Oil- and Water-Soluble Vitamins Tablets
DEFINITION
Oil- and Water-Soluble Vitamins 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; and 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. Tablets contain NLT 90.0% and NMT 165.0% 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), alpha tocopheryl acetate (C31H52O3), or alpha tocopheryl acid succinate (C33H54O5); phytonadione (C31H46O2); and beta carotene (C40H56); and NLT 90.0% and NMT 150.0% 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.
They do not contain any minerals. They may contain other labeled added substances that are generally recognized as safe, in amounts that are unobjectionable.
STRENGTH
[Note—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. ]
• 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 retinyl acetate. It is to be used where USP Vitamin A RS is specified. Use low-actinic glassware throughout this procedure. ]
Mobile phase:
n-Hexane
Standard solution:
15 µg/mL of retinyl acetate from USP Vitamin A RS in n-hexane
System suitability stock solution:
15 µg/mL of retinyl palmitate in n-hexane
System suitability solution:
Mix equal volumes of the System suitability stock solution and the Standard solution to obtain concentrations of 7.5 µg/mL each of retinyl acetate and retinyl palmitate.
Sample solution:
Finely powder NLT 20 Tablets. Transfer a 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 min 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 min, 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 min, 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. Dilute a 10-mL volume of this solution with n-hexane to obtain a solution with a concentration of 15 µg/mL of vitamin A as retinol (C20H30O).
. [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 min, 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 min, 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. Dilute a 10-mL volume of this solution with n-hexane to obtain a solution with a concentration of 15 µg/mL of vitamin A as retinol (C20H30O).
Chromatographic system
Mode:
LC
Detector:
UV 325 nm
Column:
4.6-mm × 15-cm; 3-µm packing L8
Flow rate:
1 mL/min
Injection volume:
40 µL
System suitability
Sample:
System suitability solution
Suitability requirements
Resolution:
NLT 10 between all-trans-retinyl acetate and all-trans-retinyl palmitate
Relative standard deviation:
NMT 3.0%
Analysis
Samples:
Standard solution and Sample solution
Measure the peak area for all-trans-retinyl acetate from the Standard solution and the peak area for all-trans-retinyl acetate or all-trans-retinyl palmitate from the Sample solution. For products containing vitamin A acetate or vitamin A palmitate, calculate the percentage of the labeled amount of vitamin A, as retinol (C20H30O), in the portion of Tablets taken:
Result = (rU/rS) × (CS/CU) × F × 100
| rU | = | = peak area of the all-trans-retinyl ester from the Sample solution |
| rS | = | = peak area of the all-trans-retinyl ester from the Standard solution |
| CS | = | = concentration of retinyl acetate (C22H32O2) from USP Vitamin A RS in the Standard solution (µg/mL) |
| CU | = | = nominal concentration of vitamin A, as retinol (C20H30O), in the Sample solution (µg/mL) |
| F | = | = factor used to convert retinyl acetate, the ester form present in USP Vitamin A RS, to retinol, 0.872 |
[Note—The molar responses of retinyl acetate and retinyl palmitate are equivalent. ]
Acceptance criteria:
90.0%–165.0% of the labeled amount of vitamin A, as retinol (C20H30O)
• 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 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, and dilute with methanol to volume.
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, and dilute with water to volume.
Lecithin solution:
5 mg/mL of lecithin in 2,2,4-trimethylpentane
Mobile phase:
n-Hexane and ethyl acetate (99.7: 0.3)
Standard solution:
15 µg/mL of retinyl acetate from USP Vitamin A RS in 2,2,4-trimethylpentane
System suitability stock solution:
15 µg/mL of retinyl palmitate in 2,2,4-trimethylpentane
System suitability solution:
Mix equal volumes of the System suitability stock solution and the Standard solution to obtain concentrations of 7.5 µg/mL each of retinyl acetate and retinyl palmitate.
Sample solution:
[Note—This preparation is suitable for the determination of vitamin A, vitamin D, and vitamin E, when present in the formulation. ] Finely powder NLT 20 Tablets. If vitamin D is present in the formulation, transfer a portion of the powder, equivalent to 30 µg of the labeled amount of cholecalciferol or ergocalciferol (vitamin D), 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 90 mg of the labeled amount 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 2.5 mg of the labeled amount of vitamin A, as retinyl acetate or retinyl palmitate. Add 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 min. Add 6 mL of dimethyl sulfoxide, mix on a vortex mixer to form a suspension, and shake for 12 min. Add 6 mL of 3 N methanolic sulfuric acid solution, mix on a vortex mixer to form a suspension, and shake for 12 min. Add 12.5 mL of 2,2,4-trimethylpentane, mix on a vortex mixer to form a suspension, and shake for 10 min. Centrifuge for 10 min 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 concentration close to that of the Standard solution.
Chromatographic system
Mode:
LC
Detector:
UV 325 nm
Column:
4.6-mm × 25-cm; 5-µm packing L24
Flow rate:
1.5 mL/min
Injection volume:
40 µL
System suitability
Sample:
System suitability solution
Suitability requirements
Resolution:
NLT 8.0 between all-trans-retinyl acetate and all-trans-retinyl palmitate
Relative standard deviation:
NMT 3.0%
Analysis
Samples:
Standard solution and Sample solution
Measure the peak area for all-trans-retinyl acetate from the Standard solution and the peak area of all-trans-retinyl acetate or all-trans-retinyl palmitate from the Sample solution.
Calculate the percentage of the labeled amount of vitamin A, as retinol (C20H30O), in the portion of Tablets taken:
Result = (rU/rS) × (CS/CU) × F × 100
| rU | = | = peak area of the all-trans-retinyl ester from the Sample solution |
| rS | = | = peak area of the all-trans-retinyl ester from the Standard solution |
| CS | = | = concentration of retinyl acetate (C22H32O2) from USP Vitamin A RS in the Standard solution (µg/mL) |
| CU | = | = nominal concentration of vitamin A, as retinol (C20H30O), in the Sample solution (µg/mL) |
| F | = | = factor used to convert retinyl acetate, the ester form present in the USP Vitamin A RS, to retinol, 0.872 |
[Note—Account for the initial extraction volume of 26.5 mL of 2,2,4-trimethylpentane to calculate the nominal concentration. The molar responses of retinyl acetate and retinyl palmitate are equivalent. ]
Acceptance criteria:
90.0%–165.0% of the labeled amount of vitamin A, as retinol (C20H30O)
• 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 retinyl acetate. It is to be used where USP Vitamin A RS is specified. Use low-actinic glassware throughout this procedure. ]
Extraction solvent:
n-Hexane and methylene chloride (3:1)
Potassium hydroxide solution:
800 mg/mL of potassium hydroxide in water. [Note—Cautiously add the potassium hydroxide to the water. Mix, and cool. ]
Diluent:
10 mg/mL of pyrogallol in alcohol
Mobile phase:
n-Hexane and isopropyl alcohol (92:8)
Standard stock solution:
30 µg/mL of retinyl acetate from USP Vitamin A RS in Diluent. [Note—This solution may be stored in a refrigerator for 1 week. ]
Standard solution:
Dilute a volume of the Standard stock solution with Diluent to obtain a concentration of 1 µg/mL of USP Vitamin A RS. Transfer 10.0 mL of this solution to a stoppered 125-mL flask, and add 5 mL of water, 5 mL of Diluent, and 3 mL of Potassium hydroxide solution. Insert the stopper tightly, shake for 15 min 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 s. Rinse the sides of the flask with 60 mL of water, and allow to stand for 10 min until the layers separate. Withdraw a portion of the organic layer for injection into the chromatograph. This Standard solution contains 0.34 µg/mL of retinol.
, 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 s. Rinse the sides of the flask with 60 mL of water, and allow to stand for 10 min until the layers separate. Withdraw a portion of the organic layer for injection into the chromatograph. This Standard solution contains 0.34 µg/mL of retinol.
Sample solution:
Finely powder a counted number of Tablets. Transfer a portion of the powder, equivalent to 1.5 mg of retinyl acetate, to a stoppered 125-mL flask. Add 5 mL of water, 15 mL of Diluent, and 3 mL of Potassium hydroxide solution. Insert the stopper tightly, shake for 15 min 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 s or longer, if necessary, for complete extraction. Rinse the sides of the flask with 60 mL of water, and allow to stand for 10 min until the layers separate. [Note—Do not shake, because an emulsion may form. ] Withdraw a portion of the organic layer, and dilute with Extraction solvent to obtain a concentration of 0.34 µg/mL of retinol.
, 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 s or longer, if necessary, for complete extraction. Rinse the sides of the flask with 60 mL of water, and allow to stand for 10 min until the layers separate. [Note—Do not shake, because an emulsion may form. ] Withdraw a portion of the organic layer, and dilute with Extraction solvent to obtain a concentration of 0.34 µg/mL of retinol.
Chromatographic system
Mode:
LC
Detector:
UV 335 nm
Column:
6.2-mm × 8-cm; packing L3
Column temperature:
40
Flow rate:
4 mL/min
Injection volume:
50 µL
System suitability
Sample:
Standard solution
[Note—The relative retention times for 13-cis-retinol and all-trans-retinol are 0.92 and 1.0, respectively. ]
Suitability requirements
Relative standard deviation:
NMT 5.0%
Analysis
Samples:
Standard solution and Sample solution
Measure the peak areas for all-trans-retinol and 13-cis-retinol. Calculate the percentage of the labeled amount of vitamin A, as retinol (C20H30O), in the portion of Tablets taken:
Result = (rT1/rT2) × (CS/CU) × F × 100
| rT1 | = | = sum of the areas of the all-trans-retinol and 13-cis-retinol peaks from the Sample solution |
| rT2 | = | = sum of the areas of the all-trans-retinol and 13-cis-retinol peaks from the Standard solution |
| CS | = | = concentration of retinyl acetate (C23H32O2) from USP Vitamin A RS in the Standard solution (µg/mL) |
| CU | = | = nominal concentration of vitamin A, as retinol (C20H30O), in the Sample solution (µg/mL) |
| F | = | = factor used to convert retinyl acetate, the ester form present in USP Vitamin A RS, to retinol, 0.872 |
Acceptance criteria:
90.0%–165.0% of the labeled amount of vitamin A, as retinol (C20H30O)
• 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:
n-Hexane and isopropyl alcohol (99:1)
Standard solution:
2 µg/mL of USP Cholecalciferol RS or USP Ergocalciferol RS in n-hexane
System suitability solution:
Heat a volume of the Standard solution at 60 for 1 h to partially isomerize vitamin D (cholecalciferol or ergocalciferol) to its corresponding precursor.
for 1 h to partially isomerize vitamin D (cholecalciferol or ergocalciferol) to its corresponding precursor.
Sample solution:
Proceed as directed for the Sample solution in Vitamin A, Method 1. Transfer NLT 20 mL of this solution to a suitable container, and evaporate, if necessary, under vacuum at room temperature to obtain a concentration of 2 µg/mL of cholecalciferol or ergocalciferol.
Chromatographic system
Mode:
LC
Detector:
UV 265 nm
Column:
4.6-mm × 15-cm; 3-µm packing L8
Flow rate:
1 mL/min
Injection volume:
100 µL
System suitability
Samples:
Standard solution and System suitability solution
Suitability requirements
Resolution:
NLT 10 between the vitamin D form present and its corresponding precursor, System suitability solution
Relative standard deviation:
NMT 3.0%, Standard solution
Analysis
Samples:
Standard solution and Sample solution
Measure the peak areas for vitamin D. Calculate the percentage of the labeled amount of cholecalciferol (C27H44O) or ergocalciferol (C28H44O) in the portion of Tablets taken:
Result = (rU/rS) × (CS/CU) × F × 100
| rU | = | = peak area of cholecalciferol or ergocalciferol from the Sample solution |
| rS | = | = peak area of cholecalciferol or ergocalciferol from the Standard solution |
| CS | = | = concentration of USP Cholecalciferol RS or USP Ergocalciferol RS in the Standard solution (µg/mL) |
| CU | = | = nominal concentration of cholecalciferol or ergocalciferol in the Sample solution (µg/mL) |
| F | = | = correction factor to account for the average amount of previtamin D present in the Sample solution, 1.09 |
Acceptance criteria:
90.0%–165.0% of the labeled amount of vitamin D as cholecalciferol (C27H44O) or ergocalciferol (C28H44O)
• 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 Sample solution:
Proceed as directed in Vitamin A, Method 2.
