Oil-and Water-Soluble Vitamins with Minerals Oral Solution
»Oil-and Water-Soluble Vitamins with Minerals Oral Solution contains one or more of the following oil-soluble vitamins:Vitamin A,Vitamin Das Ergocalciferol (Vitamin D2)or Cholecalciferol (Vitamin D3),and Vitamin E;one or more of the following water-soluble vitamins:Ascorbic Acid or its equivalent as Calcium Ascorbate or Sodium Ascorbate,Biotin,Cyanocobalamin,Niacin or Niacinamide,Dexpanthenol or Panthenol,Pantothenic Acid (as Calcium Pantothenate or Racemic Calcium Pantothenate),Pyridoxine Hydrochloride,Riboflavin or Riboflavin-5¢-Phosphate Sodium,and Thiamine Hydrochloride or Thiamine Mononitrate;and one or more minerals derived from substances generally recognized as safe,furnishing one or more of the following elements in ionizable form:chromium,fluorine,iodine,iron,magnesium,manganese,molybdenum,and zinc.It contains not less than 90.0percent and not more than 200.0percent of the labeled amounts of vitamin A(C20H30O)as retinol or esters of retinol in the form of retinyl acetate (C22H32O2)or retinyl palmitate (C36H60O2),vitamin Das ergocalciferol (C28H44O)or cholecalciferol (C27H44O),vitamin Eas alpha tocopherol (C29H50O2)or alpha tocopheryl acetate (C31H52O3)or alpha tocopheryl acid succinate (C33H54O5),ascorbic acid (C6H8O6)or its salts as calcium ascorbate (C12H14CaO12·2H2O)or sodium ascorbate (C6H7NaO6),and thiamine (C12H17ClN4OS)as thiamine hydrochloride or thiamine mononitrate;not less than 90.0percent and not more than 150.0percent of the labeled amounts of biotin (C10H16N2O3S),calcium pantothenate (C18H32CaN2O10),dexpanthenol (C9H19NO4)or panthenol (C9H19NO4),niacin (C6H5NO2)or niacinamide (C6H6N2O),pyridoxine hydrochloride (C8H11NO3·HCl),riboflavin (C17H20N4O6)or riboflavin-5¢-phosphate sodium (C17H20N4NaO9P);not less than 90.0percent and not more than 450.0percent of the labeled amount of cyanocobalamin (C63H88CoN14O14P);and not less than 90.0percent and not more than 125.0percent of the labeled amounts of chromium (Cr),fluorine (F),iodine (I),iron (Fe),magnesium (Mg),manganese (Mn),molybdenum (Mo),and zinc (Zn).
Packaging and storage— Preserve in tight,light-resistant containers,under an inert gas or with a minimum of headspace.
Labeling*— The label states that the product is Oil-and Water-Soluble Vitamins with Minerals Oral Solution.The label states the quantity of each vitamin and mineral in a given volume of the Oral Solution and,where necessary,the chemical form in which a vitamin is present,and states also the salt form of the mineral used as the source of each element.Where the product contains vitamin E,the label indicates whether it is the d-or dl-form.Where the product is labeled to contain panthenol,the label states the equivalent content of dexpanthenol.Where more than one Assaymethod is given for a particular vitamin or mineral the labeling states with which Assaymethod the product complies only if Method 1is not used.
Microbial enumeration á2021ñ— The total aerobic microbial count does not exceed 3000cfu per mL,and the combined molds and yeasts count does not exceed 300cfu per mL.The Oral Solution also meets the requirements of the tests for absence of Salmonellaspecies,Escherichia coli,and Staphylococcus aureus.
Alcohol content,Method Iá611ñ(if present): between 90.0%and 120.0%of the labeled amount of C2H5OHis found.
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.
Assay for vitamin A— [NOTE—Use low-actinic glassware throughout this procedure.]
Diluting solution— Prepare a mixture of tetrahydrofuran and acetonitrile (1:1),and mix.
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— [NOTE—USP Vitamin A RSis all-transretinyl acetate.Use it to analyze Oral Solution that contains vitamin Aas retinol,retinyl acetate,or retinyl palmitate.]Quantitatively dissolve an accurately weighed quantity of USP Vitamin A RSin Diluting solutionto obtain a solution having a known concentration of about 0.33mg of USP Vitamin A RSper mL.
Assay preparation— Transfer an accurately measured volume of Oral Solution,equivalent to about 3.3mg of vitamin Ato a 500-mLseparatory funnel containing 10mLof water and 20mLof dehydrated alcohol.Add 150mLof solvent hexane,insert the stopper,and shake for 1minute.Add another 150mLof solvent hexane,insert the stopper,shake,and allow the layers to separate.Discard the aqueous layer,and filter the solvent hexane extract through anhydrous sodium sulfate into a 500-mLround-bottom flask.Evaporate the solution to dryness with the aid of a rotary evaporator over a water bath maintained at about 65.Immediately add 10.0mLof Diluting solution,swirl to dissolve the residue,and filter.
Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 265-nm detector and a 4.6-mm ×50-cm column (prepared from two concatenated 4.6-mm ×25-cm columns)that contains packing L1.The column temperature is maintained at about 40,and the flow rate is about 1.5mLper 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 vitamin Aas the retinol equivalent (C20H30O)in each mLof the Oral Solution taken by the formula:
10(0.872C/V)(rU/rS),
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;Vis the volume,in mL,of Oral Solution taken for the Assay preparation;and rUand rSare the peak areas for retinol or retinyl ester obtained from the Assay preparationand the Standard preparation,respectively.
Assay for cholecalciferol or ergocalciferol (vitamin D)— [NOTE—Where vitamin D(cholecalciferol or ergocalciferol)is specified in the following procedure,use the chemical form present in the formulation and the relevant USP Reference Standard.Use low-actinic glassware throughout this procedure.]
Diluting solution and Mobile phase— Prepare as directed in the Assay for vitamin A.
Standard preparation— Dissolve an accurately weighed quantity of USP Cholecalciferol RSor USP Ergocalciferol RSin Diluting solution,and quantitatively dilute with Diluting solutionto obtain a solution having a known concentration of about 5µg per mL.
Assay preparation— Transfer an accurately measured volume of Oral Solution,equivalent to about 50µg of cholecalciferol or ergocalciferol,to a 500-mLseparatory funnel containing 10mLof water and 20mLof dehydrated alcohol.Add 150.0mLof solvent hexane,insert the stopper,and shake for 1minute.Add another 150mLof solvent hexane,insert the stopper,shake,and allow the layers to separate.Discard the aqueous layer.Drain the solvent hexane extract through anhydrous sodium sulfate into a 500-mLround-bottom flask.Evaporate the solution to dryness with the aid of a rotary evaporator over a water bath maintained at about 65.Immediately add 10.0mLof Diluting solution,swirl to dissolve the residue,and filter.
Chromatographic system (see Chromatography á621ñ)— Proceed as directed for Chromatographic systemin the Assay for vitamin A.Chromatograph the Standard preparation,and record the peak heights 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 heights for vitamin D.Calculate the quantity,in µg,of cholecalciferol (C27H44O)or of ergocalciferol (C28H44O)in each mLof the Oral Solution taken by the formula:
1.09(10C/V)(rU/rS),
in which 1.09is a correction factor to account for the average amount of previtamin Din the Oral Solution;Cis the concentration,in µg per mL,of USP Cholecalciferol RSor USP Ergocalciferol RSin the Standard preparation;Vis the volume,in mL,of Oral Solution taken for the Assay preparation;and rUand rSare the vitamin Dpeak heights obtained from the Assay preparationand the Standard preparation,respectively.
Assay for vitamin E— [NOTE—Use low-actinic glassware throughout this procedure.]
Diluting solution— Prepare a mixture of acetonitrile and ethyl acetate (1:1).
Potassium hydroxide solution— Transfer 90g of potassium hydroxide pellets to a 100-mLvolumetric flask containing about 60mLof water.Mix to dissolve,cool,and dilute with water to volume.
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 RSin Diluting solution,and dilute quantitatively,and stepwise if necessary,to obtain a solution having a known concentration of about 0.3mg per mL.
Assay preparation— Transfer an accurately measured volume of Oral Solution,equivalent to about 1.5mg of alpha tocopherol,to a 125-mLconical flask fitted with a ground-glass joint,and add 25.0mLof dehydrated alcohol.Attach a reflux condenser,and reflux in a boiling water bath for 1minute.Cautiously add 3mLof Potassium hydroxide solutionthrough the condenser,and continue to reflux for 30minutes.Remove the flask from the bath,and rinse the condenser with about 15mLof water.Cool,and transfer with a minimum volume of water to a 250-mLseparatory funnel.Rinse the flask with 50mLof n-hexane,and add the rinsings to the separatory funnel.Insert the stopper,shake vigorously for 1minute,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 n-hexane,and add the rinsing to the hexane solution in the flask.Evaporate the hexane solution to dryness with the aid of a rotary evaporator over a water bath maintained at about 50.Immediately add 5.0mLof 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 heights as directed for Procedure:the relative standard deviation for replicate injections is not more than 5%.
