Mode: LC

Detector: UV 325 nm

Column: 4.6-mm × 15-cm; 3-µm packing L8

Flow rate: 1 mL/min

Injection size: 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 obtained from the Standard solution and the peak area for all-trans-retinyl acetate or all-trans-retinyl palmitate in the chromatogram of 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 Capsules 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. ] Weigh NLT 20 Capsules in a tared weighing bottle. Using a sharp blade if necessary, carefully open the Capsules, without loss of shell material, and transfer the contents to a 100-mL beaker. Remove any contents adhering to the empty shells by washing with several portions of ether. Discard the washings, and dry the Capsule shells with the aid of a current of dry air. Weigh the empty Capsule shells in the tared weighing bottle, and calculate the net weight of the Capsule contents. Transfer a portion of the Capsule contents, equivalent to 30 µg of the labeled amount of cholecalciferol or ergocalciferol (vitamin D), to a container with a polytef-lined screw cap. If vitamin D is not present in the formulation, use a portion, equivalent to 90 mg of the labeled amount of vitamin E. If vitamin E is not present in the formulation, use a portion, equivalent to 2.5 mg of the labeled amount of vitamin A, as retinol. 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 size: 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 Capsules 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—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

Extraction solvent: n-Hexane and methylene chloride (3:1)

Potassium hydroxide solution: 800 mg/mL of potassium hydroxide in water. [Note—Cautiously add potassium hydroxide in 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 Standard stock solution with Diluent to obtain a concentration of 1 µg/mL of retinyl acetate from 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.

Sample solution: Weigh NLT 20 Capsules in a tared weighing bottle. Open the Capsules, without loss of shell material, and transfer the contents to a 100-mL beaker. Remove any contents adhering to the empty shells by washing with several portions of ether. Discard the washings, and dry the Capsule shells with the aid of a current of dry air. Weigh the empty Capsule shells in the tared weighing bottle, and calculate the net weight of the Capsule contents. Transfer a portion of the Capsule contents, 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 quantitatively, and stepwise if necessary, 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 size: 50 µL

System suitability

Sample: Standard solution

[Note—The relative retention times for 13-cis-retinol and all-trans-retinol are about 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 Capsules 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 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.

Sample solution: Transfer NLT 20 mL of a solution prepared as directed for Sample solution in Vitamin A, Method 1 to a suitable container, and concentrate, if necessary, in vacuum at room temperature to obtain a solution with an expected 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 size: 100 µL

System suitability

Sample: 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 Capsules 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

3 N methanolic sulfuric acid solution, Sodium ascorbate–pyrogallol solution, Lecithin solution, and Sample solution: Proceed as directed for 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.

Chromatographic system

Mode: LC

Detector: UV 265 nm

Column: 4.6-mm × 25-cm; 5-µm packing L24

Flow rate: 1 mL/min

Injection size: 40 µL

System suitability

Sample: 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 Capsules 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)

• Cholecalciferol or Ergocalciferol (Vitamin D), Method 3

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.

Sample solution: Proceed as directed for the Sample solution for Vitamin A, Method 3, through “calculate the net weight of the Capsule contents.” Transfer a portion of the Capsule contents, equivalent to 10 µg of ergocalciferol or cholecalciferol, 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 size: 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 Capsules 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: Transfer NLT 20 mL of the solution prepared as directed for the Sample solution in Vitamin A, Method 1 to a suitable container, and evaporate, in vacuum at room temperature to dryness. Transfer the contents of the flask to a suitable volumetric flask with the aid of methanol, and dilute with methanol to volume, to obtain a solution with an expected 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 size: 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 Capsules 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

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

3 N methanolic sulfuric acid solution: Cautiously mix sulfuric acid and methanol (9 in 100) in a 100-mL volumetric flask. [Note—Dissolve in a portion of methanol, cool, and then dilute to final volume. ]

Sodium ascorbate–pyrogallol solution: Transfer 10 g of sodium ascorbate and 5 g of pyrogallol to a 100-mL volumetric flask. 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

Sample solution: Proceed as directed for Vitamin A, Method 2, through “calculate the net weight of the Capsule contents.” Transfer a portion of the Capsule contents, equivalent to 55 mg of vitamin E, to a container having a polytef-lined screw cap. 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 layer. 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 size: 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

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: Proceed as directed for Vitamin A, Method 3, through “calculate the net weight of the Capsule contents.” Transfer a portion of the Capsule contents, equivalent to an expected amount of 8.0 mg of alpha tocopherol, to a glass-stoppered conical flask. 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.

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 size: 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 Capsules 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 alpha tocopherol equivalent (C29H50O2) of alpha tocopheryl acetate (C31H52O3) or alpha tocopheryl acid succinate (C33H54O5) by multiplying their contents by the factors 0.91 or 0.81, respectively. ]

Acceptance criteria: 90.0%–165.0% of the labeled amount of vitamin E

• Phytonadione

[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.

Standard solution: 20 µg/mL of USP Phytonadione RS from Standard stock solution diluted with methanol

System suitability solution: 0.65 mg/mL of USP Alpha Tocopheryl Acetate RS and 20 µg/mL of USP Phytonadione RS from 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. ]

Sample solution: Transfer NLT 20 mL of the solution prepared as directed for the Sample solution in Vitamin A, Method 1 to a suitable container, and evaporate, if necessary, in vacuum at room temperature to dryness. Transfer the contents of the flask to a suitable volumetric flask with the aid of methanol, and dilute with methanol to volume to obtain a solution with an expected 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: 2.0 mL/min

Injection size: 100 µL

System suitability

Samples: Standard solution and System suitability solution

[Note—The relative retention times for alpha tocopheryl acetate and phytonadione are about 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 Capsules taken:

Result = (rU/rS) × (CS/CU) × 100

rU	=	= peak area from the Sample solution
rS	=	= peak area 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)

• Beta Carotene

[Note—Use low-actinic glassware throughout this procedure. ]

Potassium hydroxide solution: Dissolve 58.8 g of potassium hydroxide in 50 mL of water.

Iodine solution: Transfer 10 mg of iodine to a 100-mL volumetric flask. Dissolve in cyclohexane, and dilute with cyclohexane to volume. Dilute 10 mL of this solution with cyclohexane to 100 mL. [Note—Prepare this solution fresh daily. ]

Sample solution A (for preparations containing beta carotene in oil solutions): Proceed as directed for Vitamin A, Method 1, except use cyclohexane instead of n-hexane as the extraction solvent, and dilute the filtered extracts with cyclohexane, to obtain a concentration of 2 µg/mL of beta carotene.

Sample solution B (for preparations containing beta carotene in dry powder): Remove the contents of NLT 20 Capsules by cutting open the Capsules. Mix, and determine the weight of the contents. Transfer a quantity of the Capsule contents, 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.

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 Capsules 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, Method 1

Sample solution: Weigh NLT 20 Capsules in a tared weighing bottle. Open the Capsules, without the loss of shell material, and transfer the contents to a 100-mL beaker. Remove any contents adhering to the empty shells by washing, if necessary, with several portions of ether. Discard the washings, and dry the Capsule shells with the aid of a current of dry air until the odor of ether is no longer perceptible. Weigh the empty Capsule shells in the tared weighing bottle, and calculate the average net weight per Capsule. Transfer a portion of the Capsule contents, equivalent to 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 Capsule.

Acceptance criteria: 90.0%–150.0% of the labeled amount of ascorbic acid (C6H8O6)

• Ascorbic Acid, Method 2: Proceed as directed in Automated Methods of Analysis

, Assay for Ascorbic Acid.

Acceptance criteria: 90.0%–150.0% of the labeled amount of ascorbic acid (C6H8O6)

• Calcium Ascorbate, Method 1: Proceed as directed for Ascorbic Acid, Method 1.

Acceptance criteria: 90.0%–150.0% of the labeled amount of calcium ascorbate (C12H14CaO12·2H2O)

• Calcium Ascorbate, Method 2: Proceed as directed in Automated Methods of Analysis

, Assay for Ascorbic Acid.

Acceptance criteria: 90.0%–150.0% of the labeled amount of calcium ascorbate (C12H14CaO12·2H2O)

• Sodium Ascorbate, Method 1: Proceed as directed for Ascorbic Acid, Method 1.

Acceptance criteria: 90.0%–150.0% of the labeled amount of sodium ascorbate (C6H7NaO6)

• Sodium Ascorbate, Method 2: Proceed as directed in Automated Methods of Analysis

, Assay for 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, and 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: Proceed as directed in Ascorbic Acid, Method 1, through “calculate the average net weight per Capsule.” Transfer a portion of the Capsule contents, 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.