Mobile phase:
n-Hexane and tertiary butyl alcohol (98.75: 1.25)
Standard solution:
1 µg/mL of USP Cholecalciferol RS or USP Ergocalciferol RS in 2,2,4-trimethylpentane
System suitability solution:
Heat a volume of the Standard solution at 60 for 1 h to partially isomerize vitamin D (cholecalciferol or ergocalciferol) to its corresponding precursor.
for 1 h to partially isomerize vitamin D (cholecalciferol or ergocalciferol) to its corresponding precursor.
Chromatographic system
Mode:
LC
Detector:
UV 265 nm
Column:
4.6-mm × 25-cm; 5-µm packing L24
Flow rate:
1 mL/min
Injection volume:
40 µL
System suitability
Samples:
Standard solution and System suitability solution
Suitability requirements
Resolution:
NLT 4.0 between the vitamin D form present and its corresponding precursor, System suitability solution
Relative standard deviation:
NMT 3.0%, Standard solution
Analysis
Samples:
Standard solution and Sample solution
Measure the peak areas for vitamin D. Calculate the percentage of the labeled amount of cholecalciferol (C27H44O) or ergocalciferol (C28H44O) in the portion of Tablets taken:
Result = (rU/rS) × (CS/CU) × 100
| rU | = | = peak area of cholecalciferol or ergocalciferol from the Sample solution |
| rS | = | = peak area of cholecalciferol or ergocalciferol from the Standard solution |
| CS | = | = concentration of USP Cholecalciferol RS or USP Ergocalciferol RS in the Standard solution (µg/mL) |
| CU | = | = nominal concentration of cholecalciferol or ergocalciferol in the Sample solution (µg/mL) |
[Note—Account for the initial extraction volume of 26.5 mL of 2,2,4-trimethylpentane to calculate the nominal concentration. ]
Acceptance criteria:
90.0%–165.0% of the labeled amount of vitamin D as cholecalciferol (C27H44O) or ergocalciferol (C28H44O)
• 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. ]
Diluted acetic acid:
Glacial acetic acid solution (1 in 10) in water
Phenolphthalein solution:
10 mg/mL of phenolphthalein in 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:
Methylene chloride and isopropyl alcohol (99.8: 0.2)
Mobile phase:
Acetonitrile and methanol (91:9)
Standard stock solution:
0.2 mg/mL of USP Cholecalciferol RS or USP Ergocalciferol RS in dehydrated alcohol. [Note—Prepare fresh every 4 weeks. Store in a freezer. ]
Standard solution:
[Note—Condition the solid-phase extraction column specified for use in the Standard solution and the Sample solution by initially washing the column with 4.0 mL of a mixture of methylene chloride and isopropyl alcohol (4:1), followed by 5.0 mL of Extraction solvent. Do not allow the column to dry. ] Dilute a volume of Standard stock solution with dehydrated alcohol to obtain a concentration of 5 µg/mL of USP Cholecalciferol RS or USP Ergocalciferol RS. 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 min 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 s, and allow to stand for 15 min 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 10 min 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 Diluted acetic acid dropwise, with shaking, until the washing is neutral. Allow to stand for 10 min 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 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.
. 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 s, and allow to stand for 15 min 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 10 min 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 Diluted acetic acid dropwise, with shaking, until the washing is neutral. Allow to stand for 10 min 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 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.
Sample solution:
Finely powder NLT 20 Tablets. Transfer a portion of the powder, equivalent to 10 µg of cholecalciferol or ergocalciferol, to a stoppered 125-mL flask, and proceed as directed for the Standard solution, beginning with “Add 15.0 mL of water and 15.0 mL of Potassium hydroxide solution”.
Chromatographic system
Mode:
LC
Detector:
UV 265 nm
Column:
4.6-mm × 25-cm; 5-µm packing L1
Column temperature:
27
Flow rate:
0.7 mL/min
Injection volume:
15 µL
System suitability
Sample:
Standard solution
Suitability requirements
Relative standard deviation:
NMT 4.0%
Analysis
Samples:
Standard solution and Sample solution
Measure the peak areas for vitamin D. Calculate the percentage of the labeled amount of cholecalciferol (C27H44O) or ergocalciferol (C28H44O) in the portion of Tablets taken:
Result = (rU/rS) × (CS/CU) × 100
| rU | = | = peak area of cholecalciferol or ergocalciferol from the Sample solution |
| rS | = | = peak area of cholecalciferol or ergocalciferol from the Standard solution |
| CS | = | = concentration of USP Cholecalciferol RS or USP Ergocalciferol RS in the Standard solution (µg/mL) |
| CU | = | = nominal concentration of cholecalciferol or ergocalciferol in the Sample solution (µg/mL) |
Acceptance criteria:
90.0%–165.0% of the labeled amount of vitamin D as cholecalciferol (C27H44O) or ergocalciferol (C28H44O)
• 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. ]
Solution A:
Phosphoric acid solution (1 in 100) in water
Mobile phase:
Methanol and Solution A (19:1)
System suitability solution:
Prepare a 0.65-mg/mL solution of USP Ergocalciferol RS in methanol. Transfer 1.0 mL of this solution to a 100-mL volumetric flask containing 100 mg of USP Alpha Tocopheryl Acetate RS. Dissolve in 30 mL of methanol, with the aid of sonication if necessary, and dilute with methanol to volume. Store this solution in a refrigerator.
Standard solution:
2 mg/mL of USP Alpha Tocopherol RS, USP Alpha Tocopheryl Acetate RS, or USP Alpha Tocopheryl Acid Succinate RS in methanol
Sample solution:
Proceed as directed for the Sample solution in Vitamin A, Method 1. Transfer NLT 20 mL of this solution to a suitable container, and evaporate if necessary, under vacuum at room temperature to dryness. Transfer the residue to a suitable volumetric flask with the aid of methanol, and dilute with methanol to volume to obtain a concentration of 2 mg/mL of alpha tocopherol, alpha tocopheryl acetate, or alpha tocopheryl acid succinate.
Chromatographic system
Mode:
LC
Detector:
UV 254 nm
Column:
8-mm × 10-cm; 5-µm packing L1
Flow rate:
2 mL/min
Injection volume:
100 µL
System suitability
Samples:
System suitability solution and Standard solution
[Note—The relative retention times for ergocalciferol and alpha tocopheryl acetate are about 0.5 and 1.0, respectively. ]
Suitability requirements
Resolution:
NLT 12 between ergocalciferol and alpha tocopheryl acetate, System suitability solution
Tailing factor:
Between 0.8 and 1.2, System suitability solution
Relative standard deviation:
NMT 3.0%, Standard solution
Analysis
Samples:
Standard solution and Sample solution
Measure the peak areas. Calculate the percentage of the labeled amount of alpha tocopherol (C29H50O2), alpha tocopheryl acetate (C31H52O3), or alpha tocopheryl acid succinate (C33H54O5) in the portion of Tablets taken:
Result = (rU/rS) × (CS/CU) × 100
| rU | = | = peak area of the relevant vitamin E form from the Sample solution |
| rS | = | = peak area of the relevant vitamin E form from the Standard solution |
| CS | = | = concentration of the corresponding USP Reference Standard in the Standard solution (mg/mL) |
| CU | = | = nominal concentration of the corresponding form of vitamin E in the Sample solution (mg/mL) |
Acceptance criteria:
90.0%–165.0% of the labeled amount of vitamin E as alpha tocopherol (C29H50O2), alpha tocopheryl acetate (C31H52O3), or alpha tocopheryl acid succinate (C33H54O5)
• 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:
Mix 240 mL of methanol with 10 mL of water, followed by 0.5 mL of 50% phosphoric acid, and dilute with acetonitrile to 1000 mL.
System suitability solution:
2 mg/mL each of USP Alpha Tocopherol RS, USP Alpha Tocopheryl Acetate RS, and USP Alpha Tocopheryl Acid Succinate RS in methanol
Standard solution:
2 mg/mL of USP Alpha Tocopherol RS, USP Alpha Tocopheryl Acetate RS, or USP Alpha Tocopheryl Acid Succinate RS in methanol
Sample solution:
Proceed as directed for the Sample solution in Vitamin A, Method 2. Transfer a volume of the supernatant 2,2,4-trimethylpentane layer to a suitable volumetric flask, the volume of the specimen withdrawn from the 2,2,4-trimethylpentane layer and the size of the volumetric flask being such that the final concentration of the Sample solution is equivalent to that of the Standard solution. Evaporate nearly to dryness, add several mL of methanol, and evaporate the remaining 2,2,4-trimethylpentane. Dilute with methanol to volume.
Chromatographic system
Mode:
LC
Detector:
UV 280 nm
Column:
4.6-mm × 25-cm; 5-µm packing L1
Flow rate:
1.5 mL/min
Injection volume:
25 µL
System suitability
Samples:
System suitability solution and Standard solution
[Note—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. ]
Suitability requirements
Resolution:
NLT 4.0 between alpha tocopheryl acid succinate and alpha tocopherol and NLT 3.0 between alpha tocopherol and alpha tocopheryl acetate, System suitability solution
Relative standard deviation:
NMT 3.0%, Standard solution
Analysis
Samples:
Standard solution and Sample solution
Measure the peak areas. Calculate the percentage of the labeled amount of alpha tocopherol (C29H50O2), alpha tocopheryl acetate (C31H52O3), or alpha tocopheryl acid succinate (C33H54O5) in the portion of Tablets taken:
Result = (rU/rS) × (CS/CU) × 100
| rU | = | = peak area of the relevant vitamin E form from the Sample solution |
| rS | = | = peak area of the relevant vitamin E form from the Standard solution |
| CS | = | = concentration of the corresponding USP Reference Standard in the Standard solution (mg/mL) |
| CU | = | = nominal concentration of the corresponding form of vitamin E in the Sample solution (mg/mL) |
[Note—Account for the initial extraction volume of 26.5 mL of 2,2,4-trimethylpentane and the dilution factor to exchange the solvent from 2,2,4-trimethylpentane to methanol to calculate the nominal concentration. ]
Acceptance criteria:
90.0%–165.0% of the labeled amount of vitamin E as alpha tocopherol (C29H50O2), alpha tocopheryl acetate (C31H52O3), or alpha tocopheryl acid succinate (C33H54O5)
• Vitamin E, Method 3
Diluent:
Acetonitrile and ethyl acetate (1:1)
Mobile phase:
Methanol, acetonitrile, and n-hexane (46.5: 46.5: 7.0)
Standard solution:
0.3 mg/mL of USP Alpha Tocopherol RS in methanol
Sample solution:
Finely powder NLT 20 Tablets. Transfer a portion of the powder, equivalent to 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 h 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 s, 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 Diluent, and swirl to dissolve the residue.
. 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 s, 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 Diluent, and swirl to dissolve the residue.
Chromatographic system
Mode:
LC
Detector:
UV 291 nm
Column:
4.6-mm × 25-cm; packing L1
Column temperature:
40
Flow rate:
3 mL/min
Injection volume:
20 µL
System suitability
Sample:
Standard solution
Suitability requirements
Relative standard deviation:
NMT 5.0%
Analysis
Samples:
Standard solution and Sample solution
Measure the peak areas. Calculate the percentage of the labeled amount of vitamin E as alpha tocopherol (C29H50O2) in the portion of Tablets taken:
Result = (rU/rS) × (CS/CU) × 100
| rU | = | = peak area of alpha tocopherol from the Sample solution |
| rS | = | = peak area of alpha tocopherol from the Standard solution |
| CS | = | = concentration of alpha tocopherol in the Standard solution (mg/mL) |
| CU | = | = nominal concentration of vitamin E as alpha tocopherol in the Sample solution (mg/mL) |
[Note—Calculate the content of alpha tocopheryl acetate (C31H52O3) or alpha tocopheryl acid succinate (C33H54O5) by dividing the content, in mg/Tablet of vitamin E as alpha tocopherol (C29H50O2), by the factor 0.91 or 0.81, respectively. ]
Acceptance criteria:
90.0%–165.0% of the labeled amount of vitamin E as alpha tocopherol (C29H50O2), alpha tocopheryl acetate (C31H52O3), or alpha tocopheryl acid succinate (C33H54O5)
• Phytonadione, Method 1
[Note—Use low-actinic glassware throughout this procedure. ]
Mobile phase:
Methanol and water (19:1)
Standard stock solution:
200 µg/mL of USP Phytonadione RS in methanol. Dissolve with the aid of sonication if necessary.
