Mobile phase— Use n-hexane.

Standard preparation— Dissolve an accurately weighed quantity of USP Vitamin A RS in n-hexane, and dilute quantitatively, and stepwise if necessary, to obtain a solution having a known concentration of about 15 µg of retinyl acetate per mL.

System suitability preparation— Dissolve an accurately weighed quantity of retinyl palmitate in n-hexane to obtain a solution having a concentration of about 15 µg of retinyl palmitate per mL. Mix equal volumes of this solution and the Standard preparation to obtain a solution having concentrations of 7.5 µg each of retinyl acetate and retinyl palmitate per mL.

Assay preparation— Weigh and finely powder not fewer than 20 Tablets. Transfer an accurately weighed portion of the powder, equivalent to 5 Tablets, to a container having a polytef-lined screw cap, add 10 mL of dimethyl sulfoxide and 15 mL of n-hexane, and shake for 45 minutes on a wrist-action shaker in a water bath maintained at 60. [note—Set up the wrist-action shaker to ensure that the contents of the container are mixed vigorously and thoroughly.] Centrifuge at 3000 rpm for 10 minutes, and transfer the hexane layer by means of a pipet to a 100-mL volumetric flask. Add 15 mL of n-hexane to the dimethyl sulfoxide layer, shake thoroughly for 5 minutes, and transfer the hexane layer by means of a pipet to the 100-mL volumetric flask. Repeat this extraction with three additional 15-mL portions of n-hexane. Dilute the extracts in the volumetric flask with n-hexane to volume, and mix. Quantitatively dilute a 10-mL volume of this solution with n-hexane to obtain a solution having a final concentration of about 15 µg of vitamin A per mL. Retain the remaining solution for use in the assays for vitamin D, vitamin E, and phytonadione.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 325-nm detector and a 4.6-mm × 15-cm column that contains 3-µm packing L8. The flow rate is about 1 mL per minute. Chromatograph the System suitability preparation, and measure the peak responses as directed for Procedure: the resolution, R, between retinyl acetate and retinyl palmitate is not less than 10; and the relative standard deviation for replicate injections is not more than 3.0%.

Procedure— Separately inject equal volumes (about 40 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the peak area for retinyl acetate obtained from the Standard preparation and the peak area for retinyl acetate or retinyl palmitate in the chromatogram of the Assay preparation. For products containing vitamin A acetate or vitamin A palmitate, calculate the quantity, in mg, of vitamin A as the retinol equivalent (C20H30O) in the portion of Tablets taken by the formula: in which 0.872 is the factor used to convert retinyl acetate, obtained from USP Vitamin A RS, to its retinol equivalent; C is the concentration, in mg per mL, of USP Vitamin A RS in the Standard preparation; D is the dilution factor, in mL, for the Assay preparation; and rU and rS are the peak areas of the all-trans retinyl ester obtained from the Assay preparation and the Standard preparation, respectively. [note—The molar responses of retinyl acetate and retinyl palmitate are equivalent.]

3 N Methanolic sulfuric acid solution— Cautiously add 9 mL of sulfuric acid to 80 mL of methanol in a 100-mL volumetric flask. Cool, dilute with methanol to volume, and mix.

Sodium ascorbate–pyrogallol solution— Transfer 10 g of sodium ascorbate and 5 g of pyrogallol to a 100-mL volumetric flask, and add sufficient water to dissolve. Add 1.7 mL of sulfuric acid, dilute with water to volume, and mix.

Lecithin solution— Transfer 0.5 g of lecithin to a 100-mL volumetric flask, dissolve in and dilute with 2,2,4-trimethylpentane to volume, and mix.

Mobile phase— Prepare a mixture of n-hexane and ethyl acetate (99.7:0.3).

Standard preparation— Dissolve an accurately weighed quantity of USP Vitamin A RS in 2,2,4-trimethylpentane, and dilute quantitatively, and stepwise if necessary, with 2,2,4-trimethylpentane to obtain a solution having a known concentration of about 15 µg of retinyl acetate per mL.

System suitability preparation— Dissolve an accurately weighed quantity of retinyl palmitate in 2,2,4-trimethylpentane to obtain a solution having a concentration of about 15 µg per mL. Mix equal volumes of this solution and the Standard preparation to obtain a solution having concentrations of 7.5 µg each of retinyl acetate and retinyl palmitate per mL.

Assay preparation— [note—This preparation is suitable for the determination of vitamin A, vitamin D, and vitamin E, when present in the formulation.] Weigh and finely powder not fewer than 20 Tablets. If vitamin D is present in the formulation, transfer an accurately weighed portion of the powder, equivalent to about 30 µg of cholecalciferol or ergocalciferol, to a container having a polytef-lined screw cap. If vitamin D is not present in the formulation, use a portion of the powder equivalent to about 90 mg of vitamin E (as alpha tocopherol, alpha tocopheryl acetate, or alpha tocopheryl hemisuccinate). If vitamin E is not present in the formulation, use a portion of the powder equivalent to about 2.5 mg of retinyl acetate or retinyl palmitate. Add about 0.5 g of sodium bicarbonate, 1.5 mL of Lecithin solution, and 12.5 mL of 2,2,4-trimethylpentane, and disperse on a vortex mixer. Add 6 mL of Sodium ascorbate–pyrogallol solution, shake slowly, and allow the solution to degas. Continue shaking until the evolution of gas has ceased, and then shake for an additional 12 minutes. Add 6 mL of dimethyl sulfoxide, mix on a vortex mixer to form a suspension, and shake for 12 minutes. Add 6 mL of 3 N Methanolic sulfuric acid solution, mix on a vortex mixer to form a suspension, and shake for 12 minutes. Add 12.5 mL of 2,2,4-trimethylpentane, mix on a vortex mixer to form a suspension, and shake for 10 minutes. Centrifuge for about 10 minutes to break up the emulsion and to clarify the supernatant. [note—The supernatant is used for the determination of vitamin A, and also vitamin D and vitamin E, if present in the formulation.] If necessary, quantitatively dilute a volume of the supernatant with 2,2,4-trimethylpentane to obtain a solution having a concentration close to that of the Standard preparation.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 325-nm detector and a 4.6-mm × 25-cm column that contains 5-µm packing L24. The flow rate is about 1.5 mL per minute. Chromatograph the System suitability preparation, and measure the peak areas as directed for Procedure: the resolution, R, between retinyl acetate and retinyl palmitate is not less than 8.0; and the relative standard deviation for replicate injections is not more than 3.0%.

Procedure— Separately inject equal volumes (about 40 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the peak area of retinyl acetate obtained from the Standard preparation and the peak area of retinyl acetate or retinyl palmitate obtained from the Assay preparation. Calculate the quantity, in mg, of vitamin A, as the retinol (C20H30O) equivalent, in the portion of Tablets taken by the formula: in which E is the factor used to convert the retinyl acetate, obtained from USP Vitamin A RS, to its retinol equivalent, the factor being 0.872; C is the concentration, in mg per mL, of USP Vitamin A RS in the Standard preparation; D is the dilution factor, in mL, used to prepare the Assay preparation from the supernatant; and rU and rS are the peak responses of the all-trans retinyl ester obtained from the Assay preparation and the Standard preparation, respectively. [note—The initial extraction into 26.5 mL of 2,2,4-trimethylpentane is already accounted for in this equation. The molar responses of retinyl acetate and retinyl palmitate are equivalent.]

Extraction solvent— Prepare a mixture of n-hexane and methylene chloride (3:1).

Potassium hydroxide solution— Cautiously add 80 g of potassium hydroxide to 100 mL of water, mix, and cool.

Diluting solution— Transfer 1.0 g of pyrogallol to a 100-mL volumetric flask, and add alcohol to dissolve. Dilute with alcohol to volume, and mix.

Mobile phase— Prepare a mixture of n-hexane and isopropyl alcohol (92:8). Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard stock solution— Dissolve an accurately weighed quantity of USP Vitamin A RS in Diluting solution, and dilute quantitatively, and stepwise if necessary, with Diluting solution to obtain a solution having a known concentration of about 30 µg per mL. This solution may be stored in a refrigerator for 1 week.

Standard preparation— Quantitatively dilute an accurately measured volume of Standard stock solution with Diluting solution to obtain a solution having a known concentration of about 1 µg of USP Vitamin A RS per mL. Transfer 10.0 mL of this solution to a stoppered 125-mL flask, and add 5 mL of water, 5 mL of Diluting solution, and 3 mL of Potassium hydroxide solution. Insert the stopper tightly, shake for 15 minutes over a water bath maintained at 60 ± 5, and cool to room temperature. Add 7 mL of water and 25.0 mL of Extraction solvent. Insert the stopper tightly, and shake vigorously for 60 seconds. Rinse the sides of the flask with about 60 mL of water, and allow to stand for about 10 minutes until the layers separate. Withdraw a portion of the organic layer for injection into the chromatograph. This Standard preparation contains about 0.34 µg of retinol per mL.

Assay preparation— Weigh and finely powder a counted number of Tablets. Transfer an accurately weighed portion of the powder, equivalent to about 1.5 mg of retinyl acetate, to a stoppered 125-mL flask. Add 5 mL of water, 15 mL of Diluting solution, and 3 mL of Potassium hydroxide solution. Insert the stopper tightly, shake for 15 minutes over a water bath maintained at 60 ± 5, and cool to room temperature. Add 7 mL of water and 25.0 mL of Extraction solvent. Insert the stopper tightly, and shake vigorously for 60 seconds or longer, if necessary, for complete extraction. Rinse the sides of the flask with about 60 mL of water, and allow to stand for about 10 minutes until the layers separate. [note—Do not shake, as an emulsion may form.] Withdraw a portion of the organic layer, and dilute quantitatively, and stepwise if necessary, with Extraction solvent to obtain a solution having a concentration of about 0.34 µg of retinol per mL.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 335-nm detector and a 6.2-mm × 8-cm column that contains packing L3. The column temperature is maintained at 40, and the flow rate is about 4 mL per minute. Chromatograph the Standard preparation, and measure the peak areas as directed for Procedure: the relative retention times are about 0.92 for 13-cis retinol and 1.0 for all-trans retinol; and the relative standard deviation for replicate injections is not more than 5.0%.

Procedure— Separately inject equal volumes (about 50 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the peak areas for all-trans retinol and 13-cis retinol. Calculate the quantity, in mg, of vitamin A, as the retinol (C20H30O) equivalent, in the portion of Tablets taken by the formula: in which 0.872 is the factor used to convert retinyl acetate, obtained from USP Vitamin A RS, to its retinol equivalent; C is the concentration, in mg per mL, of USP Vitamin A RS in the Standard preparation; D is the dilution factor, in mL, used to prepare the Assay preparation; rU is the sum of the areas of the all-trans retinol and 13-cis retinol peaks obtained from the Assay preparation; and rS is the peak area of all-trans retinyl acetate obtained from the Standard preparation.

Mobile phase— Prepare a filtered and degassed mixture of n-hexane and isopropyl alcohol (99:1). Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard preparation— Dissolve an accurately weighed quantity of USP Cholecalciferol RS or USP Ergocalciferol RS in n-hexane, and dilute quantitatively, and stepwise if necessary, to obtain a solution having a known concentration of about 2 µg per mL.

System suitability preparation— Heat a volume of the Standard preparation at 60 for 1 hour to partially isomerize vitamin D (cholecalciferol or ergocalciferol) to its corresponding precursor.

Assay preparation— Transfer not less than 20 mL, accurately measured, of the solution retained as specified in the directions for Assay preparation in the Assay for vitamin A, Method 1 to a suitable container, and evaporate, if necessary, in vacuum at room temperature to obtain a solution having a concentration of about 2 µg of cholecalciferol or ergocalciferol per mL.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 265-nm detector and a 4.6-mm × 15-cm column that contains 3-µm packing L8. The flow rate is about 1 mL per minute. Chromatograph the System suitability preparation, and record the peak heights as directed for Procedure: the resolution, R, between the vitamin D form present and its corresponding precursor is not less than 10. Chromatograph the Standard preparation, and record the peak heights as directed for Procedure: the relative standard deviation for replicate injections is not more than 3.0%.

Procedure— Separately inject equal volumes (about 100 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the peak heights for vitamin D. Calculate the quantity, in µg, of cholecalciferol (C27H44O) or ergocalciferol (C28H44O) in the portion of Tablets taken by the formula: in which 1.09 is a correction factor to account for the average amount of previtamin D present in the formulation; C is the concentration, in µg per mL, of USP Cholecalciferol RS or USP Ergocalciferol RS in the Standard preparation; D is the dilution factor, in mL, for the Assay preparation; and rU and rS are the peak heights for cholecalciferol or ergocalciferol obtained from the Assay preparation and the Standard preparation, respectively.

3 N Methanolic sulfuric acid solution, Sodium ascorbate–pyrogallol solution, Lecithin solution, and Assay preparation— Proceed as directed in the Assay for vitamin A, Method 2.

Mobile phase— Prepare a filtered and degassed mixture of n-hexane and tertiary butyl alcohol (98.75:1.25). Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard preparation— Dissolve an accurately weighed quantity of USP Cholecalciferol RS or USP Ergocalciferol RS in 2,2,4-trimethylpentane, and dilute quantitatively, and stepwise if necessary, with 2,2,4-trimethylpentane to obtain a solution having a known concentration of about 1 µg per mL.

System suitability preparation— Heat a volume of the Standard preparation at 60 for 1 hour to partially isomerize vitamin D (cholecalciferol or ergocalciferol) to its corresponding precursor.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 265-nm detector and a 4.6-mm × 25-cm column that contains 5-µm packing L24. The flow rate is about 1.0 mL per minute. Chromatograph the System suitability preparation, and record the peak areas as directed for Procedure: the resolution, R, between the vitamin D form present and its corresponding precursor is not less than 4.0. Chromatograph the Standard preparation, and record the peak areas as directed for Procedure: the relative standard deviation for replicate injections is not more than 3.0%.

