Oil- and Water-Soluble Vitamins with Minerals Tablets
» Oil- and Water-Soluble Vitamins with Minerals Tablets contain one or more of the following oil-soluble vitamins: Vitamin A, Vitamin D as Ergocalciferol (Vitamin D2) or Cholecalciferol (Vitamin D3), Vitamin E, Phytonadione (Vitamin K1), and Beta Carotene; one or more of the following water-soluble vitamins: Ascorbic Acid or its equivalent as Calcium Ascorbate or Sodium Ascorbate, Biotin, Cyanocobalamin, Folic Acid, Niacin or Niacinamide, Pantothenic Acid (as Calcium Pantothenate or Racemic Calcium Pantothenate), Pyridoxine Hydrochloride, Riboflavin, 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: calcium, chromium, copper, fluorine, iodine, iron, magnesium, manganese, molybdenum, phosphorus, potassium, selenium, and zinc. Tablets contain not less than 90.0 percent and not more than 165.0 percent 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 D as cholecalciferol (C27H44O) or ergocalciferol (C28H44O), vitamin E as alpha tocopherol (C29H50O2) or alpha tocopheryl acetate (C31H52O3) or alpha tocopheryl acid succinate (C33H54O5), phytonadione (C31H46O2), and beta carotene (C40H56); not less than 90.0 percent and not more than 150.0 percent of the labeled amounts of ascorbic acid (C6H8O6) or its salts as calcium ascorbate (C12H14CaO12·2H2O) or sodium ascorbate (C6H7NaO6), biotin (C10H16N2O3S), cyanocobalamin (C63H88CoN14O14P), folic acid (C19H19N7O6), niacin (C6H5NO2) or niacinamide (C6H6N2O), calcium pantothenate (C18H32CaN2O10), pyridoxine hydrochloride (C8H11NO3·HCl), riboflavin (C17H20N4O6), and thiamine (C12H17ClN4OS) as thiamine hydrochloride or thiamine mononitrate; not less than 90.0 percent and not more than 125.0 percent of the labeled amounts of calcium (Ca), copper (Cu), iron (Fe), manganese (Mn), magnesium (Mg), phosphorus (P), potassium (K), and zinc (Zn); and not less than 90.0 percent and not more than 160.0 percent of the labeled amounts of chromium (Cr), fluorine (F), iodine (I), molybdenum (Mo), and selenium (Se).
They may contain other labeled added substances that are generally recognized as safe, in amounts that are unobjectionable.
Packaging and storage
Preserve in tight, light-resistant containers.
Labeling1
The label states that the product is Oil- and Water-Soluble Vitamins with Minerals Tablets. The label also states the quantity of each vitamin and mineral per dosage unit and where necessary the chemical form in which a vitamin is present and also states 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 more than one Assay method is given for a particular vitamin, the labeling states with which Assay method the product complies only if Method 1 is not used.
USP Reference standards
![]() ![]() USP Alpha Tocopherol RS. USP Alpha Tocopheryl Acetate RS. USP Alpha Tocopheryl Acid Succinate RS. USP Biotin RS. USP Calcium Pantothenate RS. USP Cholecalciferol RS. USP Cyanocobalamin RS. USP Ergocalciferol RS. USP Folic Acid RS. USP Niacin RS. USP Niacinamide RS. USP Phytonadione RS. USP Pyridoxine Hydrochloride RS. USP Riboflavin RS. USP Sodium Fluoride RS. USP Thiamine Hydrochloride RS. USP Vitamin A RS.
Microbial enumeration
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Disintegration and dissolution
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Weight variation
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Assay for vitamin A, Method 1
[noteWhere the use of a vitamin A ester (retinyl acetate or retinyl palmitate) is specified in the following procedure, use the chemical form present in the formulation. USP Vitamin A RS is all-trans retinyl acetate. It is to be used where USP Vitamin A RS is specified. Use low-actinic glassware throughout this procedure.]
Mobile phase
Use n-hexane.
Standard preparation
Dissolve an accurately weighed quantity of USP Vitamin A RS in n-hexane, and dilute quantitatively, and stepwise if necessary, to obtain a solution having a known concentration of about 15 µg 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 15 µg of retinyl palmitate per mL. Mix equal volumes of this solution and the Standard preparation to obtain a solution having concentrations of 7.5 µg each of retinyl acetate and retinyl palmitate per mL.
Assay preparation
Weigh and finely powder not fewer than 20 Tablets. Transfer an accurately weighed portion of the powder, equivalent to 5 Tablets, to a container having a polytef-lined screw cap, add 10 mL of dimethyl sulfoxide and 15 mL of n-hexane, and shake for 45 minutes on a wrist-action shaker in a water bath maintained at 60
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Chromatographic system (see Chromatography
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Procedure
Separately inject equal volumes (about 40 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the peak area for retinyl acetate obtained from the Standard preparation and the peak area for retinyl acetate or retinyl palmitate in the chromatogram of the Assay preparation. For products containing vitamin A acetate or vitamin A palmitate, calculate the quantity, in mg, of vitamin A as the retinol equivalent (C20H30O) in the portion of Tablets taken by the formula:
0.872CD(rU / rS)
in which 0.872 is the factor used to convert retinyl acetate, obtained from USP Vitamin A RS, to its retinol equivalent; C is the concentration, in mg per mL, of USP Vitamin A RS in the Standard preparation; D is the dilution factor, in mL, for the Assay preparation; and rU and rS are the peak areas of the all-trans retinyl ester obtained from the Assay preparation and the Standard preparation, respectively. [noteThe molar responses of retinyl acetate and retinyl palmitate are equivalent.]
Assay for vitamin A, Method 2
[noteWhere a vitamin A ester (retinyl acetate or retinyl palmitate) is indicated in the following procedure, use the chemical form present in the formulation. USP Vitamin A RS is all-trans retinyl acetate. It is to be used where USP Vitamin A RS is specified. Use low-actinic glassware throughout this procedure.]
3 N Methanolic sulfuric acid solution
Cautiously add 9 mL of sulfuric acid to 80 mL of methanol in a 100-mL volumetric flask. Cool, dilute with methanol to volume, and mix.
Sodium ascorbatepyrogallol solution
Transfer 10 g of sodium ascorbate and 5 g of pyrogallol to a 100-mL volumetric flask, and add sufficient water to dissolve. Add 1.7 mL of sulfuric acid, dilute with water to volume, and mix.
Lecithin solution
Transfer 0.5 g of lecithin to a 100-mL volumetric 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 RS in 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 15 µg 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 15 µg per mL. Mix equal volumes of this solution and the Standard preparation to obtain a solution having concentrations of 7.5 µg each of retinyl acetate and retinyl palmitate per mL.
Assay preparation
[noteThis 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 20 Tablets. If vitamin D is present in the formulation, transfer an accurately weighed portion of the powder, equivalent to about 30 µg of cholecalciferol or ergocalciferol, to a container having a polytef-lined screw cap. If vitamin D is not present in the formulation, use a portion of the powder equivalent to about 90 mg of vitamin E (as alpha tocopherol, alpha tocopheryl acetate, or alpha tocopheryl hemisuccinate). If vitamin E is not present in the formulation, use a portion of the powder equivalent to about 2.5 mg of retinyl acetate or retinyl palmitate. Add about 0.5 g of sodium bicarbonate, 1.5 mL of Lecithin solution, and 12.5 mL of 2,2,4-trimethylpentane, and disperse on a vortex mixer. Add 6 mL of Sodium ascorbatepyrogallol solution, shake slowly, and allow the solution to degas. Continue shaking until the evolution of gas has ceased, and then shake for an additional 12 minutes. Add 6 mL of dimethyl sulfoxide, mix on a vortex mixer to form a suspension, and shake for 12 minutes. Add 6 mL of 3 N Methanolic sulfuric acid solution, mix on a vortex mixer to form a suspension, and shake for 12 minutes. Add 12.5 mL of 2,2,4-trimethylpentane, mix on a vortex mixer to form a suspension, and shake for 10 minutes. Centrifuge for about 10 minutes to break up the emulsion and to clarify the supernatant. [noteThe supernatant is used for the determination of vitamin A, and also vitamin D and 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
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Procedure
Separately inject equal volumes (about 40 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the peak area of retinyl acetate obtained from the Standard preparation and 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:
26.5ECD(rU / rS)
in which E is the factor used to convert the retinyl acetate, obtained from USP Vitamin A RS, to its retinol equivalent, the factor being 0.872; C is the concentration, in mg per mL, of USP Vitamin A RS in the Standard preparation; D is the dilution factor, in mL, used to prepare the Assay preparation from the supernatant; and rU and rS are the peak responses of the all-trans retinyl ester obtained from the Assay preparation and the Standard preparation, respectively. [noteThe initial extraction into 26.5 mL of 2,2,4-trimethylpentane is already accounted for in this equation. The molar responses of retinyl acetate and retinyl palmitate are equivalent.]
Assay for vitamin A, Method 3
[noteWhere a vitamin A ester (retinyl acetate or retinyl palmitate) is indicated in the following procedure, use the chemical form present in the formulation. USP Vitamin A RS is all-trans retinyl acetate. It is to be used where USP Vitamin A RS is specified. Use low-actinic glassware throughout this procedure.]
Extraction solvent
Prepare a mixture of n-hexane and methylene chloride (3:1).
Potassium hydroxide solution
Cautiously add 80 g of potassium hydroxide to 100 mL of water, mix, and cool.
Diluting solution
Transfer 1.0 g of pyrogallol to a 100-mL volumetric 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 Suitability under Chromatography
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Standard stock solution
Dissolve an accurately weighed quantity of USP Vitamin A RS in Diluting solution, and dilute quantitatively, and stepwise if necessary, with Diluting solution to obtain a solution having a known concentration of about 30 µg per mL. This solution may be stored in a refrigerator for 1 week.
Standard preparation
Quantitatively dilute an accurately measured volume of Standard stock solution with Diluting solution to obtain a solution having a known concentration of about 1 µg of USP Vitamin A RS per mL. Transfer 10.0 mL of this solution to a stoppered 125-mL flask, and add 5 mL of water, 5 mL of Diluting solution, and 3 mL of Potassium hydroxide solution. Insert the stopper tightly, shake for 15 minutes over a water bath maintained at 60 ± 5
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Assay preparation
Weigh and finely powder a counted number of Tablets. Transfer an accurately weighed portion of the powder, equivalent to about 1.5 mg of retinyl acetate, to a stoppered 125-mL flask. Add 5 mL of water, 15 mL of Diluting solution, and 3 mL of Potassium hydroxide solution. Insert the stopper tightly, shake for 15 minutes over a water bath maintained at 60 ± 5
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Chromatographic system (see Chromatography
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Procedure
Separately inject equal volumes (about 50 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the peak areas for all-trans retinol and 13-cis retinol. Calculate the quantity, in mg, of vitamin A, as the retinol (C20H30O) equivalent, in the portion of Tablets taken by the formula:
0.872CD(rU / rS)
in which 0.872 is the factor used to convert retinyl acetate, obtained from USP Vitamin A RS, to its retinol equivalent; C is the concentration, in mg per mL, of USP Vitamin A RS in the Standard preparation; D is the dilution factor, in mL, used to prepare the Assay preparation; rU is the sum of the areas of the all-trans retinol and 13-cis retinol peaks obtained from the Assay preparation; and rS is the peak area of all-trans retinyl acetate obtained from the Standard preparation.
Assay for cholecalciferol or ergocalciferol (vitamin D), Method 1
[noteWhere 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.]
Mobile phase
Prepare a filtered and degassed mixture of n-hexane and isopropyl alcohol (99:1). Make adjustments if necessary (see System Suitability under Chromatography
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Standard preparation
Dissolve an accurately weighed quantity of USP Cholecalciferol RS or USP Ergocalciferol RS in n-hexane, and dilute quantitatively, and stepwise if necessary, to obtain a solution having a known concentration of about 2 µg per mL.
System suitability preparation
Heat a volume of the Standard preparation at 60
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Assay preparation
Transfer not less than 20 mL, accurately measured, of the solution retained as specified in the directions for Assay preparation in the Assay for vitamin A, Method 1 to a suitable container, and evaporate, if necessary, in vacuum at room temperature to obtain a solution having a concentration of about 2 µg of cholecalciferol or ergocalciferol per mL.
Chromatographic system (see Chromatography
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Procedure
Separately inject equal volumes (about 100 µL) of the Standard preparation and the Assay preparation into 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:
1.09CD(rU / rS)
in which 1.09 is a correction factor to account for the average amount of previtamin D present in the formulation; C is the concentration, in µg per mL, of USP Cholecalciferol RS or USP Ergocalciferol RS in the Standard preparation; D is the dilution factor, in mL, for the Assay preparation; and rU and rS are the peak heights for cholecalciferol or ergocalciferol obtained from the Assay preparation and the Standard preparation, respectively.