System suitability solution:
0.65 mg/mL of USP Alpha Tocopheryl Acetate RS and 20 µg/mL of USP Phytonadione RS from the Standard stock solution diluted with methanol. [Note—Dissolve USP Alpha Tocopheryl Acetate RS in a portion of methanol, add the Standard stock solution, and then dilute with methanol to volume. ]
Standard solution:
20 µg/mL of USP Phytonadione RS from the Standard stock solution diluted with methanol
Sample solution:
Transfer NLT 20 mL of the solution retained as specified in the directions for the Sample solution in Vitamin A, Method 1 to a suitable container, and evaporate, if necessary, under vacuum at room temperature to dryness. Transfer the residue to a suitable volumetric flask with the aid of methanol, and dilute with methanol to volume to obtain a concentration of 20 µg/mL of phytonadione.
Chromatographic system
Mode:
LC
Detector:
UV 254 nm
Column:
8-mm × 10-cm; 5-µm packing L1
Flow rate:
2 mL/min
Injection volume:
100 µL
System suitability
Samples:
System suitability solution and Standard solution
[Note—The relative retention times for alpha tocopheryl acetate and phytonadione are 0.68 and 1.0, respectively. ]
Suitability requirements
Resolution:
NLT 5 between alpha tocopheryl acetate and phytonadione, System suitability solution
Relative standard deviation:
NMT 3.0%, Standard solution
Analysis
Samples:
Standard solution and Sample solution
Measure the peak areas. Calculate the percentage of the labeled amount of phytonadione (C31H46O2) in the portion of Tablets taken:
Result = (rU/rS) × (CS/CU) × 100
| rU | = | = peak area of phytonadione from the Sample solution |
| rS | = | = peak area of phytonadione from the Standard solution |
| CS | = | = concentration of USP Phytonadione RS in the Standard solution (µg/mL) |
| CU | = | = nominal concentration of phytonadione in the Sample solution (µg/mL) |
Acceptance criteria:
90.0%–165.0% of the labeled amount of phytonadione (C31H46O2)
• Phytonadione, Method 2
[Note—Use low-actinic glassware throughout this procedure. ]
Solvent:
Methanol and isopropyl alcohol (19:1)
Mobile phase:
Mix 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:
5 µg/mL of menaquinone 4 (vitamin K2) in Solvent. [Note—A concentrated stock solution of menaquinone 4 (100 µg/mL) can be stored for 2 months in a refrigerator. ]
Standard stock solution:
5 µg/mL of USP Phytonadione RS, prepared by dissolving in methylene chloride with the aid of sonication, and diluting with Solvent to final volume.
Standard solution:
Transfer 1.0 mL of the Standard stock solution and 1.0 mL of the Internal standard solution to a suitable flask, and dilute with Solvent to 5 mL. Pass through a membrane filter of 0.45-µm or finer pore size.
Sample solution:
Finely powder NLT 20 Tablets. To a centrifuge tube fitted with a cap transfer an 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 min. Remove from the bath, and again shake or mix using a vortex mixer for 1 min 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 min. Remove from the bath, and again shake or mix using a vortex mixer for 2 min while the preparation is still hot. Cool to room temperature. Add a volume of the Internal standard solution, equivalent to 1.0 mL for 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 min to thoroughly mix the contents. Centrifuge to separate the two layers. Transfer a volume of the top layer of petroleum ether, equivalent to 5–50 µg of the nominal amount of phytonadione, to an appropriate flask. Place the flask in a water bath at 35–45, 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 concentration of 1 µg/mL of phytonadione.
for 5 min. Remove from the bath, and again shake or mix using a vortex mixer for 1 min 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 min. Remove from the bath, and again shake or mix using a vortex mixer for 2 min while the preparation is still hot. Cool to room temperature. Add a volume of the Internal standard solution, equivalent to 1.0 mL for 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 min to thoroughly mix the contents. Centrifuge to separate the two layers. Transfer a volume of the top layer of petroleum ether, equivalent to 5–50 µg of the nominal amount of phytonadione, to an appropriate flask. Place the flask in a water bath at 35–45, 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 concentration of 1 µg/mL of phytonadione.
Chromatographic system
Mode:
LC
Detector:
Fluorometric detector set at 320 nm for excitation and 420 nm for emission
Column:
4.6-mm × 25-cm; 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. ]
Flow rate:
1 mL/min
Injection volume:
25 µL
System suitability
Sample:
Standard solution
[Note—The relative retention times for the internal standard and phytonadione are 1.0 and 1.4, respectively. ]
Suitability requirements
Column efficiency:
NLT 2500 theoretical plates for the phytonadione peak
Tailing factor:
NMT 1.5 for the phytonadione peak
Relative standard deviation:
NMT 3.0%
Analysis
Samples:
Standard solution and Sample solution
Calculate the percentage of the labeled amount of phytonadione (C31H46O2) in the portion of Tablets taken:
Result = (RU/RS) × (CS/CU) × 100
| RU | = | = peak response ratio of phytonadione to that of the internal standard from the Sample solution |
| RS | = | = peak response ratio of phytonadione to that of the internal standard from the Standard solution |
| CS | = | = concentration of USP Phytonadione RS in the Standard solution (µg/mL) |
| CU | = | = nominal concentration of phytonadione in the Sample solution (µg/mL) |
Acceptance criteria:
90.0%–165.0% of labeled amount of phytonadione (C31H46O2)
• 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:
0.01 mg/mL of iodine in cyclohexane. [Note—Prepare this solution fresh daily. ]
Sample solution:
Weigh NLT 20 Tablets. Grind the Tablets to a fine powder, and transfer a quantity of the powder, equivalent to 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 min with constant stirring. Cool to room temperature. Add 170 mL of solvent hexane, and stir for 30 min. Quantitatively transfer the contents of the flask to a 500-mL separatory funnel with portions of solvent hexane. Allow the layers to separate for 5–10 min, 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 min. 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 min. 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 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 min in a water bath maintained at 65. Cool rapidly, and dilute with cyclohexane to volume.
. Cool rapidly, and dilute with cyclohexane to volume.
Instrumental conditions
Mode:
Vis
Analytical wavelength:
452 nm
Blank:
Cyclohexane
Analysis
Sample:
Sample solution
Determine the absorbance against the Blank. Calculate the percentage of the labeled amount of beta carotene (C40H56) in the portion of Tablets taken:
Result = (AU/F) × (100/CU)
| AU | = | = absorbance of the Sample solution |
| F | = | = absorptivity of beta carotene at 452 nm, 223 |
| CU | = | = nominal concentration of beta carotene in the Sample solution (mg/mL) |
Acceptance criteria:
90.0%–165.0% of the labeled amount of beta carotene (C40H56)
• Ascorbic Acid
Sample solution:
Finely powder NLT 20 Tablets. Transfer a portion of the powder, equivalent of 100 mg of ascorbic acid, to a 200-mL volumetric flask, and add 75 mL of metaphosphoric–acetic acids TS. Insert a stopper into the flask, and shake by mechanical means for 30 min. Dilute with water to volume. 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, and add 5 mL of metaphosphoric–acetic acids TS.
Analysis:
Titrate with standard dichlorophenol–indophenol solution VS to a rose-pink color that persists for at least 5 s. 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.
Acceptance criteria:
90.0%–150.0% of the labeled amount of ascorbic acid (C6H8O6)
• Calcium Ascorbate:
Proceed as directed in Ascorbic Acid.
Acceptance criteria:
90.0%–150.0% of the labeled amount of calcium ascorbate (C12H14CaO12·2H2O)
• Sodium Ascorbate:
Proceed as directed in Ascorbic Acid.
Acceptance criteria:
90.0%–150.0% of the labeled amount of sodium ascorbate (C6H7NaO6)
• Biotin, Method 1
[Note—Use low-actinic glassware throughout this procedure. ]
Mobile phase:
Mix 85 mL of acetonitrile, 1 g of sodium perchlorate, 1 mL of phosphoric acid, and dilute with water to 1000 mL.
Standard stock solution:
0.333 mg/mL of USP Biotin RS in dimethyl sulfoxide
Standard solution:
5 µg/mL of USP Biotin RS prepared by diluting the Standard stock solution in water
Sample solution:
Finely powder NLT 20 Tablets. Transfer a portion of the powder, equivalent to 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 60–70 for 5 min. Sonicate for 5 min, dilute with water to volume, and filter.
–70 for 5 min. Sonicate for 5 min, dilute with water to volume, and filter.
Chromatographic system
Mode:
LC
Detector:
UV 200 nm
Column:
4.6-mm × 15-cm; 3-µm packing L7
Flow rate:
1.2 mL/min
Injection volume:
100 µL
System suitability
Sample:
Standard solution
Suitability requirements
Relative standard deviation:
NMT 3.0%
Analysis
Samples:
Standard solution and Sample solution
Measure the peak areas of biotin. Calculate the percentage of the labeled amount of biotin (C10H16N2O3S) in the portion of Tablets taken:
Result = (rU/rS) × (CS/CU) × 100
| rU | = | = peak area of biotin from the Sample solution |
| rS | = | = peak area of biotin from the Standard solution |
| CS | = | = concentration of USP Biotin RS in the Standard solution (µg/mL) |
| CU | = | = nominal concentration of biotin in the Sample solution (µg/mL) |
Acceptance criteria:
90.0%–150.0% of the labeled amount of biotin (C10H16N2O3S)
• Biotin, Method 2
[Note—Use 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:
50 µg/mL of USP Biotin RS in 50% alcohol. Store this solution in a refrigerator.
Standard solution:
0.1 ng/mL of USP Biotin RS in water, prepared by dilution of the Standard stock solution with water on the day of the assay
Sample solution:
Finely powder NLT 30 Tablets. Transfer a portion of the powder, equivalent to 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 60–70 for 5 min. Sonicate for 5 min, dilute with 50% alcohol to volume, and filter. Dilute a volume of the filtrate quantitatively, and stepwise if necessary, with water to obtain a solution with a concentration of 0.1 ng/mL.
–70 for 5 min. Sonicate for 5 min, dilute with 50% alcohol to volume, and filter. Dilute a volume of the filtrate quantitatively, and stepwise if necessary, with water to obtain a solution with a concentration of 0.1 ng/mL.
Acid-hydrolyzed casein solution:
Mix 100 g of vitamin-free casein with 500 mL of 6 N hydrochloric acid, and reflux the mixture for 8–12 h. 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 dilute with water to make 1000 mL. Add 20 g of activated charcoal, stir for 1 h, and filter. Repeat the treatment with activated charcoal. Store under toluene in a cool place at a temperature not below 10. Filter the solution if a precipitate forms during storage.
. Filter the solution if a precipitate forms during storage.
Cystine–tryptophan solution:
Suspend 4.0 g of l-cystine in a solution of 1.0 g of l-tryptophan (or 2.0 g of d,l-tryptophan) in 700–800 mL of water. Heat to 70–80, and add dilute hydrochloric acid (1 in 2) dropwise, with stirring, until the solids are dissolved. Cool, and dilute with water to make 1000 mL. Store under toluene in a cool place at a temperature NLT 10.
–80, and add dilute hydrochloric acid (1 in 2) dropwise, with stirring, until the solids are dissolved. Cool, and dilute with water to make 1000 mL. Store under toluene in a cool place at a temperature NLT 10.
Adenine–guanine–uracil 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:
100 mg/mL of polysorbate 80 in alcohol
Calcium pantothenate solution:
10 µg/mL of calcium pantothenate in 50% alcohol. Store in a refrigerator.
Riboflavin–thiamine hydrochloride solution:
20 µg/mL of riboflavin and 10 µg/mL of thiamine hydrochloride in 0.02 N acetic acid. Store under toluene, protected from light, in a refrigerator.
p-Aminobenzoic acid–niacin–pyridoxine hydrochloride solution:
10 µg/mL of p-aminobenzoic acid, 50 µg/mL of niacin, and 40 µg/mL of pyridoxine hydrochloride in a mixture of neutralized alcohol and water (1:3). Store in a refrigerator.
Salt solution A:
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. Store under toluene.
Salt solution B:
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 according to Table 1, and adjust with 1 N sodium hydroxide to a pH of 6.8. Dilute with water to 250 mL.
