(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 the 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 retinyl acetate. 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 ergocalciferol or cholecalciferol, 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— 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 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.

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

Iodine solution— Transfer about 10 mg of iodine crystals to a 100-mL volumetric flask. Dissolve in and 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 1— Proceed as directed in the Assay for vitamin A, Method 1, except to use cyclohexane instead of n-hexane as the extraction solvent, and to dilute the filtered extracts quantitatively, and stepwise if necessary, with cyclohexane to obtain a solution having a concentration of 2 µg of beta carotene per mL.

Assay preparation 2— Accurately weigh not fewer than 20 Capsules, cut open the Capsules, using a sharp blade if necessary, and combine the contents in a suitable container. Transfer an accurately weighed quantity of the Capsule contents, 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 Assay preparation 1 or Assay preparation 2 at the wavelength of maximum absorbance at 452 nm, using cyclohexane as the blank. Calculate the quantity, in mg, of beta carotene (C40H56) in the Capsules taken by the formula: in which L is the labeled amount, in mg, of beta carotene in each Capsule; D is the concentration, in mg per mL, of beta carotene in the Assay preparation, based on the labeled quantity per Capsule and the extent of dilution; AU is the absorbance of the Assay preparation; and 223 is the absorptivity of beta carotene at 452 nm.