Procedure— Separately inject equal volumes (about 40 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the peak areas for vitamin D. Calculate the quantity, in µg, of cholecalciferol (C27H44O) or ergocalciferol (C28H44O) in the portion of Tablets taken by the formula: in which C is the concentration, in µg per mL, of USP Cholecalciferol RS or USP Ergocalciferol RS in the Standard preparation; and rU and rS are the peak responses of cholecalciferol or ergocalciferol in the Assay preparation and the Standard preparation, respectively.

Dilute acetic acid— Transfer 10 mL of glacial acetic acid to a 100-mL volumetric flask, dilute with water to volume, and mix.

Phenolphthalein solution— Prepare a solution containing 1 g of phenolphthalein in 100 mL of alcohol.

Potassium hydroxide solution— Slowly dissolve 14 g of potassium hydroxide in a mixture of 31 mL of dehydrated alcohol and 5 mL of water. Prepare fresh daily.

Extraction solvent— Prepare a mixture of methylene chloride and isopropyl alcohol (99.8:0.2).

Mobile phase— Prepare a mixture of acetonitrile and methanol (91:9). Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard stock solution— Dissolve an accurately weighed quantity of USP Cholecalciferol RS or USP Ergocalciferol RS in dehydrated alcohol to obtain a solution having a known concentration of about 0.2 mg per mL. Prepare fresh every 4 weeks. Store in a freezer.

Standard preparation— [note—Condition the solid-phase extraction column specified for use in the Standard preparation and the Assay preparation by initially washing the column with 4.0 mL of a mixture of methylene chloride and isopropyl alcohol (80:20), followed by 5.0 mL of Extraction solvent. Do not allow the column to dry.] Dilute an accurately measured volume of Standard stock solution with dehydrated alcohol to obtain a solution having a known concentration of about 5 µg of USP Cholecalciferol RS or USP Ergocalciferol RS per mL. Prepare this solution fresh daily. Transfer 2.0 mL of this solution to a stoppered 125-mL flask. Add 15.0 mL of water and 15.0 mL of Potassium hydroxide solution, insert the stopper, and shake for 30 minutes in a water bath maintained at 60. Allow to cool to room temperature, and transfer the contents of the flask to a 250-mL separatory funnel. Add 15.0 mL of water to the flask, insert the stopper, shake vigorously, and transfer this solution to the separatory funnel. Rinse the flask with 60 mL of n-hexane, and transfer the rinsing to the separatory funnel. Insert the stopper, shake vigorously for 90 seconds, and allow to stand for about 15 minutes until the layers separate. Drain and discard the aqueous layer. Add 15.0 mL of water to the hexane layer in the separatory funnel, insert the stopper, and shake vigorously. Allow to stand for about 10 minutes until the layers separate, and discard the aqueous layer. Add 1 drop of Phenolphthalein solution and 15.0 mL of water to the separatory funnel. Add Dilute acetic acid dropwise, with shaking, until the washing is neutral. Allow to stand for about 10 minutes until the layers separate. Drain and discard the aqueous layer. Filter the hexane layer through anhydrous sodium sulfate supported by a small pledget of cotton into a 100-mL round-bottom flask. Rinse the funnel and sodium sulfate with a few mL of n-hexane, and collect the rinsings in the same flask. Evaporate the hexane in the flask on a rotary evaporator at 50 to dryness. Immediately add 2.0 mL of Extraction solvent to dissolve the residue. Transfer this solution to a freshly conditioned solid-phase extraction column containing silica packing with a sorbent mass to column volume ratio of 500 mg to 2.8 mL or equivalent, rinse the round-bottom flask with 1.0 mL of Extraction solvent, and transfer to the column. Elute the column with 2.0 mL of Extraction solvent, and discard this fraction. Elute the column with 7.0 mL of Extraction solvent, and collect the eluate in a suitable flask. Place the flask in a warm water bath maintained at about 42, and evaporate the solvent with the aid of a stream of nitrogen. Immediately add 2.0 mL of acetonitrile to the residue, and use the solution for injection into the chromatograph.

Assay preparation— Weigh and finely powder not fewer than 20 Tablets. Transfer an accurately weighed portion of the powder, equivalent to about 10 µg of cholecalciferol or ergocalciferol, to the stoppered 125-mL flask, and proceed as directed for the Standard preparation, beginning with “Add 15.0 mL of water and 15.0 mL of Potassium hydroxide solution”.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 265-nm detector and a 4.6-mm × 25-cm column that contains 5-µm packing L1. The column temperature is maintained at 27, and the flow rate is about 0.7 mL per minute. Chromatograph the Standard preparation, and record the peak heights as directed for Procedure: the relative standard deviation for replicate injections is not more than 4.0%.

Procedure— Separately inject equal volumes (about 15 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the peak heights for vitamin D. Calculate the quantity, in µg, of cholecalciferol (C27H44O) or ergocalciferol (C28H44O) in the portion of Tablets taken by the formula: in which 1.09 is a correction factor to account for the average amount of previtamin D present in the formulation; C is the concentration, in µg per mL, of USP Cholecalciferol RS or USP Ergocalciferol RS in the Standard preparation; and rU and rS are the peak heights for cholecalciferol or ergocalciferol obtained from the Assay preparation and the Standard preparation, respectively.

Mobile phase— Dilute 10 mL of phosphoric acid with water to 1000 mL to obtain Solution A. Prepare a filtered and degassed mixture of methanol and Solution A (95:5). Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard preparation— Dissolve an accurately weighed quantity of USP Alpha Tocopherol RS, USP Alpha Tocopheryl Acetate RS, or USP Alpha Tocopheryl Acid Succinate RS in methanol, and dilute quantitatively with methanol to obtain a solution having a known concentration of about 2 mg per mL.

System suitability preparation— Dissolve an accurately weighed quantity of USP Ergocalciferol RS in methanol, and dilute quantitatively, and stepwise if necessary, with methanol to obtain a solution having a concentration of 0.65 mg per mL. Transfer 1.0 mL of this solution to a 100-mL volumetric flask containing about 100 mg of USP Alpha Tocopheryl Acetate RS, accurately weighed. Dissolve in 30 mL of methanol, with the aid of sonication if necessary, dilute with methanol to volume, and mix. Store this solution in a refrigerator.

Assay preparation— Transfer not less than 20 mL, accurately measured, of the solution retained as specified in the directions for Assay preparation in the Assay for vitamin A, Method 1 to a suitable container, and evaporate in vacuum at room temperature to dryness. Transfer the residue with the aid of methanol to a suitable volumetric flask, and dilute with methanol to volume to obtain a solution having a concentration of about 2 mg of alpha tocopherol, alpha tocopheryl acetate, or alpha tocopheryl acid succinate per mL.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 254-nm detector and an 8-mm × 10-cm column that contains 5-µm packing L1. The flow rate is about 2 mL per minute. Chromatograph the System suitability preparation, and record the peak areas as directed for Procedure: the relative retention times are about 0.5 for ergocalciferol and 1.0 for alpha tocopheryl acetate; the resolution, R, between ergocalciferol and alpha tocopheryl acetate is not less than 12; and the tailing factor is between 0.8 and 1.2. Chromatograph the Standard preparation, and record the peak areas as directed for Procedure: the relative standard deviation for replicate injections is not more than 3.0%.

Procedure— Separately inject equal volumes (about 100 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the peak areas. Calculate the quantity, in mg, of alpha tocopherol (C29H50O2), alpha tocopheryl acetate (C31H52O3), or alpha tocopheryl acid succinate (C33H54O5) in the portion of Tablets taken by the formula: in which C is the concentration, in mg per mL, of the corrresponding USP Reference Standard in the Standard preparation; D is the dilution factor, in mL, for the Assay preparation; and rU and rS are the peak responses for the relevant vitamin E form obtained from the Assay preparation and the Standard preparation, respectively. Calculate the alpha tocopherol equivalent of alpha tocopheryl acetate or alpha tocopheryl acid succinate by multiplying the content, in mg, by the factor 0.91 or 0.81, respectively.

Mobile phase— Transfer 240 mL of methanol to a 1000-mL volumetric flask, add 10 mL of water followed by 0.5 mL of 50% phosphoric acid, dilute with acetonitrile to volume, mix, filter, and degas. Make adjustments if necessary (see System Suitability under Chromatography 621).

System suitability preparation— Dissolve accurately weighed quantities of USP Alpha Tocopherol RS, USP Alpha Tocopheryl Acetate RS, and USP Alpha Tocopheryl Acid Succinate RS in methanol, and dilute quantitatively with methanol to obtain a solution having known concentrations of about 2 mg of each USP Reference Standard per mL.

Standard preparation— Dissolve an accurately weighed quantity of USP Alpha Tocopherol RS, USP Alpha Tocopheryl Acetate RS, or USP Alpha Tocopheryl Acid Succinate RS in methanol, and dilute quantitatively with methanol to obtain a solution having a known concentration of about 2 mg per mL.

Assay preparation— Proceed as directed for Assay preparation in the Assay for vitamin A, Method 2. Transfer an accurately measured volume of the supernatant 2,2,4-trimethylpentane to a suitable volumetric flask, the volume of the specimen withdrawn from the 2,2,4-trimethylpentane and the size of the volumetric flask being such that the final concentration of the Assay preparation is equivalent to that of the Standard preparation. Evaporate nearly to dryness, add several mL of methanol, and evaporate the remaining 2,2,4-trimethylpentane. Dilute with methanol to volume, and mix.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 280-nm detector and a 4.6-mm × 25-cm column that contains 5-µm packing L1. The flow rate is about 1.5 mL per minute. Chromatograph the System suitability preparation, and record the peak areas as directed for Procedure: the relative retention times for alpha tocopheryl acid succinate, alpha tocopherol, and alpha tocopheryl acetate are about 0.6, 0.8, and 1.0, respectively; the resolution between alpha tocopheryl acid succinate and alpha tocopherol is not less than 4.0; and the resolution between alpha tocopherol and alpha tocopheryl acetate is not less than 3.0. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the relative standard deviation for replicate injections is not more than 3.0%.

Procedure— Separately inject equal volumes (about 25 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the peak areas. Calculate the quantity, in mg, of alpha tocopherol (C29H50O2), alpha tocopheryl acetate (C31H52O3), or alpha tocopheryl acid succinate (C33H54O5) in the portion of Tablets taken by the formula: in which C is the concentration, in mg per mL, of the corresponding USP Reference Standard in the Standard preparation; D is the dilution factor used in exchanging the solvent from 2,2,4-trimethylpentane to methanol; and rU and rS are the peak areas of the relevant vitamin E form obtained from the Assay preparation and the Standard preparation, respectively. [note—The initial extraction to 26.5 mL of 2,2,4-trimethylpentane has been accounted for in the calculation formula.] Calculate the alpha tocopherol equivalent of alpha tocopheryl acetate or alpha tocopheryl acid succinate by multiplying the content, in mg, by the factor 0.91 or 0.81, respectively.

Diluting solution— Prepare a mixture of acetonitrile and ethyl acetate (1:1).

Mobile phase— Prepare a filtered and degassed mixture of methanol, acetonitrile, and n-hexane (46.5:46.5:7.0). Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard preparation— Dissolve an accurately weighed quantity of USP Alpha Tocopherol RS, USP Alpha Tocopheryl Acetate RS, or USP Alpha Tocopheryl Acid Succinate RS in methanol, and quantitatively dilute with methanol to obtain a solution having a known concentration of about 0.3 mg per mL.

Assay preparation— Weigh and finely powder not fewer than 20 Tablets. Transfer an accurately weighed portion of the powder, equivalent to about 8 mg of alpha tocopherol, to a 125-mL flask fitted with a ground-glass joint. Add 25.0 mL of water, 25.0 mL of dehydrated alcohol, and 3.5 g of potassium hydroxide pellets. Shake for 1 hour in a water bath maintained at 55, cool, and transfer with the aid of a minimum volume of water to a 125-mL separatory funnel. Rinse the flask with 50 mL of n-hexane, and add the rinsing to the separatory funnel. Insert the stopper, shake vigorously for 60 seconds, and allow the layers to separate. Drain the aqueous layer into a second 250-mL separatory funnel, and repeat the extraction with 50 mL of n-hexane. Discard the aqueous layer, and combine the hexane extracts. Wash the combined extracts with 25 mL of water, allow the layers to separate, and discard the aqueous layer. Add 3 drops of glacial acetic acid, and repeat the washing procedure two more times. Filter the washed hexane layer through anhydrous sodium sulfate into a 250-mL round-bottom flask. Rinse the funnel and sodium sulfate with a few mL of n-hexane, and add the rinsing to the hexane solution in the flask. Place the flask in a water bath maintained at 50, and evaporate the hexane solution with the aid of a rotary evaporator to dryness. Immediately add 25.0 mL of Diluting solution, and swirl to dissolve the residue.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 291-nm detector and a 4.6-mm × 25-cm column that contains packing L1. The column temperature is maintained at about 40, and the flow rate is about 3 mL per minute. Chromatograph the Standard preparation, and record the peak areas as directed for Procedure: the relative standard deviation for replicate injections is not more than 5.0%.

Procedure— Separately inject equal volumes (about 20 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the peak areas. Calculate the quantity, in mg, of alpha tocopherol (C29H50O2), alpha tocopheryl acetate (C31H52O3), or alpha tocopheryl acid succinate (C33H54O5) in the portion of Tablets taken by the formula: in which C is the concentration, in mg per mL, of the corresponding USP Reference Standard in the Standard preparation; and rU and rS are the peak areas for the relevant vitamin E form obtained from the Assay preparation and the Standard preparation, respectively. Calculate the alpha tocopherol equivalent of alpha tocopheryl acetate or alpha tocopheryl acid succinate by multiplying the content, in mg, by the factor 0.91 or 0.81, respectively.