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 size: 100 µL

System suitability

Sample: Standard solution

Suitability requirements

Relative standard deviation: NMT 3.0%

Analysis

Samples: Standard solution and Sample solution

Measure the responses for the biotin peaks. Calculate the percentage of the labeled amount of biotin (C10H16N2O3S) in the portion of Capsules taken:

Result = (rU/rS) × (CS/CU) × 100

rU	=	= peak response from the Sample solution
rS	=	= peak response 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.

–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 having 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 add 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.

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 add 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 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: 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: 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.

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.

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. ] Make a transfer of cells from the Stock culture of Lactobacillus plantarum to a sterile tube containing 10 mL of Culture medium. Incubate this culture for 16–24 h at a temperature between 30

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 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 from 540 to 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.

Calculate the response, y = 2.00

SU , adding together the two transmittances for each level of the Sample solution (SU). Read from the standard curve the logarithm of the volume of the Standard solution corresponding to each of those values of y that fall within the range of lowest and highest points plotted for the Standard. Subtract from each logarithm so obtained the logarithm of the volume, in mL, of the 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 Capsules taken:

antilog M = antilog (M¢ + log R)

= number of µg of biotin that was assumed to be present in the portion of the Capsules taken

Calculate the percentage of the labeled amount of biotin in the portion of the Capsules taken

Result = [(antilog M)/N] × 100

= nominal amount of biotin (C10H16N2O3S) in the portion of Capsules 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.

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 Standard stock solution diluted with water

Sample solution: Proceed as directed for Ascorbic Acid, Method 1, through “calculate the average net weight per Capsule.” Transfer a portion of the Capsule contents, equivalent to 100 µg of cyanocobalamin, to a 250-mL flask. Add 100.0 mL of water, and carefully extract for 2 min. Filter 10 mL of the extract, and use the clear 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 size: 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 responses for cyanocobalamin. Calculate the percentage of the labeled amount of cyanocobalamin (C63H88CoN14O14P) in the portion of Capsules taken:

Result = (rU/rS) × (CS/CU) × 100

rU	=	= peak response from the Sample solution
rS	=	= peak response 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 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 the Standard solution. Prepare this solution fresh for each assay.

Sample solution: Proceed as directed for Ascorbic Acid, Method 1, through “calculate the average net weight per Capsule.” Transfer a portion of the Capsule contents, equivalent to 1.0 µg of cyanocobalamin, to an appropriate vessel containing, for each g of Capsule contents taken, 25 mL of an aqueous extracting solution prepared just before use to contain 12.9 mg/mL of dibasic sodium phosphate, 11.0 mg/mL of anhydrous citric acid, and 10 mg/mL 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 approximately equivalent to that of the Standard solution.

Acid-hydrolyzed casein solution: Prepare as directed for Calcium Pantothenate, 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 for Calcium Pantothenate, 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 add 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, and 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: 20 g of polysorbate 80 in alcohol to make 200 mL. Store in a refrigerator.

Vitamin solution A: 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: 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 of between 5.5 and 6.0, and add 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 add water to make 100 mL. Place 10-mL portions of the solution in test tubes, and plug with cotton. Sterilize the tubes and contents in an autoclave at 121

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.2 [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 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. ] Make a transfer of cells from the Stock culture of Lactobacillus leichmannii to two sterile tubes containing 10 mL of the Culture medium each. Incubate these cultures for 16–24 h at a temperature 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. The cell suspension so obtained is the Inoculum. [Note—This dilution may be altered, when necessary, to obtain the desired test response. ]

Calibration of spectrophotometer: Check the wavelength of the spectrophotometer periodically, using a standard wavelength cell or other suitable device. Before reading any tests, calibrate the spectrophotometer for 0% and 100% transmittance, using water and with the wavelength set at 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.

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.

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 between 30

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, and 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

Calculate the response, y = 2.00

SU, adding together the two transmittances for each level of the Sample solution (SU). 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 in the portion of Capsules taken:

antilog M = antilog (M¢ + log R)

= number of µg of cyanocobalamin that was assumed to be present in each mg in the portion of Capsules taken

Calculate the percentage of the labeled amount of biotin in the portion of the Capsules taken:

Result = [(antilog M)/N] × 100

= nominal amount of cyanocobalamin (C63H88CoN14O14P) in the portion of Capsules taken (µg)

111

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 (between 1% and 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 Internal standard solution

Sample solution: Proceed as directed for Ascorbic Acid, Method 1, through “calculate the average net weight per Capsule.” Transfer an amount of Capsule contents to a suitable centrifuge tube, and add a volume of Internal standard solution to obtain a concentration of 0.016 mg/mL of folic acid. 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 size: 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 Capsules 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 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.

Sample solution: Proceed as directed for the Sample solution for Ascorbic Acid, Method 1, through “calculate the net weight of the Capsule contents.” Transfer a portion of the Capsule contents, 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.

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 size: 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 Capsules 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)

• Dexpanthenol or Panthenol

[Note—The following procedure is applicable also to the determination of the dextrorotatory component of racemic panthenol in preparations containing panthenol. ]

Standard stock solution: 800 µg/mL of USP Dexpanthenol RS or 1600 µg/mL of USP Racemic Panthenol RS in water. Store in a refrigerator, protected from light, and use within 30 days.

Standard solution: On the day of the assay, prepare a dilution of 1.2 µg/mL of dexpanthenol or 2.4 µg/mL of panthenol from Standard stock solution diluted with water.

Sample solution: Weigh NLT 30 Capsules in a tared weighing bottle. Open the Capsules, without loss of shell material, and transfer the contents as completely as possible to a beaker. Remove any contents adhering to the empty Capsule shells by washing with several portions of ether. Discard the washings, and dry the Capsule shells with the aid of a current of dry air until the odor of ether is no longer perceptible. Weigh the empty Capsule shells in the tared weighing bottle, and calculate the average net weight per Capsule. Dissolve a portion of the Capsule contents, equivalent to 1.2 mg of dexpanthenol or 2.4 mg of panthenol, in 100.0 mL of water. Quantitatively dilute a portion of this solution with water to obtain a concentration of 1.2 µg/mL of dexpanthenol or 2.4 µg/mL of panthenol.

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 about 500 mL of water, adjust the solution with 1 N sodium hydroxide to a pH of 3.5 ± 0.1, and add water to make 1000 mL. Add 20 g of activated charcoal, stir for 1 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.

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 75 ± 5

, and add hydrochloric acid solution (1 in 2) dropwise, with stirring, until the solids are dissolved. Cool, and add 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. Add 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 neutral 25% alcohol. Store in a refrigerator.

Salt solution A: 50 mg/mL of monobasic potassium phosphate and 50 mg/mL of dibasic potassium phosphate in water. Add 10 drops of hydrochloric acid/L of solution. Store under toluene.

Salt solution B: 20 mg/mL of magnesium sulfate, 1 mg/mL of sodium chloride, 1 mg/mL of ferrous sulfate, and 1 mg/mL of manganese sulfate in water. Add 10 drops of hydrochloric acid/L of the solution. Store under toluene.

Pyridoxal–calcium pantothenate solution: 200 µg/mL of pyridoxal hydrochloride and 1.875 µg/mL of calcium pantothenate in 10% alcohol. Store in a refrigerator, and use within 30 days.

Polysorbate 40–oleic acid solution: 50 mg/mL of polysorbate 40 and 0.5 mg/mL of oleic acid in 20% alcohol. Store in a refrigerator, and use within 30 days.

Modified pantothenate medium: Dissolve anhydrous dextrose and sodium acetate in the solutions previously mixed according to Table 3, and adjust with 1 N sodium hydroxide to a pH of 6.8. Finally, 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
Pyridoxal–calcium pantothenate solution	5 mL
Polysorbate 40–oleic acid solution	5 mL

Double-strength modified pantothenate medium: Prepare as directed in Modified pantothenate medium, but make the final dilution to 125 mL instead of 250 mL. Prepare fresh.

