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USP36-NF31 “Oil-and Water-Soluble Vitamins with Minerals Tablets”

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 11—
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 2021— The total aerobic microbial count does not exceed 3000 cfu per g, and the combined molds and yeasts count does not exceed 300 cfu per g. Tablets also meet the requirements of the tests for absence of Salmonella species, Escherichia coli, and Staphylococcus aureus.
Disintegration and dissolution 2040: meet the requirements for Dissolution.
Weight variation 2091: meet the requirements.
NOTE— In the following Assays, where more than one Assay method is given for an individual ingredient, the requirements may be met by following any one of the specified methods, the method used being stated in the labeling only if Method 1 is not used.
Assay for vitamin A, Method 1— [NOTE—Where 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. [NOTE—Set up the wrist-action shaker to ensure that the contents of the container are mixed vigorously and thoroughly.] Centrifuge at 3000 rpm for 10 minutes, and transfer the hexane layer by means of a pipet to a 100-mL volumetric flask. Add 15 mL of n-hexane to the dimethyl sulfoxide layer, shake thoroughly for 5 minutes, and transfer the hexane layer by means of a pipet to the 100-mL volumetric flask. Repeat this extraction with three additional 15-mL portions of n-hexane. Dilute the extracts in the volumetric flask with n-hexane to volume, and mix. Quantitatively dilute a 10-mL volume of this solution with n-hexane to obtain a solution having a final concentration of about 15 μg of vitamin A per mL. Retain the remaining solution for use in the assays for vitamin D, vitamin E, and phytonadione.
Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 325-nm detector and a 4.6-mm × 15-cm column that contains 3-μm packing L8. The flow rate is about 1 mL per minute. Chromatograph the System suitability preparation, and measure the peak responses as directed for Procedure: the resolution, R, between retinyl acetate and retinyl palmitate is not less than 10; and the relative standard deviation for replicate injections is not more than 3.0%.
Procedure— Separately inject equal volumes (about 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. [NOTE—The molar responses of retinyl acetate and retinyl palmitate are equivalent.]
Assay for vitamin A, Method 2— [NOTE—Where 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 ascorbate–pyrogallol 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— [NOTE—This preparation is suitable for the determination of vitamin A, vitamin D, and vitamin E, when present in the formulation.] Weigh and finely powder not fewer than 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 ascorbate–pyrogallol solution, shake slowly, and allow the solution to degas. Continue shaking until the evolution of gas has ceased, and then shake for an additional 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. [NOTE—The 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 621)— The liquid chromatograph is equipped with a 325-nm detector and a 4.6-mm × 25-cm column that contains 5-μm packing L24. The flow rate is about 1.5 mL per minute. Chromatograph the System suitability preparation, and measure the peak areas as directed for Procedure: the resolution, R, between retinyl acetate and retinyl palmitate is not less than 8.0; and the relative standard deviation for replicate injections is not more than 3.0%.
Procedure— Separately inject equal volumes (about 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. [NOTE—The 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— [NOTE—Where 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 621).
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, and cool to room temperature. Add 7 mL of water and 25.0 mL of Extraction solvent. Insert the stopper tightly, and shake vigorously for 60 seconds. Rinse the sides of the flask with about 60 mL of water, and allow to stand for about 10 minutes until the layers separate. Withdraw a portion of the organic layer for injection into the chromatograph. This Standard preparation contains about 0.34 μg of retinol per mL.
Assay preparation— Weigh and finely powder a counted number of Tablets. Transfer an accurately weighed portion of the powder, equivalent to about 1.5 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, and cool to room temperature. Add 7 mL of water and 25.0 mL of Extraction solvent. Insert the stopper tightly, and shake vigorously for 60 seconds or longer, if necessary, for complete extraction. Rinse the sides of the flask with about 60 mL of water, and allow to stand for about 10 minutes until the layers separate. [NOTE—Do not shake, as an emulsion may form.] Withdraw a portion of the organic layer, and dilute quantitatively, and stepwise if necessary, with Extraction solvent to obtain a solution having a concentration of about 0.34 μg of retinol per mL.
Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 335-nm detector and a 6.2-mm × 8-cm column that contains packing L3. The column temperature is maintained at 40, and the flow rate is about 4 mL per minute. Chromatograph the Standard preparation, and measure the peak areas as directed for Procedure: the relative retention times are about 0.92 for 13-cis retinol and 1.0 for all-trans retinol; and the relative standard deviation for replicate injections is not more than 5.0%.
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— [NOTE—Where vitamin D (cholecalciferol or ergocalciferol) is specified in the following procedure, use the chemical form present in the formulation and the relevant USP Reference Standard. Use low-actinic glassware throughout this procedure.]
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 621).
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 for 1 hour to partially isomerize vitamin D (cholecalciferol or ergocalciferol) to its corresponding precursor.