Table 1
| Acid-hydrolyzed casein solution | 25 mL |
| Cystine–tryptophan solution | 25 mL |
| Polysorbate 80 solution | 0.25 mL |
| Dextrose, anhydrous | 10 g |
| Sodium acetate, anhydrous | 5 g |
| Adenine–guanine–uracil solution | 5 mL |
| Calcium pantothenate solution | 5 mL |
| Riboflavin–thiamine hydrochloride solution | 5 mL |
| p-Aminobenzoic acid–niacin–pyridoxine hydrochloride solution | 5 mL |
| Salt solution A | 5 mL |
| Salt solution B | 5 mL |
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 for 15 min, and allow the tubes to cool in an upright position. Prepare stab cultures in three or more of the tubes, using a pure culture of Lactobacillus plantarum,1 incubating for 16–24 h at a temperature between 30 and 37 held constant to within ±0.5. Store in a refrigerator. Prepare a fresh stab of the stock culture every week, and do not use for Inoculum if the culture is more than 1 week old.
for 15 min, and allow the tubes to cool in an upright position. Prepare stab cultures in three or more of the tubes, using a pure culture of Lactobacillus plantarum,1 incubating for 16–24 h at a temperature between 30 and 37 held constant to within ±0.5. Store in a refrigerator. Prepare a fresh stab of the stock culture every week, and do not use for Inoculum if the culture is more than 1 week old.
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 for 15 min, and cool.
for 15 min, and cool.
Inoculum:
[Note—A frozen suspension of Lactobacillus plantarum may be used as the stock culture, provided it yields an inoculum comparable to a fresh culture. ]
Transfer cells from the Stock culture of Lactobacillus plantarum to a sterile tube containing 10 mL of Culture medium. Incubate this culture for 16–24 h at a temperature between 30 and 37 held constant to within ±0.5. The cell suspension so obtained is the Inoculum.
and 37 held constant to within ±0.5. The cell suspension so obtained is the Inoculum.
Analysis
Samples:
Standard solution and Sample solution
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 solution. 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 Sample solution corresponding to three or more of the levels specified for the Standard solution, 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 sample 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 for 5 min. Cool. Add 1 drop of Inoculum to each tube, except two of the four tubes containing no Standard solution (the uninoculated blanks). Incubate the tubes at a temperature of between 30 and 37 held constant to within ±0.5 until, following 16–24 h of incubation, there has been no substantial increase in turbidity in the tubes containing the highest level of Standard during a 2-h period.
for 5 min. Cool. Add 1 drop of Inoculum to each tube, except two of the four tubes containing no Standard solution (the uninoculated blanks). Incubate the tubes at a temperature of between 30 and 37 held constant to within ±0.5 until, following 16–24 h of incubation, there has been no substantial increase in turbidity in the tubes containing the highest level of Standard during a 2-h period.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 of 540–660 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 s 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 (SS) as the difference, y = 2.00 
SS. Plot this response on the ordinate of cross-section paper against the logarithm of the mL of Standard solution per tube on the abscissa, using for the ordinate either an arithmetic or a logarithmic scale, whichever gives the better approximation to a straight line. Draw the straight line or smooth curve that best fits the plotted points.
SS. Plot this response on the ordinate of cross-section paper against the logarithm of the mL of Standard solution per tube on the abscissa, using for the ordinate either an arithmetic or a logarithmic scale, whichever gives the better approximation to a straight line. Draw the straight line or smooth curve that best fits the plotted points.
Calculate the response, y = 2.00 SU, adding together the two transmittances (SU) for each level of the Sample solution. Read from the standard curve the logarithm of the volume of the Standard solution corresponding to each of those values of y that falls 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 Sample solution 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 Sample solution. Determine the quantity, in µg, of biotin (C10H16N2O3S) in the portion of Tablets taken:
SU, adding together the two transmittances (SU) for each level of the Sample solution. Read from the standard curve the logarithm of the volume of the Standard solution corresponding to each of those values of y that falls 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 Sample solution 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 Sample solution. Determine the quantity, in µg, of biotin (C10H16N2O3S) in the portion of Tablets taken: antilog M = antilog (M¢ + log R)
| R | = | = number of µg of biotin assumed to be present in the portion of Tablets taken |
Calculate the percentage of the labeled amount of biotin (C10H16N2O3S) in the portion of the Tablets taken:
Result = [(antilog M)/N] × 100
| N | = | = nominal amount of biotin in the portion of the Tablets taken (µg) |
Replication:
Repeat the entire determination at least once, using separately prepared Sample solutions. If the difference between the two log-potencies M is NMT 0.08, their mean, M, is the assayed log-potency of the test material (see Design and Analysis of Biological Assays 
111
, The Confidence Interval and Limits of Potency). If the two determinations differ by more than 0.08, conduct one or more additional determinations. From the mean of two or more values of M that do not differ by more than 0.15, compute the mean potency of the preparation under assay.
111, The Confidence Interval and Limits of Potency). If the two determinations differ by more than 0.08, conduct one or more additional determinations. From the mean of two or more values of M that do not differ by more than 0.15, compute the mean potency of the preparation under assay.
Acceptance criteria:
90.0%–150.0% of the labeled amount of biotin (C10H16N2O3S)
• Biotin, Method 3
[Note—Use low-actinic glassware throughout this procedure. ]
Solution A:
Transfer 800 mL of water and 100 mL of triethylamine to a 1000-mL volumetric flask. Add 80 mL of 85% phosphoric acid, and dilute with water to volume.
Mobile phase:
Transfer 80 mL of acetonitrile and 10 mL of Solution A to a 1000-mL volumetric flask. Dilute with water to volume.
Standard solution:
0.6 µg/mL of USP Biotin RS in water. [Note—A portion of the Standard solution will be used to determine the percent recovery of biotin from the Solid-phase extraction procedure. ]
Sample solution:
Finely powder NLT 20 Tablets. Transfer an amount of powdered Tablets to a volumetric flask to obtain a nominal concentration of 0.6 µg/mL of biotin. Add water up to 80% of the flask capacity, and sonicate for 30–40 min, with occasional mixing, to dissolve. Dilute with water to volume, and filter. Adjust the pH of the solution with either dilute acetic acid or 0.1 N sodium hydroxide to a pH of 6.0–7.0.
Solid-phase extraction:
[Note—Condition 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 each of the Sample solution and Standard solution into freshly conditioned solid-phase extraction columns consisting of a mixed-mode packing with a sorbent-mass of 60 mg. [Note—The mixed-mode packing consists of anion-exchange and reversed-phase sorbents. The reverse-phase component is a copolymer of N-vinylpyrrolidone and divinylbenzene. The anion exchange moiety is a trialkylamino group.2 ]
Wash the column with 10 mL of 30% (v/v) methanol in water. Apply an appropriate volume (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
Mode:
LC
Detector:
UV 200 nm
Column:
4.6-mm × 25-cm; packing L1
Flow rate:
2 mL/min
Injection volume:
100 µL
System suitability
Samples:
Standard solution and a portion of the Standard solution that has undergone Solid-phase extraction
Suitability requirements
Tailing factor:
NMT 1.5, Standard solution
Relative standard deviation:
NMT 2.0%, Standard solution and Standard solution that has undergone Solid-phase extraction
Recovery:
95%–100%, Standard solution that has undergone Solid-phase extraction
Analysis
Samples:
Standard solution and Sample solution that have both undergone Solid-phase extraction
Measure the peak area of biotin. Calculate the percentage of the labeled amount of biotin (C10H16N2O3S) in the portion of Tablets taken:
Result = (rU/rS) × (CS/CU) × 100
| rU | = | = peak area of biotin from the Sample solution |
| rS | = | = peak area of biotin from the Standard solution |
| CS | = | = concentration of USP Biotin RS in the Standard solution (µg/mL) |
| CU | = | = nominal concentration of biotin in the Sample solution (µg/mL) |
Acceptance criteria:
90.0%–150.0% of the labeled amount of biotin (C10H16N2O3S)
• Cyanocobalamin, Method 1
[Note—Use low-actinic glassware throughout this procedure. ]
Mobile phase:
Methanol and water (7:13)
Standard stock solution:
10 µg/mL of USP Cyanocobalamin RS in water. [Note—Store this stock solution in a dark place, and discard after 1 week. ]
Standard solution:
1 µg/mL of USP Cyanocobalamin RS from the Standard stock solution diluted with water
Sample solution:
Finely powder NLT 30 Tablets. Transfer a portion of the powder, equivalent to 100 µg of cyanocobalamin, to a 250-mL flask. Quantitatively add 100.0 mL of water, and carefully extract for 2 min. Filter 10 mL of the extract, and use the filtrate.
Chromatographic system
Mode:
LC
Detector:
550 nm
Column:
4.6-mm × 15-cm; 5-µm packing L1
Flow rate:
0.5 mL/min
Injection volume:
200 µL
System suitability
Sample:
Standard solution
Suitability requirements
Relative standard deviation:
NMT 3.0%
Analysis
Samples:
Standard solution and Sample solution
Measure the peak area of cyanocobalamin. Calculate the percentage of the labeled amount of cyanocobalamin (C63H88CoN14O14P) in the portion of Tablets taken:
Result = (rU/rS) × (CS/CU) × 100
| rU | = | = peak area of cyanocobalamin from the Sample solution |
| rS | = | = peak area of cyanocobalamin from the Standard solution |
| CS | = | = concentration of USP Cyanocobalamin RS in the Standard solution (µg/mL) |
| CU | = | = nominal concentration of cyanocobalamin in the Sample solution (µg/mL) |
Acceptance criteria:
90.0%–150.0% of the labeled amount of cyanocobalamin (C63H88CoN14O14P)
• Cyanocobalamin, Method 2
[Note—Use low-actinic glassware throughout this procedure. ]
Standard stock solution:
1.0 µg/mL of USP Cyanocobalamin RS in 25% alcohol. Store in a refrigerator.
Standard solution:
Dilute a suitable volume of the Standard stock solution with water to a measured volume such that after the incubation period as described in the Analysis, the difference in transmittance between the inoculated blank and the 5.0-mL level of the Standard solution is NLT 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/mL of Standard solution. Prepare this solution fresh for each assay.
Sample solution:
Finely powder NLT 20 Tablets. Transfer a portion of the powdered Tablets, equivalent to 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 before 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 for 10 min. Allow any undissolved particles of the extract to settle, and filter or centrifuge if necessary. Dilute an aliquot of the clear solution with water to obtain a final solution containing vitamin B12 activity equivalent to the nominal activity of the Standard solution.
for 10 min. Allow any undissolved particles of the extract to settle, and filter or centrifuge if necessary. Dilute an aliquot of the clear solution with water to obtain a final solution containing vitamin B12 activity equivalent to the nominal activity of the Standard solution.
Acid-hydrolyzed casein solution:
Prepare as directed in Biotin, Method 2.
Asparagine solution:
Dissolve 2.0 g of l-asparagine in water to make 200 mL. Store under toluene in a refrigerator.
Adenine–guanine–uracil solution:
Prepare as directed in Biotin, Method 2.
Xanthine solution:
Suspend 0.20 g of xanthine in 30–40 mL of water, heat to 70, add 6.0 mL of 6 N ammonium hydroxide, and stir until the solid is dissolved. Cool, and dilute with water to make 200 mL. Store under toluene in a refrigerator.
, add 6.0 mL of 6 N ammonium hydroxide, and stir until the solid is dissolved. Cool, and dilute with water to make 200 mL. Store under toluene in a refrigerator.
Salt solution A:
Dissolve 10 g of monobasic potassium phosphate and 10 g of dibasic potassium phosphate in water to make 200 mL. Add 2 drops of hydrochloric acid. Store this solution under toluene.
Salt solution B:
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. 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 A:
Dissolve 10 mg of riboflavin, 10 mg of thiamine hydrochloride, 100 µg of biotin, and 20 mg of niacin in 0.02 N acetic acid to make 400 mL. Store under toluene, protected from light, in a refrigerator.
Vitamin solution B:
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 in Table 2, carefully dissolving Cystine and Tryptophan in the hydrochloric acid before adding the next eight solutions to the resulting solution. Add 100 mL of water, and dissolve the Dextrose, Sodium acetate, and Ascorbic acid. Filter, if necessary. Add the Polysorbate 80 solution, adjust with 1 N sodium hydroxide to a pH 5.5–6.0, and dilute with Purified Water to make 250 mL.
Table 2
| l-Cystine | 0.1 g |
| l-Tryptophan | 0.05 g |
| 1 N hydrochloric acid | 10 mL |
| Adenine–guanine–uracil solution | 5 mL |
| Xanthine solution | 5 mL |
| Vitamin solution A | 10 mL |
| Vitamin solution B | 10 mL |
| Salt solution A | 5 mL |
| Salt solution B | 5 mL |
| Asparagine solution | 5 mL |
| Acid-hydrolyzed casein solution | 25 mL |
| Dextrose, anhydrous | 10 g |
| Sodium acetate, anhydrous | 5 g |
| Ascorbic acid | 1 g |
| Polysorbate 80 solution | 5 mL |
Tomato juice preparation:
Centrifuge commercially canned tomato juice so that most of the pulp is removed. Suspend 5 g/L of analytical filter aid in the supernatant, and pass, with the aid of reduced pressure, through a layer of the filter aid. Repeat, if necessary, until a clear, straw-colored filtrate is obtained. Store under toluene in a refrigerator.