Assay for cholecalciferol or ergocalciferol (vitamin D), Method 2
[noteWhere 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.]
3 N Methanolic sulfuric acid solution, Sodium ascorbatepyrogallol 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 Suitability under Chromatography
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Standard preparation
Dissolve an accurately weighed quantity of USP Cholecalciferol RS or USP Ergocalciferol RS in 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 1 µg per mL.
System suitability preparation
Heat a volume of the Standard preparation at 60
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Chromatographic system (see Chromatography
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Procedure
Separately inject equal volumes (about 40 µL) of the Standard preparation and the Assay preparation into 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:
26.5C(rU / rS)
in which C is the concentration, in µg per mL, of USP Cholecalciferol RS or USP Ergocalciferol RS in the Standard preparation; and rU and rS are the peak responses of cholecalciferol or ergocalciferol in the Assay preparation and the Standard preparation, respectively.
Assay for cholecalciferol or ergocalciferol (vitamin D), Method 3
[noteWhere 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.]
Dilute acetic acid
Transfer 10 mL of glacial acetic acid to a 100-mL volumetric flask, dilute with water to volume, and mix.
Phenolphthalein solution
Prepare a solution containing 1 g of phenolphthalein in 100 mL of alcohol.
Potassium hydroxide solution
Slowly dissolve 14 g of potassium hydroxide in a mixture of 31 mL of dehydrated alcohol and 5 mL of 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 Suitability under Chromatography
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Standard stock solution
Dissolve an accurately weighed quantity of USP Cholecalciferol RS or USP Ergocalciferol RS in dehydrated alcohol to obtain a solution having a known concentration of about 0.2 mg per mL. Prepare fresh every 4 weeks. Store in a freezer.
Standard preparation
[noteCondition the solid-phase extraction column specified for use in the Standard preparation and the Assay preparation by initially washing the column with 4.0 mL of a mixture of methylene chloride and isopropyl alcohol (80:20), followed by 5.0 mL of Extraction solvent. Do not allow the column to dry.] Dilute an accurately measured volume of Standard stock solution with dehydrated alcohol to obtain a solution having a known concentration of about 5 µg of USP Cholecalciferol RS or USP Ergocalciferol RS per mL. Prepare this solution fresh daily. Transfer 2.0 mL of this solution to a stoppered 125-mL flask. Add 15.0 mL of water and 15.0 mL of Potassium hydroxide solution, insert the stopper, and shake for 30 minutes in a water bath maintained at 60
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Assay preparation
Weigh and finely powder not fewer than 20 Tablets. Transfer an accurately weighed portion of the powder, equivalent to about 10 µg of cholecalciferol or ergocalciferol, to the stoppered 125-mL flask, and proceed as directed for the Standard preparation, beginning with Add 15.0 mL of water and 15.0 mL of Potassium hydroxide solution.
Chromatographic system (see Chromatography
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Procedure
Separately inject equal volumes (about 15 µL) of the Standard preparation and the Assay preparation into 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:
1.09(2C)(rU / rS)
in which 1.09 is a correction factor to account for the average amount of previtamin D present in the formulation; C is the concentration, in µg per mL, of USP Cholecalciferol RS or USP Ergocalciferol RS in the Standard preparation; and rU and rS are the peak heights for cholecalciferol or ergocalciferol obtained from the Assay preparation and the Standard preparation, respectively.
Assay for vitamin E, Method 1
[noteWhere vitamin E (alpha tocopherol, alpha tocopheryl acetate, or alpha tocopheryl acid succinate) 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.]
Mobile phase
Dilute 10 mL of phosphoric acid with water to 1000 mL to obtain Solution A. Prepare a filtered and degassed mixture of methanol and Solution A (95:5). Make adjustments if necessary (see System Suitability under Chromatography
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Standard preparation
Dissolve an accurately weighed quantity of USP Alpha Tocopherol RS, USP Alpha Tocopheryl Acetate RS, or USP Alpha Tocopheryl Acid Succinate RS in methanol, and dilute quantitatively with methanol to obtain a solution having a known concentration of about 2 mg per mL.
System suitability preparation
Dissolve an accurately weighed quantity of USP Ergocalciferol RS in methanol, and dilute quantitatively, and stepwise if necessary, with methanol to obtain a solution having a concentration of 0.65 mg per mL. Transfer 1.0 mL of this solution to a 100-mL volumetric flask containing about 100 mg of USP Alpha Tocopheryl Acetate RS, accurately weighed. Dissolve in 30 mL of 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 20 mL, accurately measured, of the solution retained as specified in the directions for Assay preparation in the Assay for vitamin A, Method 1 to 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 2 mg of alpha tocopherol, alpha tocopheryl acetate, or alpha tocopheryl acid succinate per mL.
Chromatographic system (see Chromatography
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Procedure
Separately inject equal volumes (about 100 µL) of the Standard preparation and the Assay preparation into 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:
CD(rU / rS)
in which C is the concentration, in mg per mL, of the corrresponding USP Reference Standard in the Standard preparation; D is the dilution factor, in mL, for the Assay preparation; and rU and rS are the peak responses for the relevant vitamin E form obtained from the Assay preparation and 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 0.91 or 0.81, respectively.
Assay for vitamin E, Method 2
[noteWhere vitamin E (alpha tocopherol, alpha tocopheryl acetate, or alpha tocopheryl acid succinate) 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.]
Mobile phase
Transfer 240 mL of methanol to a 1000-mL volumetric flask, add 10 mL of water followed by 0.5 mL of 50% phosphoric acid, dilute with acetonitrile to volume, mix, filter, and degas. Make adjustments if necessary (see System Suitability under Chromatography
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System suitability preparation
Dissolve accurately weighed quantities of USP Alpha Tocopherol RS, USP Alpha Tocopheryl Acetate RS, and USP Alpha Tocopheryl Acid Succinate RS in methanol, and dilute quantitatively with methanol to obtain a solution having known concentrations of about 2 mg 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 RS in methanol, and dilute quantitatively with methanol to obtain a solution having a known concentration of about 2 mg per mL.
Assay preparation
Proceed as directed for Assay preparation in 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 preparation is equivalent to that of the Standard preparation. Evaporate nearly to dryness, add several mL of methanol, and evaporate the remaining 2,2,4-trimethylpentane. Dilute with methanol to volume, and mix.
Chromatographic system (see Chromatography
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Procedure
Separately inject equal volumes (about 25 µL) of the Standard preparation and the Assay preparation into 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:
26.5CD(rU / rS)
in which C is the concentration, in mg per mL, of the corresponding USP Reference Standard in the Standard preparation; D is the dilution factor used in exchanging the solvent from 2,2,4-trimethylpentane to methanol; and rU and rS are the peak areas of the relevant vitamin E form obtained from the Assay preparation and the Standard preparation, respectively. [noteThe initial extraction to 26.5 mL of 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 0.91 or 0.81, respectively.
Assay for vitamin E, Method 3
[noteWhere vitamin E (alpha tocopherol, alpha tocopheryl acetate, or alpha tocopheryl acid succinate) 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
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 Suitability under Chromatography
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Standard preparation
Dissolve an accurately weighed quantity of USP Alpha Tocopherol RS, USP Alpha Tocopheryl Acetate RS, or USP Alpha Tocopheryl Acid Succinate RS in methanol, and quantitatively dilute with methanol to obtain a solution having a known concentration of about 0.3 mg per mL.
Assay preparation
Weigh and finely powder not fewer than 20 Tablets. Transfer an accurately weighed portion of the powder, equivalent to about 8 mg of alpha tocopherol, to a 125-mL flask fitted with a ground-glass joint. Add 25.0 mL of water, 25.0 mL of dehydrated alcohol, and 3.5 g of potassium hydroxide pellets. Shake for 1 hour in a water bath maintained at 55
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Chromatographic system (see Chromatography
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Procedure
Separately inject equal volumes (about 20 µL) of the Standard preparation and the Assay preparation into 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:
25C(rU / rS)
in which C is the concentration, in mg per mL, of the corresponding USP Reference Standard in the Standard preparation; and rU and rS are the peak areas for the relevant vitamin E form obtained from the Assay preparation and 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 0.91 or 0.81, respectively.
Assay for phytonadione, Method 1
[noteUse low-actinic glassware throughout this procedure.]
Mobile phase
Prepare a filtered and degassed mixture of methanol and water (95:5). Make adjustments if necessary (see System Suitability under Chromatography
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Standard stock solution
Dissolve an accurately weighed quantity of USP Phytonadione RS in methanol, with the aid of sonication if necessary, and quantitatively dilute with methanol to obtain a solution having a known concentration of about 200 µg per mL.
Standard preparation
Pipet 10 mL of Standard stock solution into a 100-mL volumetric flask, dilute with methanol to volume, and mix.
System suitability preparation
Transfer 65 mg of USP Alpha Tocopheryl Acetate RS to a 100-mL volumetric flask, and dissolve in about 75 mL of methanol. Add 10 mL of Standard stock solution, dilute with methanol to volume, and mix.
Assay preparation
Transfer not less than 20 mL, accurately measured, of the solution retained as specified in the directions for Assay preparation in the Assay for vitamin A, Method 1 to 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 20 µg of phytonadione per mL.
Chromatographic system (see Chromatography
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Procedure
Separately inject equal volumes (about 100 µL) of the Standard preparation and the Assay preparation into 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:
C(L/D)(rU / rS)
in which C is the concentration, in µg per mL, of USP Phytonadione RS in the Standard preparation; L is the labeled amount, in µg, of phytonadione in each Tablet; D is 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 rU and rS are the peak areas for phytonadione obtained from the Assay preparation and the Standard preparation, respectively.
Assay for phytonadione, Method 2
[noteUse low-actinic glassware throughout this procedure.]
Solvent
Prepare a mixture of methanol and isopropanol (95:5).
Mobile phase
Prepare a filtered and degassed mixture of 800 mL of methanol, 200 mL of methylene chloride, 0.1 mL of glacial acetic acid, 1.36 g of zinc chloride, and 0.41 g of sodium acetate.
Internal standard solution
Prepare a solution of menaquinone 4 (vitamin K2) in Solvent having a concentration of about 5 µg per mL. [noteA concentrated stock solution of menaquinone 4 (100 µg per mL) can be stored for 2 months in a refrigerator.]
Standard stock solution
Dissolve an accurately weighed quantity of USP Phytonadione RS in methylene chloride with the aid of sonication. Dilute with Solvent quantitatively, and stepwise if necessary, to obtain a solution having a known concentration of about 5 µg per mL.
Standard preparation
Pipet 1.0 mL of Standard stock solution and 1.0 mL of Internal standard solution into a 5.0-mL volumetric flask, dilute with Solvent to volume, and mix. Filter through a membrane having a 0.45-µm or finer porosity.
Assay preparation
Weigh and finely powder not fewer than 20 Tablets. To a centrifuge tube fitted with a cap, transfer an accurately weighed amount of powder, not exceeding 800 mg, and equivalent to an amount of phytonadione not exceeding 50 µg. Add 4 mL of water. Insert the stopper, and mix using a vortex mixer until the sample is dispersed. Place the tube in a water bath at 60
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Chromatographic system (see Chromatography
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Procedure
Separately inject equal volumes (about 25 µL) of the Standard preparation and the Assay preparation into 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:
5CD(RU / RS)
in which C is the concentration, in µg per mL, of USP Phytonadione RS in the Standard preparation; D is the volume, in mL, of Internal standard solution used to prepare the Assay preparation; and RU and RS are the peak response ratios of phytonadione to that of the internal standard obtained from the Assay preparation and the Standard preparation, respectively.
Assay for beta carotene
[noteUse low-actinic glassware throughout this procedure.]
Potassium hydroxide solution
Dissolve 58.8 g of potassium hydroxide in 50 mL of water.
Iodine solution
Transfer about 10 mg of iodine to a 100-mL volumetric flask. Dissolve in cyclohexane, dilute with cyclohexane to volume, and mix. Dilute 10 mL of this solution with cyclohexane to 100 mL, and mix. [notePrepare this solution fresh daily.]
