit the use of Lanthanum chloride solution.

Procedure— Concomitantly determine the absorbances of the Standard preparationsand the Assay preparationat the copper emission line at 324.7nm with an atomic absorption spectrophotometer (see Spectrophotometry and Light-Scattering á851ñ)equipped with a copper hollow-cathode lamp and an air–acetylene flame,using 0.125Nhydrochloric acid as the blank.Plot the absorbances of the Standard preparationsversus concentration,in µg per mL,of copper,and draw the straight line best fitting the five plotted points.From the graph so obtained,determine the concentration,C,in µg per mL,of copper in the Assay preparation.Calculate the quantity,in mg,of copper (Cu)in the portion of Tablets taken by the formula: in which Dis the dilution factor used to prepare the Assay preparation.

3M Sodium acetate solution— Dissolve 408g of sodium acetate in about 600mLof water contained in a 1000-mLvolumetric flask.Allow the solution to equilibrate to room temperature,dilute with water to volume,and mix.Adjust with a few drops of acetic acid to a pHof 7.0.

Sodium citrate solution— Dissolve 222g of sodium citrate in 250mLof water in a 1000-mLvolumetric flask.Add 28mLof perchloric acid,dilute with water to volume,and mix.

Fluoride standard stock solution— Transfer an accurately weighed quantity of about 1.105g of sodium fluoride,previously dried at 100for 4hours and cooled in a desiccator,to a 1000-mLvolumetric flask.Dissolve in and dilute with water to volume,and mix to obtain a solution having a known concentration of about 500µg of fluoride per mL.

Intermediate stock solution 1— Pipet 20.0mLof Fluoride standard stock solutioninto a 100-mLvolumetric flask,dilute with water to volume,and mix to obtain a solution having a known concentration of about 100µg of fluoride per mL.

Intermediate stock solution 2— Transfer 2.0mLof Fluoride standard stock solutionto a 100-mLvolumetric flask,dilute with water to volume,and mix to obtain a solution having a known concentration of about 10µg of fluoride per mL.

Standard preparations— To five separate 100-mLvolumetric flasks,transfer 3.0,5.0,and 10.0mLof Intermediate stock solution 2and 5.0and 10.0mLof Intermediate stock solution 1.To each flask,add 10.0mLof 1Nhydrochloric acid,25mLof 3M Sodium acetate solution,and 25.0mLof Sodium citrate solution.Dilute the contents of each flask with water to volume,and mix to obtain solutions having known concentrations of about 0.3,0.5,1.0,5.0,and 10.0µg of fluoride per mL.

Assay preparation— Weigh and finely powder a counted number of Tablets.Transfer an accurately weighed quantity of the powder,equivalent to about 200µg of fluoride,to a 100-mLvolumetric flask.Dissolve in 10.0mLof 1Nhydrochloric acid,25.0mLof 3M Sodium acetate solution,and 25.0mLof Sodium citrate solution,dilute with water to volume,and mix.

Procedure— To separate plastic beakers,each containing a plastic-coated stirring bar,transfer 50.0mLeach of the Standard preparationsand the Assay preparation.Concomitantly measure the potentials (see pHá791ñ),in mV,of the Standard preparationsand the Assay preparation,with a pHmeter capable of a minimum reproducibility of ±0.2mVand equipped with a fluoride-specific ion-indicating electrode and a calomel reference electrode.[NOTE—When taking measurements,immerse the electrodes in the solution,stir on a magnetic stirrer having an insulated top until equilibrium is attained (1to 2minutes),and record the potential.Rinse and dry the electrodes between measurements,taking care to avoid damaging the crystal of the specific-ion electrode.]Plot the logarithms of fluoride concentrations,in µg per mL,of the Standard preparationsversus potential,in mV.From the standard response curve so obtained and the measured potential of the Assay preparation,determine the concentration,C,in µg per mL,of fluoride in the Assay preparation.Calculate the quantity,in mg,of fluoride in the portion of Tablets taken by the formula:

pH10.0Buffer— Add 214mLof 0.1Nsodium hydroxide to 1000mLof 0.05Msodium bicarbonate.