Culture medium:
[Note—A 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 60–70 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 dilute with 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 for 15 min. Cool as rapidly as possible to avoid color formation resulting from overheating the medium.
for 15 min. Cool as rapidly as possible to avoid color formation resulting from overheating the medium.
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 1.0–1.5 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 for 15 min in an autoclave, and allow the tubes to cool in an upright position. Inoculate three or more of the tubes by stab transfer of a pure culture of Lactobacillus leichmannii.3 [Note—Before first using a fresh culture in this assay, make NLT 10 successive transfers of the culture in a 2-week period. ] Incubate for 16–24 h at a temperature of between 30 and 40 held constant to within ±0.5. Store in a refrigerator.
for 15 min in an autoclave, and allow the tubes to cool in an upright position. Inoculate three or more of the tubes by stab transfer of a pure culture of Lactobacillus leichmannii.3 [Note—Before first using a fresh culture in this assay, make NLT 10 successive transfers of the culture in a 2-week period. ] Incubate for 16–24 h at a temperature of between 30 and 40 held constant to within ±0.5. Store in a refrigerator.
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 2–4 h after inoculation. A slow-growing culture seldom gives a suitable response curve and may lead to erratic results.
Inoculum:
[Note—A frozen suspension of Lactobacillus leichmannii may be used as the stock culture, provided it yields an Inoculum comparable to a fresh culture. ]
Transfer cells from the Stock culture of Lactobacillus leichmannii to two sterile tubes containing 10 mL each of the Culture medium. Incubate these cultures for 16–24 h at a temperature of between 30 and 40 held constant to within ±0.5. Under aseptic conditions centrifuge the cultures, and decant the supernatant. Suspend the cells from the culture in 5 mL of sterile Suspension medium, and combine. Using sterile Suspension medium, adjust the volume so that a 1-in-20 dilution in saline TS produces 70% transmittance when read on a suitable spectrophotometer that has been set at a wavelength of 530 nm, equipped with a 10-mm cell, and read against saline TS set at 100% transmittance. Prepare a 1-in-400 dilution of the adjusted suspension using sterile Basal medium stock solution. [Note—This dilution may be altered, when necessary, to obtain the desired test response. ] The cell suspension so obtained is the Inoculum.
and 40 held constant to within ±0.5. Under aseptic conditions centrifuge the cultures, and decant the supernatant. Suspend the cells from the culture in 5 mL of sterile Suspension medium, and combine. Using sterile Suspension medium, adjust the volume so that a 1-in-20 dilution in saline TS produces 70% transmittance when read on a suitable spectrophotometer that has been set at a wavelength of 530 nm, equipped with a 10-mm cell, and read against saline TS set at 100% transmittance. Prepare a 1-in-400 dilution of the adjusted suspension using sterile Basal medium stock solution. [Note—This dilution may be altered, when necessary, to obtain the desired test response. ] The cell suspension so obtained is the Inoculum.
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.
Analysis
Samples:
Standard solution and Sample solution
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 for 2 h, using hard-glass 20-mm × 150-mm test tubes and other necessary glassware.
for 2 h, using hard-glass 20-mm × 150-mm test tubes and other necessary glassware.To separate test tubes add, in duplicate, 1.0, 1.5, 2.0, 3.0, 4.0, and 5.0 mL of the Standard solution. 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 Sample solution. 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 sample 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 for 5 min, arranging to reach this temperature in NMT 10 min by preheating the autoclave if necessary. Cool as rapidly as possible to avoid color formation resulting from overheating the medium. Take precautions to maintain uniformity of sterilizing and cooling conditions throughout the assay, because packing the tubes too closely in the autoclave or overloading it may cause variation in the heating rate.
for 5 min, arranging to reach this temperature in NMT 10 min by preheating the autoclave if necessary. Cool as rapidly as possible to avoid color formation resulting from overheating the medium. Take precautions to maintain uniformity of sterilizing and cooling conditions throughout the assay, because packing the tubes too closely in the autoclave or overloading it may cause variation in the heating rate.Aseptically add 0.5 mL of Inoculum to each tube so prepared, except two of the four containing no Standard solution (the uninoculated blanks). Incubate the tubes at a temperature of between 30 and 40, held constant to within ±0.5 for 16–24 h.
and 40, held constant to within ±0.5 for 16–24 h.Terminate growth by heating to a temperature NLT 80 for 5 min. Cool to room temperature. After agitating its contents, read the transmittance at 530 nm when a steady state is reached. This steady state is observed a few seconds after agitation when the reading remains constant for 30 s or more. Allow approximately the same time interval for the reading on each tube.
for 5 min. Cool to room temperature. After agitating its contents, read the transmittance at 530 nm when a steady state is reached. This steady state is observed a few seconds after agitation when the reading remains constant for 30 s or more. Allow approximately the same time interval for the reading on each tube.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 (SS) as the difference, y = 2.00 SS. Plot this response on the ordinate of cross-section paper against the logarithm of the mL of the Standard solution per tube on the abscissa, using for the ordinate either an arithmetic or a logarithmic scale, whichever gives the better approximation to a straight line. Draw the straight line or smooth curve that best fits the plotted points.
SS. Plot this response on the ordinate of cross-section paper against the logarithm of the mL of the Standard solution per tube on the abscissa, using for the ordinate either an arithmetic or a logarithmic scale, whichever gives the better approximation to a straight line. Draw the straight line or smooth curve that best fits the plotted points.
Calculate the response, y = 2.00 SU, adding together the two transmittances (SU) for each level of the Sample solution. Read from the standard curve the logarithm of the volume of the Standard solution 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 Sample solution 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 Sample solution. Determine the quantity, in µg, of cyanocobalamin (C63H88CoN14O14P) in the portion of Tablets taken:
SU, adding together the two transmittances (SU) for each level of the Sample solution. Read from the standard curve the logarithm of the volume of the Standard solution 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 Sample solution 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 Sample solution. Determine the quantity, in µg, of cyanocobalamin (C63H88CoN14O14P) in the portion of Tablets taken: antilog M = antilog (M¢ + log R)
| R | = | = number of µg of cyanocobalamin assumed to be present in the portion of Tablets taken |
Calculate the percentage of the labeled amount of cyanocobalamin (C63H88CoN14O14P) in the portion of Tablets taken:
Result = [(antilog M)/N] × 100
| N | = | = nominal amount of cyanocobalamin in the portion of the Tablets taken (µg) |
Replication:
Repeat the entire determination at least once, using separately prepared Sample solutions. If the difference between the two log-potencies M is NMT 0.08, their mean, M, is the assayed log-potency of the test material (see Vitamin B12 Activity in Design and Analysis of Biological Assays 111, The Confidence Interval and Limits of Potency). If the two determinations differ by more than 0.08, conduct one or more additional determinations. From the mean of two or more values of M that do not differ by more than 0.15, compute the mean potency of the preparation under assay.
111, The Confidence Interval and Limits of Potency). If the two determinations differ by more than 0.08, conduct one or more additional determinations. From the mean of two or more values of M that do not differ by more than 0.15, compute the mean potency of the preparation under assay.
Acceptance criteria:
90.0%–150.0% of the labeled amount of cyanocobalamin (C63H88CoN14O14P)
• Folic Acid, Method 1
[Note—Use low-actinic glassware throughout this procedure. ]
Reagent A:
25% solution of tetrabutylammonium hydroxide in methanol
Reagent B:
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.
Mobile phase:
2 g of monobasic potassium phosphate in 650 mL of water. Add 12.0 mL of Reagent A, 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 1000 mL, and filter. Recheck the pH before use by adding water or methanol to the prepared Mobile phase to obtain baseline separation of folic acid and the internal standard. The pH may be increased up to 7.15 to obtain better separation. [Note—The methanol and water content may be varied (1%–3%). ]
Internal standard solution:
Transfer 40 mg of methylparaben to a 1000-mL volumetric flask, and add 220 mL of methanol to dissolve. Dissolve 2.0 g of monobasic potassium phosphate in 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 A, 7 mL of 3 N phosphoric acid, and 30 mL of Reagent B. Adjust with ammonia TS to a pH of 9.8, bubble nitrogen through the solution for 30 min, dilute with water to volume, and mix.
Standard solution:
0.016 mg/mL of USP Folic Acid RS in the Internal standard solution
Sample solution:
Finely powder NLT 30 Tablets. Transfer a portion of powder, equivalent to 0.4 mg of folic acid, to a 50-mL amber-colored centrifuge tube. Add 25.0 mL of the Internal standard solution, shake by mechanical means for 10 min, and centrifuge. Filter a portion of the clear supernatant, and use the filtrate.
Chromatographic system
Mode:
LC
Detector:
UV 280 nm
Column:
3.9-mm × 30-cm; packing L1
Flow rate:
1 mL/min
Injection volume:
15 µL
System suitability
Sample:
Standard solution
[Note—The relative retention times for folic acid and methylparaben are about 0.8 and 1.0, respectively. ]
Suitability requirements
Relative standard deviation:
NMT 3.0%
Analysis
Samples:
Standard solution and Sample solution
Measure the peak areas for folic acid and methylparaben. Calculate the percentage of the labeled amount of folic acid (C19H19N7O6) in the portion of Tablets taken:
Result = (RU/RS) × (CS/CU) × 100
| RU | = | = peak area ratio of folic acid to methylparaben from the Sample solution |
| RS | = | = peak area ratio of folic acid to methylparaben from the Standard solution |
| CS | = | = concentration of USP Folic Acid RS in the Standard solution (µg/mL) |
| CU | = | = nominal concentration of folic acid in the Sample solution (µg/mL) |
Acceptance criteria:
90.0%–150.0% of the labeled amount of folic acid (C19H19N7O6)
• Folic Acid, Method 2
[Note—Use 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.
Diluent:
60 µg/mL of ammonium hydroxide
Mobile phase:
Transfer 0.4 mL of triethylamine, 15 mL of glacial acetic acid, and 350 mL of methanol to a 2000-mL volumetric flask, and dilute with 0.008 M sodium 1-hexanesulfonate to volume.
Standard stock solution:
60 µg/mL of USP Folic Acid RS in Diluent. Prepare this solution fresh daily.
Standard solution:
Mix 5.0 mL of the Standard stock solution with 10.0 mL of methanol and 35.0 mL of Reagent. Shake for 15 min in a water bath maintained at 60, and cool. Filter, discarding the first few mL of the filtrate.
, and cool. Filter, discarding the first few mL of the filtrate.
Sample solution:
Transfer a portion of finely powdered Tablets, equivalent to 0.3 mg of folic acid, to a 125-mL stoppered flask. Add 10.0 mL of methanol and 35.0 mL of Reagent. Shake for 15 min in a water bath maintained at 60, and cool. Filter, discarding the first few mL of the filtrate.
, and cool. Filter, discarding the first few mL of the filtrate.
Chromatographic system
Mode:
LC
Detector:
UV 270 nm
Column:
4.6-mm × 25-cm; packing L7
Column temperature:
50
Flow rate:
2 mL/min
Injection volume:
5 µL
System suitability
Sample:
Standard solution
Suitability requirements
Relative standard deviation:
NMT 2.0%
Analysis
Samples:
Standard solution and Sample solution
Measure the areas of the major peaks. Calculate the percentage of the labeled amount of folic acid (C19H19N7O6) in the portion of Tablets taken:
Result = (rU/rS) × (CS/CU) × 100
| rU | = | = peak area of folic acid from the Sample solution |
| rS | = | = peak area of folic acid from the Standard solution |
| CS | = | = concentration of USP Folic Acid RS in the Standard solution (µg/mL) |
| CU | = | = nominal concentration of folic acid in the Sample solution (µg/mL) |
Acceptance criteria:
90.0%–150.0% of the labeled amount of folic acid (C19H19N7O6)
• Calcium Pantothenate, Method 1
Mobile phase:
Phosphoric acid and water (1:1000)
Internal standard solution:
80 mg of p-hydroxybenzoic acid in 3 mL of alcohol. Add 50 mL of water and 7.1 g of dibasic sodium phosphate, and dilute with water to 1000 mL. Adjust with phosphoric acid to a pH of 6.7.