Stock culture of Pediococcus acidilactici: Dissolve in 800 mL of water, with the aid of heat, 6.0 g of peptone, 4.0 g of pancreatic digest of casein, 3.0 g of yeast extract, 1.5 g of beef extract, 1.0 g of dextrose, and 15.0 g of agar. Adjust with 0.1 N sodium hydroxide or 0.1 N hydrochloric acid to a pH of 6.5–6.6, and dilute with water to 1000 mL. Add 10-mL portions of the solution to culture tubes, place caps on the tubes, and sterilize in an autoclave at 121

for 15 min. Cool on a slant, and store in a refrigerator. Prepare a stock culture of Pediococcus acidilactici3 on a slant of this medium. Incubate at 35

for 20–24 h, and store in a refrigerator. Maintain the stock culture by monthly transfer onto fresh slants.

Inoculum: Inoculate three 250-mL portions of Modified pantothenate medium from a stock culture slant, and incubate at 35

for 20–24 h. Centrifuge the suspension from the combined portions, and wash the cells with Modified pantothenate medium. Resuspend the cells in sufficient Modified pantothenate medium so that a 1-in-50 dilution, when tested in a 13-mm diameter test tube, gives 80% light transmission at 530 nm. Transfer 1.2-mL portions of this stock suspension to glass ampuls, seal, freeze in liquid nitrogen, and store in a freezer. On the day of the assay, allow the ampuls to reach room temperature, mix the contents, and dilute 1 mL of thawed culture with sterile saline TS to 150 mL. [Note—This dilution may be altered when necessary to obtain the desired test response. ]

Analysis: Prepare in triplicate a series of eight culture tubes by adding the following quantities of water to the tubes within a set: 5.0, 4.5, 4.0, 3.5, 3.0, 2.0, 1.0, and 0.0 mL. To these same tubes and in the same order, add 0.0, 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, and 5.0 mL of the Standard solution.

Prepare in duplicate a series of five culture tubes by adding the following quantities of water to the tubes within a set: 4.0, 3.5, 3.0, 2.0, and 1.0 mL. To these same tubes, and in the same order, add 1.0, 1.5, 2.0, 3.0, and 4.0 mL of the Sample solution.

Add 5.0 mL of Double-strength modified pantothenate medium to each tube. Cover the tubes with metal caps, and sterilize in an autoclave at 121

for 5 min. Cool to room temperature in a chilled water bath, and inoculate each tube with 0.5 mL of the Inoculum. Allow to incubate at 37

for 16 h. Terminate growth by heating to a temperature NLT 80

, such as by steaming at atmospheric pressure in a sterilizer for 5–10 min. Cool, and determine the percentage transmittance of the suspensions, in cells of equal pathlength, on a suitable spectrophotometer, at a wavelength of 530 nm.

Calculation: Draw a dose-response curve on arithmetic graph paper by plotting the average response, in percentage of transmittance, for each set of tubes of the standard curve against the standard level concentrations. The curve is drawn by connecting each adjacent pair of points with a straight line. From this standard curve, determine by interpolation the potency, of each tube containing portions of the Sample solution. Divide the potency of each tube by the amount of the Sample solution added to it, to obtain the individual responses. Calculate the mean response by averaging the individual responses that vary from their mean by NMT 15%, using NLT half the total number of tubes. Calculate the potency of the portion of the material taken, by multiplying the mean response by the appropriate dilution factor. Calculate the percentage of the labeled amount of dexpanthenol or panthenol in the portion of Capsules taken:

Result = (P/N) × 100

P	=	= potency of dexpanthenol or panthenol in the portion of Capsules taken (mg)
N	=	= nominal amount of dexpanthenol or panthenol in the portion of Capsules taken (mg)

Acceptance criteria: 90.0%–150.0% of the labeled amount of dexpanthenol or panthenol (C9H19NO4)

• 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: Proceed as directed for Ascorbic Acid, Method 1, through “calculate the average net weight per Capsule.” To a centrifuge tube, transfer an amount of mixed Capsule contents and a volume of Internal standard solution to obtain a concentration of 0.6 mg/mL in the Sample solution.

Chromatographic system

Mode: LC

Detector: UV 210 nm

Column: 3.9-mm × 15-cm; packing L1

Flow rate: 1.5 mL/min

Injection size: 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 responses for calcium pantothenate and the internal standard. Calculate the percentage of the labeled amount of calcium pantothenate (C18H32CaN2O10) in the portion of Capsules taken:

Result = (RU/RS) × (CS/CU) × 100

RU	=	= peak response ratio of calcium pantothenate to p-hydroxybenzoic acid from the Sample solution
RS	=	= peak response 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 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 being used, are within the linear portion of the log-concentration response curve.

Sample solution: Proceed as directed for Ascorbic Acid, Method 1, through “calculate the average net weight per Capsule.” Transfer a portion of the Capsule contents, equivalent to an amount of 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.

. 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

Polysorbate 80 solution: 100 mg/mL of polysorbate 80 in alcohol

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 4, and adjust with 1 N sodium hydroxide to a pH of 6.8. Dilute with water to 250 mL.

Table 4

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 plantarum1 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: Add 5.0 mL of water containing 0.2 µg of calcium pantothenate to each of a series of test tubes containing 5.0 mL of Basal medium stock solution. Plug the tubes with cotton, sterilize in an autoclave at 121

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. ] Make a transfer of cells from the of 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.

Analysis

Samples: Standard solution and Sample solution

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.

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 from 540 to 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.

Calculate the response, y = 2.00

SU, adding together the two transmittances for each level of the Sample solution (SU). Read from the standard curve the logarithm of the volume of the Standard solution corresponding to each of those values of y that fall within the range of the lowest and highest points plotted for the Standard. Subtract from each logarithm so obtained the logarithm of the volume, in mL, of the 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 Capsules taken:

antilog M = antilog (M¢ + log R)

= number of mg of calcium pantothenate that was assumed to be present in the portion of the Capsules taken

Calculate the percentage of calcium pantothenate in the portion of Capsules taken:

Result = [(antilog M)/N] × 100

= nominal amount of calcium pantothenate in the portion of the Capsules taken (mg)

111

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 Standard stock solution diluted with water

Sample solution: Proceed as directed for the Sample solution for Ascorbic Acid, Method 1, through “calculate the net weight of the Capsule contents.” Transfer a portion of the Capsule contents, equivalent to 10 mg of calcium pantothenate, to a 250-mL volumetric flask. Add 10 mL of methanol, and swirl the flask to disperse the Capsules contents. 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 size: 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 for calcium pantothenate. Calculate the percentage of the labeled amount of calcium pantothenate (C18H32CaN2O10) in the portion of Capsules taken:

Result = (rU/rS) × (CS/CU) × 100

rU	=	= peak area from the Sample solution
rS	=	= peak area 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.

Sample solution: Proceed as directed for Ascorbic Acid, Method 1, through “calculate the average net weight per Capsule.” Transfer a portion of the Capsule contents, 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. ]

Chromatographic system

Mode: LC

Detector: UV 280 nm

Column: 3.9-mm × 30-cm; packing L1

Flow rate: 1 mL/min

Injection size: 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 Capsules 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 Capsules 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 Capsules 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 Standard stock solution to a 25-mL volumetric flask. 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. ] Weigh NLT 20 Capsules in a tared weighing bottle. Open the Capsules, without loss of shell material, and transfer the contents to a beaker. Remove any contents adhering to the shells by washing with several portions of ether. Discard the washings, and dry the Capsule shells with the aid of a current of dry air. Weigh the empty Capsule shells in the tared weighing bottle, and calculate the net weight of the Capsule contents. Transfer a portion of the Capsule contents, equivalent to 2 mg of riboflavin, to a 200-mL volumetric flask. If riboflavin is not present in the formulation, use a portion, equivalent to a nominal amount of 2 mg of pyridoxine. If pyridoxine is not present in the formulation, use a portion, 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.

Chromatographic system

Mode: LC

Detector: UV 254 nm

Column: 4.6-mm × 25-cm; packing L1

Flow rate: 1 mL/min

Injection size: 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 for niacin. Calculate the percentage of the labeled amount of niacin (C6H5NO2) in the portion of Capsules taken:

Result = (rU/rS) × (CS/CU) × 100

rU	=	= peak area from the Sample solution
rS	=	= peak area 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 solution: Using USP Niacinamide RS in place of USP Niacin RS, proceed as directed for Niacin, Method 2.

Analysis

Samples: Standard solution and Sample solution

Measure the peak areas for niacinamide. Calculate the percentage of the labeled amount of niacinamide (C6H6N2O) in the portion of Capsules taken:

Result = (rU/rS) × (CS/CU) × 100

rU	=	= peak area from the Sample solution
rS	=	= peak area 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 for Niacin, Method 2.