Procedure— Separately inject equal volumes (about 20µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the peak heights.Calculate the quantity,in mg,of alpha tocopherol (C29H50O2)in each mLof the Oral Solution taken by the formula:
5(C/V)(rU/rS),
in which Cis the concentration,in mg per mL,of USP Alpha Tocopherol RSin the Standard preparation;Vis the volume,in mL,of Oral Solution taken;and rUand rSare the peak heights for alpha tocopherol obtained from the Assay preparationand the Standard preparation,respectively.
Assay for ascorbic acid,Method 1— Proceed as directed in the Assay for Ascorbic Acidunder Automated Methods of Analysis á16ñ.
Assay for ascorbic acid,Method 2— Transfer an accurately measured volume of Oral Solution,equivalent to about 80mg of ascorbic acid,to a conical flask.Add 50mLof water,100mLof 0.1Nsulfuric acid VS,and 15.0mLof 0.1Niodine VS.Stir the contents for 30seconds,add 5mLof starch TS,and immediately titrate with 0.1Nsodium thiosulfate VSto the disappearance of the color.Each mLof 0.1Niodine is equivalent to 8.806mg of C6H8O6.
Assay for calcium ascorbate,Method 1— Proceed as directed in the Assay for Ascorbic Acidunder Automated Methods of Analysis á16ñ.
Assay for calcium ascorbate,Method 2— Proceed as directed in the Assay for ascorbic acid,Method 2.Each mLof 0.1Niodine is equivalent to 10.66mg of C12H14CaO12·2H2O.
Assay for sodium ascorbate,Method 1— Proceed as directed in the Assay for Ascorbic Acidunder Automated Methods of Analysis á16ñ.
Assay for sodium ascorbate,Method 2— Proceed as directed in the Assay for ascorbic acid,Method 2.Each mLof 0.1Niodine is equivalent to 9.905mg of C6H7NaO6.
Change to read:
Assay for biotin,Method 1USP28— Proceed as directed in the Assay for biotin,Method 2under Oil-and Water-Soluble Vitamins with Minerals Tablets,except to read Oral Solution in place of Tablets and to use the following Assay preparation.
Assay preparation— Transfer an accurately measured volume of Oral Solution to a volumetric flask,and dilute quantitatively,and stepwise if necessary,to obtain a solution having a concentration of 0.1ng of biotin per mL.
Add the following:
Assay for biotin,Method 2— Proceed as directed in the Assay for biotin,Method 3under Oil-and Water-Soluble Vitamins with Minerals Tablets,except to read Oral Solution in place of Tablets and to use the following Assay preparation.
Assay preparation— Transfer an accurately measured volume of Oral Solution to a volumetric flask,and dilute quantitatively,and stepwise if necessary with water,to obtain a solution having a concentration of 0.6µg of biotin per mL.Adjust the solution with either dilute acetic acid or 0.1Nsodium hydroxide to a pHof between 6.0and 7.0.USP28
Assay for cyanocobalamin— Proceed as directed in the Assay for cyanocobalamin,Method 2under Oil-and Water-Soluble Vitamins with Minerals Tablets,except to read Oral Solution in place of Tablets and to use the following Assay preparation.
Assay preparation— Transfer an accurately measured volume of Oral Solution,equivalent to about 1.0µg of cyanocobalamin,to an appropriate vessel containing,for each mLof the Oral Solution 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.
Assay for calcium pantothenate,Method 1—
Dilute phosphoric acid— Dilute 11.8mLof phosphoric acid with water to 100.0mL,and mix.
Mobile phase— Mix 30mLof methanol with 970mLof 0.2Mmonobasic sodium phosphate.Adjust with Dilute phosphoric acidto a pHof 3.2±0.1,mix,and filter.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).
Standard preparation— Dissolve an accurately weighed quantity of USP Calcium Pantothenate RSin Mobile phaseto obtain a solution having a known concentration of about 80µg per mL.
System suitability solution— Dissolve an accurately weighed quantity of USP Racemic Panthenol RSin Mobile phaseto obtain a solution having a known concentration of about 80µg per mL.Transfer 5.0mLof this solution to a 10-mLvolumetric flask,dilute with Standard preparationto volume,and mix.
Assay preparation— Dilute an accurately measured volume of Oral Solution quantitatively,and stepwise if necessary,with Mobile phaseto obtain a solution having a concentration of about 80µg of calcium pantothenate per mL.
Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 210-nm detector and a 4.0-mm ×10-cm column that contains packing L1.The flow rate is about 1.0mLper minute.Chromatograph 20µLof the System suitability solution,and record the peak areas as directed for Procedure:the resolution,R,between panthenol and calcium pantothenate is not less than 1.5;and the tailing factor for both the calcium pantothenate and the panthenol peaks is not more than 2.0.Chromatograph the Standard preparation,and record the peak areas as directed for Procedure:the relative standard deviation for replicate injections is not more than 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 obtained for calcium pantothenate.Calculate the quantity,in mg,of calcium pantothenate (C18H32CaN2O10)in each mLof the Oral Solution taken by the formula:
C(L/D)(rU/rS),
in which Cis the concentration,in mg per mL,of USP Calcium Pantothenate RSin the Standard preparation;Lis the labeled quantity,in mg per mL,of calcium pantothenate in the Oral Solution taken;Dis the concentration,in mg per mL,of calcium pantothenate in the Assay preparation,on the basis of the labeled quantity and the extent of dilution;and rUand rSare the peak areas for calcium pantothenate obtained from the Assay preparationand the Standard preparation,respectively.
Assay for calcium pantothenate,Method 2— Proceed as directed in the Assay for calcium pantothenate,Method 2under Oil-and Water-Soluble Vitamins with Minerals Tablets,except to read Oral Solution in place of Tablets and to use the following Assay preparation.
Assay preparation— Transfer an accurately measured volume of Oral Solution,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,with water to obtain a solution having about the same concentration as that of the Standard preparation.
Assay for dexpanthenol or panthenol,Method 1—
Dilute phosphoric acid,Mobile phase,and Chromatographic system— Proceed as directed in the Assay for calcium pantothenate,Method 1.
Standard preparation— Dissolve an accurately weighed quantity of USP Dexpanthenol RSor USP Racemic Panthenol RSin Mobile phaseto obtain a solution having a known concentration of about 80µg per mL.[NOTE—Use USP Dexpanthenol RSto analyze Oral Solution that contains dexpanthenol and USP Racemic Panthenol RSto analyze Oral Solution that contains panthenol.]
System suitability solution— Dissolve an accurately weighed quantity of USP Calcium Pantothenate RSin Mobile phaseto obtain a solution having a known concentration of about 80µg per mL.Transfer 5.0mLof this solution to a 10-mLvolumetric flask,dilute with Standard preparationto volume,and mix.
Assay preparation— Dilute an accurately measured volume of Oral Solution quantitatively,and stepwise if necessary,with Mobile phaseto obtain a solution having a concentration of about 80µg of dexpanthenol or panthenol per mL.
Procedure— Separately inject equal volumes (about 20µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the peak areas obtained for panthenol.Calculate the quantity,in mg,of dexpanthenol (C9H19NO4)or panthenol (C9H19NO4)in each mLof the Oral Solution taken by the formula:
C(L/D)(rU/rS),
in which Cis the concentration,in mg per mL,of USP Dexpanthenol RSor USP Racemic Panthenol RSin the Standard preparation;Lis the labeled quantity,in mg per mL,of dexpanthenol or panthenol in the Oral Solution taken;Dis the concentration,in mg per mL,of dexpanthenol or panthenol in the Assay preparation,on the basis of the labeled quantity and the extent of dilution;and rUand rSare the peak areas for dexpanthenol or panthenol obtained from the Assay preparationand the Standard preparation,respectively.
Assay for dexpanthenol or panthenol,Method 2— Proceed as directed in the Assay for dexpanthenol or panthenolunder Water-Soluble Vitamins Capsules,except to read Oral Solution in place of Capsules and to use the following Assay preparation.
Assay preparation— Transfer an accurately measured volume of Oral Solution,equivalent to about 1.2mg of dexpanthenol or 2.4mg of panthenol,to a 100-mLvolumetric flask,dissolve in and dilute with water to volume,and mix.Dilute a portion of this solution quantitatively,and stepwise if necessary,with water to obtain a solution having a concentration of about 1.2µg of dexpanthenol or 2.4µg of panthenol per mL.
Assay for niacin or niacinamide— [NOTE—Use low-actinic glassware throughout this procedure.]
Diluting solution— Dissolve 25g of edetate disodium in 1000mLof water,and mix.
Mobile phase— Mix 0.4mLof triethylamine,15.0mLof glacial acetic acid,and 350mLof methanol,and dilute with 0.008Msodium 1-hexanesulfonate to 2000mL.Filter,and degas.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).
Standard preparation— [NOTE—Use USP Niacin RSfor Oral Solution that contains niacin and USP Niacinamide RSfor Oral Solution that contains niacinamide.]Dissolve an accurately weighed quantity of USP Niacin RSor USP Niacinamide RSin Diluting solutionin a volumetric flask,and dilute quantitatively,and stepwise if necessary,with Diluting solutionto obtain a solution having a known concentration of about 0.10mg per mL.
Assay preparation— Transfer an accurately measured volume of Oral Solution to a volumetric flask,dissolve in Diluting solution,and dilute quantitatively,and stepwise if necessary,with Diluting solutionto obtain a solution having a known concentration of about 0.1mg of niacin or niacinamide per mL.