Mobile phase— Prepare a filtered and degassed mixture of water,alcohol,and 0.1Nsulfuric acid (175:20:5).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard stock solution— Dissolve an accurately weighed quantity of USP Sodium Fluoride RSin water,and dilute quantitatively,and stepwise if necessary,with water to obtain a solution having a known concentration of about 220µg per mL.This solution contains about 100µg of fluoride per mL.

Standard preparation— [NOTE—Condition the solid-phase extraction column specified for use in the Standard preparationand the Assay preparationin the following manner.Using a vacuum at a pressure not exceeding 5mm of mercury,wash the column with one column volume of methanol followed by one column volume of pH10.0Buffer.Do not allow the column top to dry.If the top of the column becomes dry,recondition the column.]Transfer 10.0mLof Standard stock solutionto a 100-mLvolumetric flask.Add 75mLof water,and adjust with 0.1Nsodium hydroxide to a pHof 10.4±0.1.Dilute with water to volume,and mix.Filter,discarding the first 15mLof the filtrate.Transfer 25.0mLof the filtrate to a 50-mLvolumetric flask,add 15.0mLof water,and adjust with 0.1Nsodium hydroxide to a pHof 10.0.Dilute with pH10.0Bufferto volume,and mix.Elute a portion of this solution through a 3-mLsolid-phase extraction column containing L1packing that is connected through an adaptor to a second solid-phase extraction column containing sulfonylpropyl strong cation-exchange packing.Discard the first 3mLof the eluate,and collect the rest of the eluate in a suitable flask 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 1mg of fluoride,to a stoppered 100-mLvolumetric flask,add 15mLof water,and shake vigorously.Rinse the sides of the flask with 15mLof water,and allow to stand for 10minutes.Dilute with water to about 85mL,adjust with 1Nsodium hydroxide to a pHof 10.4±0.1,dilute with water to volume,and mix.Proceed as directed for Standard preparation,beginning with “Filter,discarding the first 15mLof the filtrate.”

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a conductivity detector,a 4.6-mm ×3-cm guard column that contains packing L17,and a 7.8-mm ×30-cm analytical column that contains packing L17.The flow rate is about 0.5mLper minute.Chromatograph the Standard preparation,and measure the peak areas as directed for Procedure:the relative standard deviation for replicate injections is not more than 2.0%.

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

Bromine water— To about 20mLof bromine in a glass-stoppered bottle,add 100mLof water.Insert the stopper into the bottle,and shake.Allow to stand for 30minutes,and use the supernatant.

Procedure— Weigh a counted number of Tablets,and grind them to a fine powder.Transfer an accurately weighed quantity of the powder,equivalent to about 3mg of iodide,to a nickel crucible.Add 5g of sodium carbonate,5mLof 50%(w/v)sodium hydroxide solution,and 10mLof alcohol,taking care that the entire specimen is moistened.Heat the crucible on a steam bath to evaporate the alcohol,then dry the crucible at about 100for about 30minutes to prevent spattering upon subsequent heating.Transfer the crucible with its contents to a furnace heated to about 500,and heat the crucible for about 15minutes.[NOTE—Heating at about 500is necessary to carbonize any organic matter present;higher temperature may be used,if necessary,to ensure complete carbonization of all organic matter.]Cool the crucible,add 25mLof water,cover the crucible with a watchglass,and boil gently for about 10minutes.Filter the solution,and wash the crucible with boiling water,collecting the filtrate and washings in a beaker.Add phosphoric acid until the solution is neutral to methyl orange,then add 1mLexcess of phosphoric acid.Add excess of Bromine water,and boil the solution gently until colorless and then for 5minutes longer.Add a few crystals of salicylic acid,and cool the solution to about 20.Add 1mLof phosphoric acid and about 0.5g of potassium iodide,and titrate the liberated iodine with 0.005Nsodium thiosulfate VS,adding starch solution when the liberated iodine color has nearly disappeared.Calculate the quantity,in µg,of iodide in the portion of Tablets taken by the formula: in which Vis the volume,in mL,of sodium thiosulfate consumed;and Nis the normality of the sodium thiosulfate solution used.