Assay preparation
Weigh accurately not fewer than 20 Tablets. Grind the Tablets to a fine powder, and transfer an accurately weighed quantity of the powder, equivalent to about 2 mg of beta carotene, to a 500-mL saponification flask. Add 100 mL of alcohol, 6 mL of Potassium hydroxide solution, and a magnetic stirring bar. Attach an air condenser to the flask, and heat under reflux for 45 minutes with constant stirring. Cool to room temperature, add 170 mL of solvent hexane, and stir for 30 minutes. Quantitatively transfer the contents of the flask to a 500-mL separatory funnel with portions of solvent hexane. Allow the layers to separate for 5 to 10 minutes, and transfer the upper organic layer to a 500-mL volumetric flask. Transfer the lower aqueous layer into the saponification flask, add 170 mL of solvent hexane, and stir for an additional 20 minutes. 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 10 minutes. 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 3 g of anhydrous sodium sulfate, shake, and allow to settle. Quantitatively transfer a volume of this solution, equivalent to about 100 µg of beta carotene, to a 50-mL volumetric flask. Evaporate under a stream of nitrogen to dryness, and immediately add cyclohexane. Add 2 mL of Iodine solution, and heat for 15 minutes in a water bath maintained at 65
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Procedure
Determine the absorbance of the Assay preparation at the wavelength of maximum absorbance at about 452 nm, using cyclohexane as the blank. Calculate the quantity, in mg, of beta carotene (C40H56) in the Tablets taken by the formula:
(L/D)(AU /223)
in which L is the labeled amount, in mg, of beta carotene in each Tablet; D is 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; AU is the absorbance of the Assay preparation; and 223 is the absorptivity of beta carotene at 452 nm.
Assay for ascorbic acid, Method 1
Weigh and powder not fewer than 20 Tablets. Transfer an accurately weighed portion of the powder, equivalent to about 100 mg of ascorbic acid, to a 200-mL volumetric flask, and add about 75 mL of metaphosphoricacetic acids TS. Insert a stopper into the flask, and shake by mechanical means for about 30 minutes. Dilute with water to volume, and mix. Transfer a portion of the solution to a centrifuge tube, and centrifuge until a clear supernatant is obtained. Pipet 4.0 mL of this solution into a 50-mL conical flask, add 5 mL of metaphosphoricacetic acids TS, and titrate with standard dichlorophenolindophenol solution VS to a rose-pink color that persists for at least 5 seconds. Correct for the volume of dichlorophenolindophenol solution consumed by a mixture of 5.5 mL of metaphosphoricacetic acids TS and 15 mL of water. From the ascorbic acid equivalent of the standard dichlorophenolindophenol solution, calculate the content of ascorbic acid in each Tablet.
Assay for ascorbic acid, Method 2
Proceed as directed in the Assay for Ascorbic Acid under Automated Methods of Analysis
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Assay for calcium ascorbate, Method 1
Proceed as directed in the Assay for ascorbic acid, Method 1.
Assay for calcium ascorbate, Method 2
Proceed as directed in the Assay for Ascorbic Acid under Automated Methods of Analysis
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Assay for sodium ascorbate, Method 1
Proceed as directed in the Assay for ascorbic acid, Method 1.
Assay for sodium ascorbate, Method 2
Proceed as directed in the Assay for Ascorbic Acid under Automated Methods of Analysis
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Assay for biotin, Method 1
[noteUse low-actinic glassware throughout this procedure.]
Mobile phase
Transfer 85 mL of acetonitrile, 1 g of sodium perchlorate, and 1 mL of phosphoric acid to a 1000-mL volumetric flask, dilute with water to volume, mix, filter, and degas. Make adjustments if necessary (see System Suitability under Chromatography
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Standard preparation
Transfer about 67 mg of USP Biotin RS, accurately weighed, to a 200-mL volumetric flask, dissolve in and dilute with dimethyl sulfoxide to volume, and mix. Transfer 3.0 mL of this solution to a 200-mL volumetric flask, dilute with water to volume, and mix to obtain a solution having a known concentration of about 5 µg of USP Biotin RS per mL.
Assay preparation
Weigh and finely powder not fewer than 20 Tablets. Transfer an accurately weighed portion of the powder, equivalent to about 1 mg of biotin, to a 200-mL volumetric flask. Add 3 mL of dimethyl sulfoxide, and swirl to wet the contents. Place the flask in a water bath at 60
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Chromatographic system (see Chromatography
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Procedure
Separately inject equal volumes (about 100 µL) of the Standard preparation and the Assay preparation into 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:
200C(rU / rS)
in which C is the concentration, in µg per mL, of USP Biotin RS in the Standard preparation; and rU and rS are the biotin peak responses obtained from the Assay preparation and the Standard preparation, respectively.
Assay for biotin, Method 2
[noteUse low-actinic glassware throughout this procedure.] Dehydrated mixtures yielding formulations similar to the media described herein may be used provided that, when constituted as directed, they have growth-promoting properties equal to or superior to those obtained with the media prepared as described herein.
Standard stock solution
Dissolve an accurately weighed quantity of USP Biotin RS in 50% alcohol, dilute with 50% alcohol to obtain a solution having a known concentration of about 50 µg per mL, and mix. Store this solution in a refrigerator.
Standard preparation
On the day of the assay, dilute a volume of Standard stock solution with water to obtain a solution having a known concentration of about 0.1 ng per mL.
Assay preparation
Weigh and finely powder not fewer than 30 Tablets. Transfer an accurately weighed quantity of the powder, equivalent to about 100 µg of biotin, to a 200-mL volumetric flask, add 3 mL of 50% alcohol, and swirl to wet the contents. Heat the flask in a water bath at 60
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Acid-hydrolyzed casein solution
Mix 100 g of vitamin-free casein with 500 mL of 6 N hydrochloric acid, and reflux the mixture for 8 to 12 hours. 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 1 N sodium hydroxide to a pH of 3.5 ± 0.1, and add water to make 1000 mL. Add 20 g of activated charcoal, stir for 1 hour, and filter. Repeat the treatment with activated charcoal. Store under toluene in a refrigerator at a temperature not below 10
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Cystinetryptophan solution
Suspend 4.0 g of l-cystine in 1.0 g of l-tryptophan (or 2.0 g of d,l-tryptophan) in 700 to 800 mL of water, heat to 70
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Adenineguanineuracil solution
Dissolve 200 mg each of adenine sulfate, guanine hydrochloride, and uracil, with the aid of heat, in 10 mL of 4 N hydrochloric acid, cool, and add water to make 200 mL. Store under toluene in a refrigerator.
Polysorbate 80 solution
Dissolve 25 g of polysorbate 80 in alcohol to make 250 mL.
Calcium pantothenate solution
Prepare a solution of calcium pantothenate in 50% alcohol containing 10 µg per mL. Store in a refrigerator.
Riboflavinthiamine hydrochloride solution
Prepare a solution of riboflavin and thiamine hydrochloride in 0.02 N acetic acid containing 20 µg of riboflavin and 10 µg of thiamine hydrochloride per mL. Store under toluene, protected from light, in a refrigerator.
p-Aminobenzoic acidniacinpyridoxine hydrochloride solution
Prepare a solution in a mixture of water and neutralized alcohol (3:1) containing 10 µg of p-aminobenzoic acid, 50 µg of niacin, and 40 µg of pyridoxine hydrochloride per mL. Store in a refrigerator.
Salt solution 1
Dissolve 25 g of monobasic potassium phosphate and 25 g of dibasic potassium phosphate in water to make 500 mL. Add 5 drops of hydrochloric acid, and mix. Store under toluene.
Salt solution 2
Dissolve 10 g of magnesium sulfate, 0.5 g of sodium chloride, 0.5 g of ferrous sulfate, and 0.5 g of manganese sulfate in water to make 500 mL. Add 5 drops of hydrochloric acid, and mix. Store under toluene.
Basal medium stock solution
Dissolve the anhydrous dextrose and anhydrous sodium acetate in the solutions previously mixed, and adjust with 1 N sodium hydroxide to a pH of 6.8. Dilute with water to 250 mL, and mix.
Stock culture of Lactobacillus plantarum
Dissolve 2.0 g of yeast extract in 100 mL of water, add 500 mg of anhydrous dextrose, 500 mg of anhydrous sodium acetate, and 1.5 g of agar, and heat the mixture on a steam bath, with stirring, until the agar dissolves. Add 10-mL portions of the hot solution to test tubes, close or cover the tubes, sterilize in an autoclave at 121
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Culture medium
To each of a series of test tubes containing 5.0 mL of Basal medium stock solution, add 5.0 mL of water containing 0.5 ng of biotin. Plug the tubes with cotton, sterilize in an autoclave at 121
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Inoculum
[noteA frozen suspension of Lactobacillus plantarum may 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 plantarum to a sterile tube containing 10 mL of culture medium. Incubate this culture for 16 to 24 hours at a temperature between 30
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Procedure
To similar separate test tubes add, in duplicate, 1.0 and/or 1.5, 2.0, 3.0, 4.0, and 5.0 mL of the Standard preparation. To each tube and to four similar empty tubes, add 5.0 mL of Basal medium stock solution and sufficient water to make 10 mL.
To similar test tubes add, in duplicate, volumes of the Assay preparation corresponding to three or more of the levels specified for the Standard preparation, including the levels of 2.0, 3.0, and 4.0 mL. To each tube add 5.0 mL of the Basal medium stock solution and sufficient water to make 10 mL. Place one complete set of Standard and Assay tubes together in one tube rack and the duplicate set in a second rack or section of a rack, preferably in random order.
Cover the tubes of both series to prevent contamination, and sterilize in an autoclave at 121
![]() ![]() ![]() ![]() Determine the transmittance of the tubes in the following manner. Mix the contents of each tube, and transfer to a spectrophotometer cell. Place the cell in a spectrophotometer that has been set at a specific wavelength between 540 and 660 nm, and read the transmittance when a steady state is reached. This steady state is observed a few seconds after agitation when the galvanometer reading remains constant for 30 seconds or more. Allow approximately the same time interval for the reading on each tube.
With the transmittance set at 1.00 for the uninoculated blank, read the transmittance of the inoculated blank. With the transmittance set at 1.00 for the inoculated blank, read the transmittance for each of the remaining tubes. If there is evidence of contamination with a foreign microorganism, disregard the result of the assay.
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
![]() Calculate the response, y, adding together the two transmittances for each level of the Assay preparation. Read from the standard curve the logarithm of the volume of the Standard preparation corresponding to each of those values of y that fall within the range of lowest and highest points plotted for the standard. Subtract from each logarithm so obtained the logarithm of the volume, in mL, of the Assay preparation to obtain the difference, x, for each dosage level. Average the values of x for each of three or more dosage levels to obtain x, which equals the log-relative potency, M ¢, of the Assay preparation. Determine the quantity, in µg, of USP Biotin RS corresponding to the biotin in the portion of Tablets taken by the equation:
antilog M = antilog (M ¢ + log R)
in which R is the weight, in µg, of biotin that was assumed to be present in each mg in the portion of the Tablets taken.
Replication
Repeat the entire determination at least once, using separately prepared Assay preparations. If the difference between the two log-potencies M is not greater than 0.08, their mean, M, is the assayed log-potency of the test material (see The Confidence Interval and Limits of Potency under Design and Analysis of Biological Assays
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Assay for biotin, Method 3
[noteUse low-actinic glassware throughout this procedure.]
Buffer
Transfer 800 mL of water and 100 mL of triethylamine to a 1000-mL volumetric flask. Add 80 mL of 85% phosphoric acid, dilute with water to volume, and mix.
Mobile phase
Transfer 80 mL of acetonitrile and 10 mL of Buffer to a 1000-mL volumetric flask. Dilute with water to volume, mix, filter, and degas. Make adjustments if necessary (see System Suitability under Chromatography
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Standard preparation
Transfer about 25 mg of USP Biotin RS, accurately weighed, to a 250-mL volumetric flask, dissolve in and dilute with water to volume, and mix. Transfer 1.5 mL of this solution to a 250-mL volumetric flask, dilute with water to volume, and mix to obtain a solution having a known concentration of about 0.6 µg per mL of USP Biotin RS. [noteA portion of the Standard preparation will be used to determine the percent recovery of biotin from the Solid-phase extraction procedure.]
Assay preparation
Weigh and finely powder not fewer than 20 Tablets. Transfer an accurately weighed portion of the powder, equivalent to about 60 µg of biotin, to a 100-mL volumetric flask. Add about 80 mL of water, and sonicate for 30 to 40 minutes with occasional mixing. Cool to room temperature, dilute with water to volume, mix, and filter. Adjust the pH of the solution with either dilute acetic acid or 0.1 N sodium hydroxide to between 6.0 and 7.0.
Solid-phase extraction
[noteCondition the extraction column specified in this procedure in the following manner. Wash the column with a 2-mL portion of methanol. Equilibrate with a 2-mL portion of water.] Separately pipet 5.0 mL of the Assay preparation and Standard preparation into freshly conditioned solid-phase extraction columns consisting of a mixed-mode packing with a sorbent-mass of 60 mg. [noteThe 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.3 ] Wash the column with 10 mL of 30% (v/v) methanol in water. Apply an appropriate volume (about 4.9 mL) of 30% (v/v) methanol in 0.1 N hydrochloric acid to the column. Collect the eluate in a 5-mL volumetric flask, containing 100 µL of 40% (w/v) sodium acetate in water, and dilute with 30% (v/v) methanol in 0.1 N hydrochloric acid to volume.