Mobile phase— Prepare a filtered and degassed mixture of methanol and water (95:5). Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard stock solution— Dissolve an accurately weighed quantity of USP Phytonadione RS in methanol, with the aid of sonication if necessary, and quantitatively dilute with methanol to obtain a solution having a known concentration of about 200 µg per mL.

Standard preparation— Pipet 10 mL of Standard stock solution into a 100-mL volumetric flask, dilute with methanol to volume, and mix.

System suitability preparation— Transfer 65 mg of USP Alpha Tocopheryl Acetate RS to a 100-mL volumetric flask, and dissolve in about 75 mL of methanol. Add 10 mL of Standard stock solution, dilute with methanol to volume, and mix.

Assay preparation— Transfer not less than 20 mL, accurately measured, of the solution retained as specified in the directions for Assay preparation in the Assay for vitamin A, Method 1 to a suitable container, and evaporate in vacuum at room temperature to dryness. Transfer the residue with the aid of methanol to a suitable volumetric flask, and dilute with methanol to volume to obtain a solution having a concentration of about 20 µg of phytonadione per mL.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 254-nm detector and an 8-mm × 10-cm column that contains 5-µm packing L1. Chromatograph the System suitability preparation, and record the peak areas as directed for Procedure: the resolution, R, between alpha tocopheryl acetate and phytonadione is not less than 5. Chromatograph the Standard preparation, and record the peak areas as directed for Procedure: the relative retention times are about 0.68 for alpha tocopheryl acetate and 1.0 for phytonadione; and the relative standard deviation for replicate injections is not more than 3.0%.

Procedure— Separately inject equal volumes (about 100 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the peak areas. Calculate the quantity, in µg, of phytonadione (C31H46O2) in the Tablets taken by the formula: in which C is the concentration, in µg per mL, of USP Phytonadione RS in the Standard preparation; L is the labeled amount, in µg, of phytonadione in each Tablet; D is the concentration, in µg per mL, of phytonadione in the Assay preparation, based on the labeled quantity per Tablet and the extent of dilution; and rU and rS are the peak areas for phytonadione obtained from the Assay preparation and the Standard preparation, respectively.

Solvent— Prepare a mixture of methanol and isopropanol (95:5).

Mobile phase— Prepare a filtered and degassed mixture of 800 mL of methanol, 200 mL of methylene chloride, 0.1 mL of glacial acetic acid, 1.36 g of zinc chloride, and 0.41 g of sodium acetate.

Internal standard solution— Prepare a solution of menaquinone 4 (vitamin K2) in Solvent having a concentration of about 5 µg per mL. [note—A concentrated stock solution of menaquinone 4 (100 µg per mL) can be stored for 2 months in a refrigerator.]

Standard stock solution— Dissolve an accurately weighed quantity of USP Phytonadione RS in methylene chloride with the aid of sonication. Dilute with Solvent quantitatively, and stepwise if necessary, to obtain a solution having a known concentration of about 5 µg per mL.

Standard preparation— Pipet 1.0 mL of Standard stock solution and 1.0 mL of Internal standard solution into a 5.0-mL volumetric flask, dilute with Solvent to volume, and mix. Filter through a membrane having a 0.45-µm or finer porosity.

Assay preparation— Weigh and finely powder not fewer than 20 Tablets. To a centrifuge tube fitted with a cap, transfer an accurately weighed amount of powder, not exceeding 800 mg, and equivalent to an amount of phytonadione not exceeding 50 µg. Add 4 mL of water. Insert the stopper, and mix using a vortex mixer until the sample is dispersed. Place the tube in a water bath at 60 for 5 minutes. Remove from the bath, and again shake or mix using a vortex mixer for 1 minute while the preparation is still hot. Add 8 mL of alcohol, and swirl the contents to mix. Place the tube in a water bath at 60 for 5 minutes. Remove from the bath, and again shake or mix using a vortex mixer for 2 minutes while the preparation is still hot. Cool to room temperature. Add an accurately measured volume of Internal standard solution, equivalent to 1.0 mL per each 5 µg of the expected amount of phytonadione in the aliquot taken. Add 20.0 mL of petroleum ether, and cap the tube tightly. Shake or mix using a vortex mixer for 15 minutes to thoroughly mix the contents. Centrifuge to separate the two layers. Transfer a volume of the top layer of petroleum ether, equivalent to 5 to 50 µg of the expected amount of phytonadione, to an appropriate flask. Place the flask in a water bath at 35 to 45, and evaporate the solvent under a stream of nitrogen until an oily residue is left. Dissolve the residue in a volume of Solvent to obtain a solution having a concentration of about 1 µg per mL of phytonadione.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a fluorometric detector set at 320 nm for excitation and 420 nm for emission, a 4.6-mm × 25-cm column that contains 5-µm, end-capped packing L1, and a postcolumn reactor constituted with a 4.6-mm × 3-cm PEEK column tightly packed with zinc powder. [note—Prepare the postcolumn reactor daily, or as necessary, to meet the system suitability requirements.] The flow rate is about 1.0 mL per minute. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the relative retention times are 1.0 for the internal standard and 1.4 for phytonadione; the column efficiency for the phytonadione peak is not less than 2500 theoretical plates; the tailing factor for the phytonadione peak is not more than 1.5; and the relative standard deviation for replicate injections is not more than 3.0%.

Procedure— Separately inject equal volumes (about 25 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the peak responses. Calculate the quantity, in µg, of phytonadione (C31H46O2) in the portion of Tablets taken by the formula: in which C is the concentration, in µg per mL, of USP Phytonadione RS in the Standard preparation; D is the volume, in mL, of Internal standard solution used to prepare the Assay preparation; and RU and RS are the peak response ratios of phytonadione to that of the internal standard obtained from the Assay preparation and the Standard preparation, respectively.

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

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

Assay preparation— Weigh accurately not fewer than 20 Tablets. Grind the Tablets to a fine powder, and transfer an accurately weighed quantity of the powder, equivalent to about 2 mg of beta carotene, to a 500-mL saponification flask. Add 100 mL of alcohol, 6 mL of Potassium hydroxide solution, and a magnetic stirring bar. Attach an air condenser to the flask, and heat under reflux for 45 minutes with constant stirring. Cool to room temperature, add 170 mL of solvent hexane, and stir for 30 minutes. Quantitatively transfer the contents of the flask to a 500-mL separatory funnel with portions of solvent hexane. Allow the layers to separate for 5 to 10 minutes, and transfer the upper organic layer to a 500-mL volumetric flask. Transfer the lower aqueous layer into the saponification flask, add 170 mL of solvent hexane, and stir for an additional 20 minutes. Quantitatively transfer the contents of the saponification flask to the separatory funnel with the aid of portions of solvent hexane. Allow the layers to separate for 10 minutes. Drain the lower aqueous layer, and discard. Transfer the organic layer to the volumetric flask containing the previously collected organic layer. Rinse the separatory funnel with small portions of solvent hexane, and transfer the washings to the volumetric flask. Dilute the hexane extracts with solvent hexane to volume, add 3 g of anhydrous sodium sulfate, shake, and allow to settle. Quantitatively transfer a volume of this solution, equivalent to about 100 µg of beta carotene, to a 50-mL volumetric flask. Evaporate under a stream of nitrogen to dryness, and immediately add cyclohexane. Add 2 mL of Iodine solution, and heat for 15 minutes in a water bath maintained at 65. Cool rapidly, dilute with cyclohexane to volume, and mix.

Procedure— Determine the absorbance of the Assay preparation at the wavelength of maximum absorbance at about 452 nm, using cyclohexane as the blank. Calculate the quantity, in mg, of beta carotene (C40H56) in the Tablets taken by the formula: in which L is the labeled amount, in mg, of beta carotene in each Tablet; D is the concentration, in mg per mL, of beta carotene in the Assay preparation, based on the labeled quantity per Tablet and the extent of dilution; AU is the absorbance of the Assay preparation; and 223 is the absorptivity of beta carotene at 452 nm.

Mobile phase— Transfer 85 mL of acetonitrile, 1 g of sodium perchlorate, and 1 mL of phosphoric acid to a 1000-mL volumetric flask, dilute with water to volume, mix, filter, and degas. Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard preparation— Transfer about 67 mg of USP Biotin RS, accurately weighed, to a 200-mL volumetric flask, dissolve in and dilute with dimethyl sulfoxide to volume, and mix. Transfer 3.0 mL of this solution to a 200-mL volumetric flask, dilute with water to volume, and mix to obtain a solution having a known concentration of about 5 µg of USP Biotin RS per mL.

Assay preparation— Weigh and finely powder not fewer than 20 Tablets. Transfer an accurately weighed portion of the powder, equivalent to about 1 mg of biotin, to a 200-mL volumetric flask. Add 3 mL of dimethyl sulfoxide, and swirl to wet the contents. Place the flask in a water bath at 60 to 70 for 5 minutes. Sonicate for 5 minutes, dilute with water to volume, mix, and filter.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 200-nm detector and a 4.6-mm × 15-cm column containing 3-µm packing L7. The flow rate is about 1.2 mL per minute. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the relative standard deviation for replicate injections is not more than 3%.

Procedure— Separately inject equal volumes (about 100 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the biotin peaks. Calculate the quantity, in µg, of biotin (C10H16N2O3S) in the portion of Tablets taken by the formula: in which C is the concentration, in µg per mL, of USP Biotin RS in the Standard preparation; and rU and rS are the biotin peak responses obtained from the Assay preparation and the Standard preparation, respectively.

Standard stock solution— Dissolve an accurately weighed quantity of USP Biotin RS in 50% alcohol, dilute with 50% alcohol to obtain a solution having a known concentration of about 50 µg per mL, and mix. Store this solution in a refrigerator.

Standard preparation— On the day of the assay, dilute a volume of Standard stock solution with water to obtain a solution having a known concentration of about 0.1 ng per mL.

Assay preparation— Weigh and finely powder not fewer than 30 Tablets. Transfer an accurately weighed quantity of the powder, equivalent to about 100 µg of biotin, to a 200-mL volumetric flask, add 3 mL of 50% alcohol, and swirl to wet the contents. Heat the flask in a water bath at 60 to 70 for 5 minutes. Sonicate for 5 minutes, dilute with 50% alcohol to volume, mix, and filter. Dilute an accurately measured volume of the filtrate, quantitatively, and stepwise if necessary, with water to obtain a solution having a concentration of about 0.1 ng per 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 to 12 hours. Remove the hydrochloric acid from the mixture by distillation under reduced pressure until a thick paste remains. Redissolve the resulting paste in water, adjust the solution with 1 N sodium hydroxide to a pH of 3.5 ± 0.1, and add water to make 1000 mL. Add 20 g of activated charcoal, stir for 1 hour, and filter. Repeat the treatment with activated charcoal. Store under toluene in a refrigerator at a temperature not below 10. Filter the solution if a precipitate forms during storage.

Cystine–tryptophan solution— Suspend 4.0 g of l-cystine in 1.0 g of l-tryptophan (or 2.0 g of d,l-tryptophan) in 700 to 800 mL of water, heat to 70 to 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 refrigerator 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— Dissolve 25 g of polysorbate 80 in alcohol to make 250 mL.

Calcium pantothenate solution— Prepare a solution of calcium pantothenate in 50% alcohol containing 10 µg per mL. Store in a refrigerator.

Riboflavin–thiamine hydrochloride solution— Prepare a solution of riboflavin and thiamine hydrochloride in 0.02 N acetic acid containing 20 µg of riboflavin and 10 µg of thiamine hydrochloride per mL. Store under toluene, protected from light, in a refrigerator.

p-Aminobenzoic acid–niacin–pyridoxine hydrochloride solution— Prepare a solution in a mixture of water and neutralized alcohol (3:1) containing 10 µg of p-aminobenzoic acid, 50 µg of niacin, and 40 µg of pyridoxine hydrochloride per mL. Store in a refrigerator.

Salt solution 1— Dissolve 25 g of monobasic potassium phosphate and 25 g of dibasic potassium phosphate in water to make 500 mL. Add 5 drops of hydrochloric acid, and mix. Store under toluene.

Salt solution 2— Dissolve 10 g of magnesium sulfate, 0.5 g of sodium chloride, 0.5 g of ferrous sulfate, and 0.5 g of manganese sulfate in water to make 500 mL. Add 5 drops of hydrochloric acid, and mix. Store under toluene.

Basal medium stock solution—

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, 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,2 incubating for 16 to 24 hours 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, 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 to 24 hours at a temperature between 30 and 37 held constant to within ±0.5. The cell suspension so obtained is the Inoculum.

Procedure— To similar separate test tubes add, in duplicate, 1.0 and/or 1.5, 2.0, 3.0, 4.0, and 5.0 mL of the Standard preparation. To each tube and to four similar empty tubes, add 5.0 mL of Basal medium stock solution and sufficient water to make 10 mL.

Calculation— Prepare a standard concentration-response curve as follows. For each level of the Standard, calculate the response from the sum of the duplicate values of the transmittance (S) as the difference, y = 2.00 S. Plot this response on the ordinate of cross-section paper against the logarithm of the mL of Standard preparation 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.

Replication— Repeat the entire determination at least once, using separately prepared Assay preparations. If the difference between the two log-potencies M is not greater than 0.08, their mean, M, is the assayed log-potency of the test material (see The Confidence Interval and Limits of Potency under Design and Analysis of Biological Assays 111). 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.