Iron standard stock solution— Transfer about 100mg of iron powder,accurately weighed,to a 1000-mLvolumetric flask,dissolve in 25mLof 6Nhydrochloric acid,dilute with water to volume,and mix.

Standard preparations— To separate 100-mLvolumetric flasks,transfer 2.0,4.0,5.0,6.0,and 8.0mLof Iron standard stock solution.Dilute the contents of each flask with water to volume,and mix to obtain solutions having known concentrations of about 2.0,4.0,5.0,6.0,and 8.0µg of iron per mL.

Assay preparation— Proceed as directed for Assay preparationin the Assay for calcium,except to prepare the Assay preparationto contain 5µg of iron per mLand to omit the use of the Lanthanum chloride solution.

Procedure— Concomitantly determine the absorbances of the Standard preparationsand the Assay preparationat the iron emission line at 248.3nm with an atomic absorption spectrophotometer (see Spectrophotometry and Light-Scattering á851ñ)equipped with an iron hollow-cathode lamp and an air–acetylene flame,using 0.125Nhydrochloric acid as the blank.Plot the absorbances of the Standard preparationsversus concentration,in µg per mL,of iron,and draw the straight line best fitting the five plotted points.From the graph so obtained,determine the concentration,C,in µg per mL,of iron in the Assay preparation.Calculate the quantity,in mg,of iron (Fe)in the portion of Tablets taken by the formula: in which Dis the dilution factor used to prepare the Assay preparation.

Lanthanum chloride solution— Prepare as directed in the Assay for calcium.

Magnesium standard stock solution— Transfer about 1.0g of magnesium ribbon,accurately weighed,to a 1000-mLvolumetric flask,dissolve in 50mLof 6Nhydrochloric acid,dilute with water to volume,and mix to obtain a solution having a known concentration of about 1000µg of magnesium per mL.

Standard preparations— Quantitatively dilute a volume of Magnesium standard stock solutionwith 0.125Nhydrochloric acid to obtain a standard solution having a concentration of 20µg of magnesium per mL.To separate 100-mLvolumetric flasks,transfer 1.0,1.5,2.0,2.5,and 3.0mLof standard solution.To each flask add 1.0mLof Lanthanum chloride solution,dilute with 0.125Nhydrochloric acid to volume,and mix to obtain solutions having known concentrations of about 0.2,0.3,0.4,0.5,and 0.6µg of magnesium per mL.

Assay preparation— Proceed as directed for Assay preparationin the Assay for calcium,except to prepare the Assay preparationto contain 0.4µg of magnesium per mL.

Procedure— Concomitantly determine the absorbances of the Standard preparationsand the Assay preparationat the magnesium emission line at 285.2nm with an atomic absorption spectrophotometer (see Spectrophotometry and Light-Scattering á851ñ)equipped with a magnesium hollow-cathode lamp and an air–acetylene flame,using 0.125Nhydrochloric acid containing 0.1%Lanthanum chloride solutionas the blank.Plot the absorbances of the Standard preparationsversus concentration,in µg per mL,of magnesium,and draw the straight line best fitting the five plotted points.From the graph so obtained,determine the concentration,C,in µg per mL,of magnesium in the Assay preparation.Calculate the quantity,in mg,of magnesium (Mg)in the portion of Tablets taken by the formula: in which Dis the dilution factor used to prepare the Assay preparation.