Chromatographic system (see Chromatography
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Procedure
Separately inject equal volumes (about 100 µL) of the Standard preparation and the Assay preparation that 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:
100C(rU / rS)
in which C is the concentration, in µg per mL, of USP Biotin RS in the Standard preparation; and rU and rS are the biotin peak responses obtained from the Assay preparation and the Standard preparation, respectively.
Assay for cyanocobalamin, Method 1
[noteUse low-actinic glassware throughout this procedure.]
Mobile phase
Prepare a filtered and degassed mixture of water and methanol (65:35). Make adjustments if necessary (see System Suitability under Chromatography
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Standard preparation
Dissolve an accurately weighed quantity of USP Cyanocobalamin RS in water to obtain a stock solution having a known concentration of about 10 µg per mL. [noteStore this stock solution in a dark place, and discard after 1 week.] Dilute a portion of this stock solution quantitatively with water to obtain a solution having a known concentration of about 1 µg per mL.
Assay preparation
Weigh and finely powder not fewer than 30 Tablets. Transfer an accurately weighed portion of the powder, equivalent to about 100 µg of cyanocobalamin, to a 250-mL flask. Quantitatively add 100.0 mL of water, and carefully extract for 2 minutes. Filter about 10 mL of the extract, and use the filtrate.
Chromatographic system (see Chromatography
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Procedure
Separately inject equal volumes (about 200 µL) of the Standard preparation and the Assay preparation into 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:
100C(rU / rS)
in which C is the concentration, in µg per mL, of USP Cyanocobalamin RS in the Standard preparation; and rU and rS are the peak responses for cyanocobalamin obtained from the Assay preparation and the Standard preparation, respectively.
Assay for cyanocobalamin, Method 2
[noteUse low-actinic glassware throughout this procedure.]
Standard cyanocobalamin stock solution
Dissolve an accurately weighed quantity of USP Cyanocobalamin RS in 25% alcohol to obtain a solution having a known concentration of about 1.0 µg of USP Cyanocobalamin RS per mL. Store in a refrigerator.
Standard preparation
Dilute a suitable volume of Standard cyanocobalamin stock solution with 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-mL level of the Standard preparation is not less than that which corresponds to a difference of 1.25 mg in dried cell weight. This concentration usually falls between 0.01 and 0.04 ng per mL of Standard preparation. Prepare this solution fresh for each assay.
Assay preparation
Weigh and finely powder not fewer than 20 Tablets. Transfer an accurately weighed portion of the powder, equivalent to about 1.0 µg of cyanocobalamin, to an appropriate vessel containing, for each g of powdered Tablets taken, 25 mL of an aqueous extracting solution prepared just prior to use to contain, in each 100 mL, 1.29 g of dibasic sodium phosphate, 1.1 g of anhydrous citric acid, and 1.0 g of sodium metabisulfite. Autoclave the mixture at 121
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Acid-hydrolyzed casein solution
Prepare as directed in the Assay for calcium pantothenate, Method 2.
Asparagine solution
Dissolve 2.0 of l-asparagine in water to make 200 mL. Store under toluene in a refrigerator.
Adenineguanineuracil solution
Prepare as directed in the Assay for calcium pantothenate, Method 2.
Xanthine solution
Suspend 0.20 g of xanthine in 30 to 40 mL of water, heat to about 70
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Salt solution 1
Dissolve 10 g of monobasic potassium phosphate and 10 g of dibasic potassium phosphate in water to make 200 mL, and add 2 drops of hydrochloric acid. Store this solution under toluene.
Salt solution 2
Dissolve 4.0 g of magnesium sulfate, 0.20 g of sodium chloride, 0.20 g of ferrous sulfate, and 0.20 g of manganese sulfate in water to make 200 mL, and add 2 drops of hydrochloric acid. Store this solution under toluene.
Polysorbate 80 solution
Dissolve 20 g of polysorbate 80 in alcohol to make 200 mL. Store in a refrigerator.
Vitamin solution 1
Dissolve 10 mg of riboflavin, 10 mg of thiamine hydrochloride, 100 µg of biotin, and 20 mg of niacin in 0.02 N glacial acetic acid to make 400 mL. Store under toluene, protected from light, in a refrigerator.
Vitamin solution 2
Dissolve 20 mg of p-aminobenzoic acid, 10 mg of calcium pantothenate, 40 mg of pyridoxine hydrochloride, 40 mg of pyridoxal hydrochloride, 8 mg of pyridoxamine dihydrochloride, and 2 mg of folic acid in a mixture of water and neutralized alcohol (3:1) to make 400 mL. Store, protected from light, in a refrigerator.
Basal medium stock solution
Prepare the medium according to the following formula and directions. A dehydrated mixture containing the same ingredients may be used provided that, when constituted as directed in the labeling, it yields a medium comparable to that obtained from the formula given herein.
Add the ingredients in the order listed, carefully dissolving cystine and tryptophan in the hydrochloric acid before adding the next eight solutions in the resulting solution. Add 100 mL of water, mix, and dissolve dextrose, sodium acetate, and ascorbic acid. Filter, if necessary, add the Polysorbate 80 solution, adjust with 1 N sodium hydroxide to a pH of between 5.5 and 6.0, and add Purified Water to make 250 mL.
Tomato juice preparation
Centrifuge commercially canned tomato juice so that most of the pulp is removed. Suspend about 5 g per L 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
[noteA dehydrated 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 0.75 g of yeast extract, 0.75 g of dried peptone, 1.0 g of anhydrous dextrose, and 0.20 g of monobasic potassium phosphate in 60 to 70 mL of water. Add 10 mL of Tomato juice preparation and 1 mL of Polysorbate 80 solution. Adjust with 1 N sodium hydroxide to a pH of 6.8, and add water to make 100 mL. Place 10-mL portions of the solution in test tubes, and plug with cotton. Sterilize the tubes and contents in an autoclave at 121
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Suspension medium
Dilute a measured volume of Basal medium stock solution with an equal volume of water. Place 10-mL portions of the diluted medium in test tubes. Sterilize, and cool as directed for Culture medium.
Stock culture of Lactobacillus leichmannii
To 100 mL of Culture medium, add 1.0 to 1.5 g of agar, and heat the mixture on a steam bath, with stirring, until the agar dissolves. Place 10-mL portions of the hot solution in test tubes, cover the tubes, sterilize at 121
![]() ![]() ![]() ![]() Prepare fresh stab cultures at least three times each week, and do not use them for preparing the Inoculum if more than 4 days old. The activity of the microorganism can be increased by daily or twice-daily transfer of the stab culture, to the point where definite turbidity in the liquid Inoculum can be observed 2 to 4 hours after inoculation. A slow-growing culture seldom gives a suitable response curve and may lead to erratic results.
Inoculum
[noteA frozen suspension of Lactobacillus leichmannii may 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 leichmannii to two sterile tubes containing 10 mL of the Culture medium each. Incubate these cultures for 16 to 24 hours at a temperature between 30
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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 530 nm.
Procedure
Because of the high sensitivity of the test organism to minute amounts of vitamin B12 activity and to traces of many cleansing agents, cleanse meticulously by suitable means, followed preferably by heating at 250
![]() To separate test tubes add, in duplicate, 1.0, 1.5, 2.0, 3.0, 4.0, and 5.0 mL of the Standard preparation. To each of these tubes and to four similar empty tubes, add 5.0 mL of Basal medium stock solution and sufficient water to make 10 mL.
To similar separate test tubes add, in duplicate, 1.0, 1.5, 2.0, 3.0, and 4.0 mL of the Assay preparation. To each tube add 5.0 mL of Basal medium stock solution and sufficient water to make 10 mL. Place one complete set of standard and assay tubes together in one tube rack and the duplicate set in a second rack or section of a rack, preferably in random order.
Cover the tubes to prevent bacterial contamination, and sterilize in an autoclave at 121
![]() Aseptically add 0.5 mL of Inoculum to each tube so prepared, except two of the four containing no Standard preparation (the uninoculated blanks). Incubate the tubes at a temperature between 30
![]() ![]() ![]() Terminate growth by heating to a temperature not lower than 80
![]() With the transmittance set at 100% for the uninoculated blank, read the transmittance of the inoculated blank. If the difference is greater than 5% or if there is evidence of contamination with a foreign microorganism, disregard the results of the assay.
With the transmittance set at 100% for the uninoculated blank, read the transmittance of each of the remaining tubes. Disregard the results of the assay if the slope of the standard curve indicates a problem with sensitivity.
Calculation
Prepare a standard concentration-response curve by the following procedure. Test for and replace any aberrant individual transmittances. For each level of the Standard, calculate the response from the sum of the duplicate values of the transmittances (S) as the difference, y = 2.00
![]() Calculate the response, y, adding together the two transmittances for each level of the Assay preparation. Read from the standard curve the logarithm of the volume of the Standard preparation corresponding to each of those values of y that falls within the range of the lowest and highest points plotted for the standard. Subtract from each logarithm so obtained the logarithm of the volume, in mL, of the Assay preparation to obtain the difference, x, for each dosage level. Average the values of x for each of three or more dosage levels to obtain x, which equals the log-relative potency, M ¢, of the Assay preparation. Determine the quantity, in µg, of USP Cyanocobalamin RS corresponding to the cyanocobalamin in the portion of Tablets taken by the equation:
antilog M = antilog (M ¢ + log R)
in which R is the number of µg of cyanocobalamin that was assumed to be present in each mg in the portion of Tablets taken.
Replication
Repeat the entire determination at least once, using separately prepared Assay preparations. If the difference between the two log-potencies M is not greater than 0.08, their mean, M, is the assayed log-potency of the test material (see Vitamin B12 Activity under Design and Analysis of Biological Assays
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Assay for folic acid, Method 1
[noteUse low-actinic glassware throughout this procedure.]
Reagent 1
Use a 25% solution of tetrabutylammonium hydroxide in methanol.
Reagent 2
Transfer 5.0 g of pentetic acid to a 50-mL volumetric flask. Using sonication if necessary, dissolve in and dilute with 1 N sodium hydroxide to volume, and mix.
Mobile phase
Transfer 2 g of monobasic potassium phosphate to a 1-L volumetric flask, and dissolve in 650 mL of water. Add 12.0 mL of Reagent 1, 7.0 mL of 3 N phosphoric acid, and 240 mL of methanol. Cool to room temperature, adjust with phosphoric acid or ammonia TS to a pH of 7.0, dilute with water to volume, mix, and filter. Recheck the pH before use. Make adjustments if necessary (see System Suitability under Chromatography
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Internal standard preparation
Transfer about 40 mg of methylparaben, accurately weighed, to a 1000-mL volumetric flask, and add 220 mL of methanol to dissolve. Dissolve 2.0 g of monobasic potassium phosphate in about 300 mL of water in a separate beaker, quantitatively transfer this solution to the flask containing the methylparaben solution, and add an additional 300 mL of water. Add 19 mL of Reagent 1, 7 mL of 3 N phosphoric acid, and 30 mL of Reagent 2. Adjust with ammonia TS to a pH of 9.8, bubble nitrogen through the solution for 30 minutes, dilute with water to volume, and mix.
Standard preparation
Dissolve an accurately weighed quantity of USP Folic Acid RS in Internal standard preparation to obtain a solution having a known concentration of about 0.2 mg per mL. Transfer 2.0 mL of this solution to a 25-mL volumetric flask, dilute with Internal standard preparation to volume, and mix.
Assay preparation
Weigh and finely powder not fewer than 30 Tablets. Transfer an accurately weighed portion of the powder, equivalent to about 0.4 mg of folic acid, to a 50-mL amber-colored centrifuge tube. Add 25.0 mL of Internal standard preparation, insert a stopper, shake by mechanical means for 10 minutes, and centrifuge. Filter a portion of the clear supernatant, and use the filtrate.
Chromatographic system (see Chromatography
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Procedure
Separately inject equal volumes (about 15 µL) of the Standard preparation and the Assay preparation into 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:
25C(RU / RS)
in which C is the concentration, in mg per mL, of USP Folic Acid RS in the Standard preparation; and RU and RS are the peak area ratios of folic acid to methylparaben obtained from the Assay preparation and the Standard preparation, respectively.
Assay for folic acid, Method 2
[noteUse low-actinic glassware throughout this procedure.]
Reagent
Dissolve 7.5 g of edetate disodium, with stirring, in 500 mL of water containing 10 mL of ammonium hydroxide.
Diluting solution
Prepare a solution of ammonium hydroxide containing 60 µg per mL.