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 621)— The liquid chromatograph is equipped with a 265-nm detector and a 4.6-mm × 15-cm column that contains 3-μm packing L8. The flow rate is about 1 mL per minute. Chromatograph the System suitability preparation, and record the peak heights as directed for Procedure: the resolution, R, between the vitamin D form present and its corresponding precursor is not less than 10. Chromatograph the Standard preparation, and record the peak heights as directed for Procedure: the relative standard deviation for replicate injections is not more than 3.0%.
Procedure— Separately inject equal volumes (about 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— [NOTE—Where vitamin D (cholecalciferol or ergocalciferol) is specified in the following procedure, use the chemical form present in the formulation and the relevant USP Reference Standard. Use low-actinic glassware throughout this procedure.]
3 N Methanolic sulfuric acid solution, Sodium ascorbate–pyrogallol solution, Lecithin solution, and Assay preparation— Proceed as directed in the Assay for vitamin A, Method 2.
Mobile phase— Prepare a filtered and degassed mixture of n-hexane and tertiary butyl alcohol (98.75:1.25). Make adjustments if necessary (see System Suitability under Chromatography 621).
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 for 1 hour to partially isomerize vitamin D (cholecalciferol or ergocalciferol) to its corresponding precursor.
Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 265-nm detector and a 4.6-mm × 25-cm column that contains 5-μm packing L24. The flow rate is about 1.0 mL per minute. Chromatograph the System suitability preparation, and record the peak areas as directed for Procedure: the resolution, R, between the vitamin D form present and its corresponding precursor is not less than 4.0. Chromatograph the Standard preparation, and record the peak areas as directed for Procedure: the relative standard deviation for replicate injections is not more than 3.0%.
Procedure— Separately inject equal volumes (about 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— [NOTE—Where vitamin D (cholecalciferol or ergocalciferol) is specified in the following procedure, use the chemical form present in the formulation and the relevant USP Reference Standard. Use low-actinic glassware throughout this procedure.]
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 621).
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— [NOTE—Condition 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. Allow to cool to room temperature, and transfer the contents of the flask to a 250-mL separatory funnel. Add 15.0 mL of water to the flask, insert the stopper, shake vigorously, and transfer this solution to the separatory funnel. Rinse the flask with 60 mL of n-hexane, and transfer the rinsing to the separatory funnel. Insert the stopper, shake vigorously for 90 seconds, and allow to stand for about 15 minutes until the layers separate. Drain and discard the aqueous layer. Add 15.0 mL of water to the hexane layer in the separatory funnel, insert the stopper, and shake vigorously. Allow to stand for about 10 minutes until the layers separate, and discard the aqueous layer. Add 1 drop of Phenolphthalein solution and 15.0 mL of water to the separatory funnel. Add Dilute acetic acid dropwise, with shaking, until the washing is neutral. Allow to stand for about 10 minutes until the layers separate. Drain and discard the aqueous layer. Filter the hexane layer through anhydrous sodium sulfate supported by a small pledget of cotton into a 100-mL round-bottom flask. Rinse the funnel and sodium sulfate with a few mL of n-hexane, and collect the rinsings in the same flask. Evaporate the hexane in the flask on a rotary evaporator at 50 to dryness. Immediately add 2.0 mL of Extraction solvent to dissolve the residue. Transfer this solution to a freshly conditioned solid-phase extraction column containing silica packing with a sorbent mass to column volume ratio of 500 mg to 2.8 mL or equivalent, rinse the round-bottom flask with 1.0 mL of Extraction solvent, and transfer to the column. Elute the column with 2.0 mL of Extraction solvent, and discard this fraction. Elute the column with 7.0 mL of Extraction solvent, and collect the eluate in a suitable flask. Place the flask in a warm water bath maintained at about 42, and evaporate the solvent with the aid of a stream of nitrogen. Immediately add 2.0 mL of acetonitrile to the residue, and use the solution 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 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 621)— The liquid chromatograph is equipped with a 265-nm detector and a 4.6-mm × 25-cm column that contains 5-μm packing L1. The column temperature is maintained at 27, and the flow rate is about 0.7 mL per minute. Chromatograph the Standard preparation, and record the peak heights as directed for Procedure: the relative standard deviation for replicate injections is not more than 4.0%.
Procedure— Separately inject equal volumes (about 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— [NOTE—Where 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 621).
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 621)— The liquid chromatograph is equipped with a 254-nm detector and an 8-mm × 10-cm column that contains 5-μm packing L1. The flow rate is about 2 mL per minute. Chromatograph the System suitability preparation, and record the peak areas as directed for Procedure: the relative retention times are about 0.5 for ergocalciferol and 1.0 for alpha tocopheryl acetate; the resolution, R, between ergocalciferol and alpha tocopheryl acetate is not less than 12; and the tailing factor is between 0.8 and 1.2. Chromatograph the Standard preparation, and record the peak areas as directed for Procedure: the relative standard deviation for replicate injections is not more than 3.0%.
Procedure— Separately inject equal volumes (about 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— [NOTE—Where 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 621).