Manganese standard stock solution— Transfer 1.00g of manganese,accurately weighed,to a 1000-mLvolumetric flask,dissolve in 20mLof nitric acid,dilute with 6Nhydrochloric acid to volume,and mix to obtain a solution containing 1000µg of manganese per mL.

Standard preparations— Quantitatively dilute 10.0mLof the Manganese standard stock solutionwith 0.125Nhydrochloric acid to 200.0mLto obtain a standard solution having a concentration of 50µg of manganese per mL.To separate 100-mLvolumetric flasks,transfer 1.0,1.5,2.0,3.0,and 4.0mLof standard solution,dilute the contents of each flask with 0.125Nhydrochloric acid to volume,and mix to obtain solutions having known concentrations of about 0.5,0.75,1.0,1.5,and 2.0µg of manganese per mL.

Assay preparation— Proceed as directed for Assay preparationin the Assay for calcium,except to prepare the Assay preparationto contain 1µg of manganese per mLand to omit the use of the Lanthanum chloride solution.

Procedure— Concomitantly determine the absorbances of the Standard preparationsand the Assay preparationat the manganese emission line at 279.5nm with an atomic absorption spectrophotometer (see Spectrophotometry and Light-Scattering á851ñ)equipped with a manganese hollow-cathode lamp and an air–acetylene flame,using 0.125Nhydrochloric acid as the blank.Plot the absorbances of the Standard preparationsversus concentration,in µg per mL,of manganese,and draw the straight line best fitting the five plotted points.From the graph so obtained,determine the concentration,C,in µg per mL,of manganese in the Assay preparation.Calculate the quantity,in mg,of manganese (Mn)in the portion of Tablets taken by the formula: in which Dis the dilution factor used to prepare the Assay preparation.

Diluting solution— Dissolve 40g of ammonium chloride in 2000mLof water.

Molybdenum standard stock solution— Transfer about 1.0g of molybdenum wire,accurately weighed,to a 1000-mLvolumetric flask,and dissolve in 50mLof nitric acid,warming if necessary.Dilute with water to volume,and mix to obtain a solution containing 1000µg of molybdenum per mL.

Standard preparations— Quantitatively dilute 10.0mLof the Molybdenum standard stock solutionwith water to 100.0mLto obtain a standard solution having a known concentration of about 100µg of molybdenum per mL.To separate 100-mLvolumetric flasks,transfer 2.0,10.0,and 25.0mLof the standard solution,and add 5.0mLof perchloric acid to each flask.Gently boil the solution in each flask for 15minutes,cool to room temperature,dilute each with Diluting solutionto volume,and mix to obtain solutions having known concentrations of about 5.0,10.0,and 25.0µg of molybdenum per mL.

Assay preparation— Weigh and finely powder a counted number of Tablets.Transfer an accurately weighed portion of the powder,equivalent to about 1000µg of molybdenum,to a suitable flask,and add about 12mLof nitric acid.[NOTE—The volume of nitric acid may be varied to ensure that the powder is uniformly dispersed.]Carefully swirl the flask to disperse the test specimen.Sonicate for about 10minutes,or until the test specimen is completely dissolved.Gently boil the solution for about 15minutes,and cool to room temperature.Carefully add about 8mLof perchloric acid,heat until perchloric acid fumes appear,and swirl the flask to dissipate the fumes.Repeat the heating and swirling until the fumes persist.Cool to room temperature.Quantitatively transfer the contents of the flask to a 100-mLvolumetric flask with the aid of the Diluting solution,dilute with Diluting solutionto volume,and mix.

Procedure— Concomitantly determine the absorbances of the Standard preparationsand the Assay preparationat the molybdenum emission line at 313nm with an atomic absorption spectrophotometer (see Spectrophotometry and Light-Scattering á851ñ)equipped with a molybdenum hollow-cathode lamp and a nitrous oxide–acetylene flame,using a mixture of Diluting solutionand perchloric acid (20:1)as the blank.Plot the absorbances of the Standard preparationsversus concentration,in µg per mL,of molybdenum,and draw the straight line best fitting the three plotted points.From the graph so obtained,determine the concentration,C,in µg per mL,of molybdenum in the Assay preparation.Calculate the quantity,in mg,of molybdenum (Mo)in the portion of Tablets taken by the formula: in which Dis the diluting factor used to prepare the Assay preparation.