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 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 Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the peak areas.Calculate the quantity,in mg,of niacin (C6H5NO2)or niacinamide (C6H6N2O)in each mLof the Oral Solution taken by the formula:
C(L/D)(rU/rS),
in which Cis the concentration,in mg per mL,of USP Niacin RSor USP Niacinamide RSin the Standard preparation;Lis the labeled amount,in mg per mL,of niacin or niacinamide in the Oral Solution taken;Dis the concentration,in mg per mL,of niacin or niacinamide in the Assay preparation,based on the labeled quantity and the extent of dilution;and rUand rSare the peak areas for niacin or niacinamide obtained from the Assay preparationand the Standard preparation,respectively.
Assay for pyridoxine hydrochloride—
Diluting solution,Mobile phase,and Chromatographic system— Proceed as directed in the Assay for niacin or niacinamide.
Standard preparation— Dissolve an accurately weighed quantity of USP Pyridoxine Hydrochloride RSin Diluting solution,and dilute quantitatively,and stepwise if necessary,with Diluting solutionto obtain a solution having a known concentration of about 0.024mg per mL.
Assay preparation— Transfer an accurately measured volume of Oral Solution to a volumetric flask,dissolve in Diluting solution,and dilute quantitatively,and stepwise if necessary,with Diluting solutionto obtain a solution having a concentration of about 0.024mg of pyridoxine hydrochloride per mL.
Procedure— Separately inject equal volumes (about 5µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the areas for the major peaks.Calculate the quantity,in mg,of pyridoxine hydrochloride (C8H11NO3·HCl)in each mLof the Oral Solution taken by the formula:
C(L/D)(rU/rS),
in which Cis the concentration,in mg per mL,of USP Pyridoxine Hydrochloride RSin the Standard preparation;Lis the labeled amount,in mg per mL,of pyridoxine hydrochloride in the Oral Solution taken;Dis the concentration,in mg per mL,of pyridoxine hydrochloride in the Assay preparation,based on the labeled quantity and the extent of dilution;and rUand rSare the peak areas for pyridoxine hydrochloride obtained from the Assay preparationand Standard preparation,respectively.
Assay for riboflavin or riboflavin-5¢-phosphate sodium,Method 1— [NOTE—Riboflavin-5¢-phosphate sodium is quantitated against USP Riboflavin RSin this procedure.In the chromatogram of the Assay preparation,the riboflavin-5¢-phosphate peak is the only peak measured for calculation.]
Diluting solution,Mobile phase,and Chromatographic system— Proceed as directed in the Assay for niacin or niacinamide.
Standard preparation— Dissolve an accurately weighed quantity of USP Riboflavin RSin Diluting solution,by heating if necessary,in a volumetric flask,and dilute quantitatively,and stepwise if necessary,with Diluting solutionto obtain a solution having a known concentration of about 8µg per mL.
Assay preparation— Transfer a volume of Oral Solution,accurately measured,to a volumetric flask,dissolve in Diluting solution,and dilute quantitatively,and stepwise if necessary,with Diluting solutionto obtain a solution having a known concentration of about 8µg of riboflavin per mL.
Procedure— Separately inject equal volumes (about 5µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the peak areas for riboflavin and riboflavin-5¢-phosphate.The relative retention times are about 0.18for riboflavin-5¢-phosphate and 1.0for riboflavin.Calculate the quantity,in mg,of riboflavin (C17H20N4O6)in each mLof the Oral Solution taken by the formula:
1.493C(L/D)(rU/rS),
in which 1.493is the factor for converting riboflavin-5¢-phosphate to riboflavin;Cis the concentration,in mg per mL,of USP Riboflavin RSin the Standard preparation;Lis the labeled amount,in mg per mL,of riboflavin in the Oral Solution taken;Dis the concentration,in mg per mL,of riboflavin in the Assay preparation,based on the labeled quantity and the extent of dilution;rUis the peak area for riboflavin-5¢-phosphate obtained from the Assay preparation;and rSis the peak area for riboflavin obtained from the Standard preparation.
Assay for riboflavin or riboflavin-5¢-phosphate sodium,Method 2— [NOTE—Use low-actinic glassware throughout this procedure.]
Solvent blank— Transfer 1mLof 1Nhydrochloric acid and 2mLof 2.5Msodium acetate to a 100-mLvolumetric flask,dilute with water to volume,and mix.
Riboflavin stock solution— Transfer about 16mg of USP Riboflavin RS,accurately weighed,to a 100-mLvolumetric flask,dissolve in 1.0mLof 1Nhydrochloric acid and 2.0mLof 2.5Msodium acetate,dilute with water to volume,and mix.Transfer 10.0mLof this solution to a 100-mLvolumetric flask,dilute with water to volume,and mix.