Mobile phase
Transfer 0.4 mL of triethylamine, 15.0 mL of glacial acetic acid, and 350 mL of methanol to a 2000-mL volumetric flask, dilute with 0.008 M sodium 1-hexanesulfonate to volume, and mix. Filter, and degas. Make adjustments if necessary (see System Suitability under Chromatography
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Standard stock solution
Dissolve an accurately weighed quantity of USP Folic Acid RS in Diluting solution to obtain a solution having a known concentration of about 60 µg per mL. Prepare this solution fresh daily.
Standard preparation
Transfer 5.0 mL of Standard stock solution to a stoppered 125-mL flask, and add 10.0 mL of methanol and 35.0 mL of Reagent. Insert the stopper, shake for 15 minutes in a water bath maintained at 60
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Assay preparation
Weigh and finely powder a counted number of Tablets. Transfer an accurately weighed portion of the powder, equivalent to about 0.3 mg of folic acid, to a stoppered 125-mL flask. Add 10.0 mL of methanol and 35.0 mL of Reagent, insert the stopper, shake for 15 minutes in a water bath maintained at 60
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Chromatographic system (see Chromatography
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Procedure
Separately inject equal volumes (about 5 µL) of the Assay preparation and the Standard preparation into 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:
45C(rU / rS)
in which C is the concentration, in mg per mL, of USP Folic Acid RS in the Standard preparation; and rU and rS are the peak areas of folic acid obtained from the Assay preparation and the Standard preparation, respectively.
Assay for calcium pantothenate, Method 1
Mobile phase
Prepare a filtered and degassed mixture of water and phosphoric acid (1000:1). Make adjustments if necessary (see System Suitability under Chromatography
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Internal standard preparation
Transfer about 80 mg of p-hydroxybenzoic acid, accurately weighed, to a 1000-mL volumetric flask, and dissolve in 3 mL of alcohol. Add about 50 mL of water and 7.1 g of dibasic sodium phosphate, dilute with water to volume, and mix. Adjust with phosphoric acid to a pH of 6.7, and mix.
Standard preparation
Dissolve an accurately weighed quantity of USP Calcium Pantothenate RS in Internal standard preparation to obtain a solution having a known concentration of about 0.6 mg per mL.
Assay preparation
Weigh and finely powder not fewer than 30 Tablets. Transfer an accurately weighed portion of the powder, equivalent to about 15 mg of calcium pantothenate, to a centrifuge tube. Add 25.0 mL of the Internal standard preparation, and shake vigorously for 10 minutes. Centrifuge, filter, and use the clear filtrate.
Chromatographic system (see Chromatography
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Procedure
Separately inject equal volumes (about 10 µL) of the Standard preparation and the Assay preparation into 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:
25C(RU / RS)
in which C is the concentration, in mg per mL, of USP Calcium Pantothenate RS in the Standard preparation; and RU and RS are the peak response ratios of calcium pantothenate to p-hydroxybenzoic acid obtained from the Assay preparation and the Standard preparation, respectively.
Assay for calcium pantothenate, Method 2
Standard stock solution
Dissolve about 50 mg 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 500 mL of water in a 1000-mL volumetric flask. Add 10 mL of 0.2 N acetic acid and 100 mL of sodium acetate solution (1 in 60), and dilute with water to volume to obtain a solution containing 50 µg of USP Calcium Pantothenate RS per mL. Store under toluene in a refrigerator.
Standard preparation
On the day of the assay, dilute an accurately measured volume of Standard stock solution with water to obtain a solution having a known concentration of 0.01 to 0.04 µg of calcium pantothenate per mL, the exact concentration being such that the responses obtained as directed for Procedure, 2.0 and 4.0 mL of the Standard preparation being used, are within the linear portion of the log-concentration response curve.
Assay preparation
Weigh and powder not fewer than 30 Tablets. Transfer an accurately weighed portion of the powder, equivalent to about 50 mg of calcium pantothenate, to a 1000-mL volumetric flask containing 500 mL of water. Add 10 mL of 0.2 N acetic acid and 100 mL of sodium acetate solution (1 in 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 100 g of vitamin-free casein with 500 mL of 6 N hydrochloric acid, and reflux the mixture for 8 to 12 hours. 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 1 N sodium hydroxide to a pH of 3.5 ± 0.1, and add water to make 1000 mL. Add 20 g of activated charcoal, stir for 1 hour, and filter. Repeat the treatment with activated charcoal. Store under toluene in a refrigerator at a temperature not below 10
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Cystinetryptophan solution
Suspend 4.0 g of l-cystine and 1.0 g of l-tryptophan (or 2.0 g of d,l-tryptophan) in 700 to 800 mL of water, heat to 70
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Adenineguanineuracil solution
Dissolve 200 mg each of adenine sulfate, guanine hydrochloride, and uracil, with the aid of heat, in 10 mL of 4 N hydrochloric acid, cool, and add water to make 200 mL. Store under toluene in a refrigerator.
Polysorbate 80 solution
Dissolve 25 g of polysorbate 80 in alcohol to make 250 mL.
Riboflavinthiamine hydrochloridebiotin solution
Prepare a solution of riboflavin, thiamine hydrochloride, and biotin in 0.02 N acetic acid containing 20 µg of riboflavin, 10 µg of thiamine hydrochloride, and 0.04 µg of biotin per mL. Store under toluene, protected from light, in a refrigerator.
p-Aminobenzoic acidniacinpyridoxine hydrochloride solution
Prepare a solution in a mixture of water and neutralized alcohol (3:1) containing 10 µg of p-aminobenzoic acid, 50 µg of niacin, and 40 µg of pyridoxine hydrochloride per mL. Store in a refrigerator.
Salt solution 1
Dissolve 25 g of monobasic potassium phosphate and 25 g of dibasic potassium phosphate in water to make 500 mL. Add 5 drops of hydrochloric acid, and mix. Store under toluene.
Salt solution 2
Dissolve 10 g of magnesium sulfate, 0.5 g of sodium chloride, 0.5 g of ferrous sulfate, and 0.5 g of manganese sulfate in water to make 500 mL. Add 5 drops of hydrochloric acid, and mix. Store under toluene.
Basal medium stock solution
Dissolve the anhydrous dextrose and anhydrous sodium acetate in the solutions previously mixed, and adjust with 1 N sodium hydroxide to a pH of 6.8. Dilute with water to 250 mL, and mix.
Stock culture of Lactobacillus plantarum
Dissolve 2.0 g of yeast extract in 100 mL of water, add 500 mg of anhydrous dextrose, 500 mg of anhydrous sodium acetate, and 1.5 g of agar, and heat the mixture on a steam bath, with stirring, until the agar dissolves. Add 10-mL portions of the hot solution to the test tubes, close or cover the tubes, sterilize in an autoclave at 121
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Culture medium
To each of a series of test tubes containing 5.0 mL of Basal medium stock solution, add 5.0 mL of water containing 0.2 µg of calcium pantothenate. Plug the tubes with cotton, sterilize in an autoclave at 121
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Inoculum
[noteA frozen suspension of Lactobacillus plantarum may 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 plantarum to a sterile tube containing 10 mL of culture medium. Incubate this culture for 16 to 24 hours at a temperature between 30
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Procedure
To similar separate test tubes add, in duplicate, 1.0 and/or 1.5, 2.0, 3.0, 4.0, and 5.0 mL of the Standard preparation. To each tube and to four similar empty tubes, add 5.0 mL of Basal medium stock solution and sufficient water to make 10 mL.
To similar separate test tubes add, in duplicate, volumes of the Assay preparation corresponding to three or more of the levels specified for the Standard preparation, including the levels of 2.0, 3.0, and 4.0 mL. To each tube add 5.0 mL of the Basal medium stock solution and sufficient water to make 10 mL. Place one complete set of standard and assay tubes together in one tube rack and the duplicate set in a second rack or section of a rack, preferably in random order.
Cover the tubes of both series to prevent contamination, and sterilize in an autoclave at 121
![]() ![]() ![]() ![]() Determine the transmittance of the tubes in the following manner. Mix the contents of each tube, and transfer to an optical container if necessary. Place the container in a spectrophotometer that has been set at a specific wavelength between 540 and 660 nm, and read the transmittance when a steady state is reached. This steady state is observed a few seconds after agitation when the galvanometer reading remains constant for 30 seconds or more. Allow approximately the same time interval for the reading on each tube.
With the transmittance set at 1.00 for the uninoculated blank, read the transmittance of the inoculated blank. With the transmittance set at 1.00 for the inoculated blank, read the transmittance for each of the remaining tubes. If there is evidence of contamination with a foreign microorganism, disregard the result of the assay.
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
![]() Calculate the response, y, adding together the two transmittances for each level of the Assay preparation. Read from the standard curve the logarithm of the volume of the Standard preparation corresponding to each of those values of y that fall within the range of the lowest and highest points plotted for the standard. Subtract from each logarithm so obtained the logarithm of the volume, in mL, of the Assay preparation to obtain the difference, x, for each dosage level. Average the values of x for each of three or more dosage levels to obtain x, which equals the log-relative potency, M ¢, of the Assay preparation. Determine the quantity, in mg, of USP Calcium Pantothenate RS corresponding to the calcium pantothenate in the portion of Tablets taken by the equation:
antilog M = antilog (M ¢ + log R)
in which R is the number of mg of calcium pantothenate that was assumed to be present in each mg in the portion of Tablets taken.
Replication
Repeat the entire determination at least once, using separately prepared Assay preparations. If the difference between the two log-potencies M is not greater than 0.08, their mean, M, is the assayed log-potency of the test material (see The Confidence Interval and Limits of Potency under Design and Analysis of Biological Assays
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Assay for calcium pantothenate, Method 3
Buffer solution
Dissolve 10.0 g of monobasic potassium phosphate in 2000 mL of water, and adjust with phosphoric acid to a pH of 3.5.
Mobile phase
Prepare a filtered and degassed mixture of Buffer solution and methanol (90:10). Make adjustments if necessary (see System Suitability under Chromatography
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Standard stock solution
Dissolve an accurately weighed quantity of USP Calcium Pantothenate RS in water to obtain a solution having a known concentration of about 0.25 mg per mL. Prepare fresh every 4 weeks. Store in a refrigerator.
Standard preparation
Quantitatively dilute an accurately measured volume of Standard stock solution with water to obtain a solution having a known concentration of about 40 µg of USP Calcium Pantothenate RS per mL.
Assay preparation
Weigh and finely powder not fewer than 20 Tablets. Transfer an accurately weighed portion of the powder, equivalent to about 10 mg of calcium pantothenate, to a 250-mL volumetric flask. Add 10 mL of methanol, and swirl to disperse the test specimen. Dilute with water to volume, mix, and filter.
Chromatographic system (see Chromatography
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Procedure
Separately inject equal volumes (about 25 µL) of the Standard preparation and the Assay preparation into 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:
250C(rU / rS)
in which C is the concentration, in mg per mL, of USP Calcium Pantothenate RS in the Standard preparation; and rU and rS are the peak areas obtained from the Assay preparation and the Standard preparation, respectively.
Assay for niacin or niacinamide, pyridoxine hydrochloride, riboflavin, and thiamine, Method 1
[noteUse low-actinic glassware throughout this procedure.]
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 140 mg of sodium 1-hexanesulfonate per 100 mL. Make adjustments if necessary (see System Suitability under Chromatography
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Standard preparation
[noteUse USP Niacin RS in place of USP Niacinamide RS for formulations containing Niacin.] Transfer about 80 mg of USP Niacinamide RS, 20 mg of USP Pyridoxine Hydrochloride RS, 20 mg of USP Riboflavin RS, and 20 mg of USP Thiamine Hydrochloride RS, each accurately weighed, to a 200-mL volumetric flask, and add about 180 mL of Diluting solution. Immerse the flask in a hot water bath maintained at 65
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Assay preparation
Weigh and finely powder not fewer than 30 Tablets, and transfer an accurately weighed portion of the powder, equivalent to about 10 mg of niacinamide and 2.5 mg each of pyridoxine hydrochloride, riboflavin, and thiamine hydrochloride, to a 50-mL centrifuge tube. Add 25.0 mL of Diluting solution, and mix, using a vortex mixer, for 30 seconds to completely suspend the powder. Immerse the centrifuge tube in a hot water bath maintained at 65
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Chromatographic system (see Chromatography
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Procedure
Separately inject equal volumes (about 10 µL) of the Standard preparation and the Assay preparation into 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:
25C(rU / rS)
in which C is the concentration, in mg per mL, of USP Niacinamide RS in the Standard preparation; and rU and rS are the peak responses of niacinamide obtained from the Assay preparation and the Standard preparation, respectively. For formulations containing niacin, C is the concentration, in mg per mL, of USP Niacin RS in the Standard preparation; and rU and rS are the peak areas for niacin obtained from the Assay preparation and 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 C is the concentration, in mg per mL, of the relevant USP Reference Standard in the Standard preparation; and rU and rS are the peak areas for the corresponding vitamin obtained from the Assay preparation and 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:
(327.36/337.27)(25C)(rU / rS)
in which 327.36 and 337.27 are the molecular weights of thiamine mononitrate and thiamine hydrochloride, respectively; and the other terms are as previously defined.