Standard solution:
0.6 mg/mL of USP Calcium Pantothenate RS in Internal standard solution
Sample solution:
Finely powder NLT 30 Tablets. Transfer a portion of the powder, equivalent to 15 mg of calcium pantothenate, to a centrifuge tube. Add 25.0 mL of the Internal standard solution, and shake vigorously for 10 min. Centrifuge, filter, and use the clear filtrate.
Chromatographic system
Mode:
LC
Detector:
UV 210 nm
Column:
3.9-mm × 15-cm; packing L1
Flow rate:
1.5 mL/min
Injection volume:
10 µL
System suitability
Sample:
Standard solution
[Note—The relative retention times for calcium pantothenate and p-hydroxybenzoic acid are about 0.5 and 1.0, respectively. ]
Suitability requirements
Relative standard deviation:
NMT 3.0%
Analysis
Samples:
Standard solution and Sample solution
Measure the peak areas of calcium pantothenate and the internal standard. Calculate the percentage of the labeled amount of calcium pantothenate (C18H32CaN2O10) in the portion of Tablets taken:
Result = (RU/RS) × (CS/CU) × 100
| RU | = | = peak area ratio of calcium pantothenate to p-hydroxybenzoic acid from the Sample solution |
| RS | = | = peak area ratio of calcium pantothenate to p-hydroxybenzoic acid from the Standard solution |
| CS | = | = concentration of USP Calcium Pantothenate RS in the Standard solution (mg/mL) |
| CU | = | = nominal concentration of calcium pantothenate in the Sample solution (mg/mL) |
Acceptance criteria:
90.0%–150.0% of the labeled amount of calcium pantothenate (C18H32CaN2O10)
• Calcium Pantothenate, Method 2
Standard stock solution:
Dissolve 50 mg of USP Calcium Pantothenate RS, previously dried and stored in the dark over phosphorus pentoxide and protected from absorption of moisture while weighing, in 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 concentration of 50 µg/mL of USP Calcium Pantothenate RS. Store under toluene in a refrigerator.
Standard solution:
On the day of the assay, dilute a volume of the Standard stock solution with water to obtain a concentration of 0.01–0.04 µg/mL of calcium pantothenate, the exact concentration being such that the responses obtained as directed in the Analysis, 2.0 and 4.0 mL of the Standard solution, are within the linear portion of the log-concentration response curve.
Sample solution:
Finely powder NLT 30 Tablets. Transfer a portion of the powder, equivalent to 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 a volume of this solution to obtain a solution having approximately the same concentration as that of the Standard solution.
Acid-hydrolyzed casein solution:
Mix 100 g of vitamin-free casein with 500 mL of 6 N hydrochloric acid, and reflux the mixture for 8–12 h. 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 dilute with water to make 1000 mL. Add 20 g of activated charcoal, stir for 1 h, and filter. Repeat the treatment with activated charcoal. Store under toluene in a cool place at a temperature not below 10. Filter the solution if a precipitate forms during storage.
. Filter the solution if a precipitate forms during storage.
Cystine–tryptophan solution:
Suspend 4.0 g of l-cystine in a solution of 1.0 g of l-tryptophan (or 2.0 g of d,l-tryptophan) in 700–800 mL of water, heat to 70–80, and add dilute hydrochloric acid (1 in 2) dropwise, with stirring, until the solids are dissolved. Cool, and dilute with water to make 1000 mL. Store under toluene in a cool place at a temperature not below 10.
–80, and add dilute hydrochloric acid (1 in 2) dropwise, with stirring, until the solids are dissolved. Cool, and dilute with water to make 1000 mL. Store under toluene in a cool place at a temperature not below 10.
Adenine–guanine–uracil 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 dilute with water to make 200 mL. Store under toluene in a refrigerator.
Polysorbate 80 solution:
100 mg/mL of polysorbate 80 in alcohol
Riboflavin–thiamine hydrochloride–biotin solution:
20 µg/mL of riboflavin, 10 µg/mL of thiamine hydrochloride, and 0.04 µg/mL of biotin in 0.02 N acetic acid. Store under toluene, protected from light, in a refrigerator.
p-Aminobenzoic acid–niacin–pyridoxine hydrochloride solution:
10 µg/mL of p-aminobenzoic acid, 50 µg/mL of niacin, and 40 µg/mL of pyridoxine hydrochloride in a mixture of neutralized alcohol and water (1:3). Store in a refrigerator.
Salt solution A:
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. Store under toluene.
Salt solution B:
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. Store under toluene.
Basal medium stock solution:
Dissolve the anhydrous Dextrose and anhydrous Sodium acetate in the solutions previously mixed according to Table 3, and adjust with 1 N sodium hydroxide to a pH of 6.8. Dilute with water to 250 mL.
Table 3
| Acid-hydrolyzed casein solution | 25 mL |
| Cystine–tryptophan solution | 25 mL |
| Polysorbate 80 solution | 0.25 mL |
| Dextrose, anhydrous | 10 g |
| Sodium acetate, anhydrous | 5 g |
| Adenine–guanine–uracil solution | 5 mL |
| Riboflavin–thiamine hydrochloride–biotin solution | 5 mL |
| p-Aminobenzoic acid–niacin–pyridoxine hydrochloride solution | 5 mL |
| Salt solution A | 5 mL |
| Salt solution B | 5 mL |
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 for 15 min, and allow the tubes to cool in an upright position. Prepare stab cultures in three or more of the tubes, using a pure culture of Lactobacillus plantarum,1 incubating for 16–24 h at a temperature between 30 and 37 held constant to within ±0.5. Store in a refrigerator. Prepare a fresh stab of the stock culture every week, and do not use for Inoculum if the culture is more than 1 week old.
for 15 min, and allow the tubes to cool in an upright position. Prepare stab cultures in three or more of the tubes, using a pure culture of Lactobacillus plantarum,1 incubating for 16–24 h at a temperature between 30 and 37 held constant to within ±0.5. Store in a refrigerator. Prepare a fresh stab of the stock culture every week, and do not use for Inoculum if the culture is more than 1 week old.
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 for 15 min, and cool.
for 15 min, and cool.
Inoculum:
[Note—A frozen suspension of Lactobacillus plantarum may be used as the stock culture, provided it yields an Inoculum comparable to a fresh culture. ]
Transfer cells from the Stock culture of Lactobacillus plantarum to a sterile tube containing 10 mL of Culture medium. Incubate this culture for 16–24 h at a temperature between 30 and 37 held constant to within ±0.5. The cell suspension so obtained is the Inoculum.
and 37 held constant to within ±0.5. The cell suspension so obtained is the Inoculum.
Analysis
Samples:
Standard solution and Sample solution
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 solution. 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 Sample solution corresponding to three or more of the levels specified for the Standard solution, 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 sample 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 for 5 min. Cool, and add 1 drop of Inoculum to each tube, except two of the four tubes containing no Standard solution (the uninoculated blanks). Incubate the tubes at a temperature between 30 and 37, held constant to within ±0.5 until, following 16–24 h of incubation, there has been no substantial increase in turbidity in the tubes containing the highest level of Standard during a 2-h period.
for 5 min. Cool, and add 1 drop of Inoculum to each tube, except two of the four tubes containing no Standard solution (the uninoculated blanks). Incubate the tubes at a temperature between 30 and 37, held constant to within ±0.5 until, following 16–24 h of incubation, there has been no substantial increase in turbidity in the tubes containing the highest level of Standard during a 2-h period.Determine the transmittance of the tubes in the following manner. Mix the contents of each tube, and transfer to an optical container if necessary. Read the transmittance between 540 and 660 nm when a steady state is reached. This steady state is observed a few seconds after agitation when the galvanometer reading remains constant for 30 s 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 (SS) as the difference, y = 2.00 SS. Plot this response on the ordinate of cross-section paper against the logarithm of the mL of Standard solution per tube on the abscissa, using for the ordinate either an arithmetic or a logarithmic scale, whichever gives the better approximation to a straight line. Draw the straight line or smooth curve that best fits the plotted points.
SS. Plot this response on the ordinate of cross-section paper against the logarithm of the mL of Standard solution per tube on the abscissa, using for the ordinate either an arithmetic or a logarithmic scale, whichever gives the better approximation to a straight line. Draw the straight line or smooth curve that best fits the plotted points.
Calculate the response, y = 2.00 SU, adding together the two transmittances (SU) for each level of the Sample solution. Read from the standard curve the logarithm of the volume of the Standard solution 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 Sample solution 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 Sample solution. Determine the quantity, in mg, of calcium pantothenate (C18H32CaN2O10) in the portion of Tablets taken:
SU, adding together the two transmittances (SU) for each level of the Sample solution. Read from the standard curve the logarithm of the volume of the Standard solution 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 Sample solution 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 Sample solution. Determine the quantity, in mg, of calcium pantothenate (C18H32CaN2O10) in the portion of Tablets taken: antilog M = antilog (M¢ + log R)
| R | = | = number of mg of calcium pantothenate assumed to be present in the portion of the Tablets taken |
Calculate the percentage of calcium pantothenate (C18H32CaN2O10) in the portion of Tablets taken:
Result = [(antilog M)/N] × 100
| N | = | = nominal amount of calcium pantothenate in the portion of Tablets taken (mg) |
Replication:
Repeat the entire determination at least once, using separately prepared Sample solutions. If the difference between the two log-potencies M is NMT 0.08, their mean, M, is the assayed log-potency of the test material (see Design and Analysis of Biological Assays 111, The Confidence Interval and Limits of Potency). If the two determinations differ by more than 0.08, conduct one or more additional determinations. From the mean of two or more values of M that do not differ by more than 0.15, compute the mean potency of the preparation under assay.
111, The Confidence Interval and Limits of Potency). If the two determinations differ by more than 0.08, conduct one or more additional determinations. From the mean of two or more values of M that do not differ by more than 0.15, compute the mean potency of the preparation under assay.
Acceptance criteria:
90.0%–150.0% of the labeled amount of calcium pantothenate (C18H32CaN2O10)
• 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:
Methanol and Buffer solution (1:9)
Standard stock solution:
0.25 mg/mL of USP Calcium Pantothenate RS in water. Prepare fresh every 4 weeks. Store in a refrigerator.
Standard solution:
40 µg/mL of USP Calcium Pantothenate RS from the Standard stock solution diluted with water
Sample solution:
Finely powder NLT 20 Tablets. Transfer a portion of the powder, equivalent to 10 mg of calcium pantothenate, to a 250-mL volumetric flask. Add 10 mL of methanol, and swirl the flask to disperse. Dilute with water to volume, mix, and filter.
Chromatographic system
Mode:
LC
Detector:
UV 205 nm
Column:
3.9-mm × 30-cm; 5-µm packing L1
Column temperature:
50
Flow rate:
2 mL/min
Injection volume:
25 µL
System suitability
Sample:
Standard solution
Suitability requirements
Relative standard deviation:
NMT 3.0%
Analysis
Samples:
Standard solution and Sample solution
Measure the peak areas of calcium pantothenate. Calculate the percentage of the labeled amount of calcium pantothenate (C18H32CaN2O10) in the portion of Tablets taken:
Result = (rU/rS) × (CS/CU) × 100
| rU | = | = peak area of calcium pantothenate from the Sample solution |
| rS | = | = peak area of calcium pantothenate from the Standard solution |
| CS | = | = concentration of USP Calcium Pantothenate RS in the Standard solution (mg/mL) |
| CU | = | = nominal concentration of calcium pantothenate in the Sample solution (mg/mL) |
Acceptance criteria:
90.0%–150.0% of the labeled amount of calcium pantothenate (C18H32CaN2O10)
• Niacin or Niacinamide, Pyridoxine Hydrochloride, Riboflavin, and Thiamine, Method 1
[Note—Use low-actinic glassware throughout this procedure. ]
Diluent:
Acetonitrile, glacial acetic acid, and water (5:1:94)
Mobile phase:
A mixture of methanol, glacial acetic acid, and water (27:1:73) containing 140 mg of sodium 1-hexanesulfonate per 100 mL
Standard solution:
[Note—Use USP Niacin RS in place of USP Niacinamide RS for formulations containing niacin. ] Transfer 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, to a 200-mL volumetric flask, and add 180 mL of Diluent. Immerse the flask in a hot water bath maintained at 65–70 for 10 min with regular shaking or using a vortex mixer, until all the solid materials are dissolved. Chill rapidly in a cold water bath for 10 min to room temperature, and dilute with Diluent to volume.