Standard stock solution: 0.1 mg/mL of USP Pyridoxine Hydrochloride RS in Extraction solvent

Standard solution: 20 µg/mL of USP Pyridoxine Hydrochloride RS from Standard stock solution diluted with Extraction solvent

Chromatographic system

Mode: LC

Detector: UV 254 nm

Column: 4.6-mm × 25-cm; packing L1

Flow rate: 1 mL/min

Injection size: 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 pyridoxine. Calculate the percentage of the labeled amount of pyridoxine hydrochloride (C8H11NO3·HCl) in the portion of Capsules taken:

Result = (rU/rS) × (CS/CU) × 100

rU	=	= peak area from the Sample solution
rS	=	= peak area 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 for Niacin, Method 2.

Solution A: 6.8 g/L of sodium acetate in water

Mobile phase: Prepare a mixture of Solution A 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.

Standard solution: Dilute 5.0 mL of 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 size: 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 riboflavin. Calculate the percentage of the labeled amount of riboflavin (C17H20N4O6) in the portion of Capsules taken:

Result = (rU/rS) × (CS/CU) × 100

rU	=	= peak area from the Sample solution
rS	=	= peak area 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 g/L 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 Standard stock solution diluted with 0.2 N hydrochloric acid

Sample solution: Proceed as directed for the Sample solution for Ascorbic Acid, Method 1, through “calculate the net weight of the Capsule contents.” Mix a portion of the Capsule contents 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 size: 20 µL

System suitability

Sample: Standard solution

Suitability requirements

Relative standard deviation: NMT 3.0%

Analysis

Samples: Standard solution and Sample solution

Measure the 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 Capsules 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 Capsules 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 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 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.

Sample solution: Proceed as directed for the Sample solution for Ascorbic Acid, Method 1, through “calculate the net weight of the Capsule contents.” Transfer a portion of the Capsule contents, 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.

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 size: 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 peaks. Calculate the percentage of the labeled amount of niacin (C6H5NO2) or niacinamide (C6H6N2O) in the portion of Capsules 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 (C12H17ClN4OS) (for products containing thiamine hydrochloride) in the portion of Capsules 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 Capsules 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

[Note—Commercially available atomic absorption standard solutions for the minerals, where applicable, may be used where preparation of a Standard stock solution is described in the following assays. Use deionized water where water is specified. Where atomic absorption spectrophotometry is specified in the assay, the Standard solutions and the Sample solution may be diluted quantitatively with the solvent specified, if necessary, to yield solutions of suitable concentrations adaptable to the linear or working range of the instrument. ]

• Calcium, Method 1

Lanthanum chloride solution: 267 mg/mL of lanthanum chloride heptahydrate in 0.125 N hydrochloric acid

Calcium standard solution: 400 µg/mL of calcium. Dissolve 1.001 g of calcium carbonate, previously dried at 300

for 3 h and cooled in a desiccator for 2 h, in 25 mL of 1 N hydrochloric acid. Boil to expel carbon dioxide, and dilute with water to 1000 mL.

Standard stock solution: 100 µg/mL of calcium from Calcium standard solution diluted with 0.125 N hydrochloric acid

Standard solutions: Into separate 100-mL volumetric flasks pipet 1.0, 1.5, 2.0, 2.5, and 3.0 mL of the Standard stock solution. To each flask add 1.0 mL of Lanthanum chloride solution, and dilute with water to volume to obtain concentrations of 1.0, 1.5, 2.0, 2.5, and 3.0 µg/mL of calcium.

Polysorbate 80 solution: Polysorbate 80 and alcohol (1:10)

Sample solution: Transfer 5 Capsules to a 100-mL volumetric flask. [Note—For hard gelatin Capsules, weigh NLT 20 Capsules. Open the Capsules, without loss of shell material, and transfer the contents to a suitable container. Remove any contents adhering to the empty shells by washing with several portions of ether. Discard the washings, and allow the Capsule shells to dry. Weigh the empty Capsule shells, calculate the net weight of the Capsule contents, and transfer a portion of the Capsule contents, equivalent to 5 Capsules, to a 100-mL volumetric flask. ] Add 15 mL of water, 10 mL of 6 N hydrochloric acid, and 1 mL of Polysorbate 80 solution to the flask. Heat on a hot plate or steam bath, with intermittent swirling, until the Capsules are completely disintegrated or the contents are dissolved. Boil gently for an additional 15 min. Cool, dilute with water to volume, and filter, discarding the first 5 mL of the filtrate. Dilute this solution with 0.125 N hydrochloric acid, to obtain a concentration of 2 µg/mL of calcium, adding 1 mL of Lanthanum chloride solution per 100 mL of the final volume.

Instrumental conditions

Mode: Atomic absorption spectrophotometry

Analytical wavelength: Calcium emission line at 422.7 nm

Lamp: Calcium hollow-cathode

Flame: Nitrous oxide–acetylene

Blank: 0.125 N hydrochloric acid containing 1 mL of Lanthanum chloride solution per 100 mL

Analysis

Samples: Standard solutions and Sample solution

Determine the absorbances of the solutions against the Blank. Plot the absorbances of the Standard solutions versus the concentration, in µg/mL, of calcium, and draw the straight line best fitting the five plotted points. From the graph so obtained, determine the concentration, C, in mg/mL, of calcium in the Sample solution.

Calculate the percentage of of the labeled amount of calcium (Ca) in the portion of Capsules taken:

Result = (C/CU) × 100

C	=	= measured concentration of calcium in the Sample solution (µg/mL)
CU	=	= nominal concentration of calcium in the Sample solution (µg/mL)

Acceptance criteria: 90.0%–125.0% of the labeled amount of calcium (Ca)

• Chromium, Method 1

Chromium standard solution: 1000 µg/mL of chromium from potassium dichromate, previously dried at 120

for 4 h in water. Store in a polyethylene bottle.

Standard stock solution: 10 µg/mL of chromium from Chromium standard solution diluted with 6 N hydrochloric acid and water (1 in 20)

Standard solutions: Transfer 10.0 and 20.0 mL of the Standard stock solution to separate 100-mL volumetric flasks, and transfer 15.0 and 20.0 mL of the Standard stock solution to separate 50-mL volumetric flasks. Dilute the contents of each of the four flasks with 0.125 N hydrochloric acid to volume to obtain concentrations of 1.0, 2.0, 3.0, and 4.0 µg/mL of chromium.

Sample solution: Proceed as directed for Calcium, Method 1, except to prepare the Sample solution to contain 1 µg/mL of chromium and to omit the use of the Lanthanum chloride solution.

Instrumental conditions

Mode: Atomic absorption spectrophotometry

Analytical wavelength: Chromium emission line at 357.9 nm

Lamp: Chromium hollow-cathode

Flame: Air–acetylene

Blank: 0.125 N hydrochloric acid

Analysis

Samples: Standard solutions and Sample solution

Determine the absorbances of the solutions against the Blank. Plot the absorbances of the Standard solutions versus the concentration, in µg/mL, of chromium, and draw the straight line best fitting the four plotted points. From the graph so obtained, determine the concentration, C, in µg/mL, of chromium in the Sample solution.

Calculate the percentage of the labeled amount of chromium (Cr) in the portion of Capsules taken:

Result = (C/CU) × 100

C	=	= measured concentration of chromium in the Sample solution (µg/mL)
CU	=	= nominal concentration of chromium in the Sample solution (µg/mL)

Acceptance criteria: 90.0%–160.0% of the labeled amount of chromium (Cr)

• Copper, Method 1

Copper standard solution: Dissolve 1.00 g of copper foil in a minimum volume of a 50% solution of nitric acid, and dilute with a 1% solution of nitric acid to 1000 mL. This solution contains 1000 µg/mL of copper.

Standard stock solution: 100 µg/mL of copper from Copper standard solution diluted with 0.125 N hydrochloric acid

Standard solutions: To separate 200-mL volumetric flasks transfer 1.0, 2.0, 4.0, 6.0, and 8.0 mL of the Standard stock solution. Dilute with water to volume to obtain concentrations of 0.5, 1.0, 2.0, 3.0, and 4.0 µg/mL of copper.

Sample solution: Proceed as directed for Calcium, Method 1, except to prepare the Sample solution to contain 2 µg/mL of copper and to omit the use of the Lanthanum chloride solution.