Buffer— Transfer 800 mL of water and 100 mL of triethylamine to a 1000-mL volumetric flask. Add 80 mL of 85% phosphoric acid, dilute with water to volume, and mix.

Standard preparation— Transfer about 25 mg of USP Biotin RS, accurately weighed, to a 250-mL volumetric flask, dissolve in and dilute with water to volume, and mix. Transfer 1.5 mL of this solution to a 250-mL volumetric flask, dilute with water to volume, and mix to obtain a solution having a known concentration of about 0.6 µg per mL of USP Biotin RS. [note—A portion of the Standard preparation will be used to determine the percent recovery of biotin from the Solid-phase extraction procedure.]

Assay preparation— Weigh and finely powder not fewer than 20 Tablets. Transfer an accurately weighed portion of the powder, equivalent to about 60 µg of biotin, to a 100-mL volumetric flask. Add about 80 mL of water, and sonicate for 30 to 40 minutes with occasional mixing. Cool to room temperature, dilute with water to volume, mix, and filter. Adjust the pH of the solution with either dilute acetic acid or 0.1 N sodium hydroxide to between 6.0 and 7.0.

Solid-phase extraction— [note—Condition the extraction column specified in this procedure in the following manner. Wash the column with a 2-mL portion of methanol. Equilibrate with a 2-mL portion of water.] Separately pipet 5.0 mL of the Assay preparation and Standard preparation into freshly conditioned solid-phase extraction columns consisting of a mixed-mode packing with a sorbent-mass of 60 mg. [note—The mixed-mode packing consists of anion-exchange and reversed-phase sorbents. The reverse-phase component is a polymer of copolymer N-vinylpyrrolidone and divinylbenzene. The anion exchange moiety is a trialkyamino group.3 ] Wash the column with 10 mL of 30% (v/v) methanol in water. Apply an appropriate volume (about 4.9 mL) of 30% (v/v) methanol in 0.1 N hydrochloric acid to the column. Collect the eluate in a 5-mL volumetric flask, containing 100 µL of 40% (w/v) sodium acetate in water, and dilute with 30% (v/v) methanol in 0.1 N hydrochloric acid to volume.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 200-nm detector and a 4.6-mm × 25-cm column containing packing L1. The flow rate is about 2.0 mL per minute. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the tailing factor is not more than 1.5; and the relative standard deviation for replicate injections is not more than 2%. Chromatograph the portion of Standard preparation that has undergone solid-phase extraction, and record the peak responses as directed for Procedure: the relative standard deviation for replicate injections is not more than 2%; and the recovery is between 95% and 100%.

Procedure— Separately inject equal volumes (about 100 µL) of the Standard preparation and the Assay preparation that have undergone solid-phase extraction, into the chromatograph, record the chromatograms, and measure the responses for the biotin peak. Calculate the quantity, in µg, of biotin (C10H16N2O3S) in the portion of Tablets taken by the formula: in which C is the concentration, in µg per mL, of USP Biotin RS in the Standard preparation; and rU and rS are the biotin peak responses obtained from the Assay preparation and the Standard preparation, respectively.

Mobile phase— Prepare a filtered and degassed mixture of water and methanol (65:35). Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard preparation— Dissolve an accurately weighed quantity of USP Cyanocobalamin RS in water to obtain a stock solution having a known concentration of about 10 µg per mL. [note—Store this stock solution in a dark place, and discard after 1 week.] Dilute a portion of this stock solution quantitatively with water to obtain a solution having a known concentration of about 1 µg per mL.

Assay preparation— Weigh and finely powder not fewer than 30 Tablets. Transfer an accurately weighed portion of the powder, equivalent to about 100 µg of cyanocobalamin, to a 250-mL flask. Quantitatively add 100.0 mL of water, and carefully extract for 2 minutes. Filter about 10 mL of the extract, and use the filtrate.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 550-nm detector and a 4.6-mm × 15-cm column containing 5-µm packing L1. The flow rate is about 0.5 mL per minute. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the relative standard deviation for replicate injections is not more than 3.0%.

Procedure— Separately inject equal volumes (about 200 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the peak responses for cyanocobalamin. Calculate the quantity, in µg, of cyanocobalamin (C63H88CoN14O14P) in the portion of Tablets taken by the formula: in which C is the concentration, in µg per mL, of USP Cyanocobalamin RS in the Standard preparation; and rU and rS are the peak responses for cyanocobalamin obtained from the Assay preparation and the Standard preparation, respectively.

Standard cyanocobalamin stock solution— Dissolve an accurately weighed quantity of USP Cyanocobalamin RS in 25% alcohol to obtain a solution having a known concentration of about 1.0 µg of USP Cyanocobalamin RS per mL. Store in a refrigerator.

Standard preparation— Dilute a suitable volume of Standard cyanocobalamin stock solution with water to a measured volume such that after the incubation period as described for Procedure, the difference in transmittance between the inoculated blank and the 5.0-mL level of the Standard preparation is not less than that which corresponds to a difference of 1.25 mg in dried cell weight. This concentration usually falls between 0.01 and 0.04 ng per mL of Standard preparation. Prepare this solution fresh for each assay.

Assay preparation— Weigh and finely powder not fewer than 20 Tablets. Transfer an accurately weighed portion of the powder, equivalent to about 1.0 µg of cyanocobalamin, to an appropriate vessel containing, for each g of powdered Tablets taken, 25 mL of an aqueous extracting solution prepared just prior to use to contain, in each 100 mL, 1.29 g of dibasic sodium phosphate, 1.1 g of anhydrous citric acid, and 1.0 g of sodium metabisulfite. Autoclave the mixture at 121 for 10 minutes. 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 preparation.

Acid-hydrolyzed casein solution— Prepare as directed in the Assay for calcium pantothenate, Method 2.

Asparagine solution— Dissolve 2.0 of l-asparagine in water to make 200 mL. Store under toluene in a refrigerator.

Adenine–guanine–uracil solution— Prepare as directed in the Assay for calcium pantothenate, Method 2.

Xanthine solution— Suspend 0.20 g of xanthine in 30 to 40 mL of water, heat to about 70, 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 1— Dissolve 10 g of monobasic potassium phosphate and 10 g of dibasic potassium phosphate in water to make 200 mL, and add 2 drops of hydrochloric acid. Store this solution under toluene.

Salt solution 2— Dissolve 4.0 g of magnesium sulfate, 0.20 g of sodium chloride, 0.20 g of ferrous sulfate, and 0.20 g of manganese sulfate in water to make 200 mL, and add 2 drops of hydrochloric acid. Store this solution under toluene.

Polysorbate 80 solution— Dissolve 20 g of polysorbate 80 in alcohol to make 200 mL. Store in a refrigerator.

Vitamin solution 1— Dissolve 10 mg of riboflavin, 10 mg of thiamine hydrochloride, 100 µg of biotin, and 20 mg of niacin in 0.02 N glacial acetic acid to make 400 mL. Store under toluene, protected from light, in a refrigerator.

Vitamin solution 2— Dissolve 20 mg of p-aminobenzoic acid, 10 mg of calcium pantothenate, 40 mg of pyridoxine hydrochloride, 40 mg of pyridoxal hydrochloride, 8 mg of pyridoxamine dihydrochloride, and 2 mg of folic acid in a mixture of water and neutralized alcohol (3:1) to make 400 mL. Store, protected from light, in a refrigerator.

Basal medium stock solution— Prepare the medium according to the following formula and directions. A dehydrated mixture containing the same ingredients may be used provided that, when constituted as directed in the labeling, it yields a medium comparable to that obtained from the formula given herein.

Tomato juice preparation— Centrifuge commercially canned tomato juice so that most of the pulp is removed. Suspend about 5 g per L of analytical filter-aid in the supernatant, and filter, with the aid of reduced pressure, through a layer of the filter-aid. Repeat, if necessary, until a clear, straw-colored filtrate is obtained. Store under toluene in a refrigerator.

Culture medium— [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 to 70 mL of water. Add 10 mL of Tomato juice preparation and 1 mL of Polysorbate 80 solution. Adjust with 1 N sodium hydroxide to a pH of 6.8, and add water to make 100 mL. Place 10-mL portions of the solution in test tubes, and plug with cotton. Sterilize the tubes and contents in an autoclave at 121 for 15 minutes. 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 to 1.5 g of agar, and heat the mixture on a steam bath, with stirring, until the agar dissolves. Place 10-mL portions of the hot solution in test tubes, cover the tubes, sterilize at 121 for 15 minutes in an autoclave (exhaust line temperature), 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.4 [note—Before first using a fresh culture in this assay, make not fewer than 10 successive transfers of the culture in a 2-week period.] Incubate for 16 to 24 hours at a temperature between 30 and 40 held constant to within ±0.5. Store in a refrigerator.

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 to 24 hours 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 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.

Procedure— Because of the high sensitivity of the test organism to minute amounts of vitamin B12 activity and to traces of many cleansing agents, cleanse meticulously by suitable means, followed preferably by heating at 250 for 2 hours, using hard-glass 20-mm × 150-mm test tubes, and other necessary glassware.

Calculation— Prepare a standard concentration-response curve by the following procedure. Test for and replace any aberrant individual transmittances. For each level of the Standard, calculate the response from the sum of the duplicate values of the transmittances (S) as the difference, y = 2.00 S. Plot this response on the ordinate of cross-section paper against the logarithm of the mL of Standard preparation 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.

Replication— Repeat the entire determination at least once, using separately prepared Assay preparations. If the difference between the two log-potencies M is not greater than 0.08, their mean, M, is the assayed log-potency of the test material (see Vitamin B12 Activity under Design and Analysis of Biological Assays 111). 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.

Reagent 1— Use a 25% solution of tetrabutylammonium hydroxide in methanol.

Reagent 2— Transfer 5.0 g of pentetic acid to a 50-mL volumetric flask. Using sonication if necessary, dissolve in and dilute with 1 N sodium hydroxide to volume, and mix.

Mobile phase— Transfer 2 g of monobasic potassium phosphate to a 1-L volumetric flask, and dissolve in 650 mL of water. Add 12.0 mL of Reagent 1, 7.0 mL of 3 N phosphoric acid, and 240 mL of methanol. Cool to room temperature, adjust with phosphoric acid or ammonia TS to a pH of 7.0, dilute with water to volume, mix, and filter. Recheck the pH before use. Make adjustments if necessary (see System Suitability under Chromatography 621). [note—The methanol and water content may be varied (between 1% and 3%) 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.]

Internal standard preparation— Transfer about 40 mg of methylparaben, accurately weighed, to a 1000-mL volumetric flask, and add 220 mL of methanol to dissolve. Dissolve 2.0 g of monobasic potassium phosphate in about 300 mL of water in a separate beaker, quantitatively transfer this solution to the flask containing the methylparaben solution, and add an additional 300 mL of water. Add 19 mL of Reagent 1, 7 mL of 3 N phosphoric acid, and 30 mL of Reagent 2. Adjust with ammonia TS to a pH of 9.8, bubble nitrogen through the solution for 30 minutes, dilute with water to volume, and mix.

Standard preparation— Dissolve an accurately weighed quantity of USP Folic Acid RS in Internal standard preparation to obtain a solution having a known concentration of about 0.2 mg per mL. Transfer 2.0 mL of this solution to a 25-mL volumetric flask, dilute with Internal standard preparation to volume, and mix.

Assay preparation— Weigh and finely powder not fewer than 30 Tablets. Transfer an accurately weighed portion of the powder, equivalent to about 0.4 mg of folic acid, to a 50-mL amber-colored centrifuge tube. Add 25.0 mL of Internal standard preparation, insert a stopper, shake by mechanical means for 10 minutes, and centrifuge. Filter a portion of the clear supernatant, and use the filtrate.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 280-nm detector and a 3.9-mm × 30-cm column that contains packing L1. The flow rate is about 1 mL per minute. Chromatograph the Standard preparation, and record the peak areas as directed for Procedure: the relative retention times are about 0.8 for folic acid and 1.0 for methylparaben; and the relative standard deviation for replicate injections is not more than 3.0%.

Procedure— Separately inject equal volumes (about 15 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the peak areas for folic acid and methylparaben. Calculate the quantity, in mg, of folic acid (C19H19N7O6) in the portion of Tablets taken by the formula: in which C is the concentration, in mg per mL, of USP Folic Acid RS in the Standard preparation; and RU and RS are the peak area ratios of folic acid to methylparaben obtained from the Assay preparation and the Standard preparation, respectively.

Reagent— Dissolve 7.5 g of edetate disodium, with stirring, in 500 mL of water containing 10 mL of ammonium hydroxide.

Diluting solution— Prepare a solution of ammonium hydroxide containing 60 µg per mL.

Mobile phase— Transfer 0.4 mL of triethylamine, 15.0 mL of glacial acetic acid, and 350 mL of methanol to a 2000-mL volumetric flask, dilute with 0.008 M sodium 1-hexanesulfonate to volume, and mix. Filter, and degas. Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard stock solution— Dissolve an accurately weighed quantity of USP Folic Acid RS in Diluting solution to obtain a solution having a known concentration of about 60 µg per mL. Prepare this solution fresh daily.

Standard preparation— Transfer 5.0 mL of Standard stock solution to a stoppered 125-mL flask, and add 10.0 mL of methanol and 35.0 mL of Reagent. Insert the stopper, shake for 15 minutes in a water bath maintained at 60, and cool. Filter, discarding the first few mL of the filtrate.