Standard preparation— Transfer 5.0mLof Riboflavin stock solutionto a 500-mLvolumetric flask,add 5mLof 1Nhydrochloric acid and 10mLof 2.5Msodium acetate,dilute with water to volume,and mix.
Assay preparation— Transfer an accurately measured volume of Oral Solution,equivalent to about 0.8mg of riboflavin,to a 100-mLvolumetric flask,dilute with water to volume,and mix.Transfer 10.0mLof this solution to a 500-mLvolumetric flask,add 5mLof 1Nhydrochloric acid and 10mLof 2.5Msodium acetate,dilute with water to volume,and mix.
Procedure— Using a spectrofluorometer that has been set to zero with the Solvent blank,determine the maximum fluorescence intensities,ISand IU,of the Standard preparationand the Assay preparation,respectively,at an emission wavelength of about 530nm and an excitation wavelength of about 440nm.Calculate the quantity,in mg,of riboflavin (C17H20N4O6)in each mLof the Oral Solution taken by the formula:
C(L/D)(IU/IS),
in which Cis the concentration,in µg per mL,of USP Riboflavin RSin the Standard preparation;Lis the labeled amount,in mg per mL,of riboflavin in the Oral Solution taken;Dis the concentration,in µg per mL,of riboflavin in the Assay preparation,based on the labeled amount in a given volume and the extent of dilution;and IUand ISare the fluorescence values obtained from the Assay preparationand the Standard preparation,respectively.
Assay for thiamine—
Diluting solution,Mobile phase,and Chromatographic system— Proceed as directed in the Assay for niacin or niacinamide.
Standard preparation— Dissolve an accurately weighed quantity of USP Thiamine Hydrochloride RSin Diluting solution,and dilute quantitatively,and stepwise if necessary,with Diluting solutionto obtain a solution having a known concentration of about 0.024mg of USP Thiamine Hydrochloride RSper mL.
Assay preparation— Dissolve an accurately measured volume of Oral Solution in Diluting solution,and dilute quantitatively,and stepwise if necessary,with Diluting solutionto obtain a solution having a concentration of about 0.024mg of thiamine hydrochloride per mL.
Procedure— Separately inject equal volumes (about 5µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the peak areas.Calculate the quantity,in mg,of thiamine hydrochloride (C12H17ClN4OS·HCl)in each mLof the Oral Solution taken by the formula:
C(L/D)(rU/rS),
in which Cis the concentration,in mg per mL,of USP Thiamine Hydrochloride RSin the Standard preparation;Lis the labeled amount,in mg per mL,of thiamine hydrochloride in the Oral Solution taken;Dis the concentration,in mg per mL,of thiamine hydrochloride in the Assay preparation,based on the labeled quantity and the extent of dilution;and rUand rSare the peak areas for thiamine hydrochloride obtained from the Assay preparationand Standard preparation,respectively.
NOTE—Commercially available atomic absorption standard solutions for the minerals,where applicable,may be used where preparation of a standard stock solution is described in the following Assays.Use deionized water where water is specified.Where atomic absorption spectrophotometry is specified in the Assay,the Standard preparationsand the Assay preparationmay be diluted quantitatively with the solvent specified,if necessary,to yield solutions of suitable concentrations adaptable to the linear or working range of the instrument.
Assay for chromium—
Chromium standard stock solution and Standard preparations— Proceed as directed in the Assay for chromiumunder Oil-and Water-Soluble Vitamins with Minerals Tablets.
Assay preparation— Dilute an accurately measured volume of Oral Solution quantitatively,and stepwise if necessary,with 0.125Nhydrochloric acid to obtain a solution having a concentration of about 1µg of chromium per mL.
Procedure— Proceed as directed for Procedurein the Assay for chromiumunder Oil-and Water-Soluble Vitamins with Minerals Tablets,except to calculate the quantity,in µg,of chromium (Cr)in each mLof the Oral Solution taken by the formula:
C(L/D),
in which Lis the labeled quantity,in µg per mL,of chromium in the Oral Solution;and Dis the concentration,in µg per mL,of chromium in the Assay preparation,on the basis of the labeled quantity and the extent of dilution.
Assay for fluoride— [NOTE—Use plastic containers throughout this procedure.]
Ascorbic acid solution— Dissolve 7g of ascorbic acid in 100mLof water,and mix.