Instrumental conditions

Mode: Atomic absorption spectrophotometry

Analytical wavelength: Copper emission line at 324.7 nm

Lamp: Copper hollow-cathode

Flame: Air–acetylene

Blank: 0.125 N hydrochloric acid

Analysis

Samples: Standard solutions and Sample solution

Determine the absorbances of the solutions against the Blank. Plot the absorbances of the Standard solutions versus the concentration, in µg/mL, of copper, and draw the straight line best fitting the five plotted points. From the graph so obtained, determine the concentration, C, in µg/mL, of copper in the Sample solution.

Calculate the percentage of the labeled amount of copper (Cu) in the portion of Capsules taken:

Result = (C/CU) × 100

C	=	= measured concentration of copper in the Sample solution (µg/mL)
CU	=	= nominal concentration of copper in the Sample solution (µg/mL)

Acceptance criteria: 90.0%–125.0% of the labeled amount of copper (Cu)

• Fluoride, Method 1

[Note—Store all solutions in plastic containers. ]

3 M sodium acetate solution: Dissolve 408 g of sodium acetate in 600 mL of water contained in a 1000-mL volumetric flask. Allow the solution to equilibrate to room temperature, and dilute with water to volume. Adjust with a few drops of acetic acid to a pH of 7.0.

Sodium citrate solution: Dissolve 222 g of sodium citrate in 250 mL of water in a 1000-mL volumetric flask. Add 28 mL of perchloric acid, and dilute with water to volume.

Fluoride standard stock solution: 500 µg/mL of fluoride from a quantity of sodium fluoride, previously dried at 100

for 4 h and cooled in a desiccator, in water

Intermediate stock solution A: 100 µg/mL of fluoride from Fluoride standard stock solution diluted with water

Intermediate stock solution B: 10 µg/mL of fluoride from Fluoride standard stock solution diluted with water

Standard solutions: To five separate 100-mL volumetric flasks transfer 3.0, 5.0, and 10.0 mL of Intermediate stock solution B and 5.0 and 10.0 mL of Intermediate stock solution A. To each flask add 10.0 mL of 1 N hydrochloric acid, 25 mL of 3 M sodium acetate solution, and 25.0 mL of Sodium citrate solution. Dilute the contents of each flask with water to volume to obtain concentrations of 0.3, 0.5, 1.0, 5.0, and 10.0 µg/mL of fluoride.

Sample solution: Remove the contents of Capsules by cutting open the Capsules. Mix, and determine the weight of the contents. Transfer a quantity of the mixed Capsule contents, equivalent to 200 µg of fluoride, to a 100-mL volumetric flask. Add 10.0 mL of 1 N hydrochloric acid, 25.0 mL of 3 M sodium acetate solution, and 25.0 mL of Sodium citrate solution. Dilute with water to volume.

Analysis

Samples: Standard solutions and Sample solution

To separate plastic beakers, each containing a plastic-coated stirring bar, transfer 50.0 mL each of the Standard solutions and the Sample solution. Measure the potentials (see pH

791

), in mV, of the Standard solutions and the Sample solution, with a pH meter capable of a minimum reproducibility of ±0.2 mV and equipped with a fluoride-specific ion-indicating electrode and a calomel reference electrode. [Note—When taking measurements, immerse the electrodes in the solution, stir on a magnetic stirrer having an insulated top until equilibrium is attained (1–2 min), and record the potential. Rinse and dry the electrodes between measurements, taking care to avoid damaging the crystal of the specific-ion electrode. ]

Plot the logarithms of fluoride concentrations, in µg/mL, of the Standard solutions versus potential, in mV. From the standard response curve so obtained and the measured potential of the Sample solution, determine the concentration, C, in mg/mL, of fluoride in the Sample solution.

Calculate the percentage of the labeled amount of fluorine (F) in the portion of Capsules taken:

Result = (C/CU) × 100

C	=	= measured concentration of fluoride in the Sample solution (µg/mL)
CU	=	= nominal concentration of fluorine in the Sample solution (µg/mL)

Acceptance criteria: 90.0%–160.0% of the labeled amount of fluorine (F)

• Fluoride, Method 2

[Note—Use plastic containers and deionized water throughout this procedure. ]

pH 10.0 buffer: Add 214 mL of 0.1 N sodium hydroxide to 1000 mL of 0.05 M sodium bicarbonate.

Mobile phase: Alcohol, 0.1 N sulfuric acid, and water (20:5:175)

Standard stock solution: 220 µg/mL of USP Sodium Fluoride RS in water. This solution contains 100 µg/mL of fluoride.

Standard solution: [Note—Condition the solid-phase extraction column specified for use in the Standard solution and the Sample solution in the following manner. Using a vacuum at a pressure not exceeding 5 mm of mercury, wash the column with one column volume of methanol followed by one column volume of pH 10.0 buffer. Do not allow the column top to dry. If the top of the column becomes dry, recondition the column. ] Transfer 10.0 mL of Standard stock solution to a 100-mL volumetric flask. Add 75 mL of water, and adjust with 0.1 N sodium hydroxide to a pH of 10.4 ± 0.1. Dilute with water to volume. Filter, discarding the first 15 mL of the filtrate. Transfer 25.0 mL of the filtrate to a 50-mL volumetric flask, add 15.0 mL of water, and adjust with 0.1 N sodium hydroxide to a pH of 10.0. Dilute with pH 10.0 buffer to volume. Elute a portion of this solution through a 3-mL solid-phase extraction column containing L1 packing that is connected through an adaptor to a second solid-phase extraction column containing sulfonylpropyl strong cation-exchange packing. Discard the first 3 mL of the eluate, and collect the rest of the eluate in a suitable flask for injection into the chromatograph.

Sample solution: Weigh NLT 20 Capsules in a tared weighing bottle. Open the Capsules, without loss of shell material, and transfer the contents to a 100-mL container. If necessary, remove any contents adhering to the empty shells by washing with several portions of ether. Discard the washings, and dry the Capsule shells with the aid of a current of dry air. Weigh the empty Capsule shells in the tared weighing bottle, and calculate the net weight of the Capsule contents. Transfer a portion of the Capsule contents, equivalent to 1 mg of fluorine, to a 100-mL volumetric flask. Add 15 mL of water, and shake vigorously. Rinse the sides of the flask with 15 mL of water, and allow to stand for 10 min. Dilute with water to 85 mL, adjust with 1 N sodium hydroxide to a pH of 10.4 ± 0.1, and dilute with water to 100 mL. Proceed as directed for the Standard solution, beginning with “Filter, discarding the first 15 mL of the filtrate.”

Chromatographic system

Mode: LC

Detector: Conductivity detector

Guard column: 4.6-mm × 3-cm; packing L17

Analytical column: 7.8-mm × 30-cm; packing L17

Flow rate: 0.5 mL/min

Injection size: 100 µL

System suitability

Sample: Standard solution

Suitability requirements

Relative standard deviation: NMT 2.0%

Analysis

Samples: Sample solution and Standard solution

Measure the peak areas for fluoride. Calculate the percentage of the labeled amount of fluorine (F) in the portion of Capsules taken:

Result = (rU/rS) × (CS/CU) × 100

rU	=	= peak area from the Sample solution
rS	=	= peak area from the Standard solution
CS	=	= concentration of fluoride in the Standard solution (µg/mL)
CU	=	= nominal concentration of fluorine in the Sample solution (µg/mL)

Acceptance criteria: 90.0%–160.0% of the labeled amount of fluorine (F)

• Iodide, Method 1

Bromine water: To 20 mL of bromine in a glass-stoppered bottle add 100 mL of water. Insert the stopper into the bottle, and shake. Allow to stand for 30 min, and use the supernatant.

Analysis

Sample: Capsules

Remove the contents of Capsules by cutting open the Capsules. Mix, and determine the weight of the contents. Transfer a quantity of the contents, equivalent to 3 mg of iodine, to a nickel crucible. Add 5 g of sodium carbonate, 5 mL of 50% (w/v) sodium hydroxide solution, and 10 mL of alcohol, taking care that the entire specimen is moistened. Heat the crucible on a steam bath to evaporate the alcohol, then dry the crucible at 100

for 30 min to prevent spattering upon subsequent heating. Transfer the crucible with its contents to a furnace heated to 500

, and heat the crucible for 15 min. [Note—Heating at 500

is necessary to carbonize any organic matter present; a higher temperature may be used, if necessary, to ensure complete carbonization of all organic matter. ]

Cool the crucible, add 25 mL of water, cover the crucible with a watchglass, and boil gently for 10 min. Filter the solution, and wash the crucible with boiling water, collecting the filtrate and washings in a beaker. Add phosphoric acid until the solution is neutral to methyl orange, then add 1 mL excess of phosphoric acid. Add excess of Bromine water, and boil the solution gently until colorless and then for 5 min longer. Add a few crystals of salicylic acid, and cool the solution to 20

. Add 1 mL of phosphoric acid and 0.5 g of potassium iodide, and titrate the liberated iodine with 0.005 N sodium thiosulfate VS, adding starch TS when the liberated iodine color has nearly disappeared.