Assay preparation— Weigh and finely powder a counted number of Tablets. Transfer an accurately weighed portion of the powder, equivalent to about 0.3 mg of folic acid, to a stoppered 125-mL flask. Add 10.0 mL of methanol and 35.0 mL of Reagent, insert the stopper, shake for 15 minutes in a water bath maintained at 60, and cool. Filter, discarding the first few mL of the filtrate.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 270-nm detector and a 4.6-mm × 25-cm column that contains packing L7. The column temperature is maintained at 50, and the flow rate is about 2.0 mL per minute. Chromatograph the Standard preparation, and record the peak areas as directed for Procedure: the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 5 µL) of the Assay preparation and the Standard preparation into the chromatograph, record the chromatograms, and measure the areas of the major peaks. Calculate the quantity, in mg, of folic acid (C19H19N7O6) in the portion of Tablets taken by the formula: in which C is the concentration, in mg per mL, of USP Folic Acid RS in the Standard preparation; and rU and rS are the peak areas of folic acid obtained from the Assay preparation and the Standard preparation, respectively.

Mobile phase— Prepare a filtered and degassed mixture of water and phosphoric acid (1000:1). Make adjustments if necessary (see System Suitability under Chromatography 621).

Internal standard preparation— Transfer about 80 mg of p-hydroxybenzoic acid, accurately weighed, to a 1000-mL volumetric flask, and dissolve in 3 mL of alcohol. Add about 50 mL of water and 7.1 g of dibasic sodium phosphate, dilute with water to volume, and mix. Adjust with phosphoric acid to a pH of 6.7, and mix.

Standard preparation— Dissolve an accurately weighed quantity of USP Calcium Pantothenate RS in Internal standard preparation to obtain a solution having a known concentration of about 0.6 mg per mL.

Assay preparation— Weigh and finely powder not fewer than 30 Tablets. Transfer an accurately weighed portion of the powder, equivalent to about 15 mg of calcium pantothenate, to a centrifuge tube. Add 25.0 mL of the Internal standard preparation, and shake vigorously for 10 minutes. Centrifuge, filter, and use the clear filtrate.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 210-nm detector and a 3.9-mm × 15-cm column that contains packing L1. The flow rate is about 1.5 mL per minute. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the relative retention times are 0.5 for calcium pantothenate and 1.0 for p-hydroxybenzoic acid; and the relative standard deviation for replicate injections is not more than 3.0%.

Procedure— Separately inject equal volumes (about 10 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the peak responses for calcium pantothenate and the internal standard. Calculate the quantity, in mg, of calcium pantothenate (C18H32CaN2O10) in the portion of Tablets taken by the formula: in which C is the concentration, in mg per mL, of USP Calcium Pantothenate RS in the Standard preparation; and RU and RS are the peak response ratios of calcium pantothenate to p-hydroxybenzoic acid obtained from the Assay preparation and the Standard preparation, respectively.

Standard stock solution— Dissolve about 50 mg of USP Calcium Pantothenate RS, previously dried and stored in the dark over phosphorus pentoxide and accurately weighed while protected from absorption of moisture during the weighing, in about 500 mL of water in a 1000-mL volumetric flask. Add 10 mL of 0.2 N acetic acid and 100 mL of sodium acetate solution (1 in 60), and dilute with water to volume to obtain a solution containing 50 µg of USP Calcium Pantothenate RS per mL. Store under toluene in a refrigerator.

Standard preparation— On the day of the assay, dilute an accurately measured volume of Standard stock solution with water to obtain a solution having a known concentration of 0.01 to 0.04 µg of calcium pantothenate per mL, the exact concentration being such that the responses obtained as directed for Procedure, 2.0 and 4.0 mL of the Standard preparation being used, are within the linear portion of the log-concentration response curve.

Assay preparation— Weigh and powder not fewer than 30 Tablets. Transfer an accurately weighed portion of the powder, equivalent to about 50 mg of calcium pantothenate, to a 1000-mL volumetric flask containing 500 mL of water. Add 10 mL of 0.2 N acetic acid and 100 mL of sodium acetate solution (1 in 60), dilute with water to volume, and filter. Dilute an accurately measured volume of this solution quantitatively, and stepwise if necessary, to obtain a solution having approximately the same concentration as that of the Standard preparation.

Acid-hydrolyzed casein solution— Mix 100 g of vitamin-free casein with 500 mL of 6 N hydrochloric acid, and reflux the mixture for 8 to 12 hours. Remove the hydrochloric acid from the mixture by distillation under reduced pressure until a thick paste remains. Redissolve the resulting paste in water, adjust the solution with 1 N sodium hydroxide to a pH of 3.5 ± 0.1, and add water to make 1000 mL. Add 20 g of activated charcoal, stir for 1 hour, and filter. Repeat the treatment with activated charcoal. Store under toluene in a refrigerator at a temperature not below 10. Filter the solution if a precipitate forms during storage.

Cystine–tryptophan solution— Suspend 4.0 g of l-cystine and 1.0 g of l-tryptophan (or 2.0 g of d,l-tryptophan) in 700 to 800 mL of water, heat to 70 to 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 refrigerator 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— Dissolve 25 g of polysorbate 80 in alcohol to make 250 mL.

Riboflavin–thiamine hydrochloride–biotin solution— Prepare a solution of riboflavin, thiamine hydrochloride, and biotin in 0.02 N acetic acid containing 20 µg of riboflavin, 10 µg of thiamine hydrochloride, and 0.04 µg of biotin per mL. Store under toluene, protected from light, in a refrigerator.

p-Aminobenzoic acid–niacin–pyridoxine hydrochloride solution— Prepare a solution in a mixture of water and neutralized alcohol (3:1) containing 10 µg of p-aminobenzoic acid, 50 µg of niacin, and 40 µg of pyridoxine hydrochloride per mL. Store in a refrigerator.

Salt solution 1— Dissolve 25 g of monobasic potassium phosphate and 25 g of dibasic potassium phosphate in water to make 500 mL. Add 5 drops of hydrochloric acid, and mix. Store under toluene.

Salt solution 2— Dissolve 10 g of magnesium sulfate, 0.5 g of sodium chloride, 0.5 g of ferrous sulfate, and 0.5 g of manganese sulfate in water to make 500 mL. Add 5 drops of hydrochloric acid, and mix. Store under toluene.

Basal medium stock solution—

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, 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 plantarum5 incubating for 16 to 24 hours at a temperature between 30 and 37 held constant to within ±0.5. Store in a refrigerator. Prepare a fresh stab of the stock culture every week, and do not use for inoculum if the culture is more than 1 week old.

Culture medium— To each of a series of test tubes containing 5.0 mL of Basal medium stock solution, add 5.0 mL of water containing 0.2 µg of calcium pantothenate. Plug the tubes with cotton, sterilize in an autoclave at 121, 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 to 24 hours at a temperature between 30 and 37 held constant to within ±0.5. The cell suspension so obtained is the Inoculum.

Procedure— To similar separate test tubes add, in duplicate, 1.0 and/or 1.5, 2.0, 3.0, 4.0, and 5.0 mL of the Standard preparation. To each tube and to four similar empty tubes, add 5.0 mL of Basal medium stock solution and sufficient water to make 10 mL.

Calculation— Prepare a standard concentration-response curve as follows. For each level of the Standard, calculate the response from the sum of the duplicate values of the transmittance (S) as the difference, y = 2.00 S. Plot this response on the ordinate of cross-section paper against the logarithm of the mL of Standard preparation 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.

Replication— Repeat the entire determination at least once, using separately prepared Assay preparations. If the difference between the two log-potencies M is not greater than 0.08, their mean, M, is the assayed log-potency of the test material (see The Confidence Interval and Limits of Potency under Design and Analysis of Biological Assays 111). 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.

Buffer solution— Dissolve 10.0 g of monobasic potassium phosphate in 2000 mL of water, and adjust with phosphoric acid to a pH of 3.5.

Mobile phase— Prepare a filtered and degassed mixture of Buffer solution and methanol (90:10). Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard stock solution— Dissolve an accurately weighed quantity of USP Calcium Pantothenate RS in water to obtain a solution having a known concentration of about 0.25 mg per mL. Prepare fresh every 4 weeks. Store in a refrigerator.

Standard preparation— Quantitatively dilute an accurately measured volume of Standard stock solution with water to obtain a solution having a known concentration of about 40 µg of USP Calcium Pantothenate RS per mL.

Assay preparation— Weigh and finely powder not fewer than 20 Tablets. Transfer an accurately weighed portion of the powder, equivalent to about 10 mg of calcium pantothenate, to a 250-mL volumetric flask. Add 10 mL of methanol, and swirl to disperse the test specimen. Dilute with water to volume, mix, and filter.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 205-nm detector and a 3.9-mm × 30-cm column that contains 5-µm packing L1. The column temperature is maintained at 50, and the flow rate is about 2 mL per minute. Chromatograph the Standard preparation, and record the peak areas as directed for Procedure: the relative standard deviation for replicate injections is not more than 3.0%.

Procedure— Separately inject equal volumes (about 25 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the peak areas for calcium pantothenate. Calculate the quantity, in mg, of calcium pantothenate (C18H32CaN2O10) in the portion of Tablets taken by the formula: in which C is the concentration, in mg per mL, of USP Calcium Pantothenate RS in the Standard preparation; and rU and rS are the peak areas obtained from the Assay preparation and the Standard preparation, respectively.

Diluting solution— Prepare a mixture of water, acetonitrile, and glacial acetic acid (94:5:1).

Mobile phase— Prepare a mixture of water, methanol, and glacial acetic acid (73:27:1) containing 140 mg of sodium 1-hexanesulfonate per 100 mL. Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard preparation— [note—Use USP Niacin RS in place of USP Niacinamide RS for formulations containing Niacin.] Transfer about 80 mg of USP Niacinamide RS, 20 mg of USP Pyridoxine Hydrochloride RS, 20 mg of USP Riboflavin RS, and 20 mg of USP Thiamine Hydrochloride RS, each accurately weighed, to a 200-mL volumetric flask, and add about 180 mL of Diluting solution. Immerse the flask in a hot water bath maintained at 65 to 70 for about 10 minutes with regular shaking or using a vortex mixer, until all the solid materials are dissolved. Chill rapidly in a cold water bath for about 10 minutes to room temperature, dilute with Diluting solution to volume, and mix.

Assay preparation— Weigh and finely powder not fewer than 30 Tablets, and transfer an accurately weighed portion of the powder, equivalent to about 10 mg of niacinamide and 2.5 mg each of pyridoxine hydrochloride, riboflavin, and thiamine hydrochloride, to a 50-mL centrifuge tube. Add 25.0 mL of Diluting solution, and mix, using a vortex mixer, for 30 seconds to completely suspend the powder. Immerse the centrifuge tube in a hot water bath maintained at 65 to 70, heat for 5 minutes, and mix on a vortex mixer for 30 seconds. Return the tube to the hot water bath, heat for another 5 minutes, and mix on a vortex mixer for 30 seconds. Filter a portion of the solution, cool to room temperature, and use the clear filtrate. [note—Use the filtrate within 3 hours of filtration.]

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 280-nm detector and a 3.9-mm × 30-cm column that contains packing L1. The flow rate is about 1 mL per minute. Chromatograph the Standard preparation, and record the peak areas as directed for Procedure: the relative retention times of niacinamide, pyridoxine, riboflavin, and thiamine are about 0.3, 0.5, 0.8, and 1.0, respectively; and the relative standard deviation for replicate injections is not more than 3.0%.

Procedure— Separately inject equal volumes (about 10 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the peak areas for niacin or niacinamide, pyridoxine, riboflavin, and thiamine. Calculate the quantity, in mg, of niacinamide (C6H6N2O) in the portion of Tablets taken by the formula: in which C is the concentration, in mg per mL, of USP Niacinamide RS in the Standard preparation; and rU and rS are the peak responses of niacinamide obtained from the Assay preparation and the Standard preparation, respectively. For formulations containing niacin, C is the concentration, in mg per mL, of USP Niacin RS in the Standard preparation; and rU and rS are the peak areas for niacin obtained from the Assay preparation and the Standard preparation, respectively. Separately calculate the quantities, in mg, of pyridoxine hydrochloride (C8H11NO3·HCl), riboflavin (C17H20N4O6), and thiamine hydrochloride (C12H17ClN4OS·HCl) in the portion of Tablets taken by the same formula, in which C is the concentration, in mg per mL, of the relevant USP Reference Standard in the Standard preparation; and rU and rS are the peak areas for the corresponding vitamin obtained from the Assay preparation and the Standard preparation, respectively. For products containing thiamine mononitrate, calculate the quantity, in mg, of thiamine mononitrate (C12H17N5O4S), in the portion of Tablets taken by the formula: in which 327.36 and 337.27 are the molecular weights of thiamine mononitrate and thiamine hydrochloride, respectively; and the other terms are as previously defined.

Extraction solvent— Transfer 1 mL of glacial acetic acid and 2.5 g of edetate disodium to a 100-mL volumetric flask, dissolve in and dilute with water to volume, and mix. Prepare a mixture of this solution and methanol (75:25).

Mobile phase— Prepare a 0.1 M sodium acetate solution by dissolving 13.6 g of sodium acetate in 1000 mL of water. Adjust with acetic acid to a pH of 5.4, and mix. Make adjustments if necessary (see System Suitability under Chromatography 621). [note—A small amount of methanol (up to 1%) may be added to the Mobile phase to improve resolution.]

Standard stock solution— Dissolve an accurately weighed quantity of USP Niacin RS in Extraction solvent, and dilute quantitatively, and stepwise if necessary, with Extraction solvent to obtain a solution having a known concentration of about 1 mg per mL.

Standard preparation— Transfer 5.0 mL of Standard stock solution to a 25-mL volumetric flask, dilute with Extraction solvent to volume, and mix.