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 621)— The liquid chromatograph is equipped with a 280-nm detector and a 4.6-mm × 25-cm column that contains 5-μm packing L1. The flow rate is about 1.5 mL per minute. Chromatograph the System suitability preparation, and record the peak areas as directed for Procedure: the relative retention times for alpha tocopheryl acid succinate, alpha tocopherol, and alpha tocopheryl acetate are about 0.6, 0.8, and 1.0, respectively; the resolution between alpha tocopheryl acid succinate and alpha tocopherol is not less than 4.0; and the resolution between alpha tocopherol and alpha tocopheryl acetate is not less than 3.0. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the relative standard deviation for replicate injections is not more than 3.0%.
Procedure— Separately inject equal volumes (about 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. [NOTE—The 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— [NOTE—Where 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 621).
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, cool, and transfer with the aid of a minimum volume of water to a 125-mL separatory funnel. Rinse the flask with 50 mL of n-hexane, and add the rinsing to the separatory funnel. Insert the stopper, shake vigorously for 60 seconds, and allow the layers to separate. Drain the aqueous layer into a second 250-mL separatory funnel, and repeat the extraction with 50 mL of n-hexane. Discard the aqueous layer, and combine the hexane extracts. Wash the combined extracts with 25 mL of water, allow the layers to separate, and discard the aqueous layer. Add 3 drops of glacial acetic acid, and repeat the washing procedure two more times. Filter the washed hexane layer through anhydrous sodium sulfate into a 250-mL round-bottom flask. Rinse the funnel and sodium sulfate with a few mL of n-hexane, and add the rinsing to the hexane solution in the flask. Place the flask in a water bath maintained at 50, and evaporate the hexane solution with the aid of a rotary evaporator to dryness. Immediately add 25.0 mL of Diluting solution, and swirl to dissolve the residue.
Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 291-nm detector and a 4.6-mm × 25-cm column that contains packing L1. The column temperature is maintained at about 40, and the flow rate is about 3 mL per minute. Chromatograph the Standard preparation, and record the peak areas as directed for Procedure: the relative standard deviation for replicate injections is not more than 5.0%.
Procedure— Separately inject equal volumes (about 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— [NOTE—Use 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 621).
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 621)— The liquid chromatograph is equipped with a 254-nm detector and an 8-mm × 10-cm column that contains 5-μm packing L1. Chromatograph the System suitability preparation, and record the peak areas as directed for Procedure: the resolution, R, between alpha tocopheryl acetate and phytonadione is not less than 5. Chromatograph the Standard preparation, and record the peak areas as directed for Procedure: the relative retention times are about 0.68 for alpha tocopheryl acetate and 1.0 for phytonadione; and the relative standard deviation for replicate injections is not more than 3.0%.
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— [NOTE—Use 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. [NOTE—A 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 for 5 minutes. Remove from the bath, and again shake or mix using a vortex mixer for 1 minute while the preparation is still hot. Add 8 mL of alcohol, and swirl the contents to mix. Place the tube in a water bath at 60 for 5 minutes. Remove from the bath, and again shake or mix using a vortex mixer for 2 minutes while the preparation is still hot. Cool to room temperature. Add an accurately measured volume of Internal standard solution, equivalent to 1.0 mL per each 5 μg of the expected amount of phytonadione in the aliquot taken. Add 20.0 mL of petroleum ether, and cap the tube tightly. Shake or mix using a vortex mixer for 15 minutes to thoroughly mix the contents. Centrifuge to separate the two layers. Transfer a volume of the top layer of petroleum ether, equivalent to 5 to 50 μg of the expected amount of phytonadione, to an appropriate flask. Place the flask in a water bath at 35 to 45, and evaporate the solvent under a stream of nitrogen until an oily residue is left. Dissolve the residue in a volume of Solvent to obtain a solution having a concentration of about 1 μg per mL of phytonadione.
Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a fluorometric detector set at 320 nm for excitation and 420 nm for emission, a 4.6-mm × 25-cm column that contains 5-μm, end-capped packing L1, and a postcolumn reactor constituted with a 4.6-mm × 3-cm PEEK column tightly packed with zinc powder. [NOTE—Prepare the postcolumn reactor daily, or as necessary, to meet the system suitability requirements.] The flow rate is about 1.0 mL per minute. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the relative retention times are 1.0 for the internal standard and 1.4 for phytonadione; the column efficiency for the phytonadione peak is not less than 2500 theoretical plates; the tailing factor for the phytonadione peak is not more than 1.5; and the relative standard deviation for replicate injections is not more than 3.0%.
Procedure— Separately inject equal volumes (about 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— [NOTE—Use 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. [NOTE—Prepare 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. Cool rapidly, dilute with cyclohexane to volume, and mix.
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 metaphosphoric–acetic 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 metaphosphoric–acetic acids TS, and titrate with standard dichlorophenol–indophenol solution VS to a rose-pink color that persists for at least 5 seconds. Correct for the volume of dichlorophenol–indophenol solution consumed by a mixture of 5.5 mL of metaphosphoric–acetic acids TS and 15 mL of water. From the ascorbic acid equivalent of the standard dichlorophenol–indophenol 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 16.
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 16.
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 16.
Assay for biotin, Method 1— [NOTE—Use low-actinic glassware throughout this procedure.]