Sodium fluoride solution— Add 200mLof water to about 10g of sodium fluoride,stir until the solution is saturated,and filter.Store in a polyethylene bottle.

Ferrous sulfate solution— Dissolve 498mg of ferrous sulfate in water to make 100mL.

Potassium thiocyanate solution— Dissolve 20g of potassium thiocyanate in water,and dilute with water to 100mL.

20%Stannous chloride solution— Transfer 40g of stannous chloride to a beaker,and add 20mLof 6.5Nhydrochloric acid solution.Heat the solution until the stannous chloride is dissolved.Cool,dilute with water to 100mL,and mix.

Dilute stannous chloride solution— Dilute 4mLof 20%Stannous chloride solutionwith water to 100mL.Prepare this solution fresh at the time of use.

Standard preparation— Transfer about 92mg of ammonium molybdate,accurately weighed,to a 500-mLvolumetric flask,dissolve in and dilute with water to volume,and mix.Quantitatively dilute 20.0mLof this solution with water to 100.0mLto obtain a solution having a known concentration of about 20µg of molybdenum per mL.

Procedure— Weigh a counted number of Tablets,and grind the Tablets to a fine powder.Transfer an accurately weighed quantity of the powder,equivalent to about 40µg of molybdenum,to a 200-mLbeaker marked I.To another 200-mLbeaker marked II,transfer 2.0mLof the Standard preparation.Add about 20mLof nitric acid to each beaker.Cover each beaker with a watchglass,and boil slowly on a hot plate for about 45minutes.Cool to room temperature,add 6mLof perchloric acid to each beaker,cover each beaker with a watchglass,and continue the heating until digestion is complete,as indicated when the liquid becomes colorless or pale yellow.If necessary,add further portions of nitric acid and perchloric acid to beaker I,and digest further.Evaporate the solutions in each of the beakers to dryness.Rinse the sides of the beakers and the watchglasses with water,and add more water to each beaker to a volume of about 50mL.Gently boil the water solutions for a few minutes.Cool to room temperature.Add 2drops of methyl orange TSto each beaker,and neutralize with ammonium hydroxide.Add 8.2mLof hydrochloric acid to each beaker.Quantitatively transfer the contents of the beakers to separate 100-mLvolumetric flasks,rinse the beakers with water,transfer the rinsings to the respective volumetric flasks,dilute with water to volume,and mix.

Sulfuric acid solution— Cautiously add 37.5mLof sulfuric acid to 100mLof water,and mix.

Ammonium molybdate solution— Dissolve 12.5g of ammonium molybdate in 150mLof water.Add 100mLof Sulfuric acid solution,and mix.

Hydroquinone solution— Dissolve 0.5g of hydroquinone in 100mLof water,and add one drop of sulfuric acid.

Sodium bisulfite solution— Dissolve 20g of sodium bisulfite in 100mLof water.

Phosphorus standard stock solution— Accurately weigh about 4.395g of monobasic potassium phosphate,previously dried at 105for 2hours and stored in a desiccator,and transfer to a 1000-mLvolumetric flask.Dissolve in water,add 6mLof sulfuric acid as a preservative,dilute with water to volume,and mix to obtain a solution containing 1000µg of phosphorus per mL.

Standard preparation— Dissolve an accurately measured volume ofPhosphorus standard stock solutionin water,and dilute quantitatively,and stepwise if necessary,with water to obtain a solution having a known concentration of about 20µg of phosphorus per mL.