Assay preparation— [note—This preparation is suitable for the determination of niacin or niacinamide, pyridoxine, and riboflavin, when present in the formulation.] Weigh and finely powder not fewer than 20 Tablets, and transfer an accurately weighed portion of the powder, equivalent to about 2 mg of riboflavin, to a 200-mL volumetric flask. If riboflavin is not present in the formulation, transfer a portion equivalent to about 2 mg of pyridoxine. If pyridoxine is not present in the formulation, transfer a portion equivalent to about 20 mg of niacin or niacinamide. Add 100.0 mL of Extraction solvent, and mix for 20 minutes, using a wrist-action shaker. Immerse the flask in a water bath maintained at 70 to 75, and heat for 20 minutes. Mix on a vortex mixer for 30 seconds, cool to room temperature, and filter. Use the clear filtrate.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 254-nm detector and a 4.6-mm × 25-cm column that contains packing L1. The flow rate is about 1 mL per minute. Chromatograph the Standard preparation, and record the peak areas as directed for Procedure: the relative standard deviation for replicate injections is not more than 3.0%. If necessary, flush the column with methanol between injections.

Procedure— Separately inject equal volumes (about 20 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the peak areas for niacin. Calculate the quantity, in mg, of niacin (C6H5NO2) in the portion of Tablets taken by the formula: in which C is the concentration, in mg per mL, of USP Niacin RS in the Standard preparation; and rU and rS are the peak responses of niacin obtained from the Assay preparation and the Standard preparation, respectively.

Extraction solvent, Mobile phase, Standard stock solution, Standard preparation, Assay preparation, and Chromatographic system— Using USP Niacinamide RS in place of USP Niacin RS, proceed as directed in the Assay for niacin, Method 2.

Procedure— Separately inject equal volumes (about 20 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the peak areas for niacinamide. Calculate the quantity, in mg, of niacinamide (C6H6N2O) in the portion of Tablets taken by the formula: in which C is the concentration, in mg per mL, of USP Niacinamide RS in the Standard preparation; and rU and rS are the peak responses of niacinamide obtained from the Assay preparation and the Standard preparation, respectively.

Extraction solvent and Mobile phase— Prepare as directed in the Assay for niacin, Method 2.

Standard stock solution— Dissolve an accurately weighed quantity of USP Pyridoxine Hydrochloride RS in Extraction solvent, and dilute quantitatively, and stepwise if necessary, with Extraction solvent to obtain a solution having a known concentration of about 0.1 mg per mL.

Standard preparation— Transfer 5.0 mL of Standard stock solution to a 25-mL volumetric flask, dilute with Extraction solvent to volume, and mix.

Assay preparation— Prepare as directed for Assay preparation in the Assay for niacin, Method 2.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 254-nm detector and a 4.6-mm × 25-cm column that contains packing L1. The flow rate is about 1 mL per minute. Chromatograph the Standard preparation, and record the peak areas as directed for Procedure: the relative standard deviation for replicate injections is not more than 3.0%.

Procedure— Separately inject equal volumes (about 20 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the peak areas for pyridoxine. Calculate the quantity, in mg, of pyridoxine hydrochloride (C8H11NO3·HCl) in the portion of Tablets taken by the formula: in which C is the concentration, in mg per mL, of USP Pyridoxine Hydrochloride RS in the Standard preparation; and rU and rS are the peak areas obtained from the Assay preparation and the Standard preparation, respectively.

Extraction solvent— Prepare as directed in the Assay for niacin, Method 2.

Mobile phase— Dissolve 6.8 g of sodium acetate in 1000 mL of water, and mix. Prepare a mixture of this solution and methanol (65:35). Add 2 mL of triethylamine for every 1000 mL of the mixture, adjust with glacial acetic acid to a pH of 5.2, and mix. Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard stock solution— Transfer about 20 mg of USP Riboflavin RS, accurately weighed, to a 200-mL volumetric flask. Add about 180 mL of Extraction solvent, and immerse the flask for about 5 minutes in a water bath maintained at 65 to 75. Mix well, and repeat if necessary until dissolved. Chill rapidly in a cold water bath to room temperature, dilute with Extraction solvent to volume, and mix.

Standard preparation— Quantitatively dilute 5.0 mL of Standard stock solution with Extraction solvent to 25.0 mL, and mix.

Assay preparation— Prepare as directed for Assay preparation in the Assay for niacin, Method 2.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 254-nm detector and a 4.6-mm × 25-cm column that contains packing L1. The flow rate is about 1 mL per minute. Chromatograph the Standard preparation, and record the peak areas as directed for Procedure: the relative standard deviation for replicate injections is not more than 3.0%.

Procedure— Separately inject equal volumes (about 20 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the peak areas for riboflavin. Calculate the quantity, in mg, of riboflavin (C17H20N4O6) in the portion of Tablets taken by the formula: in which C is the concentration, in mg per mL, of USP Riboflavin RS in the Standard preparation; and rU and rS are the peak areas obtained from the Assay preparation and the Standard preparation, respectively.

Mobile phase— Dissolve 1.88 g of sodium 1-hexanesulfonate in 1 L of 0.1% phosphoric acid. Prepare a mixture of this solution and acetonitrile (92:18). Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard stock solution— Dissolve an accurately weighed quantity of USP Thiamine Hydrochloride RS in 0.2 N hydrochloric acid, and dilute quantitatively, and stepwise if necessary, with 0.2 N hydrochloric acid to obtain a solution having a known concentration of about 0.1 mg per mL.

Standard preparation— Transfer 5.0 mL of Standard stock solution to a 25-mL volumetric flask, dilute with 0.2 N hydrochloric acid to volume, and mix.

Assay preparation— Weigh and finely powder not fewer than 20 Tablets. Transfer an accurately weighed portion of the powder, equivalent to about 2 mg of thiamine, to a 200-mL volumetric flask. Add 100.0 mL of 0.2 N hydrochloric acid, shake for 15 minutes with a wrist-action shaker, and heat to boiling for 30 minutes. Cool to room temperature, and filter. Use the clear filtrate.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 254-nm detector and a 4.6-mm × 25-cm column that contains packing L1. The flow rate is about 2 mL per minute. Chromatograph the Standard preparation, and record the peak areas as directed for Procedure: the relative standard deviation for replicate injections is not more than 3.0%.

Procedure— Separately inject equal volumes (about 20 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the areas for the major peaks. For products containing thiamine hydrochloride, calculate the quantity, in mg, of thiamine hydrochloride (C12H17ClN4OS·HCl) in the portion of Tablets taken by the formula: in which C is the concentration, in mg per mL, of USP Thiamine Hydrochloride RS in the Standard preparation; and rU and rS are the peak areas for thiamine obtained from the Assay preparation and the Standard preparation, respectively. For products containing thiamine mononitrate, calculate the quantity, in mg, of thiamine mononitrate (C12H17N5O4S) in the portion of Tablets taken by the formula: in which 327.36 and 337.27 are the molecular weights of thiamine mononitrate and thiamine hydrochloride, respectively; and the other terms are as previously defined.

Reagent— Prepare a solution containing 25 g of edetate disodium in 1000 mL of water.

Mobile phase— Transfer 0.4 mL of triethylamine, 15.0 mL of glacial acetic acid, and 350 mL of methanol to a 2000-mL volumetric flask, dilute with 0.008 M sodium 1-hexanesulfonate to volume, and mix. Filter, and degas. Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard stock solution— Dissolve accurately weighed quantities of USP Niacin RS or USP Niacinamide RS, USP Pyridoxine Hydrochloride RS, USP Riboflavin RS, and USP Thiamine Hydrochloride RS in Reagent, with heating if necessary, to obtain a solution having known concentrations of about 1.5 mg of USP Niacin RS or USP Niacinamide RS per mL, 0.24 mg of USP Pyridoxine Hydrochloride RS per mL, 0.08 mg of USP Riboflavin RS per mL, and 0.24 mg of USP Thiamine Hydrochloride RS per mL.

Standard preparation— Transfer 5.0 mL of Standard stock solution to a stoppered 125-mL flask. Add 10.0 mL of a mixture of methanol and glacial acetic acid (9:1) and 30.0 mL of a mixture of methanol and ethylene glycol (1:1). Insert the stopper, shake for 15 minutes in a water bath maintained at 60, and cool. Filter, discarding the first few mL of the filtrate.

Assay preparation— Weigh and finely powder not fewer than 20 Tablets. Transfer an accurately weighed portion of the powder, equivalent to about 7.5 mg of niacin or niacinamide, 1.2 mg of pyridoxine hydrochloride, 0.4 mg of riboflavin, and 1.2 mg of thiamine hydrochloride, to a stoppered 125-mL flask. Add 10.0 mL of a mixture of methanol and glacial acetic acid (9:1) and 30.0 mL of a mixture of methanol and ethylene glycol (1:1). Insert the stopper, shake for 15 minutes in a water bath maintained at 60, and cool. Filter, discarding the first few mL of the filtrate.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 270-nm detector and a 4.6-mm × 25-cm column that contains packing L7. The column is maintained at 50, and the flow rate is about 2.0 mL per minute. Chromatograph the Standard preparation, and record the peak areas as directed for Procedure: the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 5 µL) of the Assay preparation and the Standard preparation into the chromatograph, record the chromatograms, and measure the areas of the peak responses. Calculate the quantity, in mg, of niacin (C6H5NO2) or niacinamide (C6H6N2O) in the portion of Tablets taken by the formula: in which C is the concentration, in mg per mL, of USP Niacin RS or USP Niacinamide RS in the Standard preparation; and rU and rS are the peak areas for niacin or niacinamide obtained from the Assay preparation and the Standard preparation, respectively. Separately calculate the quantities, in mg, of pyridoxine hydrochloride (C8H11NO3·HCl) and riboflavin (C17H20N4O6) in the portion of Tablets taken by the same formula, in which the terms are as defined therein. For products containing thiamine hydrochloride, calculate the quantity, in mg, of thiamine (C12H17ClN4OS) in the portion of Tablets taken by the same formula. For products containing thiamine mononitrate, calculate the quantity, in mg, of thiamine mononitrate (C12H17N5O4S) in the portion of Tablets taken by the formula: in which 327.36 and 337.27 are the molecular weights of thiamine mononitrate and thiamine hydrochloride, respectively; and the other terms are as previously defined.

Lanthanum chloride solution— Dissolve 26.7 g of lanthanum chloride heptahydrate in 0.125 N hydrochloric acid to make 100.0 mL.

Calcium standard stock solution— Weigh accurately about 1.001 g of calcium carbonate, previously dried at 300 for 3 hours and cooled in a desiccator for 2 hours, and dissolve in 25 mL of 1 N hydrochloric acid. Boil to expel carbon dioxide, and dilute with water to 1000 mL to obtain a solution having a known concentration of about 400 µg of calcium per mL.

Standard preparations— Quantitatively dilute a volume of Calcium standard stock solution with 0.125 N hydrochloric acid to obtain a standard solution having a known concentration of about 100 µg of calcium per mL. Into separate 100-mL volumetric flasks, pipet 1.0, 1.5, 2.0, 2.5, and 3.0 mL of the standard solution. To each flask add 1.0 mL of Lanthanum chloride solution, dilute with water to volume, and mix to obtain solutions having known concentrations of about 1.0, 1.5, 2.0, 2.5, and 3.0 µg of calcium per mL.

Assay preparation— Weigh and finely powder not fewer than 20 Tablets. Transfer an accurately weighed portion of the powder, equivalent to about 5 Tablets, to a porcelain crucible. Heat the crucible in a muffle furnace maintained at about 550 for 6 to 12 hours, and cool. Add about 60 mL of hydrochloric acid, and boil gently on a hot plate or steam bath for 30 minutes, intermittently rinsing the inner surface of the crucible with 6 N hydrochloric acid. Cool, and quantitatively transfer the contents of the crucible to a 100-mL volumetric flask. Rinse the crucible with small portions of 6 N hydrochloric acid, and add the rinsings to the flask. Dilute with water to volume, mix, and filter, discarding the first 5 mL of the filtrate. Dilute this solution quantitatively, and stepwise if necessary, with 0.125 N hydrochloric acid to obtain a solution having a concentration of about 2 µg of calcium per mL, adding 1 mL of Lanthanum chloride solution per 100 mL of the final volume.

Procedure— Concomitantly determine the absorbances of the Standard preparations and the Assay preparation at the calcium emission line at 422.7 nm with an atomic absorption spectrophotometer (see Spectrophotometry and Light-Scattering 851) equipped with a calcium hollow-cathode lamp and a nitrous oxide–acetylene flame, using 0.125 N hydrochloric acid containing 0.1% Lanthanum chloride solution as the blank. Plot the absorbances of the Standard preparations versus concentration, in µg per mL, of calcium, and draw the straight line best fitting the five plotted points. From the graph so obtained, determine the concentration, C, in µg per mL, of calcium in the Assay preparation. Calculate the weight, in mg, of calcium (Ca) in the portion of Tablets taken by the formula: in which D is the dilution factor used to prepare the Assay preparation.

Chromium standard stock solution— Transfer about 2.829 g of potassium dichromate, previously dried at 120 for 4 hours and accurately weighed, to a 1000-mL volumetric flask, dissolve in and dilute with water to volume, and mix to obtain a solution having a known concentration of about 1000 µg of chromium per mL. Store in a polyethylene bottle.

Standard preparations— Transfer 10.0 mL of the Chromium standard stock solution to a 1000-mL volumetric flask, add 50.0 mL of 6 N hydrochloric acid, dilute with water to volume, and mix to obtain a standard solution having a known concentration of about 10 µg of chromium per mL. Transfer 10.0 mL and 20.0 mL of the standard solution to separate 100-mL volumetric flasks, and transfer 15.0 mL and 20.0 mL of the standard solution to separate 50-mL volumetric flasks. Dilute the contents of each of the four flasks with 0.125 N hydrochloric acid to volume, and mix to obtain solutions having known concentrations of about 1.0, 2.0, 3.0, and 4.0 µg of chromium per mL.