Calculate the percentage of the labeled amount of iodine (I) in the portion of Capsules taken:

Result = V × NA × F × Ime × (Aw/W) × (100/L)

V	=	= volume of sodium thiosulfate consumed (mL)
NA	=	= actual normality of the sodium thiosulfate solution used
F	=	= correction factor to convert mg to µg (1000 µg/mL)
Ime	=	= milliequivalent of I (21.16 mg/meq)
Aw	=	= average weight of the Capsules content
W	=	= weight of the sample of Capsules content taken
L	=	= labeled amount of iodine (µg/Capsule)

Acceptance criteria: 90.0%–160.0% of the labeled amount of iodine (I)

• Iodide, Method 2: Proceed as directed in Automated Methods of Analysis

, Assay for Iodide.

Acceptance criteria: 90.0%–160.0% of the labeled amount of iodine (I)

• Iron, Method 1

Iron standard stock solution: Transfer 100 mg of iron powder to a 1000-mL volumetric flask. Dissolve in 25 mL of 6 N hydrochloric acid, dilute with water to volume, and mix.

Standard solutions: To separate 100-mL volumetric flasks transfer 2.0, 4.0, 5.0, 6.0, and 8.0 mL of Iron standard stock solution. Dilute the contents of each flask with water to volume to obtain concentrations of 2.0, 4.0, 5.0, 6.0, and 8.0 µg/mL of iron.

Polysorbate 80 solution: Prepare as directed for Calcium, Method 1.

Sample solution: Proceed as directed for Calcium, Method 1, except to prepare the Sample solution to contain a concentration of 5 µg/mL of iron and to omit the use of the Lanthanum chloride solution.

Instrumental conditions

Mode: Atomic absorption spectrophotometry

Analytical wavelength: Iron emission line at 248.3 nm

Lamp: Iron hollow-cathode

Flame: Air–acetylene

Blank: 0.125 N hydrochloric acid

Analysis

Samples: Standard solutions and Sample solution

Determine the absorbances of the solutions against the Blank. Plot the absorbances of the Standard solutions versus the concentration, in µg/mL, of iron, and draw the straight line best fitting the five plotted points. From the graph so obtained, determine the concentration, C, in µg/mL, of iron in the Sample solution.

Calculate the percentage of the labeled amount of iron (Fe) in the portion of Capsules taken:

Result = (C/CU) × 100

C	=	= measured concentration of iron in the Sample solution (µg/mL)
CU	=	= nominal concentration of iron in the Sample solution (µg/mL)

Acceptance criteria: 90.0%–125.0% of the labeled amount of iron (Fe)

• Magnesium, Method 1

Lanthanum chloride solution: Prepare as directed for Calcium, Method 1.

Magnesium standard solution: Transfer 1.0 g of magnesium ribbon to a 1000-mL volumetric flask, dissolve in 50 mL of 6 N hydrochloric acid, dilute with water to volume, and mix to obtain a solution with a known concentration of 1000 µg/mL of magnesium.

Standard stock solution: 20 µg/mL of magnesium from Magnesium standard solution diluted with 0.125 N hydrochloric acid

Standard solutions: To separate 100-mL volumetric flasks transfer 1.0, 1.5, 2.0, 2.5, and 3.0 mL of the Standard stock solution. To each flask add 1.0 mL of Lanthanum chloride solution, and dilute with 0.125 N hydrochloric acid to volume to obtain concentrations of 0.2, 0.3, 0.4, 0.5, and 0.6 µg/mL of magnesium.

Polysorbate 80 solution: Prepare as directed for Calcium, Method 1.

Sample solution: Proceed as directed for Calcium, Method 1, except prepare the Sample solution to contain 0.4 µg/mL of magnesium.

Instrumental conditions

Mode: Atomic absorption spectrophotometry

Analytical wavelength: Magnesium emission line at 285.2 nm

Lamp: Magnesium hollow-cathode

Flame: Air–acetylene

Blank: 0.125 N hydrochloric acid containing 1 mL of Lanthanum chloride solution per 100 mL

Analysis

Samples: Standard solutions and Sample solution

Determine the absorbances of the solutions against the Blank. Plot the absorbances of the Standard solutions versus the concentration, in µg/mL, of magnesium, and draw the straight line best fitting the five plotted points. From the graph so obtained, determine the concentration, C, in µg/mL, of magnesium in the Sample solution.

Calculate the percentage of the labeled amount of magnesium (Mg) in the portion of Capsules taken:

Result = (C/CU) × 100

C	=	= measured concentration of magnesium in the Sample solution (µg/mL)
CU	=	= nominal concentration of magnesium in the Sample solution (µg/mL)

Acceptance criteria: 90.0%–125.0% of the labeled amount of magnesium (Mg)

• Manganese, Method 1

Manganese standard stock solution: Transfer 1.00 g of manganese, to a 1000-mL volumetric flask. Dissolve in 20 mL of nitric acid, dilute with 6 N hydrochloric acid to volume, and mix to obtain a solution with a concentration of 1000 µg/mL of manganese.

Standard stock solution: 50 µg/mL of manganese from Manganese standard stock solution diluted with 0.125 N hydrochloric acid

Standard solutions: To separate 100-mL volumetric flasks transfer 1.0, 1.5, 2.0, 3.0, and 4.0 mL of Standard stock solution. Dilute the contents of each flask with 0.125 N hydrochloric acid to volume to obtain solutions with known concentrations of 0.5, 0.75, 1.0, 1.5, and 2.0 µg/mL of manganese.

Polysorbate 80 solution: Prepare as directed for Calcium, Method 1.

Sample solution: Proceed as directed for Calcium, Method 1, except prepare the Sample solution to contain to 1 µg/mL of manganese and to omit the use of the Lanthanum chloride solution.

Instrumental conditions

Mode: Atomic absorption spectrophotometry

Analytical wavelength: Manganese emission line at 279.5 nm

Lamp: Manganese hollow-cathode

Flame: Air–acetylene

Blank: 0.125 N hydrochloric acid

Analysis

Samples: Standard solutions and the Sample solution

Determine the absorbances of the solutions against the Blank. Plot the absorbances of the Standard solutions versus the concentration, in µg/mL, of manganese, and draw the straight line best fitting the five plotted points. From the graph so obtained, determine the concentration, C, in µg/mL, of manganese in the Sample solution.

Calculate the percentage of the labeled amount of manganese (Mn) in the portion of Capsules taken:

Result = (C/CU) × 100

C	=	= measured concentration of manganese in the Sample solution (µg/mL)
CU	=	= nominal concentration of manganese in the Sample solution (µg/mL)

Acceptance criteria: 90.0%–125.0% of the labeled amount of manganese (Mn)

• Molybdenum, Method 1

Diluent: 20 mg/mL of ammonium chloride in water

Molybdenum standard solution: Transfer 1.0 g of molybdenum wire to a 1000-mL volumetric flask, and dissolve in 50 mL of nitric acid, warming if necessary. Dilute with water to volume, and mix to obtain a solution with a concentration of 1000 µg/mL of molybdenum.

Standard stock solution: 100 µg/mL of molybdenum from Molybdenum standard solution diluted with water

Standard solutions: To separate 100-mL volumetric flasks transfer 2.0, 10.0, and 25.0 mL of the Standard stock solution, and add 5.0 mL of perchloric acid to each flask. Gently boil the solution in each flask for 15 min. Cool to room temperature, and dilute each with Diluent to volume to obtain concentrations of 5.0, 10.0, and 25.0 µg/mL of molybdenum.

Polysorbate 80 solution: Prepare as directed for Calcium, Method 1.

Sample solution: Proceed as directed for Calcium, Method 1, except take a number of Capsules or a portion of Capsule contents equivalent to 1000 µg of molybdenum and make appropriate dilutions to obtain a final concentration of 10 µg/mL of molybdenum, omitting the addition of the Lanthanum chloride solution.