Assay preparation— Weigh a counted number of Tablets,and grind the Tablets to a fine powder.Transfer an accurately weighed portion of the powder,equivalent to about 100mg of phosphorus,to a suitable flask,add 25mLnitric acid,and digest on a hot plate for about 30minutes.Add 15mLof hydrochloric acid,and continue the digestion to the cessation of brown fumes.Cool,and quantitatively transfer the contents of the flask to a 500-mLvolumetric flask with the aid of small portions of water.Dilute with water to volume,and mix.Transfer 10.0mLof this solution to a 100-mLvolumetric flask,dilute with water to volume,and mix.

Procedure— To three separate 25-mLvolumetric flasks,transfer 5.0mLeach of the Standard preparation,the Assay preparation,and water to provide the blank.To each of the three flasks,add 1.0mLeach of Ammonium molybdate solution,Hydroquinone solution,and Sodium bisulfite solution,and swirl to mix.Dilute the contents of each flask with water to volume,mix,and allow the flasks to stand for 30minutes.Concomitantly determine the absorbances of the solutions from the Assay preparationand the Standard preparationin 1-cm cells at the wavelength of maximum absorbance at about 650nm,against the blank.Calculate the quantity,in mg,of phosphorus (P)in the portion of Tablets taken by the formula: in which Cis the concentration,in µg per mL,of phosphorus in the Standard preparation;and AUand ASare the absorbances of the solutions obtained from the Assay preparationand the Standard preparation,respectively.

Potassium standard stock solution— Transfer about 190.7mg of potassium chloride,previously dried at 105for 2hours and accurately weighed,to a 1000-mLvolumetric flask,dissolve in and dilute with water to volume,and mix to obtain a solution having a known concentration of about 100µg of potassium per mL.

Standard preparations— Dilute an accurately measured volume of Potassium standard stock solutionwith 0.125Nhydrochloric acid to obtain a standard solution having a known concentration of about 10µg of potassium per mL.Transfer 5.0,10.0,15.0,20.0,and 25.0mLof the standard solution to separate 100-mLvolumetric flasks.Dilute the contents of each flask with 0.125Nhydrochloric acid to volume,and mix to obtain solutions containing 0.5,1.0,1.5,2.0,and 2.5µg of potassium per mL.

Assay preparation— Proceed as directed for Assay preparationin the Assay for calcium,except to prepare the Assay preparationto contain 1µg of potassium per mLand to omit the use of the Lanthanum chloride solution.

Procedure— Concomitantly determine the absorbances of the Standard preparationsand the Assay preparationat the potassium emission line at 766.5nm with an atomic absorption spectrophotometer (see Spectrophotometry and Light-Scattering á851ñ)equipped with a potassium hollow-cathode lamp and an air–acetylene flame,using water as the blank.Plot the absorbances of the Standard preparationsversus concentration,in µg per mL,of potassium,and draw the straight line best fitting the five plotted points.From the graph so obtained,determine the concentration,C,in µg per mL,of potassium in the Assay preparation.Calculate the quantity,in mg,of potassium (K)in the portion of Tablets taken by the formula: in which Dis the dilution factor used to prepare the Assay preparation.

Diluting solution— Prepare as directed in the Assay for molybdenum,Method 1.

Selenium standard stock solution— [Caution—Selenium is toxic;handle it with care. ]Dissolve about 1g of metallic selenium,accurately weighed,in a minimum volume of nitric acid.Evaporate to dryness,add 2mLof water,and evaporate to dryness.Repeat the addition of water and the evaporation to dryness three times.Dissolve the residue in 3Nhydrochloric acid,transfer to a 1000-mLvolumetric flask,dilute with 3Nhydrochloric acid to volume,and mix to obtain a solution having a known concentration of about 1000µg of selenium per mL.

Standard preparations— Quantitatively dilute 10mLof the Selenium standard stock solutionwith water to 100.0mLto obtain a standard solution having a known concentration of about 100µg of selenium per mL.To separate 100-mLvolumetric flasks,transfer 5.0,10.0,and 25.0mLof the standard solution,and add 5.0mLof perchloric acid to each flask.Gently boil the solutions for 15minutes,cool to room temperature,dilute each with Diluting solutionto volume,and mix to obtain solutions containing 5.0,10.0,and 25.0µg of selenium per mL.