Assay preparation— Proceed as directed for Assay preparation in the Assay for calcium, except to prepare the Assay preparation to contain about 1 µg of chromium per mL and to omit the use of the Lanthanum chloride solution.

Procedure— Concomitantly determine the absorbances of the Standard preparations and the Assay preparation at the chromium emission line at 357.9 nm with an atomic absorption spectrophotometer (see Spectrophotometry and Light-Scattering 851) equipped with a chromium hollow-cathode lamp and an air–acetylene flame, using 0.125 N hydrochloric acid as the blank. Plot the absorbances of the Standard preparations versus concentration, in µg per 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 per mL, of chromium in the Assay preparation. Calculate the quantity, in µg, of chromium (Cr) in the portion of Tablets taken by the formula: in which D is the dilution factor used to prepare the Assay preparation.

Copper standard stock solution— Dissolve about 1.00 g of copper foil in a minimum volume of a 50% (v/v) solution of nitric acid, and dilute with a 1% (v/v) solution of nitric acid to 1000 mL. This solution contains 1000 µg of copper per mL.

Standard preparations— Transfer 10.0 mL of Copper standard stock solution to a 100-mL volumetric flask, and dilute quantitatively with 0.125 N hydrochloric acid to volume to obtain a standard solution having a concentration of 100 µg of copper per mL. To separate 200-mL volumetric flasks, transfer 1.0, 2.0, 4.0, 6.0, and 8.0 mL of the standard solution, dilute with water to volume, and mix to obtain solutions having known concentrations of about 0.5, 1.0, 2.0, 3.0, and 4.0 µg of copper per mL.

Assay preparation— Proceed as directed for Assay preparation in the Assay for calcium, except to prepare the Assay preparation to contain about 2 µg of copper per mL and to omit the use of Lanthanum chloride solution.

Procedure— Concomitantly determine the absorbances of the Standard preparations and the Assay preparation at the copper emission line at 324.7 nm with an atomic absorption spectrophotometer (see Spectrophotometry and Light-Scattering 851) equipped with a copper hollow-cathode lamp and an air–acetylene flame, using 0.125 N hydrochloric acid as the blank. Plot the absorbances of the Standard preparations versus concentration, in µg per 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 per mL, of copper in the Assay preparation. Calculate the quantity, in mg, of copper (Cu) in the portion of Tablets taken by the formula: in which D is the dilution factor used to prepare the Assay preparation.

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

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

Fluoride standard stock solution— Transfer an accurately weighed quantity of about 1.105 g of sodium fluoride, previously dried at 100 for 4 hours and cooled in a desiccator, to a 1000-mL volumetric flask. Dissolve in and dilute with water to volume, and mix to obtain a solution having a known concentration of about 500 µg of fluoride per mL.

Intermediate stock solution 1— Pipet 20.0 mL of Fluoride standard stock solution into a 100-mL volumetric flask, dilute with water to volume, and mix to obtain a solution having a known concentration of about 100 µg of fluoride per mL.

Intermediate stock solution 2— Transfer 2.0 mL of Fluoride standard stock solution to a 100-mL volumetric flask, dilute with water to volume, and mix to obtain a solution having a known concentration of about 10 µg of fluoride per mL.

Standard preparations— To five separate 100-mL volumetric flasks, transfer 3.0, 5.0, and 10.0 mL of Intermediate stock solution 2 and 5.0 and 10.0 mL of Intermediate stock solution 1. To each flask, add 10.0 mL of 1 N hydrochloric acid, 25 mL of 3 M Sodium acetate solution, and 25.0 mL of Sodium citrate solution. Dilute the contents of each flask with water to volume, and mix to obtain solutions having known concentrations of about 0.3, 0.5, 1.0, 5.0, and 10.0 µg of fluoride per mL.

Assay preparation— Weigh and finely powder a counted number of Tablets. Transfer an accurately weighed quantity of the powder, equivalent to about 200 µg of fluoride, to a 100-mL volumetric flask. Dissolve in 10.0 mL of 1 N hydrochloric acid, 25.0 mL of 3 M Sodium acetate solution, and 25.0 mL of Sodium citrate solution, dilute with water to volume, and mix.

Procedure— To separate plastic beakers, each containing a plastic-coated stirring bar, transfer 50.0 mL each of the Standard preparations and the Assay preparation. Concomitantly measure the potentials (see pH 791), in mV, of the Standard preparations and the Assay preparation, 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 to 2 minutes), 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 per mL, of the Standard preparations versus potential, in mV. From the standard response curve so obtained and the measured potential of the Assay preparation, determine the concentration, C, in µg per mL, of fluoride in the Assay preparation. Calculate the quantity, in mg, of fluoride in the portion of Tablets taken by the formula:

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

Mobile phase— Prepare a filtered and degassed mixture of water, alcohol, and 0.1 N sulfuric acid (175:20:5). Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard stock solution— Dissolve an accurately weighed quantity of USP Sodium Fluoride RS in water, and dilute quantitatively, and stepwise if necessary, with water to obtain a solution having a known concentration of about 220 µg per mL. This solution contains about 100 µg of fluoride per mL.

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

Assay preparation— Weigh and finely powder not fewer than 20 Tablets. Transfer an accurately weighed portion of the powder, equivalent to about 1 mg of fluoride, to a stoppered 100-mL volumetric flask, add 15 mL of water, and shake vigorously. Rinse the sides of the flask with 15 mL of water, and allow to stand for 10 minutes. Dilute with water to about 85 mL, adjust with 1 N sodium hydroxide to a pH of 10.4 ± 0.1, dilute with water to volume, and mix. Proceed as directed for Standard preparation, beginning with “Filter, discarding the first 15 mL of the filtrate.”

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a conductivity detector, a 4.6-mm × 3-cm guard column that contains packing L17, and a 7.8-mm × 30-cm analytical column that contains packing L17. The flow rate is about 0.5 mL per minute. Chromatograph the Standard preparation, and measure the peak areas as directed for Procedure: the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 100 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the peak areas for fluoride. Calculate the quantity, in mg, of fluoride in the portion of Tablets taken by the formula: in which C is the concentration, in µg per mL, of fluoride in the Standard preparation; and rU and rS are the peak areas obtained from the Assay preparation and the Standard preparation, respectively.

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

Procedure— Weigh a counted number of Tablets, and grind them to a fine powder. Transfer an accurately weighed quantity of the powder, equivalent to about 3 mg of iodide, to a nickel crucible. Add 5 g of sodium carbonate, 5 mL of 50% (w/v) sodium hydroxide solution, and 10 mL of alcohol, taking care that the entire specimen is moistened. Heat the crucible on a steam bath to evaporate the alcohol, then dry the crucible at about 100 for about 30 minutes to prevent spattering upon subsequent heating. Transfer the crucible with its contents to a furnace heated to about 500, and heat the crucible for about 15 minutes. [note—Heating at about 500 is necessary to carbonize any organic matter present; 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 about 10 minutes. 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 minutes longer. Add a few crystals of salicylic acid, and cool the solution to about 20. Add 1 mL of phosphoric acid and about 0.5 g of potassium iodide, and titrate the liberated iodine with 0.005 N sodium thiosulfate VS, adding starch solution when the liberated iodine color has nearly disappeared. Calculate the quantity, in µg, of iodide in the portion of Tablets taken by the formula: in which V is the volume, in mL, of sodium thiosulfate consumed; and N is the normality of the sodium thiosulfate solution used.

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

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

Assay preparation— Proceed as directed for Assay preparation in the Assay for calcium, except to prepare the Assay preparation to contain 5 µg of iron per mL and to omit the use of the Lanthanum chloride solution.

Procedure— Concomitantly determine the absorbances of the Standard preparations and the Assay preparation at the iron emission line at 248.3 nm with an atomic absorption spectrophotometer (see Spectrophotometry and Light-Scattering 851) equipped with an iron hollow-cathode lamp and an air–acetylene flame, using 0.125 N hydrochloric acid as the blank. Plot the absorbances of the Standard preparations versus concentration, in µg per mL, of iron, and draw the straight line best fitting the five plotted points. From the graph so obtained, determine the concentration, C, in µg per mL, of iron in the Assay preparation. Calculate the quantity, in mg, of iron (Fe) in the portion of Tablets taken by the formula: in which D is the dilution factor used to prepare the Assay preparation.

Lanthanum chloride solution— Prepare as directed in the Assay for calcium.

Magnesium standard stock solution— Transfer about 1.0 g of magnesium ribbon, accurately weighed, to a 1000-mL volumetric flask, dissolve in 50 mL of 6 N hydrochloric acid, dilute with water to volume, and mix to obtain a solution having a known concentration of about 1000 µg of magnesium per mL.

Standard preparations— Quantitatively dilute a volume of Magnesium standard stock solution with 0.125 N hydrochloric acid to obtain a standard solution having a concentration of 20 µg of magnesium per mL. To separate 100-mL volumetric flasks, transfer 1.0, 1.5, 2.0, 2.5, and 3.0 mL of standard solution. To each flask add 1.0 mL of Lanthanum chloride solution, dilute with 0.125 N hydrochloric acid to volume, and mix to obtain solutions having known concentrations of about 0.2, 0.3, 0.4, 0.5, and 0.6 µg of magnesium per mL.

Assay preparation— Proceed as directed for Assay preparation in the Assay for calcium, except to prepare the Assay preparation to contain 0.4 µg of magnesium per mL.

Procedure— Concomitantly determine the absorbances of the Standard preparations and the Assay preparation at the magnesium emission line at 285.2 nm with an atomic absorption spectrophotometer (see Spectrophotometry and Light-Scattering 851) equipped with a magnesium hollow-cathode lamp and an air–acetylene flame, using 0.125 N hydrochloric acid containing 0.1% Lanthanum chloride solution as the blank. Plot the absorbances of the Standard preparations versus concentration, in µg per mL, of magnesium, and draw the straight line best fitting the five plotted points. From the graph so obtained, determine the concentration, C, in µg per mL, of magnesium in the Assay preparation. Calculate the quantity, in mg, of magnesium (Mg) in the portion of Tablets taken by the formula: in which D is the dilution factor used to prepare the Assay preparation.

Manganese standard stock solution— Transfer 1.00 g of manganese, accurately weighed, to a 1000-mL volumetric flask, dissolve in 20 mL of nitric acid, dilute with 6 N hydrochloric acid to volume, and mix to obtain a solution containing 1000 µg of manganese per mL.

Standard preparations— Quantitatively dilute 10.0 mL of the Manganese standard stock solution with 0.125 N hydrochloric acid to 200.0 mL to obtain a standard solution having a concentration of 50 µg of manganese per mL. To separate 100-mL volumetric flasks, transfer 1.0, 1.5, 2.0, 3.0, and 4.0 mL of standard solution, dilute the contents of each flask with 0.125 N hydrochloric acid to volume, and mix to obtain solutions having known concentrations of about 0.5, 0.75, 1.0, 1.5, and 2.0 µg of manganese per mL.

Assay preparation— Proceed as directed for Assay preparation in the Assay for calcium, except to prepare the Assay preparation to contain 1 µg of manganese per mL and to omit the use of the Lanthanum chloride solution.

Procedure— Concomitantly determine the absorbances of the Standard preparations and the Assay preparation at the manganese emission line at 279.5 nm with an atomic absorption spectrophotometer (see Spectrophotometry and Light-Scattering 851) equipped with a manganese hollow-cathode lamp and an air–acetylene flame, using 0.125 N hydrochloric acid as the blank. Plot the absorbances of the Standard preparations versus concentration, in µg per mL, of manganese, and draw the straight line best fitting the five plotted points. From the graph so obtained, determine the concentration, C, in µg per mL, of manganese in the Assay preparation. Calculate the quantity, in mg, of manganese (Mn) in the portion of Tablets taken by the formula: in which D is the dilution factor used to prepare the Assay preparation.

Diluting solution— Dissolve 40 g of ammonium chloride in 2000 mL of water.

Molybdenum standard stock solution— Transfer about 1.0 g of molybdenum wire, accurately weighed, to a 1000-mL volumetric flask, and dissolve in 50 mL of nitric acid, warming if necessary. Dilute with water to volume, and mix to obtain a solution containing 1000 µg of molybdenum per mL.

Standard preparations— Quantitatively dilute 10.0 mL of the Molybdenum standard stock solution with water to 100.0 mL to obtain a standard solution having a known concentration of about 100 µg of molybdenum per mL. To separate 100-mL volumetric flasks, transfer 2.0, 10.0, and 25.0 mL of the standard solution, and add 5.0 mL of perchloric acid to each flask. Gently boil the solution in each flask for 15 minutes, cool to room temperature, dilute each with Diluting solution to volume, and mix to obtain solutions having known concentrations of about 5.0, 10.0, and 25.0 µg of molybdenum per mL.

Assay preparation— Weigh and finely powder a counted number of Tablets. Transfer an accurately weighed portion of the powder, equivalent to about 1000 µg of molybdenum, to a suitable flask, and add about 12 mL of nitric acid. [note—The volume of nitric acid may be varied to ensure that the powder is uniformly dispersed.] Carefully swirl the flask to disperse the test specimen. Sonicate for about 10 minutes, or until the test specimen is completely dissolved. Gently boil the solution for about 15 minutes, and cool to room temperature. Carefully add about 8 mL of perchloric acid, heat until perchloric acid fumes appear, and swirl the flask to dissipate the fumes. Repeat the heating and swirling until the fumes persist. Cool to room temperature. Quantitatively transfer the contents of the flask to a 100-mL volumetric flask with the aid of the Diluting solution, dilute with Diluting solution to volume, and mix.