Instrumental conditions

Mode: Atomic absorption spectrophotometry

Analytical wavelength: Molybdenum emission line at 313.3 nm

Lamp: Molybdenum hollow-cathode

Flame: Nitrous oxide–acetylene

Blank: Diluent and perchloric acid (20:1)

Analysis

Samples: Standard solutions and the Sample solution

Determine the absorbances of the solutions against the Blank. Plot the absorbances of the Standard solutions versus the concentration, in µg/mL, of molybdenum, and draw the straight line best fitting the three plotted points. From the graph so obtained, determine the concentration, C, in µg/mL, of molybdenum in the Sample solution.

Calculate the percentage of the labeled amount of molybdenum (Mo) in the portion of Capsules taken:

Result = (C/CU) × 100

C	=	= measured concentration of molybdenum in the Sample solution (µg/mL)
CU	=	= nominal concentration of molybdenum in the Sample solution (µg/mL)

Acceptance criteria: 90.0%–160.0% of the labeled amount of molybdenum (Mo)

• Molybdenum, Method 2

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

Ferrous sulfate solution: 4.98 mg/mL of ferrous sulfate in water

Potassium thiocyanate solution: 200 mg/mL of potassium thiocyanate in water

20% Stannous chloride solution: Transfer 40 mg of stannous chloride to a beaker, add 20 mL of 6.5 N hydrochloric acid solution, and heat the solution until the stannous chloride is dissolved. Cool and dilute with water to 100 mL.

Diluted stannous chloride solution: 20% stannous chloride solution diluted with water (1 in 25). Prepare this solution fresh at the time of use.

Standard solution: 20 µg/mL of molybdenum in water

Sample: Remove the contents of a counted number of Capsules by cutting open the Capsules. Mix, and determine the weight of the contents. Use a quantity of the Capsule contents, equivalent to 40 µg of molybdenum.

Instrumental conditions

Mode: UV-Vis

Analytical wavelength: 465 nm

Cell: 1 cm

Blank: Amyl alcohol

Analysis

Samples: Standard solution and Sample

Transfer the Sample and 2.0 mL of the Standard solution to separate 200-mL beakers. Add 20 mL of nitric acid to each beaker. Cover each beaker with a watchglass, and boil slowly on a hot plate for 45 min. Cool to room temperature. Add 6 mL of perchloric acid, cover the beakers with a watchglass, and continue the heating until digestion is complete, as indicated when the liquid becomes colorless or pale yellow. Evaporate the solutions in the beakers to dryness. Rinse the sides of the beakers and the watchglasses with water, and add more water to complete 50 mL in each beaker. Gently boil the water solution for a few min. Cool to room temperature. Add 2 drops of methyl orange TS, and neutralize with ammonium hydroxide. Add 8.2 mL of hydrochloric acid. Quantitatively transfer the contents of the beakers to separate 100-mL volumetric flasks, rinse the beakers with water, transfer the rinsings to the corresonding volumetric flasks, and dilute with water to volume. Transfer 50.0 mL of each solution to separatory funnels. To each separatory funnel add 1.0 mL of Sodium fluoride solution, 0.5 mL of Ferrous sulfate solution, 4.0 mL of Potassium thiocyanate solution, 1.5 mL of 20% Stannous chloride solution, and 15.0 mL of amyl alcohol, and shake the separatory funnel for 1 min. Allow the layers to separate, and discard the aqueous layers. Add 25 mL of Diluted stannous chloride solution to each separatory funnel, and shake gently for 15 s. Allow the layers to separate, and discard the aqueous layers. Transfer the organic layer from each separatory funnel to a centrifuge tube, and centrifuge at 2000 rpm for 10 min. Determine the absorbances of the organic phases obtained from the Standard solution and the Sample, and correct with the Blank.

Calculate the percentage of the labeled amount of molybdenum (Mo) in the portion of Capsules taken:

Result = (AU/AS) × [(V × CS)/MU] × 100

AU	=	= absorbance of the Sample
AS	=	= absorbance of the Standard solution
V	=	= volume of the Standard solution analyzed, 2.0 mL
CS	=	= concentration of molybdenum in the Standard solution (µg/mL)
MU	=	= nominal amount of molybdenum in the Sample (µg)

Acceptance criteria: 90.0%–160.0% of the labeled amount of molybdenum (Mo)

• Phosphorus, Method 1

Sulfuric acid solution: Cautiously add sulfuric acid to water (37.5:100), and mix.

Ammonium molybdate solution: 50 mg/mL of ammonium molybdate in Sulfuric acid solution and water (2:3). [Note—Dissolve in water first, and then dilute with Sulfuric acid solution to volume. ]

Hydroquinone solution: 5 mg/mL of hydroquinone in water. Add one drop of sulfuric acid per 100 mL of solution.

Sodium bisulfite solution: 200 mg/mL of sodium bisulfite in water

Phosphorus standard stock solution: Weigh 4.395 g of monobasic potassium phosphate, previously dried at 105

for 2 h and stored in a desiccator, and transfer to a 1000-mL volumetric flask. Dissolve in water, add 6 mL of sulfuric acid as a preservative, dilute with water to volume, and mix to obtain a solution with a concentration of 1000 µg/mL of phosphorus.

Standard solution: 20 µg/mL of phosphorus from Phosphorus standard stock solution diluted with water

Sample solution: Remove the contents of Capsules by cutting open the Capsules. Mix, and determine the weight of the contents. Transfer a quantity of the Capsule contents, equivalent to 100 mg of phosphorus, to 25 mL of nitric acid, and digest on a hot plate for 30 min. Add 15 mL of hydrochloric acid, and continue the digestion to the cessation of brown fumes. Cool, and transfer the contents of the flask to a 500-mL volumetric flask with the aid of small portions of water. Dilute with water to volume. Transfer 10.0 mL of this solution to a 100-mL volumetric flask, and dilute with water to volume.

Instrumental conditions

Mode: Vis

Analytical wavelength: 650 nm

Cell: 1 cm

Analysis

Samples: Standard solution and Sample solution

To three separate 25-mL volumetric flasks transfer 5.0 mL each of the Standard solution, the Sample solution, and water to provide the blank. To each of the three flasks add 1.0 mL each of Ammonium molybdate solution, Hydroquinone solution, and Sodium bisulfite solution, and swirl to mix. Dilute the contents of each flask with water to volume, and allow the flasks to stand for 30 min. Determine the absorbances of the solutions against the blank.

Calculate the percentage of the labeled amount of phosphorus (P) in the portion of Capsules taken:

Result = (AU/AS) × (CS/CU) × 100

AU	=	= absorbance of the Sample solution
AS	=	= absorbance of the Standard solution
CS	=	= concentration of phosphorus in the Standard solution (µg/mL)
CU	=	= nominal concentration of phosphorus in the Sample solution (µg/mL)

Acceptance criteria: 90.0%–125.0% of the labeled amount of phosphorus (P)

• Potassium

Potassium standard solution: 100 µg/mL of potassium from potassium chloride, previously dried at 105

for 2 h, in water

Standard stock solution: 10 µg/mL of potassium from Potassium standard solution diluted with 0.125 N hydrochloric acid

Standard solutions: Transfer 5.0, 10.0, 15.0, 20.0, and 25.0 mL of the Standard stock solution to separate 100-mL volumetric flasks. Dilute the contents of each flask with 0.125 N hydrochloric acid to volume to obtain solutions containing 0.5, 1.0, 1.5, 2.0, and 2.5 µg/mL of potassium.

Polysorbate 80 solution: Prepare as directed for Calcium, Method 1.

Sample solution: Proceed as directed for Calcium, Method 1, except to prepare the Sample solution to contain 1 µg/mL of potassium and to omit the use of the Lanthanum chloride solution.

Instrumental conditions

Mode: Atomic absorption spectrophotometry

Analytical wavelength: Potassium emission line at 766.5 nm

Lamp: Potassium hollow-cathode

Flame: Air–acetylene

Blank: Water

Analysis

Samples: Standard solutions and the Sample solution

Determine the absorbances of the solutions against the Blank. Plot the absorbances of the Standard solutions versus the concentration, in µg/mL, of potassium, and draw the straight line best fitting the five plotted points. From the graph so obtained, determine the concentration, C, in µg/mL, of potassium in the Sample solution.