Assay for niacin, Method 2
[noteUse low-actinic glassware throughout this procedure.]
Extraction solvent
Transfer 1 mL of glacial acetic acid and 2.5 g of edetate disodium to a 100-mL volumetric 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 0.1 M sodium acetate solution by dissolving 13.6 g of sodium acetate in 1000 mL of water. Adjust with acetic acid to a pH of 5.4, and mix. Make adjustments if necessary (see System Suitability under Chromatography
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Standard stock solution
Dissolve an accurately weighed quantity of USP Niacin RS in Extraction solvent, and dilute quantitatively, and stepwise if necessary, with Extraction solvent to obtain a solution having a known concentration of about 1 mg per mL.
Standard preparation
Transfer 5.0 mL of Standard stock solution to a 25-mL volumetric flask, dilute with Extraction solvent to volume, and mix.
Assay preparation
[noteThis 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 20 Tablets, and transfer an accurately weighed portion of the powder, equivalent to about 2 mg of riboflavin, to a 200-mL volumetric flask. If riboflavin is not present in the formulation, transfer a portion equivalent to about 2 mg of pyridoxine. If pyridoxine is not present in the formulation, transfer a portion equivalent to about 20 mg of niacin or niacinamide. Add 100.0 mL of Extraction solvent, and mix for 20 minutes, using a wrist-action shaker. Immerse the flask in a water bath maintained at 70
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Chromatographic system (see Chromatography
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Procedure
Separately inject equal volumes (about 20 µL) of the Standard preparation and the Assay preparation into 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:
100C(rU / rS)
in which C is the concentration, in mg per mL, of USP Niacin RS in the Standard preparation; and rU and rS are the peak responses of niacin obtained from the Assay preparation and the Standard preparation, respectively.
Assay for niacinamide, Method 2
[noteUse low-actinic glassware throughout this procedure.]
Extraction solvent, Mobile phase, Standard stock solution, Standard preparation, Assay preparation, and Chromatographic system
Using USP Niacinamide RS in place of USP Niacin RS, proceed as directed in the Assay for niacin, Method 2.
Procedure
Separately inject equal volumes (about 20 µL) of the Standard preparation and the Assay preparation into 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:
100C(rU / rS)
in which C is the concentration, in mg per mL, of USP Niacinamide RS in the Standard preparation; and rU and rS are the peak responses of niacinamide obtained from the Assay preparation and the Standard preparation, respectively.
Assay for pyridoxine hydrochloride, Method 2
[noteUse low-actinic glassware throughout this procedure.]
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 RS in Extraction solvent, and dilute quantitatively, and stepwise if necessary, with Extraction solvent to obtain a solution having a known concentration of about 0.1 mg per mL.
Standard preparation
Transfer 5.0 mL of Standard stock solution to a 25-mL volumetric flask, dilute with Extraction solvent to volume, and mix.
Assay preparation
Prepare as directed for Assay preparation in the Assay for niacin, Method 2.
Chromatographic system (see Chromatography
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Procedure
Separately inject equal volumes (about 20 µL) of the Standard preparation and the Assay preparation into 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:
100C(rU / rS)
in which C is the concentration, in mg per mL, of USP Pyridoxine Hydrochloride RS in the Standard preparation; and rU and rS are the peak areas obtained from the Assay preparation and the Standard preparation, respectively.
Assay for riboflavin, Method 2
[noteUse low-actinic glassware throughout this procedure.]
Extraction solvent
Prepare as directed in the Assay for niacin, Method 2.
Mobile phase
Dissolve 6.8 g of sodium acetate in 1000 mL of water, and mix. Prepare a mixture of this solution and methanol (65:35). Add 2 mL of triethylamine for every 1000 mL of the mixture, adjust with glacial acetic acid to a pH of 5.2, and mix. Make adjustments if necessary (see System Suitability under Chromatography
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Standard stock solution
Transfer about 20 mg of USP Riboflavin RS, accurately weighed, to a 200-mL volumetric flask. Add about 180 mL of Extraction solvent, and immerse the flask for about 5 minutes in a water bath maintained at 65
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Standard preparation
Quantitatively dilute 5.0 mL of Standard stock solution with Extraction solvent to 25.0 mL, and mix.
Assay preparation
Prepare as directed for Assay preparation in the Assay for niacin, Method 2.
Chromatographic system (see Chromatography
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Procedure
Separately inject equal volumes (about 20 µL) of the Standard preparation and the Assay preparation into 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:
100C(rU / rS)
in which C is the concentration, in mg per mL, of USP Riboflavin RS in the Standard preparation; and rU and rS are the peak areas obtained from the Assay preparation and the Standard preparation, respectively.
Assay for thiamine, Method 2
[noteUse low-actinic glassware throughout this procedure.]
Mobile phase
Dissolve 1.88 g of sodium 1-hexanesulfonate in 1 L of 0.1% phosphoric acid. Prepare a mixture of this solution and acetonitrile (92:18). Make adjustments if necessary (see System Suitability under Chromatography
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Standard stock solution
Dissolve an accurately weighed quantity of USP Thiamine Hydrochloride RS in 0.2 N hydrochloric acid, and dilute quantitatively, and stepwise if necessary, with 0.2 N hydrochloric acid to obtain a solution having a known concentration of about 0.1 mg per mL.
Standard preparation
Transfer 5.0 mL of Standard stock solution to a 25-mL volumetric flask, dilute with 0.2 N hydrochloric acid to volume, and mix.
Assay preparation
Weigh and finely powder not fewer than 20 Tablets. Transfer an accurately weighed portion of the powder, equivalent to about 2 mg of thiamine, to a 200-mL volumetric flask. Add 100.0 mL of 0.2 N hydrochloric acid, shake for 15 minutes with a wrist-action shaker, and heat to boiling for 30 minutes. Cool to room temperature, and filter. Use the clear filtrate.
Chromatographic system (see Chromatography
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Procedure
Separately inject equal volumes (about 20 µL) of the Standard preparation and the Assay preparation into 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:
100C(rU / rS)
in which C is the concentration, in mg per mL, of USP Thiamine Hydrochloride RS in the Standard preparation; and rU and rS are the peak areas for thiamine obtained from the Assay preparation and 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:
(327.36/337.27)(100C)(rU / rS)
in which 327.36 and 337.27 are the molecular weights of thiamine mononitrate and thiamine hydrochloride, respectively; and the other terms are as previously defined.
Assay for niacin or niacinamide, pyridoxine hydrochloride, riboflavin, and thiamine, Method 3
[noteUse low-actinic glassware throughout this procedure.]
Reagent
Prepare a solution containing 25 g of edetate disodium in 1000 mL of water.
Mobile phase
Transfer 0.4 mL of triethylamine, 15.0 mL of glacial acetic acid, and 350 mL of methanol to a 2000-mL volumetric flask, dilute with 0.008 M sodium 1-hexanesulfonate to volume, and mix. Filter, and degas. Make adjustments if necessary (see System Suitability under Chromatography
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Standard stock solution
Dissolve accurately weighed quantities of USP Niacin RS or USP Niacinamide RS, USP Pyridoxine Hydrochloride RS, USP Riboflavin RS, and USP Thiamine Hydrochloride RS in Reagent, with heating if necessary, to obtain a solution having known concentrations of about 1.5 mg of USP Niacin RS or USP Niacinamide RS per mL, 0.24 mg of USP Pyridoxine Hydrochloride RS per mL, 0.08 mg of USP Riboflavin RS per mL, and 0.24 mg of USP Thiamine Hydrochloride RS per mL.
Standard preparation
Transfer 5.0 mL of Standard stock solution to a stoppered 125-mL flask. Add 10.0 mL of a mixture of methanol and glacial acetic acid (9:1) and 30.0 mL of a mixture of methanol and ethylene glycol (1:1). Insert the stopper, shake for 15 minutes in a water bath maintained at 60
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Assay preparation
Weigh and finely powder not fewer than 20 Tablets. Transfer an accurately weighed portion of the powder, equivalent to about 7.5 mg of niacin or niacinamide, 1.2 mg of pyridoxine hydrochloride, 0.4 mg of riboflavin, and 1.2 mg of thiamine hydrochloride, to a stoppered 125-mL flask. Add 10.0 mL of a mixture of methanol and glacial acetic acid (9:1) and 30.0 mL of a mixture of methanol and ethylene glycol (1:1). Insert the stopper, shake for 15 minutes in a water bath maintained at 60
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Chromatographic system (see Chromatography
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Procedure
Separately inject equal volumes (about 5 µL) of the Assay preparation and the Standard preparation into 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:
40C(rU / rS)
in which C is the concentration, in mg per mL, of USP Niacin RS or USP Niacinamide RS in the Standard preparation; and rU and rS are the peak areas for niacin or niacinamide obtained from the Assay preparation and 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:
(327.36/337.27)(40C)(rU / rS)
in which 327.36 and 337.27 are the molecular weights of thiamine mononitrate and thiamine hydrochloride, respectively; and the other terms are as previously defined.
noteCommercially 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 preparations and the Assay preparation may 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 calcium
Lanthanum chloride solution
Dissolve 26.7 g of lanthanum chloride heptahydrate in 0.125 N hydrochloric acid to make 100.0 mL.
Calcium standard stock solution
Weigh accurately about 1.001 g of calcium carbonate, previously dried at 300
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Standard preparations
Quantitatively dilute a volume of Calcium standard stock solution with 0.125 N hydrochloric acid to obtain a standard solution having a known concentration of about 100 µg of calcium per mL. Into separate 100-mL volumetric flasks, pipet 1.0, 1.5, 2.0, 2.5, and 3.0 mL of the standard solution. To each flask add 1.0 mL of 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 3.0 µg of calcium per mL.
Assay preparation
Weigh and finely powder not fewer than 20 Tablets. Transfer an accurately weighed portion of the powder, equivalent to about 5 Tablets, to a porcelain crucible. Heat the crucible in a muffle furnace maintained at about 550
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Procedure
Concomitantly determine the absorbances of the Standard preparations and the Assay preparation at the calcium emission line at 422.7 nm with an atomic absorption spectrophotometer (see Spectrophotometry and Light-Scattering
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in which D is the dilution factor used to prepare the Assay preparation.
Assay for chromium
Chromium standard stock solution
Transfer about 2.829 g of potassium dichromate, previously dried at 120
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Standard preparations
Transfer 10.0 mL of the Chromium standard stock solution to a 1000-mL volumetric flask, add 50.0 mL of 6 N hydrochloric acid, dilute with water to volume, and mix to obtain a standard solution having a known concentration of about 10 µg of chromium per mL. Transfer 10.0 mL and 20.0 mL of the standard solution to separate 100-mL volumetric flasks, and transfer 15.0 mL and 20.0 mL of the standard solution to separate 50-mL volumetric flasks. Dilute the contents of each of the four flasks with 0.125 N hydrochloric acid to volume, and mix to obtain solutions having known concentrations of about 1.0, 2.0, 3.0, and 4.0 µg of chromium per mL.
Assay preparation
Proceed as directed for Assay preparation in the Assay for calcium, except to prepare the Assay preparation to contain about 1 µg of chromium per mL and to omit the use of the Lanthanum chloride solution.
Procedure
Concomitantly determine the absorbances of the Standard preparations and the Assay preparation at the chromium emission line at 357.9 nm with an atomic absorption spectrophotometer (see Spectrophotometry and Light-Scattering
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CD
in which D is the dilution factor used to prepare the Assay preparation.
Assay for copper
Copper standard stock solution
Dissolve about 1.00 g 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 1000 mL. This solution contains 1000 µg of copper per mL.
Standard preparations
Transfer 10.0 mL of Copper standard stock solution to a 100-mL volumetric flask, and dilute quantitatively with 0.125 N hydrochloric acid to volume to obtain a standard solution having a concentration of 100 µg of copper per mL. To separate 200-mL volumetric flasks, transfer 1.0, 2.0, 4.0, 6.0, and 8.0 mL of 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 4.0 µg of copper per mL.
Assay preparation
Proceed as directed for Assay preparation in the Assay for calcium, except to prepare the Assay preparation to contain about 2 µg of copper per mL and to omit the use of Lanthanum chloride solution.
Procedure
Concomitantly determine the absorbances of the Standard preparations and the Assay preparation at the copper emission line at 324.7 nm with an atomic absorption spectrophotometer (see Spectrophotometry and Light-Scattering
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in which D is the dilution factor used to prepare the Assay preparation.