Assay preparation— Weigh and finely powder a counted number of Tablets.Transfer an accurately weighed portion of the powder,equivalent to about 1000µg of selenium,to a suitable flask,and add about 12mLof nitric acid.[NOTE—The volume of nitric acid may be varied to ensure that the powder is uniformly dispersed.]Carefully swirl the flask to disperse the test specimen.Sonicate for about 10minutes or until the test specimen is completely dissolved.Gently boil the solution for about 15minutes,and cool to room temperature.Carefully add about 8mLof perchloric acid to the flask,heat the flask until perchloric acid fumes appear,and swirl the flask to dissipate the fumes.Repeat the heating and swirling until the fumes persist.Cool to room temperature.Quantitatively transfer the contents of the flask to a 50-mLvolumetric flask with the aid of the Diluting solution,dilute with Diluting solutionto volume,and mix.

Procedure— Concomitantly determine the absorbances of the Standard preparationsand the Assay preparationat the selenium emission line at 196nm,with an atomic absorption spectrophotometer (see Spectrophotometry and Light-Scattering á851ñ)equipped with a selenium hollow-cathode lamp and an air–acetylene flame,using a mixture of Diluting solutionand perchloric acid (20:1)as the blank.Plot the absorbances of the Standard preparationsversus concentration,in µg per mL,of selenium,and draw the straight line best fitting the three plotted points.From the graph so obtained,determine the concentration,C,in µg per mL,of selenium in the Assay preparation.Calculate the quantity,in mg,of selenium (Se)in the portion of Tablets taken by the formula: in which Dis the dilution factor used to prepare the Assay preparation.

Hydrochloric acid solution— Dilute 50mLof hydrochloric acid with water to 500mL,and mix.

50%Ammonium hydroxide solution— Dilute 250mLof ammonium hydroxide with water to 500mL,and mix.

Reagent 1— Dissolve 4.5g of edetate disodium in 400mLof water in a 500-mLvolumetric flask.Add 12.5g of hydroxylamine hydrochloride,dilute with water to volume,and mix.

Reagent 2— Transfer 200mg of 2,3-diaminonaphthalene to a 250-mLseparatory funnel,and add 200mLof 0.1Nhydrochloric acid.Wash the solution with three 40-mLportions of cyclohexane,and discard the cyclohexane layer.Filter the solution into a brown bottle,and cover the solution with a 1-cm layer of cyclohexane.This solution is stable for 1week if stored in a refrigerator.

Standard stock preparation— [Caution—Selenium is toxic;handle it with care. ]Dissolve about 1g of metallic selenium,accurately weighed,in a minimum volume of nitric acid.Evaporate to dryness,add 2mLof water,and evaporate to dryness.Repeat the addition of water and evaporation to dryness three times.Dissolve the residue in 3Nhydrochoric acid,transfer to a 1000-mLvolumetric flask,dilute with 3Nhydrochloric acid to volume,and mix to obtain a solution containing about 1000µg of selenium per mL.Dilute a volume of the solution quantitatively,and stepwise if necessary,with 0.125Nhydrochloric acid to obtain a solution having a known concentration of about 2.0µg of selenium per mL.

Assay preparation— Weigh a counted number of Tablets,and grind to a fine powder.Transfer an accurately weighed portion of the powder,equivalent to about 20µg of selenium,to a suitable flask.Add 10mLof nitric acid,and warm gently on a hot plate.Continue heating until the initial nitric acid reaction has subsided,then add 3mLof perchloric acid.[Caution—Exercise care at this stage as perchloric acid reaction becomes vigorous. ]Continue heating on the hot plate until the appearance of white fumes of perchloric acid or until the digest begins to darken.Add 0.5mLof nitric acid,and resume heating,adding additional amounts of nitric acid if further darkening occurs.Digest for about 10minutes after the first appearance of perchloric acid fumes or until the digest becomes colorless.Cool the flask,add 2.5mLof Hydrochloric acid solution,and return the flask to the hot plate to expel residual nitric acid.Heat the mixture for 3minutes after it begins to boil.Cool the flask to room temperature,and dilute with water to about 20mL.

