(Official January 1, 2007)

Assay preparation— Transfer the contents of not fewer than 20 Capsules to a suitable container, mix, and weigh. Transfer an accurately weighed portion of the mixture, equivalent to 5 Capsules, to a container having a polytef-lined screw cap. [NOTE—For hard gelatin capsules, remove, as completely as possible, the contents of not fewer than 20 Capsules by cutting open the Capsule shells, using a sharp blade if necessary, transferring the shells and their contents to a suitable container, and triturating to a homogeneous mass. Transfer an accurately weighed portion of the mass, equivalent to 5 Capsules, 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 equivalent to about 15 µg of retinyl acetate per mL. Retain the remaining solution for use in the assays for vitamin D, vitamin E, and phytonadione.

Assay preparation— [NOTE—This preparation is suitable for the determination of vitamin A, vitamin D, and vitamin E, when present in the formulation.] Accurately weigh not fewer than 20 Capsules in a tared weighing bottle. Using a sharp blade if necessary, carefully open the Capsules, without loss of shell material, and transfer the contents to a 100-mL beaker. Remove any contents adhering to the empty shells by washing with several portions of ether. Discard the washings, and dry the Capsule shells with the aid of a current of dry air. Weigh the empty Capsule shells in the tared weighing bottle, and calculate the net weight of the Capsule contents. Transfer an accurately weighed portion of the Capsule contents, equivalent to about 30 µg of cholecalciferol or ergocalciferol (vitamin D), to a container having a polytef-lined screw cap. If vitamin D is not present in the formulation, use a portion equivalent to about 90 mg of vitamin E. If vitamin E is not present in the formulation, use a portion equivalent to about 2.5 mg of vitamin A. Proceed as directed for Assay preparation in the Assay for vitamin A, Method 2 under Oil- and Water-Soluble Vitamins with Minerals Tablets, beginning with “Add about 0.5 g of sodium bicarbonate.”

Assay preparation— Accurately weigh not fewer than 20 Capsules in a tared weighing bottle. Using a sharp blade if necessary, carefully open the Capsules, without loss of shell material, and transfer the contents to a 100-mL beaker. Remove any contents adhering to the empty shells by washing with several portions of ether. Discard the washings, and dry the Capsule shells with the aid of a current of dry air. Weigh the empty Capsule shells in the tared weighing bottle, and calculate the net weight of the Capsule contents. Transfer an accurately weighed portion of the Capsule contents, equivalent to about 1.5 mg of retinyl acetate, to a stoppered 125-mL flask, and proceed as directed for Assay preparation in the Assay for vitamin A, Method 3 under Oil- and Water-Soluble Vitamins with Minerals Tablets, beginning with “Add 5 mL of water.”

Assay preparation— Transfer an accurately measured volume 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.

Assay preparation— Proceed as directed for Assay preparation in the Assay for vitamin A, Method 2.

Assay preparation— Prepare as directed for Assay preparation in the Assay for vitamin A, Method 3, through “calculate the net weight of the Capsule contents.” Transfer an accurately weighed portion of the Capsule contents, equivalent to about 10 µg of cholecalciferol or ergocalciferol, to a stoppered 125-mL flask, and proceed as directed for Standard preparation, beginning with “Add 15.0 mL of water and 15.0 mL of Potassium hydroxide solution.”

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 dryness. Transfer the contents of the flask to a suitable volumetric flask with the aid of methanol, and dilute with methanol to volume to obtain a solution having a concentration of about 2 mg of alpha tocopherol, alpha tocopheryl acetate, or alpha tocopheryl acid succinate per mL.

3 N Methanolic sulfuric acid solution, Sodium ascorbate-pyrogallol solution, and Lecithin solution— Proceed as directed in the Assay for vitamin A, Method 2 under Oil-and Water-Soluble Vitamins with Minerals Tablets.

Assay preparation— Proceed as directed for Assay preparation in the Assay for vitamin A, Method 3, through “calculate the net weight of the Capsule contents.” Transfer an accurately weighed portion of the Capsule contents, equivalent to about 55 mg of vitamin E, to a container having a polytef-lined screw cap. 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 layer. Transfer an accurately measured volume of the supernatant 2,2,4-trimethylpentane layer to a suitable volumetric flask, the volume of the specimen withdrawn from the 2,2,4-trimethylpentane layer and the size of the volumetric flask being such that the final concentration of the 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.

Assay preparation— Proceed as directed for Assay preparation in the Assay for vitamin A, Method 3, through “calculate the net weight of the Capsule contents.” Transfer an accurately weighed portion of the Capsule contents, equivalent to about 8.0 mg of alpha tocopherol, to a glass-stoppered conical flask, and proceed as directed for Assay preparation in the Assay for vitamin E, Method 3 under Oil- and Water-Soluble Vitamins with Minerals Tablets, beginning with “Add 25.0 mL of water.”

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 dryness. Transfer the contents of the flask to a suitable volumetric flask with the aid of methanol, and dilute with methanol to volume to obtain a solution having a concentration of about 20 µg of phytonadione per mL.