Assay for fluoride, Method 1
[noteStore all solutions in plastic containers.]
3 M Sodium acetate solution
Dissolve 408 g of sodium acetate in about 600 mL of water contained in a 1000-mL volumetric 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 pH of 7.0.
Sodium citrate solution
Dissolve 222 g of sodium citrate in 250 mL of water in a 1000-mL volumetric flask. Add 28 mL of perchloric acid, dilute with water to volume, and mix.
Fluoride standard stock solution
Transfer an accurately weighed quantity of about 1.105 g of sodium fluoride, previously dried at 100
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Intermediate stock solution 1
Pipet 20.0 mL of Fluoride standard stock solution into a 100-mL volumetric flask, dilute with water to volume, and mix to obtain a solution having a known concentration of about 100 µg of fluoride per mL.
Intermediate stock solution 2
Transfer 2.0 mL of Fluoride standard stock solution to a 100-mL volumetric flask, dilute with water to volume, and mix to obtain a solution having a known concentration of about 10 µg of fluoride per mL.
Standard preparations
To five separate 100-mL volumetric flasks, transfer 3.0, 5.0, and 10.0 mL of Intermediate stock solution 2 and 5.0 and 10.0 mL of Intermediate stock solution 1. To each flask, add 10.0 mL of 1 N hydrochloric acid, 25 mL of 3 M Sodium acetate solution, and 25.0 mL of 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 10.0 µg 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 200 µg of fluoride, to a 100-mL volumetric flask. Dissolve in 10.0 mL of 1 N hydrochloric acid, 25.0 mL of 3 M Sodium acetate solution, and 25.0 mL of Sodium citrate solution, dilute with water to volume, and mix.
Procedure
To separate plastic beakers, each containing a plastic-coated stirring bar, transfer 50.0 mL each of the Standard preparations and the Assay preparation. Concomitantly measure the potentials (see pH
![]() ![]() 0.1C.
Assay for fluoride, Method 2
[noteUse plastic containers and deionized water throughout this procedure.]
pH 10.0 Buffer
Add 214 mL of 0.1 N sodium hydroxide to 1000 mL of 0.05 M sodium bicarbonate.
Mobile phase
Prepare a filtered and degassed mixture of water, alcohol, and 0.1 N sulfuric acid (175:20:5). Make adjustments if necessary (see System Suitability under Chromatography
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Standard stock solution
Dissolve an accurately weighed quantity of USP Sodium Fluoride RS in water, and dilute quantitatively, and stepwise if necessary, with water to obtain a solution having a known concentration of about 220 µg per mL. This solution contains about 100 µg of fluoride per mL.
Standard preparation
[noteCondition the solid-phase extraction column specified for use in the Standard preparation and the Assay preparation in the following manner. Using a vacuum at a pressure not exceeding 5 mm of mercury, wash the column with one column volume of methanol followed by one column volume of pH 10.0 Buffer. Do not allow the column top to dry. If the top of the column becomes dry, recondition the column.] Transfer 10.0 mL of Standard stock solution to a 100-mL volumetric flask. Add 75 mL of water, and adjust with 0.1 N sodium hydroxide to a pH of 10.4 ± 0.1. Dilute with water to volume, and mix. Filter, discarding the first 15 mL of the filtrate. Transfer 25.0 mL of the filtrate to a 50-mL volumetric flask, add 15.0 mL of water, and adjust with 0.1 N sodium hydroxide to a pH of 10.0. Dilute with pH 10.0 Buffer to volume, and mix. Elute a portion of this solution through a 3-mL solid-phase extraction column containing L1 packing that is connected through an adaptor to a second solid-phase extraction column containing sulfonylpropyl strong cation-exchange packing. Discard the first 3 mL of 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 20 Tablets. Transfer an accurately weighed portion of the powder, equivalent to about 1 mg of fluoride, to a stoppered 100-mL volumetric flask, add 15 mL of water, and shake vigorously. Rinse the sides of the flask with 15 mL of water, and allow to stand for 10 minutes. Dilute with water to about 85 mL, adjust with 1 N sodium hydroxide to a pH of 10.4 ± 0.1, dilute with water to volume, and mix. Proceed as directed for Standard preparation, beginning with Filter, discarding the first 15 mL of the filtrate.
Chromatographic system (see Chromatography
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Procedure
Separately inject equal volumes (about 100 µL) of the Standard preparation and the Assay preparation into 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:
0.2C(rU / rS)
in which C is the concentration, in µg per mL, of fluoride in the Standard preparation; and rU and rS are the peak areas obtained from the Assay preparation and the Standard preparation, respectively.
Assay for iodide, Method 1
Bromine water
To about 20 mL of bromine in a glass-stoppered bottle, add 100 mL of water. Insert the stopper into the bottle, and shake. Allow to stand for 30 minutes, 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 3 mg of iodide, to a nickel crucible. Add 5 g of sodium carbonate, 5 mL of 50% (w/v) sodium hydroxide solution, and 10 mL of 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 100
![]() ![]() ![]() ![]() 105.8VN/0.005
in which V is the volume, in mL, of sodium thiosulfate consumed; and N is the normality of the sodium thiosulfate solution used.
Assay for iodide, Method 2
Proceed as directed in the Assay for Iodide under Automated Methods of Analysis
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Assay for iron
Iron standard stock solution
Transfer about 100 mg of iron powder, accurately weighed, to a 1000-mL volumetric flask, dissolve in 25 mL of 6 N hydrochloric acid, dilute with water to volume, and mix.
Standard preparations
To separate 100-mL volumetric flasks, transfer 2.0, 4.0, 5.0, 6.0, and 8.0 mL of 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 8.0 µg of iron per mL.
Assay preparation
Proceed as directed for Assay preparation in the Assay for calcium, except to prepare the Assay preparation to contain 5 µg of iron per mL and to omit the use of the Lanthanum chloride solution.
Procedure
Concomitantly determine the absorbances of the Standard preparations and the Assay preparation at the iron emission line at 248.3 nm with an atomic absorption spectrophotometer (see Spectrophotometry and Light-Scattering
![]() ![]() 0.001CD
in which D is the dilution factor used to prepare the Assay preparation.
Assay for magnesium
Lanthanum chloride solution
Prepare as directed in the Assay for calcium.
Magnesium standard stock solution
Transfer about 1.0 g of magnesium ribbon, accurately weighed, to a 1000-mL volumetric flask, dissolve in 50 mL of 6 N hydrochloric acid, dilute with water to volume, and mix to obtain a solution having a known concentration of about 1000 µg of magnesium per mL.
Standard preparations
Quantitatively dilute a volume of Magnesium standard stock solution with 0.125 N hydrochloric acid to obtain a standard solution having a concentration of 20 µg of magnesium per mL. To separate 100-mL volumetric flasks, transfer 1.0, 1.5, 2.0, 2.5, and 3.0 mL of standard solution. To each flask add 1.0 mL of Lanthanum chloride solution, dilute with 0.125 N hydrochloric acid to volume, and mix to obtain solutions having known concentrations of about 0.2, 0.3, 0.4, 0.5, and 0.6 µg of magnesium per mL.
Assay preparation
Proceed as directed for Assay preparation in the Assay for calcium, except to prepare the Assay preparation to contain 0.4 µg of magnesium per mL.
Procedure
Concomitantly determine the absorbances of the Standard preparations and the Assay preparation at the magnesium emission line at 285.2 nm with an atomic absorption spectrophotometer (see Spectrophotometry and Light-Scattering
![]() ![]() 0.001CD
in which D is the dilution factor used to prepare the Assay preparation.
Assay for manganese
Manganese standard stock solution
Transfer 1.00 g of manganese, accurately weighed, to a 1000-mL volumetric flask, dissolve in 20 mL of nitric acid, dilute with 6 N hydrochloric acid to volume, and mix to obtain a solution containing 1000 µg of manganese per mL.
Standard preparations
Quantitatively dilute 10.0 mL of the Manganese standard stock solution with 0.125 N hydrochloric acid to 200.0 mL to obtain a standard solution having a concentration of 50 µg of manganese per mL. To separate 100-mL volumetric flasks, transfer 1.0, 1.5, 2.0, 3.0, and 4.0 mL of standard solution, dilute the contents of each flask with 0.125 N hydrochloric acid to volume, and mix to obtain solutions having known concentrations of about 0.5, 0.75, 1.0, 1.5, and 2.0 µg of manganese per mL.
Assay preparation
Proceed as directed for Assay preparation in the Assay for calcium, except to prepare the Assay preparation to contain 1 µg of manganese per mL and to omit the use of the Lanthanum chloride solution.
Procedure
Concomitantly determine the absorbances of the Standard preparations and the Assay preparation at the manganese emission line at 279.5 nm with an atomic absorption spectrophotometer (see Spectrophotometry and Light-Scattering
![]() ![]() 0.001CD
in which D is the dilution factor used to prepare the Assay preparation.
Assay for molybdenum, Method 1
Diluting solution
Dissolve 40 g of ammonium chloride in 2000 mL of water.
Molybdenum standard stock solution
Transfer about 1.0 g of molybdenum wire, accurately weighed, to a 1000-mL volumetric flask, and dissolve in 50 mL of nitric acid, warming if necessary. Dilute with water to volume, and mix to obtain a solution containing 1000 µg of molybdenum per mL.
Standard preparations
Quantitatively dilute 10.0 mL of the Molybdenum standard stock solution with water to 100.0 mL to obtain a standard solution having a known concentration of about 100 µg of molybdenum per mL. To separate 100-mL volumetric flasks, transfer 2.0, 10.0, and 25.0 mL of the standard solution, and add 5.0 mL of perchloric acid to each flask. Gently boil the solution in each flask for 15 minutes, cool to room temperature, dilute each with Diluting solution to volume, and mix to obtain solutions having known concentrations of about 5.0, 10.0, and 25.0 µg 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 1000 µg of molybdenum, to a suitable flask, and add about 12 mL of nitric acid. [noteThe 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 10 minutes, or until the test specimen is completely dissolved. Gently boil the solution for about 15 minutes, and cool to room temperature. Carefully add about 8 mL of 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-mL volumetric flask with the aid of the Diluting solution, dilute with Diluting solution to volume, and mix.
Procedure
Concomitantly determine the absorbances of the Standard preparations and the Assay preparation at the molybdenum emission line at 313 nm with an atomic absorption spectrophotometer (see Spectrophotometry and Light-Scattering
![]() ![]() 0.001CD
in which D is the diluting factor used to prepare the Assay preparation.
Assay for molybdenum, Method 2
Sodium fluoride solution
Add 200 mL of water to about 10 g of sodium fluoride, stir until the solution is saturated, and filter. Store in a polyethylene bottle.
Ferrous sulfate solution
Dissolve 498 mg of ferrous sulfate in water to make 100 mL.
Potassium thiocyanate solution
Dissolve 20 g of potassium thiocyanate in water, and dilute with water to 100 mL.
20% Stannous chloride solution
Transfer 40 g of stannous chloride to a beaker, and add 20 mL of 6.5 N hydrochloric acid solution. Heat the solution until the stannous chloride is dissolved. Cool, dilute with water to 100 mL, and mix.
Dilute stannous chloride solution
Dilute 4 mL of 20% Stannous chloride solution with water to 100 mL. Prepare this solution fresh at the time of use.
Standard preparation
Transfer about 92 mg of ammonium molybdate, accurately weighed, to a 500-mL volumetric flask, dissolve in and dilute with water to volume, and mix. Quantitatively dilute 20.0 mL of this solution with water to 100.0 mL to obtain a solution having a known concentration of about 20 µg 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 40 µg of molybdenum, to a 200-mL beaker marked I. To another 200-mL beaker marked II, transfer 2.0 mL of the Standard preparation. Add about 20 mL of nitric acid to each beaker. Cover each beaker with a watchglass, and boil slowly on a hot plate for about 45 minutes. Cool to room temperature, add 6 mL of 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 50 mL. Gently boil the water solutions for a few minutes. Cool to room temperature. Add 2 drops of methyl orange TS to each beaker, and neutralize with ammonium hydroxide. Add 8.2 mL of hydrochloric acid to each beaker. Quantitatively transfer the contents of the beakers to separate 100-mL volumetric flasks, rinse the beakers with water, transfer the rinsings to the respective volumetric flasks, dilute with water to volume, and mix.
Transfer 50.0 mL each of the solutions from beakers I and II to separate separatory funnels. To each separatory funnel add 1.0 mL of Sodium fluoride solution, 0.5 mL of Ferrous sulfate solution, 4.0 mL of Potassium thiocyanate solution, 1.5 mL of 20% Stannous chloride solution, and 15.0 mL of amyl alcohol, and shake the separatory funnels for 1 minute. Allow the layers to separate, and discard the aqueous layers. Add 25 mL of Dilute stannous chloride solution to each separatory funnel, and shake gently for 15 seconds. Allow the layers to separate, and discard the aqueous layers.