–70 for 10 min with regular shaking or using a vortex mixer, until all the solid materials are dissolved. Chill rapidly in a cold water bath for 10 min to room temperature, and dilute with Diluent to volume.
Sample solution:
Finely powder NLT 30 Tablets. Transfer a portion of the powder, equivalent to 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 Diluent, and mix using a vortex mixer for 30 s to completely suspend the powder. Immerse the centrifuge tube in a hot water bath maintained at 65–70, heat for 5 min, and mix on a vortex mixer for 30 s. Return the tube to the hot water bath, heat for another 5 min, and mix on a vortex mixer for 30 s. Filter a portion of the solution, cool to room temperature, and use the clear filtrate. [Note—Use the filtrate within 3 h of filtration. ]
–70, heat for 5 min, and mix on a vortex mixer for 30 s. Return the tube to the hot water bath, heat for another 5 min, and mix on a vortex mixer for 30 s. Filter a portion of the solution, cool to room temperature, and use the clear filtrate. [Note—Use the filtrate within 3 h of filtration. ]
Chromatographic system
Mode:
LC
Detector:
UV 280 nm
Column:
3.9-mm × 30-cm; packing L1
Flow rate:
1 mL/min
Injection volume:
10 µL
System suitability
Sample:
Standard solution
[Note—The relative retention times for niacinamide, pyridoxine, riboflavin, and thiamine are about 0.3, 0.5, 0.8, and 1.0, respectively. ]
Suitability requirements
Relative standard deviation:
NMT 3.0%
Analysis
Samples:
Standard solution and Sample solution
Measure the peak areas for niacin or niacinamide, pyridoxine, riboflavin, and thiamine. Calculate the percentage of the labeled amount of niacinamide (C6H6N2O) in the portion of Tablets taken:
Result = (rU/rS) × (CS/CU) × 100
| rU | = | = peak area of niacinamide from the Sample solution |
| rS | = | = peak area of niacinamide from the Standard solution |
| CS | = | = concentration of USP Niacinamide RS in the Standard solution (mg/mL) |
| CU | = | = nominal concentration of niacinamide in the Sample solution (mg/mL) |
For formulations containing niacin:
Result = (rU/rS) × (CS/CU) × 100
| rU | = | = peak area of niacin from the Sample solution |
| rS | = | = peak area of niacin from the Standard solution |
| CS | = | = concentration of USP Niacin RS in the Standard solution (mg/mL) |
| CU | = | = nominal concentration of niacin in the Sample solution (mg/mL) |
Separately calculate the percentage of the labeled amount of pyridoxine hydrochloride (C8H11NO3·HCl), riboflavin (C17H20N4O6), and thiamine hydrochloride (C12H17ClN4OS·HCl) in the portion of Tablets taken:
Result = (rU/rS) × (CS/CU) × 100
| rU | = | = peak area of the corresponding vitamin from the Sample solution |
| rS | = | = peak area of the corresponding vitamin from the Standard solution |
| CS | = | = concentration of the relevant USP Reference Standard in the Standard solution (mg/mL) |
| CU | = | = nominal concentration of the corresponding vitamin in the Sample solution (mg/mL) |
For products containing thiamine mononitrate, calculate the percentage of the labeled amount of thiamine mononitrate (C12H17N5O4S) in the portion of Tablets taken:
Result = (rU/rS) × (CS/CU) × (Mr1/Mr2) × 100
| rU | = | = peak area of thiamine from the Sample solution |
| rS | = | = peak area of thiamine from the Standard solution |
| CS | = | = concentration of USP Thiamine Hydrochloride RS in the Standard solution (mg/mL) |
| CU | = | = nominal concentration of thiamine mononitrate in the Sample solution (mg/mL) |
| Mr1 | = | = molecular weight of thiamine mononitrate, 327.36 |
| Mr2 | = | = molecular weight of thiamine hydrochloride, 337.27 |
Acceptance criteria:
90.0%–150.0% of the labeled amount of niacinamide (C6H6N2O) or niacin (C6H5NO2), pyridoxine hydrochloride (C8H11NO3·HCl), riboflavin (C17H20N4O6), and thiamine as thiamine hydrochloride (C12H17ClN4OS·HCl) or thiamine mononitrate (C12H17N5O4S)
• Niacin, Method 2
[Note—Use low-actinic glassware throughout this procedure. ]
Solution A:
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.
Extraction solvent:
Solution A and methanol (3:1)
Mobile phase:
0.1 M sodium acetate solution (13.6 mg/mL of sodium acetate in water). Adjust with acetic acid to a pH of 5.4. [Note—A small amount of methanol (up to 1%) may be added to the Mobile phase to improve resolution. ]
Standard stock solution:
1 mg/mL of USP Niacin RS in Extraction solvent
Standard solution:
Transfer 5.0 mL of the Standard stock solution to a 25-mL volumetric flask, and dilute with Extraction solvent to volume.
Sample solution:
[Note—This preparation is suitable for the determination of niacin or niacinamide, pyridoxine, and riboflavin, when present in the formulation. ] Finely powder NLT 20 Tablets. Transfer a portion of the powder, equivalent to a nominal amount of 2 mg of riboflavin, to a 200-mL volumetric flask. If riboflavin is not present in the formulation, use a portion of the powder equivalent to 2 mg of pyridoxine. If pyridoxine is not present in the formulation, use a portion of the powder equivalent to 20 mg of niacin or niacinamide. Add 100.0 mL of Extraction solvent, and mix for 20 min, using a wrist-action shaker. Immerse the flask in a water bath maintained at 70–75, and heat for 20 min. Mix on a vortex mixer for 30 s, cool to room temperature, and filter. Use the clear filtrate.
–75, and heat for 20 min. Mix on a vortex mixer for 30 s, cool to room temperature, and filter. Use the clear filtrate.
Chromatographic system
Mode:
LC
Detector:
UV 254 nm
Column:
4.6-mm × 25-cm; packing L1
Flow rate:
1 mL/min
Injection volume:
20 µL
System suitability
Sample:
Standard solution
Suitability requirements
Relative standard deviation:
NMT 3.0%
[Note—If necessary, flush the column with methanol between injections. ]
Analysis
Samples:
Standard solution and Sample solution
Measure the peak areas of niacin. Calculate the percentage of the labeled amount of niacin (C6H5NO2) in the portion of Tablets taken:
Result = (rU/rS) × (CS/CU) × 100
| rU | = | = peak area of niacin from the Sample solution |
| rS | = | = peak area of niacin from the Standard solution |
| CS | = | = concentration of USP Niacin RS in the Standard solution (mg/mL) |
| CU | = | = nominal concentration of niacin in the Sample solution (mg/mL) |
Acceptance criteria:
90.0%–150.0% of the labeled amount of niacin (C6H5NO2)
• Niacinamide, Method 2
[Note—Use low-actinic glassware throughout this procedure. ]
Solution A, Extraction solvent, Mobile phase, Standard stock solution, Standard solution, Sample solution, Chromatographic system, and System suitability:
Using USP Niacinamide RS in place of USP Niacin RS, proceed as directed in Niacin, Method 2.
Analysis
Samples:
Standard solution and Sample solution
Measure the peak areas of niacinamide. Calculate the percentage of the labeled amount of niacinamide (C6H6N2O) in the portion of Tablets taken:
Result = (rU/rS) × (CS/CU) × 100
| rU | = | = peak area of niacinamide from the Sample solution |
| rS | = | = peak area of niacinamide from the Standard solution |
| CS | = | = concentration of USP Niacinamide RS in the Standard solution (mg/mL) |
| CU | = | = nominal concentration of niacinamide in the Sample solution (mg/mL) |
Acceptance criteria:
90.0%–150.0% of the labeled amount of niacinamide (C6H6N2O)
• Pyridoxine Hydrochloride, Method 2
[Note—Use low-actinic glassware throughout this procedure. ]
Extraction solvent, Mobile phase, and Sample solution:
Prepare as directed in Niacin, Method 2.
Standard stock solution:
0.1 mg/mL of USP Pyridoxine Hydrochloride RS in Extraction solvent
Standard solution:
Transfer 5.0 mL of the Standard stock solution to a 25-mL volumetric flask, and dilute with Extraction solvent to volume.
Chromatographic system
Mode:
LC
Detector:
UV 254 nm
Column:
4.6-mm × 25-cm; packing L1
Flow rate:
1 mL/min
Injection volume:
20 µL
System suitability
Sample:
Standard solution
Suitability requirements
Relative standard deviation:
NMT 3.0%
Analysis
Samples:
Standard solution and Sample solution
Measure the peak areas of pyridoxine. Calculate the percentage of the labeled amount of pyridoxine hydrochloride (C8H11NO3·HCl) in the portion of Tablets taken:
Result = (rU/rS) × (CS/CU) × 100
| rU | = | = peak area of pyridoxine from the Sample solution |
| rS | = | = peak area of pyridoxine from the Standard solution |
| CS | = | = concentration of USP Pyridoxine Hydrochloride RS in the Standard solution (mg/mL) |
| CU | = | = nominal concentration of pyridoxine hydrochloride in the Sample solution (mg/mL) |
Acceptance criteria:
90.0%–150.0% of the labeled amount of pyridoxine hydrochloride (C8H11NO3·HCl)
• Riboflavin, Method 2
[Note—Use low-actinic glassware throughout this procedure. ]
Extraction solvent and Sample solution:
Prepare as directed in Niacin, Method 2.
Solution B:
6.8 mg/mL of sodium acetate in water
Mobile phase:
Prepare a mixture of Solution B and methanol (13:7). Add 2 mL of triethylamine per L of the mixture, and adjust with glacial acetic acid to a pH of 5.2.
Standard stock solution:
Transfer 20 mg of USP Riboflavin RS to a 200-mL volumetric flask, and add 180 mL of Extraction solvent. Immerse the flask for 5 min in a water bath maintained at 65–75. Mix well, and repeat if necessary until dissolved. Chill rapidly in a cold water bath to room temperature, and dilute with Extraction solvent to volume.
–75. Mix well, and repeat if necessary until dissolved. Chill rapidly in a cold water bath to room temperature, and dilute with Extraction solvent to volume.
Standard solution:
Dilute 5.0 mL of the Standard stock solution with Extraction solvent to 25.0 mL.
Chromatographic system
Mode:
LC
Detector:
UV 254 nm
Column:
4.6-mm × 25-cm; packing L1
Flow rate:
1 mL/min
Injection volume:
20 µL
System suitability
Sample:
Standard solution
Suitability requirements
Relative standard deviation:
NMT 3.0%
Analysis
Samples:
Standard solution and Sample solution
Measure the peak areas of riboflavin. Calculate the percentage of the labeled amount of riboflavin (C17H20N4O6) in the portion of Tablets taken:
Result = (rU/rS) × (CS/CU) × 100
| rU | = | = peak area of riboflavin from the Sample solution |
| rS | = | = peak area of riboflavin from the Standard solution |
| CS | = | = concentration of USP Riboflavin RS in the Standard solution (mg/mL) |
| CU | = | = nominal concentration of riboflavin in the Sample solution (mg/mL) |
Acceptance criteria:
90.0%–150.0% of the labeled amount of riboflavin (C17H20N4O6)
• Thiamine, Method 2
[Note—Use low-actinic glassware throughout this procedure. ]
Solution A:
1.88 mg/mL of sodium 1-hexanesulfonate in 0.1% phosphoric acid
Mobile phase:
Solution A and acetonitrile (46:9)
Standard stock solution:
0.1 mg/mL of USP Thiamine Hydrochloride RS in 0.2 N hydrochloric acid
Standard solution:
0.02 mg/mL of USP Thiamine Hydrochloride RS from the Standard stock solution diluted with 0.2 N hydrochloric acid
Sample solution:
Weigh and finely powder NLT 20 Tablets. Mix a portion of the powder with a volume of 0.2 N hydrochloric acid to obtain a concentration of 0.02 mg/mL of thiamine. Shake for 15 min with a wrist-action shaker, and heat to boiling for 30 min. Cool to room temperature, and filter. Use the clear filtrate.