Mobile phase— Prepare a filtered and degassed mixture of water,alcohol,and 1Nsulfuric acid (898:100:2).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— Transfer 5.0mLof Standard stock solutionto a 100-mLvolumetric flask.Add 2mLof Ascorbic acid solution,10mLof alcohol,and about 70mLof water,and mix.Adjust with 1Nsodium hydroxide to a pHof 4.25±0.05.Dilute with water to volume,and mix to obtain a solution having a known concentration of about 5µg of fluoride per mL.
Assay preparation— Transfer an accurately measured volume of Oral Solution,equivalent to about 0.5mg of fluoride,to a 100-mLvolumetric flask.Add 1drop of hydrochloric acid,10mLof alcohol,and about 75mLof water,and mix.Adjust with 1Nsodium hydroxide to a pHof 4.25±0.05.Dilute with water to volume,and mix.
Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a conductivity detector and a combination 4.6-mm ×3-cm guard column and 7.8-mm ×30-cm analytical column that contains packing L17.The flow rate is about 0.6mLper 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 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 (F)in the portion of Oral Solution taken by the formula:
100C(rU/rS),
in which Cis the concentration,in mg per mL,of fluoride in the Standard preparation;and rUand rSare the peak areas for fluoride obtained from the Assay preparationand the Standard preparation,respectively.
Assay for iodide,Method 1—
Mobile phase— Dissolve 5.15g of tetrabutylammonium bromide in 320mLof acetonitrile.Dilute with water to 2000mL,mix,filter,and degas.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).
Standard stock solution— Transfer about 0.13g of potassium iodide,accurately weighed,to a 100-mLvolumetric flask.Dissolve in and dilute with Mobile phaseto volume,and mix.This solution has a concentration of about 1mg of iodide per mL.
Standard preparation— Dilute an accurately measured volume of the Standard stock solutionwith Mobile phasequantitatively,and stepwise if necessary,to obtain a solution having a known concentration of about 2.5µg of iodide per mL.
System suitability solution— Transfer about 0.13g of potassium iodide and 0.5g of potassium iodate,accurately weighed,to a 100-mLvolumetric flask.Dissolve in Mobile phase,using sonication if necessary,dilute with Mobile phaseto volume,and mix.Transfer 1.0mLof this solution to a 100-mLvolumetric flask,dilute with Mobile phaseto volume,and mix.Transfer 25.0mLof this solution to a 100-mLvolumetric flask,dilute with Mobile phaseto volume,and mix.
Assay preparation— Dissolve an accurately measured volume of Oral Solution in Mobile phase,and dilute quantitatively,and stepwise if necessary,with Mobile phaseto obtain a solution having a concentration of about 2.5µg of iodide per mL.
Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 225-nm detector and a 4.6-mm ×15-cm column that contains packing L1.The flow rate is about 1.5mLper minute.Chromatograph about 30µLof the System suitability solution,and record the peak areas as directed for Procedure:the relative retention times are about 0.32for iodate and 1.0for iodide;and the resolution,R,between iodate and iodide is not less than 2.5.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%for the iodide peak.
Procedure— Separately inject equal volumes (about 30µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the peak areas for iodide.Calculate the quantity,in µg,of iodide in each mLof Oral Solution taken by the formula:
(126.9/166.0)(C)(L/D)(rU/rS),
in which 126.9is the atomic weight of iodine;166.0is the molecular weight of potassium iodide;Cis the concentration,in µg per mL,of potassium iodide,calculated on the dried basis,in the Standard preparation;Lis the labeled quantity,in µg per mL,of iodide in the Oral Solution;Dis the concentration,in µg per mL,of iodide in the Assay preparation,based on the labeled quantity and the extent of dilution;and rUand rSare the peak areas for iodide obtained from the Assay preparationand the Standard preparation,respectively.
Assay for iodide,Method 2— Proceed as directed in the Assay for Iodideunder Automated Methods of Analysis á16ñ.
Assay for iron—
Iron standard stock solution— Transfer about 100mg of iron powder,accurately weighed,to a 1000-mLvolumetric flask,dissolve in 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 0.125Nhydrochloric acid to volume,and mix to obtain the solutions having known concentrations of about 2.0,4.0,5.0,6.0,and 8.0µg of iron per mL.
Assay preparation— Dilute an accurately measured volume of Oral Solution quantitatively,and stepwise if necessary,with 0.125Nhydrochloric acid to obtain a solution having a concentration of about 6µg of iron per mL.
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 each mLof the Oral Solution taken by the formula:
C(L/D),
in which Lis the labeled quantity,in mg per mL,of iron in the Oral Solution taken;and Dis the concentration,in µg per mL,of iron in the Assay preparation,on the basis of the labeled quantity and the extent of dilution.
Assay for magnesium—
Magnesium standard stock solution— Transfer about 1.00g of magnesium ribbon,accurately weighed,to a 1000-mLvolumetric flask,dissolve in 50mLof 6Nhydrochloric acid,dilute with water to volume,and mix.