Procedure— Transfer 10.0mLof the Standard stock preparationto a glass-stoppered flask.Add 1mLof perchloric acid and 1mLof Hydrochloric acid solution,and dilute with water to about 20mL(Standard preparation).Prepare a blank by adding 1mLof perchloric acid and 1mLof Hydrochloric acid solutionto a glass-stoppered flask,and diluting with water to about 20mL.Treat the Assay preparation,the Standard preparation,and the blank as follows.Add 5mLof Reagent 1to each flask,and swirl gently to mix.Adjust the solution in each flask with 50%Ammonium hydroxide solutionto a pHof 1.1±0.1.Add 5mLof Reagent 2to each flask,and swirl gently to mix.Place the flasks in a water bath maintained at 50,and equilibrate for 30minutes,taking care that the flasks are covered to protect them from light.Cool to room temperature,and transfer the contents of each flask to separate separatory funnels.Transfer 10.0mLof cyclohexane to each separatory funnel,and extract vigorously for 1minute.Discard the aqueous layer.Transfer the cyclohexane layer to a centrifuge tube,and centrifuge at 1000rpm for 1minute to remove any remaining water.Concomitantly determine the absorbances of the solutions from the Assay preparationand the Standard preparationin 1-cm cells at the wavelength of maximum absorbance at about 380nm against the solution from the blank.Calculate the quantity,in µg,of selenium (Se)in the portion of Tablets taken by the formula: in which Cis the concentration,in µg per mL,of selenium in the Standard stock preparation;Dis the dilution factor used to prepare the Assay preparation;and AUand ASare the absorbances of the solutions from the Assay preparationand the Standard preparation,respectively.

Zinc standard stock solution— Transfer about 311mg of zinc oxide,accurately weighed,to a 250-mLvolumetric flask,and add 80mLof 5Mhydrochloric acid,warming if necessary to dissolve.Cool,dilute with water to volume,and mix to obtain a solution having a known concentration of about 1000µg of zinc per mL.

Standard preparations— Dilute a volume of the Zinc standard stock solutionquantitatively,and stepwise if necessary,with 0.125Nhydrochloric acid to obtain a standard solution having a known concentration of about 50µg of zinc per mL.Transfer 1.0,2.0,3.0,4.0,and 5.0mLof this solution to separate 100-mLvolumetric flasks.Dilute the contents of each flask with 0.125Nhydrochloric acid to volume,and mix to obtain solutions having known concentrations of about 0.5,1.0,1.5,2.0,and 2.5µg of zinc per mL.

Assay preparation— Proceed as directed for Assay preparationin the Assay for calcium,except to prepare the Assay preparationto contain 2µg of zinc per mLand to omit the use of the Lanthanum chloride solution.

Procedure— Concomitantly determine the absorbances of the Standard preparationsand the Assay preparationat the zinc emission line at 213.8nm with an atomic absorption spectrophotometer (see Spectrophotometry and Light-Scattering á851ñ)equipped with a zinc hollow-cathode lamp and an air–acetylene flame,using 0.125Nhydrochloric acid as the blank.Plot the absorbances of the Standard preparationsversus concentration,in µg per mL,of zinc,and draw the straight line best fitting the five plotted points.From the graph so obtained,determine the concentration,C,in µg per mL,of zinc in the Assay preparation.Calculate the quantity,in mg,of zinc (Zn)in the portion of Tablets taken by the formula: in which Dis the dilution factor used to prepare the Assay preparation.