Procedure— Concomitantly determine the absorbances of the Standard preparations and the Assay preparation at the molybdenum emission line at 313 nm with an atomic absorption spectrophotometer (see Spectrophotometry and Light-Scattering 851) equipped with a molybdenum hollow-cathode lamp and a nitrous oxide–acetylene flame, using a mixture of Diluting solution and perchloric acid (20:1) as the blank. Plot the absorbances of the Standard preparations versus concentration, in µg per 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 per mL, of molybdenum in the Assay preparation. Calculate the quantity, in mg, of molybdenum (Mo) in the portion of Tablets taken by the formula: in which D is the diluting factor used to prepare the Assay preparation.

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

Ferrous sulfate solution— Dissolve 498 mg of ferrous sulfate in water to make 100 mL.

Potassium thiocyanate solution— Dissolve 20 g of potassium thiocyanate in water, and dilute with water to 100 mL.

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

Dilute stannous chloride solution— Dilute 4 mL of 20% Stannous chloride solution with water to 100 mL. Prepare this solution fresh at the time of use.

Standard preparation— Transfer about 92 mg of ammonium molybdate, accurately weighed, to a 500-mL volumetric flask, dissolve in and dilute with water to volume, and mix. Quantitatively dilute 20.0 mL of this solution with water to 100.0 mL to obtain a solution having a known concentration of about 20 µg of molybdenum per mL.

Procedure— Weigh a counted number of Tablets, and grind the Tablets to a fine powder. Transfer an accurately weighed quantity of the powder, equivalent to about 40 µg of molybdenum, to a 200-mL beaker marked I. To another 200-mL beaker marked II, transfer 2.0 mL of the Standard preparation. Add about 20 mL of nitric acid to each beaker. Cover each beaker with a watchglass, and boil slowly on a hot plate for about 45 minutes. Cool to room temperature, add 6 mL of perchloric acid to each beaker, cover each beaker with a watchglass, and continue the heating until digestion is complete, as indicated when the liquid becomes colorless or pale yellow. If necessary, add further portions of nitric acid and perchloric acid to beaker I, and digest further. Evaporate the solutions in each of the beakers to dryness. Rinse the sides of the beakers and the watchglasses with water, and add more water to each beaker to a volume of about 50 mL. Gently boil the water solutions for a few minutes. Cool to room temperature. Add 2 drops of methyl orange TS to each beaker, and neutralize with ammonium hydroxide. Add 8.2 mL of hydrochloric acid to each beaker. Quantitatively transfer the contents of the beakers to separate 100-mL volumetric flasks, rinse the beakers with water, transfer the rinsings to the respective volumetric flasks, dilute with water to volume, and mix.

Sulfuric acid solution— Cautiously add 37.5 mL of sulfuric acid to 100 mL of water, and mix.

Ammonium molybdate solution— Dissolve 12.5 g of ammonium molybdate in 150 mL of water. Add 100 mL of Sulfuric acid solution, and mix.

Hydroquinone solution— Dissolve 0.5 g of hydroquinone in 100 mL of water, and add one drop of sulfuric acid.

Sodium bisulfite solution— Dissolve 20 g of sodium bisulfite in 100 mL of water.

Phosphorus standard stock solution— Accurately weigh about 4.395 g of monobasic potassium phosphate, previously dried at 105 for 2 hours 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 containing 1000 µg of phosphorus per mL.

Standard preparation— Dissolve an accurately measured volume of Phosphorus standard stock solution in water, and dilute quantitatively, and stepwise if necessary, with water to obtain a solution having a known concentration of about 20 µg of phosphorus per mL.

Assay preparation— Weigh a counted number of Tablets, and grind the Tablets to a fine powder. Transfer an accurately weighed portion of the powder, equivalent to about 100 mg of phosphorus, to a suitable flask, add 25 mL nitric acid, and digest on a hot plate for about 30 minutes. Add 15 mL of hydrochloric acid, and continue the digestion to the cessation of brown fumes. Cool, and quantitatively transfer the contents of the flask to a 500-mL volumetric flask with the aid of small portions of water. Dilute with water to volume, and mix. Transfer 10.0 mL of this solution to a 100-mL volumetric flask, dilute with water to volume, and mix.

Procedure— To three separate 25-mL volumetric flasks, transfer 5.0 mL each of the Standard preparation, the Assay preparation, and water to provide the blank. To each of the three flasks, add 1.0 mL each of Ammonium molybdate solution, Hydroquinone solution, and Sodium bisulfite solution, and swirl to mix. Dilute the contents of each flask with water to volume, mix, and allow the flasks to stand for 30 minutes. Concomitantly determine the absorbances of the solutions from the Assay preparation and the Standard preparation in 1-cm cells at the wavelength of maximum absorbance at about 650 nm, against the blank. Calculate the quantity, in mg, of phosphorus (P) in the portion of Tablets taken by the formula: in which C is the concentration, in µg per mL, of phosphorus in the Standard preparation; and AU and AS are the absorbances of the solutions obtained from the Assay preparation and the Standard preparation, respectively.

Potassium standard stock solution— Transfer about 190.7 mg of potassium chloride, previously dried at 105 for 2 hours and accurately weighed, to a 1000-mL volumetric flask, dissolve in and dilute with water to volume, and mix to obtain a solution having a known concentration of about 100 µg of potassium per mL.

Standard preparations— Dilute an accurately measured volume of Potassium standard stock solution with 0.125 N hydrochloric acid to obtain a standard solution having a known concentration of about 10 µg of potassium per mL. Transfer 5.0, 10.0, 15.0, 20.0, and 25.0 mL of the standard solution to separate 100-mL volumetric flasks. Dilute the contents of each flask with 0.125 N hydrochloric acid to volume, and mix to obtain solutions containing 0.5, 1.0, 1.5, 2.0, and 2.5 µg of potassium per mL.

Assay preparation— Proceed as directed for Assay preparation in the Assay for calcium, except to prepare the Assay preparation to contain 1 µg of potassium per mL and to omit the use of the Lanthanum chloride solution.

Procedure— Concomitantly determine the absorbances of the Standard preparations and the Assay preparation at the potassium emission line at 766.5 nm with an atomic absorption spectrophotometer (see Spectrophotometry and Light-Scattering 851) equipped with a potassium hollow-cathode lamp and an air–acetylene flame, using water as the blank. Plot the absorbances of the Standard preparations versus concentration, in µg per 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 per mL, of potassium in the Assay preparation. Calculate the quantity, in mg, of potassium (K) in the portion of Tablets taken by the formula: in which D is the dilution factor used to prepare the Assay preparation.

Diluting solution— Prepare as directed in the Assay for molybdenum, Method 1.

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

Standard preparations— Quantitatively dilute 10 mL of the Selenium standard stock solution with water to 100.0 mL to obtain a standard solution having a known concentration of about 100 µg of selenium per mL. To separate 100-mL volumetric flasks, transfer 5.0, 10.0, and 25.0 mL of the standard solution, and add 5.0 mL of perchloric acid to each flask. Gently boil the solutions for 15 minutes, cool to room temperature, dilute each with Diluting solution to volume, and mix to obtain solutions containing 5.0, 10.0, and 25.0 µg of selenium per mL.

Assay preparation— Weigh and finely powder a counted number of Tablets. Transfer an accurately weighed portion of the powder, equivalent to about 1000 µg of selenium, to a suitable flask, and add about 12 mL of nitric acid. [note—The volume of nitric acid may be varied to ensure that the powder is uniformly dispersed.] Carefully swirl the flask to disperse the test specimen. Sonicate for about 10 minutes or until the test specimen is completely dissolved. Gently boil the solution for about 15 minutes, and cool to room temperature. Carefully add about 8 mL of perchloric acid to the flask, heat the flask until perchloric acid fumes appear, and swirl the flask to dissipate the fumes. Repeat the heating and swirling until the fumes persist. Cool to room temperature. Quantitatively transfer the contents of the flask to a 50-mL volumetric flask with the aid of the Diluting solution, dilute with Diluting solution to volume, and mix.

Procedure— Concomitantly determine the absorbances of the Standard preparations and the Assay preparation at the selenium emission line at 196 nm, with an atomic absorption spectrophotometer (see Spectrophotometry and Light-Scattering 851) equipped with a selenium hollow-cathode lamp and an air–acetylene flame, using a mixture of Diluting solution and perchloric acid (20:1) as the blank. Plot the absorbances of the Standard preparations versus concentration, in µg per 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 per mL, of selenium in the Assay preparation. Calculate the quantity, in mg, of selenium (Se) in the portion of Tablets taken by the formula: in which D is the dilution factor used to prepare the Assay preparation.

Hydrochloric acid solution— Dilute 50 mL of hydrochloric acid with water to 500 mL, and mix.

50% Ammonium hydroxide solution— Dilute 250 mL of ammonium hydroxide with water to 500 mL, and mix.

Reagent 1— Dissolve 4.5 g of edetate disodium in 400 mL of water in a 500-mL volumetric flask. Add 12.5 g of hydroxylamine hydrochloride, dilute with water to volume, and mix.

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

Standard stock preparation— [Caution—Selenium is toxic; handle it with care. ] Dissolve about 1 g of metallic selenium, accurately weighed, in a minimum volume of nitric acid. Evaporate to dryness, add 2 mL of water, and evaporate to dryness. Repeat the addition of water and evaporation to dryness three times. Dissolve the residue in 3 N hydrochoric acid, transfer to a 1000-mL volumetric flask, dilute with 3 N hydrochloric acid to volume, and mix to obtain a solution containing about 1000 µg of selenium per mL. Dilute a volume of the solution quantitatively, and stepwise if necessary, with 0.125 N hydrochloric acid to obtain a solution having a known concentration of about 2.0 µg of selenium per mL.

Assay preparation— Weigh a counted number of Tablets, and grind to a fine powder. Transfer an accurately weighed portion of the powder, equivalent to about 20 µg of selenium, to a suitable flask. Add 10 mL of nitric acid, and warm gently on a hot plate. Continue heating until the initial nitric acid reaction has subsided, then add 3 mL of perchloric acid. [Caution—Exercise care at this stage as perchloric acid reaction becomes vigorous. ] Continue heating on the hot plate until the appearance of white fumes of perchloric acid or until the digest begins to darken. Add 0.5 mL of nitric acid, and resume heating, adding additional amounts of nitric acid if further darkening occurs. Digest for about 10 minutes after the first appearance of perchloric acid fumes or until the digest becomes colorless. Cool the flask, add 2.5 mL of Hydrochloric acid solution, and return the flask to the hot plate to expel residual nitric acid. Heat the mixture for 3 minutes after it begins to boil. Cool the flask to room temperature, and dilute with water to about 20 mL.

Procedure— Transfer 10.0 mL of the Standard stock preparation to a glass-stoppered flask. Add 1 mL of perchloric acid and 1 mL of Hydrochloric acid solution, and dilute with water to about 20 mL (Standard preparation). Prepare a blank by adding 1 mL of perchloric acid and 1 mL of Hydrochloric acid solution to a glass-stoppered flask, and diluting with water to about 20 mL. Treat the Assay preparation, the Standard preparation, and the blank as follows. Add 5 mL of Reagent 1 to each flask, and swirl gently to mix. Adjust the solution in each flask with 50% Ammonium hydroxide solution to a pH of 1.1 ± 0.1. Add 5 mL of Reagent 2 to each flask, and swirl gently to mix. Place the flasks in a water bath maintained at 50, and equilibrate for 30 minutes, 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 minute. Discard the aqueous layer. Transfer the cyclohexane layer to a centrifuge tube, and centrifuge at 1000 rpm for 1 minute to remove any remaining water. Concomitantly determine the absorbances of the solutions from the Assay preparation and the Standard preparation in 1-cm cells at the wavelength of maximum absorbance at about 380 nm against the solution from the blank. Calculate the quantity, in µg, of selenium (Se) in the portion of Tablets taken by the formula: in which C is the concentration, in µg per mL, of selenium in the Standard stock preparation; D is the dilution factor used to prepare the Assay preparation; and AU and AS are the absorbances of the solutions from the Assay preparation and the Standard preparation, respectively.

Zinc standard stock solution— Transfer about 311 mg of zinc oxide, accurately weighed, to a 250-mL volumetric flask, and add 80 mL of 5 M hydrochloric acid, warming if necessary to dissolve. Cool, dilute with water to volume, and mix to obtain a solution having a known concentration of about 1000 µg of zinc per mL.

Standard preparations— Dilute a volume of the Zinc standard stock solution quantitatively, and stepwise if necessary, with 0.125 N hydrochloric acid to obtain a standard solution having a known concentration of about 50 µg of zinc per mL. Transfer 1.0, 2.0, 3.0, 4.0, and 5.0 mL of this solution to separate 100-mL volumetric flasks. Dilute the contents of each flask with 0.125 N hydrochloric acid to volume, and mix to obtain solutions having known concentrations of about 0.5, 1.0, 1.5, 2.0, and 2.5 µg of zinc per mL.

Assay preparation— Proceed as directed for Assay preparation in the Assay for calcium, except to prepare the Assay preparation to contain 2 µg of zinc per mL and to omit the use of the Lanthanum chloride solution.

Procedure— Concomitantly determine the absorbances of the Standard preparations and the Assay preparation at the zinc emission line at 213.8 nm with an atomic absorption spectrophotometer (see Spectrophotometry and Light-Scattering 851) equipped with a zinc hollow-cathode lamp and an air–acetylene flame, using 0.125 N hydrochloric acid as the blank. Plot the absorbances of the Standard preparations versus concentration, in µg per mL, of zinc, and draw the straight line best fitting the five plotted points. From the graph so obtained, determine the concentration, C, in µg per mL, of zinc in the Assay preparation. Calculate the quantity, in mg, of zinc (Zn) in the portion of Tablets taken by the formula: in which D is the dilution factor used to prepare the Assay preparation.