Calculate the percentage of the labeled amount of potassium (K) in the portion of Capsules taken:

Result = (C/CU) × 100

C	=	= measured concentration of potassium in the Sample solution (µg/mL)
CU	=	= nominal concentration of potassium in the Sample solution (µg/mL)

Acceptance criteria: 90.0%–125.0% of the labeled amount of potassium (K)

• Selenium, Method 1

Diluent: Prepare as directed for Molybdenum, Method 1.

Selenium standard solution: [Caution—Selenium is toxic; handle it with care. ] Dissolve 1 g of metallic selenium in a minimum volume of nitric acid. Evaporate to dryness, add 2 mL of water, and evaporate to dryness. Repeat the addition of water and the evaporation to dryness three times. Dissolve the residue in 3 N hydrochloric acid, transfer to a 1000-mL volumetric flask, and dilute with 3 N hydrochloric acid to volume to obtain a concentration of 1000 µg/mL of selenium.

Standard stock solution: 100 µg/mL of selenium from Selenium standard solution diluted with water

Standard solutions: To separate 100-mL volumetric flasks transfer 5.0, 10.0, and 25.0 mL of the Standard stock solution, and add 5.0 mL of perchloric acid to each flask. Gently boil the solutions for 15 min, cool to room temperature, and dilute each with Diluent to volume to obtain solutions with concentrations of 5.0, 10.0, and 25.0 µg/mL of selenium.

Sample solution: Remove the contents of Capsules by cutting open the Capsules. Mix, and determine the weight of the contents. Transfer a quantity of the Capsule contents, equivalent to 1000 µg of selenium, to a suitable flask, and add 12 mL of nitric acid. [Note—The volume of nitric acid may be varied to ensure that the powder is uniformly dispersed. ] Carefully swirl the flask to disperse the sample specimen. Sonicate for 10 min or until the sample specimen is completely dissolved. Gently boil the solution for 15 min, and cool to room temperature. Carefully add 8 mL of perchloric acid to the flask, heat the flask until perchloric acid fumes appear, and swirl the flask to dissipate the fumes. Repeat the heating and swirling until the fumes appear again. Cool to room temperature. Transfer the contents of the flask to a 50-mL volumetric flask with the aid of the Diluent, and dilute with Diluent to volume.

Instrumental conditions

Mode: Atomic absorption spectrophotometry

Analytical wavelength: Selenium emission line at 196.0 nm

Lamp: Selenium hollow-cathode

Flame: Air–acetylene

Blank: Diluent and perchloric acid (20:1)

Analysis

Samples: Standard solutions and the Sample solution

Determine the absorbances of the solutions against the Blank. Plot the absorbances of the Standard solutions versus the concentration, in µg/mL, of selenium, and draw the straight line best fitting the three plotted points. From the graph so obtained, determine the concentration, C, in µg/mL, of selenium in the Sample solution.

Calculate the percentage of the labeled amount of selenium (Se) in the portion of Capsules taken:

Result = (C/CU) × 100

C	=	= measured concentration of selenium in the Sample solution (µg/mL)
CU	=	= nominal concentration of selenium in the Sample solution (µg/mL)

Acceptance criteria: 90.0%–160.0% of the labeled amount of selenium (Se)

• Selenium, Method 2

Hydrochloric acid solution: Hydrochloric acid diluted with water (1 in 10)

50% ammonium hydroxide solution: Ammonium hydroxide diluted with water (1 in 2)

Reagent A: 9 mg/mL of edetate disodium and 25 mg/mL of hydroxylamine hydrochloride in water. [Note—Dissolve edetate disodium in a portion of water first, then add hydroxylamine hydrochloride, and dilute with water to volume. ]

Reagent B: Transfer 200 mg of 2,3-diaminonaphthalene to a 250-mL separatory funnel, and add 200 mL of 0.1 N hydrochloric acid. Wash the solution with three 40-mL portions of cyclohexane, and discard the cyclohexane layer. Filter the solution into a brown bottle, and cover the solution with a 1-cm layer of cyclohexane. This solution is stable for 1 week if stored in a refrigerator.

Standard stock solution: [Caution—Selenium is toxic; handle it with care. ] Dissolve 1 g of metallic selenium in a minimum volume of nitric acid. Evaporate to dryness, add 2 mL of water, and evaporate to dryness. Repeat the addition of water and evaporation to dryness three times. Dissolve the residue in 3 N hydrochloric acid, transfer to a 1000-mL volumetric flask, and dilute with 3 N hydrochloric acid to volume to obtain a solution with a concentration of 1000 µg/mL of selenium. Dilute a volume of the solution with 0.125 N hydrochloric acid to obtain a concentration of 2.0 µg/mL of selenium.

Standard solution: Transfer 10 mL of the Standard stock solution to a glass-stoppered flask. Add 1 mL of perchloric acid and 1 mL of Hydrochloric acid solution, and dilute with water to 20 mL.

Sample solution: Remove the contents of Capsules by cutting open the Capsules. Mix, and determine the weight of the contents. Transfer a quantity of the Capsule contents, equivalent to 20 µg of selenium, to a suitable flask. Add 10 mL of nitric acid, and warm gently on a hot plate. Continue heating until the initial nitric acid reaction has subsided, then add 3 mL of perchloric acid.

[Caution—Exercise care at this stage, because perchloric acid reaction becomes vigorous. ]

Continue heating on the hot plate until the appearance of white fumes of perchloric acid or until the digest begins to darken. Add 0.5 mL of nitric acid, and resume heating, adding additional amounts of nitric acid if further darkening occurs. Digest for 10 min after the first appearance of perchloric acid fumes or until the digest becomes colorless. Cool the flask. Add 2.5 mL of Hydrochloric acid solution, and return the flask to the hot plate to expel residual nitric acid. Heat the mixture for 3 min after it begins to boil. Cool the flask to room temperature, and dilute with water to 20 mL.

Instrumental conditions

Mode: UV

Analytical wavelength: 380 nm

Cell: 1 cm

Blank: 1 mL of perchloric acid and 1 mL of Hydrochloric acid solution diluted with water to 20 mL

Analysis

Samples: Standard solution and Sample solution

Treat the Sample solution, the Standard solution, and the Blank as follows. Add 5 mL of Reagent A to each flask, and swirl gently to mix. Adjust the solution in each flask with 50% Ammonium hydroxide solution to a pH of 1.1 ± 0.1. Add 5 mL of Reagent B to each flask, and swirl gently to mix. Place the flasks in a water bath maintained at 50

, and equilibrate for 30 min, taking care that the flasks are covered to protect them from light. Cool to room temperature, and transfer the contents of each flask to separate separatory funnels. Transfer 10.0 mL of cyclohexane to each separatory funnel, and extract vigorously for 1 min. Discard the aqueous layer. Transfer the cyclohexane layer to a centrifuge tube, and centrifuge at 1000 rpm for 1 min to remove any remaining water. Determine the absorbances of the solutions obtained from the Samples against the solution obtained from the Blank.

Calculate the percentage of the labeled amount of selenium (Se) in the portion of Capsules taken:

Result = (AU/AS) × [(V × CS)/MU] × 100

AU	=	= absorbances of the cyclohexane layer from the Sample solution
AS	=	= absorbances of the cyclohexane layer from the Standard solution
V	=	= volume of the Standard stock solution used to prepare the Standard solution, 10 mL
CS	=	= concentration of selenium in the Standard stock solution (µg/mL)
MU	=	= nominal amount of selenium in the Sample solution (µg)

Acceptance criteria: 90.0%–160.0% of the labeled amount of selenium (Se)

• Zinc, Method 1

Zinc standard solution: 1000 µg/mL of zinc from zinc oxide in 5 M hydrochloric acid (3.89 mg/mL) and diluted with water to final volume. [Note—Dissolve in 5 M hydrochloric acid by warming, if necessary, cool, and then dilute to final volume. ]

Standard stock solutions: 50 µg/mL of zinc from Zinc standard solution diluted with 0.125 N hydrochloric acid

Standard solutions: Transfer 1.0, 2.0, 3.0, 4.0, and 5.0 mL of Standard stock solution to separate 100-mL volumetric flasks. Dilute the contents of each flask with 0.125 N hydrochloric acid to volume to obtain concentrations of 0.5, 1.0, 1.5, 2.0, and 2.5 µg/mL of zinc.

Polysorbate 80 solution: Prepare as directed for Calcium, Method 1.

Sample solution: Proceed as directed for Calcium, Method 1, except to prepare the Sample solution to contain 2 µg/mL of zinc and to omit the use of the Lanthanum chloride solution.

Instrumental conditions