Standard preparations— Quantitatively dilute a volume of Magnesium standard stock solutionwith water to obtain a standard solution having a concentration of 20µg of magnesium per mL.To separate 100-mLvolumetric flasks,transfer 5.0,7.5,10.0,12.5,and 15.0mLof this solution.Dilute the contents of each flask with 0.125Nhydrochloric acid to volume,and mix to obtain the solutions having known concentrations of about 1.0,1.5,2.0,2.5,and 3.0µg of magnesium per mL.
Assay preparation— Dilute an accurately measured volume of Oral Solution quantitatively,and stepwise if necessary,with 0.125Nhydrochloric acid to obtain a solution having a concentration of about 2.5µg 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 as 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 each mLof the Oral Solution taken by the formula:
C(L/D),
in which Lis the labeled quantity,in mg per mL,of magnesium in the Oral Solution taken;and Dis the concentration,in µg per mL,of magnesium in the Assay preparation,on the basis of the labeled quantity and the extent of dilution.
Assay for manganese—
Manganese standard stock solution— Transfer 1.0g of manganese,accurately weighed,to a 1000-mLvolumetric flask,dissolve in 20mLof nitric acid,dilute with 6Nhydrochloric acid to volume,and mix.
Standard preparations— Quantitatively dilute 10.0mLof Manganese standard stock solutionwith water to 200.0mLto obtain a standard solution having a known concentration of about 50µg of manganese per mL.To separate 100-mLvolumetric flasks,transfer 1.0,1.5,2.0,3.0,and 4.0mLof this 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— Dilute an accurately measured volume of Oral Solution quantitatively,and stepwise if necessary,with 0.125Nhydrochloric acid to obtain a solution having a concentration of about 1.5µg of manganese per mL.
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 each mLof the Oral Solution taken by the formula:
C(L/D),
in which Lis the labeled quantity,in mg per mL,of manganese in the Oral Solution taken;and Dis the concentration,in µg per mL,of manganese in the Assay preparation,on the basis of the labeled quantity and the extent of dilution.
Assay for molybdenum—
Molybdenum standard stock solution— Transfer 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.
Standard preparations— Quantitatively dilute 10.0mLof Molybdenum standard stock solutionwith water to obtain a standard solution having a known concentration of about 100µg of molybdenum per mL.To separate 100-mLvolumetric flasks,transfer 0.5,1.0,1.5,2.0,and 3.0mLof this solution.Dilute the contents of each flask with 0.125Nhydrochloric acid to volume,and mix to obtain the solutions having known concentrations of about 0.5,1.0,1.5,2.0,and 3.0µg of molybdenum per mL.
Assay preparation— Dilute an accurately measured volume of Oral Solution quantitatively,and stepwise if necessary,with 0.125Nhydrochloric acid to obtain a solution having a concentration of about 1µg of molybdenum per mL.
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 0.125Nhydrochloric acid 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 five 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 µg,of molybdenum (Mo)in each mLof the Oral Solution taken by the formula:
C(L/D),
in which Lis the labeled quantity,in µg per mL,of molybdenum in the Oral Solution taken;and Dis the concentration,in µg per mL,of molybdenum in the Assay preparation,on the basis of the labeled quantity and the extent of dilution.
Assay for zinc—
Zinc standard stock solution— Transfer about 311mg of zinc oxide,accurately weighed,to a 250-mLvolumetric flask,and add 80mLof 6Nhydrochloric 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 Zinc standard stock solutionquantitatively,and stepwise if necessary,with water 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 the solutions having known concentrations of about 0.5,1.0,1.5,2.0,and 2.5µg of zinc per mL.
Assay preparation— Dilute an accurately measured volume of Oral Solution quantitatively,and stepwise if necessary,with 0.125Nhydrochloric acid to obtain a solution having a concentration of about 1µg of zinc per mL.
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,C,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 each mLof the Oral Solution taken by the formula:
C(L/D),
in which Lis the labeled quantity,in mg per mL,of zinc in the Oral Solution taken;and Dis the concentration,in µg per mL,of zinc in the Assay preparation,on the basis of the labeled quantity and the extent of dilution.
*  For information on USP Units of activity for vitamins and labeling in terms of units,see the footnote to the Labelingsection under Oil-and Water-Soluble Vitamins with Minerals Tablets.
Auxiliary Information— Staff Liaison:Lawrence Evans,III,Ph.D.,Scientist
Expert Committee:(DSN)Dietary Supplements:Non-Botanicals
USP28–NF23Page 2149
Pharmacopeial Forum:Volume No.30(2)Page 585
Phone Number:1-301-816-8389