Assay preparation— Accurately weigh not fewer than 20 Capsules in a tared weighing bottle. Using a sharp blade if necessary, carefully cut open the Capsules, without the loss of shell material, and transfer the contents to a 100-mL beaker. Remove any contents adhering to the empty shells by washing if necessary with several portions of ether. Discard the washings, and dry the Capsule shells with the aid of a current of dry air until the odor of ether is no longer perceptible. Weigh the empty Capsule shells in the tared weighing bottle, and calculate the average net weight per Capsule. Transfer an accurately weighed portion of the Capsule contents, equivalent to about 100 mg of ascorbic acid, to a 200-mL volumetric flask, and proceed as directed in the Assay for ascorbic acid, Method 1 under Oil- and Water-Soluble Vitamins with Minerals Tablets, beginning with “and add about 75 mL of metaphosphoric-acetic acids TS.”

Assay preparation— Accurately weigh not fewer than 20 Capsules in a tared weighing bottle. Using a sharp blade if necessary, carefully open the Capsules, without loss of shell material, and transfer the contents to a 100-mL beaker. Remove any contents adhering to the empty shells by washing with several portions of ether. Discard the washings, and dry the Capsule shells with the aid of a current of dry air until the odor of ether is no longer perceptible. Weigh the empty Capsule shells in the tared weighing bottle, and calculate the net weight of the Capsule contents. Transfer an accurately weighed portion of the mixed Capsule contents, 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.

Assay preparation— Proceed as directed for Assay preparation in the Assay for biotin, Method 1, from the beginning through calculation of the net weight of the Capsule contents. Transfer an accurately weighed quantity of the mixed Capsule contents, 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 percent 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.

Assay preparation— Weigh and combine the contents of not fewer than 30 Capsules. Transfer an accurately weighed portion of the Capsule contents, equivalent to about 100 µg of cyanocobalamin, to a 250-mL flask, add 100.0 mL of water, and shake by hand for 2 minutes. Filter a portion of this solution, and use the clear filtrate.

Assay preparation— Proceed as directed for Assay preparation in the Assay for biotin, Method 1, through calculation of the net weight of the Capsule contents. Transfer an accurately weighed portion of the mixed Capsule contents, equivalent to about 1.0 µg of cyanocobalamin, to an appropriate vessel containing, for each g of Capsule contents taken, 25 mL of an aqueous extracting solution prepared just 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.

Assay preparation— Proceed as directed for Assay preparation in the Assay for biotin, Method 1, from the beginning through calculation of the net weight of the Capsule contents. Transfer an accurately weighed portion of the mixed Capsule contents, equivalent to about 0.4 mg of folic acid, to a 50-mL 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.

Assay preparation— Proceed as directed for Assay preparation in the Assay for biotin, Method 1, through “calculate the net weight of the Capsule contents.” Transfer an accurately weighed portion of the Capsule contents, equivalent to about 0.3 mg of folic acid, 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— Proceed as directed under Assay for biotin, Method 1, from the beginning through calculation of the net weight of the Capsule contents. Transfer an accurately weighed portion of the mixed Capsule contents, equivalent to about 15 mg of calcium pantothenate, to a centrifuge tube. Transfer 25.0 mL of Internal standard preparation to the centrifuge tube, and shake vigorously for 10 minutes. Centrifuge, and filter, using the clear filtrate.

Assay preparation— Proceed as directed in the Assay for biotin, Method 1, from the beginning through calculation of the net weight of the Capsule contents. Transfer an accurately weighed portion of the mixed Capsule contents, 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 about the same concentration as that of the Standard preparation.

Assay preparation— Proceed as directed for Assay preparation in the Assay for biotin, Method 1, through “calculate the net weight of the Capsule contents.” Transfer an accurately weighed portion of the Capsule contents, equivalent to about 10 mg of calcium pantothenate, to a 250-mL volumetric flask, add 10 mL of methanol, and swirl the flask to disperse the Capsule contents. Dilute with water to volume, mix, and filter.

Assay preparation— Proceed as directed in the Assay for biotin, Method 1, from the beginning through the calculation of the net weight of the Capsule contents. Transfer an accurately weighed portion of the mixed Capsule contents, 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. Proceed as directed for Assay preparation in the Assay for niacin or niacinamide, pyridoxine hydrochloride, riboflavin, and thiamine, Method 1 under Oil- and Water-Soluble Vitamins with Minerals Tablets, beginning with “Add 25.0 mL of Diluting solution”.

Assay preparation— [NOTE—This preparation is suitable for the determination of niacin or niacinamide, pyridoxine hydrochloride, and riboflavin, when present in the formulation.] Accurately weigh not fewer than 20 Capsules in a tared weighing bottle. Using a sharp blade if necessary, carefully open the Capsules, without loss of shell material, and transfer the contents to a beaker. Remove any contents adhering to the empty shells by washing with several portions of ether. Discard the washings, and dry the Capsule shells with the aid of a current of dry air. Weigh the empty Capsule shells in the tared weighing bottle, and calculate the net weight of the Capsule contents. Transfer an accurately weighed portion of the Capsule contents, equivalent to about 2 mg of riboflavin, to a 250-mL volumetric flask. If riboflavin is not present in the formulation, use a portion equivalent to about 2 mg of pyridoxine. If pyridoxine is not present in the formulation, use 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.

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

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

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

Assay preparation— Proceed as directed for Assay preparation in the Assay for vitamin A, Method 3, through “calculate the net weight of the Capsule contents.” Transfer an accurately weighed portion of the contents, equivalent to about 2 mg of thiamine, to a 250-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.

Assay preparation— Proceed as directed for Assay preparation in the Assay for biotin, Method 1, through “calculate the net weight of the Capsule contents.” Transfer an accurately weighed portion of the Capsule contents, 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.