Chromatographic system
Mode:
LC
Detector:
UV 254 nm
Column:
4.6-mm × 25-cm; packing L1
Flow rate:
2 mL/min
Injection volume:
20 µL
System suitability
Sample:
Standard solution
Suitability requirements
Relative standard deviation:
NMT 3.0%
Analysis
Samples:
Standard solution and Sample solution
Measure the peak areas for the major peaks. For products containing thiamine hydrochloride, calculate the percentage of the labeled amount of thiamine hydrochloride (C12H17ClN4OS·HCl) in the portion of Tablets taken:
Result = (rU/rS) × (CS/CU) × 100
| rU | = | = peak area of thiamine from the Sample solution |
| rS | = | = peak area of thiamine from the Standard solution |
| CS | = | = concentration of USP Thiamine Hydrochloride RS in the Standard solution (mg/mL) |
| CU | = | = nominal concentration of thiamine hydrochloride in the Sample solution (mg/mL) |
For products containing thiamine mononitrate, calculate the percentage of the labeled amount of thiamine mononitrate (C12H17N5O4S) in the portion of Tablets taken:
Result = (rU/rS) × (CS/CU) × (Mr1/Mr2) × 100
| rU | = | = peak area of thiamine from the Sample solution |
| rS | = | = peak area of thiamine from the Standard solution |
| CS | = | = concentration of USP Thiamine Hydrochloride RS in the Standard solution (mg/mL) |
| CU | = | = nominal concentration of thiamine mononitrate in the Sample solution (mg/mL) |
| Mr1 | = | = molecular weight of thiamine mononitrate, 327.36 |
| Mr2 | = | = molecular weight of thiamine hydrochloride, 337.27 |
Acceptance criteria:
90.0%–150.0% of the labeled amount of thiamine as thiamine hydrochloride (C12H17ClN4OS·HCl) or thiamine mononitrate (C12H17N5O4S)
• Niacin or Niacinamide, Pyridoxine Hydrochloride, Riboflavin, and Thiamine, Method 3
[Note—Use low-actinic glassware throughout this procedure. ]
Reagent:
25 mg/mL of edetate disodium in 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.
Standard stock solution:
1.5 mg/mL of USP Niacin RS or USP Niacinamide RS, 0.24 mg/mL of USP Pyridoxine Hydrochloride RS, 0.08 mg/mL of USP Riboflavin RS, and 0.24 mg/mL of USP Thiamine Hydrochloride RS in the Reagent, with heating if necessary
Standard solution:
Transfer 5.0 mL of the 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 min in a water bath maintained at 60, and cool. Filter, discarding the first few mL of the filtrate.
, and cool. Filter, discarding the first few mL of the filtrate.
Sample solution:
Weigh and finely powder NLT 20 Tablets. Transfer a portion of the powder, equivalent to 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 min in a water bath maintained at 60, and cool. Filter, discarding the first few mL of the filtrate.
, and cool. Filter, discarding the first few mL of the filtrate.
Chromatographic system
Mode:
LC
Detector:
UV 270 nm
Column:
4.6-mm × 25-cm; packing L7
Column temperature:
50
Flow rate:
2.0 mL/min
Injection volume:
5 µL
System suitability
Sample:
Standard solution
Suitability requirements
Relative standard deviation:
NMT 2.0%
Analysis
Samples:
Standard solution and Sample solution
Measure the peak areas for niacin or niacinamide. Calculate the percentage of the labeled amount of niacin (C6H5NO2) or niacinamide (C6H6N2O) in the portion of Tablets taken:
Result = (rU/rS) × (CS/CU) × 100
| rU | = | = peak area of niacin or niacinamide from the Sample solution |
| rS | = | = peak area of niacin or niacinamide from the Standard solution |
| CS | = | = concentration of USP Niacin RS or USP Niacinamide RS in the Standard solution (mg/mL) |
| CU | = | = nominal concentration of niacin or niacinamide in the Sample solution (mg/mL) |
Separately calculate the percentage of the labeled amount of pyridoxine hydrochloride (C8H11NO3·HCl), riboflavin (C17H20N4O6), and thiamine hydrochloride (C12H17ClN4OS·HCl) in the portion of Tablets taken:
Result = (rU/rS) × (CS/CU) × 100
| rU | = | = peak area of the corresponding vitamin from the Sample solution |
| rS | = | = peak area of the corresponding vitamin from the Standard solution |
| CS | = | = concentration of the relevant USP Reference Standard in the Standard solution (mg/mL) |
| CU | = | = nominal concentration of the corresponding vitamin in the Sample solution (mg/mL) |
For products containing thiamine mononitrate, calculate the percentage of the labeled amount of thiamine mononitrate (C12H17N5O4S) in the portion of Tablets taken:
Result = (rU/rS) × (CS/CU) × (Mr1/Mr2) × 100
| rU | = | = peak area of thiamine from the Sample solution |
| rS | = | = peak area of thiamine from the Standard solution |
| CS | = | = concentration of USP Thiamine Hydrochloride RS in the Standard solution (mg/mL) |
| CU | = | = nominal concentration of thiamine mononitrate in the Sample solution (mg/mL) |
| Mr1 | = | = molecular weight of thiamine mononitrate, 327.36 |
| Mr2 | = | = molecular weight of thiamine hydrochloride, 337.27 |
Acceptance criteria:
90.0%–150.0% of the labeled amount of niacin (C6H5NO2) or niacinamide (C6H6N2O), pyridoxine hydrochloride (C8H11NO3·HCl), riboflavin (C17H20N4O6), and thiamine as thiamine hydrochloride (C12H17ClN4OS·HCl) or thiamine mononitrate (C12H17N5O4S)
PERFORMANCE TESTS
• Disintegration and Dissolution of Dietary Supplements 2040:
Meet the requirements for Dissolution
2040:
Meet the requirements for Dissolution
• Weight Variation of Dietary Supplements 2091:
Meet the requirements
2091:
Meet the requirements
CONTAMINANTS
• Microbial Enumeration Tests 2021:
The total aerobic microbial count does not exceed 3 × 103 cfu/g, and the combined molds and yeasts count does not exceed 3 × 102 cfu/g.
2021:
The total aerobic microbial count does not exceed 3 × 103 cfu/g, and the combined molds and yeasts count does not exceed 3 × 102 cfu/g.
• Absence of Specified Microorganisms 2022:
Meet the requirements of the tests for absence of Salmonella species, Escherichia coli, and Staphylococcus aureus
2022:
Meet the requirements of the tests for absence of Salmonella species, Escherichia coli, and Staphylococcus aureus
ADDITIONAL REQUIREMENTS
• Packaging and Storage:
Preserve in tight, light-resistant containers.
• Labeling:4
The label states that the product is Oil- and Water-Soluble Vitamins Tablets. The label also states the quantity of each vitamin per dosage unit and, where necessary, the chemical form in which it is present. 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
11
USP Alpha Tocopherol RS 
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USP Alpha Tocopheryl Acetate RS
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USP Alpha Tocopheryl Acid Succinate RS
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USP Biotin RS
1H-Thieno[3,4-d]imidazole-4-pentanoic acid, hexahydro-2-oxo-, 3aS-[(3a
,4
,6a)]-;
(3aS,4S,6aR)-Hexahydro-2-oxo-1H-thieno[3,4-d]imidazole-4-valeric acid.
C10H16N2O3S 244.31
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1H-Thieno[3,4-d]imidazole-4-pentanoic acid, hexahydro-2-oxo-, 3aS-[(3a
,4,6a)]-;
(3aS,4S,6aR)-Hexahydro-2-oxo-1H-thieno[3,4-d]imidazole-4-valeric acid.
C10H16N2O3S 244.31
USP Calcium Pantothenate RS
-Alanine, N-(2,4-dihydroxy-3,3-dimethyl-1-oxobutyl)-, calcium salt (2:1), (R)-;
Calcium d-pantothenate (1:2).
C18H32CaN2O10 476.53
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-Alanine, N-(2,4-dihydroxy-3,3-dimethyl-1-oxobutyl)-, calcium salt (2:1), (R)-;
Calcium d-pantothenate (1:2).
C18H32CaN2O10 476.53
USP Cholecalciferol RS
9,10-Secocholesta-5,7,10(19)-trien-3-ol, (3,5Z,7E)-;
Cholecalciferol.
C27H44O 384.64
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9,10-Secocholesta-5,7,10(19)-trien-3-ol, (3
,5Z,7E)-;
Cholecalciferol.
C27H44O 384.64
USP Cyanocobalamin RS
Vitamin B12.
C63H88CoN14O14P 1355.37
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Vitamin B12.
C63H88CoN14O14P 1355.37
USP Ergocalciferol RS
9,10-Secoergosta-5,7,10 (19),22-tetraen-3-ol, (3,5Z,7E,22E)-;
Ergocalciferol.
C28H44O 396.65
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9,10-Secoergosta-5,7,10 (19),22-tetraen-3-ol, (3
,5Z,7E,22E)-;
Ergocalciferol.
C28H44O 396.65
USP Folic Acid RS
l-Glutamic acid, N-[4-[[(2-amino-1,4-dihydro-4-oxo-6-pteridinyl)methyl]amino]benzoyl]-;
Folic acid;
N-[p-[[(2-Amino-4-hydroxy-6-pteridinyl)methyl]amino]-benzoyl]-l-glutamic acid.
C19H19N7O6 441.40
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l-Glutamic acid, N-[4-[[(2-amino-1,4-dihydro-4-oxo-6-pteridinyl)methyl]amino]benzoyl]-;
Folic acid;
N-[p-[[(2-Amino-4-hydroxy-6-pteridinyl)methyl]amino]-benzoyl]-l-glutamic acid.
C19H19N7O6 441.40
USP Niacin RS
3-Pyridinecarboxylic acid;
Nicotinic acid.
C6H5NO2 123.11
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3-Pyridinecarboxylic acid;
Nicotinic acid.
C6H5NO2 123.11
USP Niacinamide RS
3-Pyridinecarboxamide;
Nicotinamide.
C6H6N2O 122.12
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3-Pyridinecarboxamide;
Nicotinamide.
C6H6N2O 122.12
USP Phytonadione RS
1,4-Naphthalenedione, 2-methyl-3-(3,7,11,15-tetramethyl-2-hexadecenyl)-, [R-[R*,R*-(E)]]-;
Phylloquinone.
C31H46O2 450.70
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1,4-Naphthalenedione, 2-methyl-3-(3,7,11,15-tetramethyl-2-hexadecenyl)-, [R-[R*,R*-(E)]]-;
Phylloquinone.
C31H46O2 450.70
USP Pyridoxine Hydrochloride RS
3,4-Pyridinedimethanol, 5-hydroxy-6-methyl-, hydrochloride;
Pyridoxol hydrochloride.
C8H11NO3·HCl 205.64
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3,4-Pyridinedimethanol, 5-hydroxy-6-methyl-, hydrochloride;
Pyridoxol hydrochloride.
C8H11NO3·HCl 205.64
USP Riboflavin RS
Riboflavine.
C17H20N4O6 376.36
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Riboflavine.
C17H20N4O6 376.36
USP Thiamine Hydrochloride RS
Thiazolium, 3-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-5-(2-hydroxyethyl)-4-methyl-, chloride, monohydrochloride;
Thiamine monohydrochloride.
C12H17ClN4OS·HCl 337.27
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Thiazolium, 3-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-5-(2-hydroxyethyl)-4-methyl-, chloride, monohydrochloride;
Thiamine monohydrochloride.
C12H17ClN4OS·HCl 337.27
USP Vitamin A RS
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1
ATCC No. 8014 is suitable. This strain was formerly known as Lactobacillus arabinosus 17-5.
2
A suitable cartridge is the Waters Oasis MAX Vac RC cartridge, particle size 30 µm, part 186000371.
3
Pure cultures of Lactobacillus leichmannii (listed as Lactobacillus delbrueckii) may be obtained as No. 7830 from ATCC, 10801 University Blvd., Manassas, VA 20110-2209 (www.atcc.org).
4
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 cholecalciferol or ergocalciferol; and 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, and 1 mg of d-alpha tocopheryl acetate = 1.36 former USP Vitamin E Units, and 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.
Auxiliary Information—
Please check for your question in the FAQs before contacting USP.
| Topic/Question | Contact | Expert Committee |
|---|---|---|
| Monograph | Natalia Davydova
Scientific Liaison (301) 816-8328 |
(DS2010) Monographs - Dietary Supplements and Herbal Medicines |
| 2021 |
Radhakrishna S Tirumalai, Ph.D.
Principal Scientific Liaison (301) 816-8339 |
(GCM2010) General Chapters - Microbiology |
| 2022 |
Radhakrishna S Tirumalai, Ph.D.
Principal Scientific Liaison (301) 816-8339 |
(GCM2010) General Chapters - Microbiology |
| Reference Standards | RS Technical Services 1-301-816-8129 rstech@usp.org |
USP38–NF33 Page 6326
Pharmacopeial Forum: Volume No. 40(5)