Transfer the organic layers from the separatory funnels to separate centrifuge tubes, and centrifuge at 2000 rpm for 10 minutes. Concomitantly determine the absorbances of the solutions obtained from the Tablets and from the Standard preparation in 1-cm cells at the wavelength of maximum absorbance at about 465 nm, using amyl alcohol as the blank. Calculate the quantity, in µg, of molybdenum (Mo) in the portion of Tablets taken by the formula:
2C(AU / AS)
in which C is the concentration, in µg per mL, of molybdenum in the Standard preparation; and AU and AS are the absorbances of the solutions obtained from the Tablets and from the Standard preparation, respectively.
Assay for phosphorus
Sulfuric acid solution
Cautiously add 37.5 mL of sulfuric acid to 100 mL of water, and mix.
Ammonium molybdate solution
Dissolve 12.5 g of ammonium molybdate in 150 mL of water. Add 100 mL of Sulfuric acid solution, and mix.
Hydroquinone solution
Dissolve 0.5 g of hydroquinone in 100 mL of water, and add one drop of sulfuric acid.
Sodium bisulfite solution
Dissolve 20 g of sodium bisulfite in 100 mL of water.
Phosphorus standard stock solution
Accurately weigh about 4.395 g of monobasic potassium phosphate, previously dried at 105
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Standard preparation
Dissolve an accurately measured volume of Phosphorus standard stock solution in water, and dilute quantitatively, and stepwise if necessary, with water to obtain a solution having a known concentration of about 20 µg 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 100 mg of phosphorus, to a suitable flask, add 25 mL nitric acid, and digest on a hot plate for about 30 minutes. Add 15 mL of hydrochloric acid, and continue the digestion to the cessation of brown fumes. Cool, and quantitatively transfer the contents of the flask to a 500-mL volumetric flask with the aid of small portions of water. Dilute with water to volume, and mix. Transfer 10.0 mL of this solution to a 100-mL volumetric flask, dilute with water to volume, and mix.
Procedure
To three separate 25-mL volumetric flasks, transfer 5.0 mL each of the Standard preparation, the Assay preparation, and water to provide the blank. To each of the three flasks, add 1.0 mL each 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 30 minutes. Concomitantly determine the absorbances of the solutions from the Assay preparation and the Standard preparation in 1-cm cells at the wavelength of maximum absorbance at about 650 nm, against the blank. Calculate the quantity, in mg, of phosphorus (P) in the portion of Tablets taken by the formula:
5C(AU / AS)
in which C is the concentration, in µg per mL, of phosphorus in the Standard preparation; and AU and AS are the absorbances of the solutions obtained from the Assay preparation and the Standard preparation, respectively.
Assay for potassium
Potassium standard stock solution
Transfer about 190.7 mg of potassium chloride, previously dried at 105
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Standard preparations
Dilute an accurately measured volume of Potassium standard stock solution with 0.125 N hydrochloric acid to obtain a standard solution having a known concentration of about 10 µg of potassium per mL. Transfer 5.0, 10.0, 15.0, 20.0, and 25.0 mL of the standard solution to separate 100-mL volumetric flasks. Dilute the contents of each flask with 0.125 N hydrochloric acid to volume, and mix to obtain solutions containing 0.5, 1.0, 1.5, 2.0, and 2.5 µg of potassium per mL.
Assay preparation
Proceed as directed for Assay preparation in the Assay for calcium, except to prepare the Assay preparation to contain 1 µg of potassium per mL and to omit the use of the Lanthanum chloride solution.
Procedure
Concomitantly determine the absorbances of the Standard preparations and the Assay preparation at the potassium emission line at 766.5 nm with an atomic absorption spectrophotometer (see Spectrophotometry and Light-Scattering
![]() ![]() 0.001CD
in which D is the dilution factor used to prepare the Assay preparation.
Assay for selenium, Method 1
Diluting solution
Prepare as directed in the Assay for molybdenum, Method 1.
Selenium standard stock solution
[CautionSelenium is toxic; handle it with care.
] Dissolve about 1 g of metallic selenium, accurately weighed, in a minimum volume of nitric acid. Evaporate to dryness, add 2 mL of water, and evaporate to dryness. Repeat the addition of water and the evaporation to dryness three times. Dissolve the residue in 3 N hydrochloric acid, transfer to a 1000-mL volumetric flask, dilute with 3 N hydrochloric acid to volume, and mix to obtain a solution having a known concentration of about 1000 µg of selenium per mL.
Standard preparations
Quantitatively dilute 10 mL of the Selenium standard stock solution with water to 100.0 mL to obtain a standard solution having a known concentration of about 100 µg of selenium per mL. To separate 100-mL volumetric flasks, transfer 5.0, 10.0, and 25.0 mL of the standard solution, and add 5.0 mL of perchloric acid to each flask. Gently boil the solutions for 15 minutes, cool to room temperature, dilute each with Diluting solution to volume, and mix to obtain solutions containing 5.0, 10.0, and 25.0 µg 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 1000 µg of selenium, to a suitable flask, and add about 12 mL of nitric acid. [noteThe 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 10 minutes or until the test specimen is completely dissolved. Gently boil the solution for about 15 minutes, and cool to room temperature. Carefully add about 8 mL of 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-mL volumetric flask with the aid of the Diluting solution, dilute with Diluting solution to volume, and mix.
Procedure
Concomitantly determine the absorbances of the Standard preparations and the Assay preparation at the selenium emission line at 196 nm, with an atomic absorption spectrophotometer (see Spectrophotometry and Light-Scattering
![]() ![]() 0.001CD
in which D is the dilution factor used to prepare the Assay preparation.
Assay for selenium, Method 2
Hydrochloric acid solution
Dilute 50 mL of hydrochloric acid with water to 500 mL, and mix.
50% Ammonium hydroxide solution
Dilute 250 mL of ammonium hydroxide with water to 500 mL, and mix.
Reagent 1
Dissolve 4.5 g of edetate disodium in 400 mL of water in a 500-mL volumetric flask. Add 12.5 g of hydroxylamine hydrochloride, dilute with water to volume, and mix.
Reagent 2
Transfer 200 mg of 2,3-diaminonaphthalene to a 250-mL separatory funnel, and add 200 mL of 0.1 N hydrochloric acid. Wash the solution with three 40-mL portions 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 1 week if stored in a refrigerator.
Standard stock preparation
[CautionSelenium is toxic; handle it with care.
] Dissolve about 1 g of metallic selenium, accurately weighed, in a minimum volume of nitric acid. Evaporate to dryness, add 2 mL of water, and evaporate to dryness. Repeat the addition of water and evaporation to dryness three times. Dissolve the residue in 3 N hydrochoric acid, transfer to a 1000-mL volumetric flask, dilute with 3 N hydrochloric acid to volume, and mix to obtain a solution containing about 1000 µg of selenium per mL. Dilute a volume of the solution quantitatively, and stepwise if necessary, with 0.125 N hydrochloric acid to obtain a solution having a known concentration of about 2.0 µg 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 20 µg of selenium, to a suitable flask. Add 10 mL of nitric acid, and warm gently on a hot plate. Continue heating until the initial nitric acid reaction has subsided, then add 3 mL of perchloric acid. [CautionExercise 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 0.5 mL of nitric acid, and resume heating, adding additional amounts of nitric acid if further darkening occurs. Digest for about 10 minutes after the first appearance of perchloric acid fumes or until the digest becomes colorless. Cool the flask, add 2.5 mL of Hydrochloric acid solution, and return the flask to the hot plate to expel residual nitric acid. Heat the mixture for 3 minutes after it begins to boil. Cool the flask to room temperature, and dilute with water to about 20 mL.
Procedure
Transfer 10.0 mL of the Standard stock preparation to a glass-stoppered flask. Add 1 mL of perchloric acid and 1 mL of Hydrochloric acid solution, and dilute with water to about 20 mL (Standard preparation). Prepare a blank by adding 1 mL of perchloric acid and 1 mL of Hydrochloric acid solution to a glass-stoppered flask, and diluting with water to about 20 mL. Treat the Assay preparation, the Standard preparation, and the blank as follows. Add 5 mL of Reagent 1 to each flask, and swirl gently to mix. Adjust the solution in each flask with 50% Ammonium hydroxide solution to a pH of 1.1 ± 0.1. Add 5 mL of Reagent 2 to each flask, and swirl gently to mix. Place the flasks in a water bath maintained at 50
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CD(AU / AS)
in which C is the concentration, in µg per mL, of selenium in the Standard stock preparation; D is the dilution factor used to prepare the Assay preparation; and AU and AS are the absorbances of the solutions from the Assay preparation and the Standard preparation, respectively.
Assay for zinc
Zinc standard stock solution
Transfer about 311 mg of zinc oxide, accurately weighed, to a 250-mL volumetric flask, and add 80 mL of 5 M hydrochloric acid, warming if necessary to dissolve. Cool, dilute with water to volume, and mix to obtain a solution having a known concentration of about 1000 µg of zinc per mL.
Standard preparations
Dilute a volume of the Zinc standard stock solution quantitatively, and stepwise if necessary, with 0.125 N hydrochloric acid to obtain a standard solution having a known concentration of about 50 µg of zinc per mL. Transfer 1.0, 2.0, 3.0, 4.0, and 5.0 mL of this solution to separate 100-mL volumetric flasks. Dilute the contents of each flask with 0.125 N hydrochloric acid to volume, and mix to obtain solutions having known concentrations of about 0.5, 1.0, 1.5, 2.0, and 2.5 µg of zinc per mL.
Assay preparation
Proceed as directed for Assay preparation in the Assay for calcium, except to prepare the Assay preparation to contain 2 µg of zinc per mL and to omit the use of the Lanthanum chloride solution.
Procedure
Concomitantly determine the absorbances of the Standard preparations and the Assay preparation at the zinc emission line at 213.8 nm with an atomic absorption spectrophotometer (see Spectrophotometry and Light-Scattering
![]() ![]() 0.001CD
in which D is the dilution factor used to prepare the Assay preparation.
1
USP Units of activity for vitamins, where such exist or formerly existed, are equivalent to the corresponding international units, where such formerly existed. The USP Unit for Vitamin E has been discontinued. International units (IU) for vitamins also have been discontinued; however, the use of IU on the labels of vitamin products continues. Where articles are labeled in terms of Units in addition to the required labeling, the relationship of the USP Units or IU to mass is as follows. One USP Vitamin A Unit = 0.3 µg of all-trans retinol (vitamin A alcohol) or 0.344 µg of all-trans retinyl acetate (vitamin A acetate) or 0.55 µg of all-trans retinyl palmitate (vitamin A palmitate), and 1 µg of retinol (3.3 USP Vitamin A Units) = 1 retinol equivalent (RE); 1 IU of beta carotene = 0.6 µg of all-trans beta carotene; 1 USP Vitamin D Unit = 0.025 µg of ergocalciferol or cholecalciferol; 1 mg of dl-alpha tocopherol = 1.1 former USP Vitamin E Units, 1 mg of dl-alpha tocopheryl acetate = 1 former USP Vitamin E Unit, 1 mg of dl-alpha tocopheryl acid succinate = 0.89 former USP Vitamin E Unit, 1 mg of d-alpha tocopherol = 1.49 former USP Vitamin E Units, 1 mg of d-alpha tocopheryl acetate = 1.36 former USP Vitamin E Units, 1 mg of d-alpha tocopheryl acid succinate = 1.21 former USP Vitamin E Units. In terms of d-alpha tocopherol equivalents, 1 mg of d-alpha tocopheryl acetate = 0.91, 1 mg of d-alpha tocopheryl acid succinate = 0.81, 1 mg of dl-alpha tocopherol = 0.74, 1 mg of dl-alpha tocopheryl acetate = 0.67, and 1 mg of dl-alpha tocopheryl acid succinate = 0.60.
2
ATCC No. 8014 is suitable. This strain was formerly known as Lactobacillus arabinosus 17-5.
3
A suitable cartridge is the Waters, Oasis MAX Vac RC cartridge, particle size 30 µm, part 186000371.
4
Pure cultures of Lactobacillus leichmannii may be obtained as No. 7830 from ATCC, 10801 University Blvd., Manassas, VA 20110-2209.
5
ATCC No. 8014 is suitable. This strain was formerly known as Lactobacillus arabinosus 17-5.
Auxiliary Information
Please check for your question in the FAQs before contacting USP.
Chromatographic Column
USP32NF27 Page 1104
Pharmacopeial Forum: Volume No. 34(6) Page 1500
Chromatographic columns text is not derived from, and not part of, USP 32 or NF 27.
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