Assay preparation— Transfer the contents of not fewer than 20Capsules to a suitable container,mix,and weigh.Transfer an accurately weighed portion of the mixture,equivalent to 5Capsules,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 20Capsules 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 5Capsules,to a container having a polytef-lined screw cap.]Add 10mLof dimethyl sulfoxide and 15mLof n-hexane,and shake for 45minutes 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 3000rpm for 10minutes,and transfer the hexane layer by means of a pipet to a 100-mLvolumetric flask.Add 15mLof n-hexane to the dimethyl sulfoxide layer,shake thoroughly for 5minutes,and transfer the hexane layer by means of a pipet to the 100-mLvolumetric flask.Repeat this extraction with three additional 15-mLportions of n-hexane.Dilute the extracts in the volumetric flask with n-hexane to volume,and mix.Quantitatively dilute a 10-mLvolume of this solution with n-hexane to obtain a solution having a final concentration equivalent to about 15µg 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 20Capsules 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-mLbeaker.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 30µg of the cholecalciferol or ergocalciferol (vitamin D),to a container having a polytef-lined screw cap.If vitamin Dis not present in the formulation,use a portion equivalent to about 90mg of vitamin E.If vitamin Eis not present in the formulation,use a portion equivalent to about 2.5mg of retinyl acetate.Proceed as directed for Assay preparationin the Assay for vitamin A,Method 2under Oil-and Water-Soluble Vitamins with Minerals Tablets,beginning with “Add about 0.5g of sodium bicarbonate.”

Assay preparation— Accurately weigh not fewer than 20Capsules 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-mLbeaker.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 1.5mg of retinyl acetate,to a stoppered 125-mLflask,and proceed as directed for Assay preparationin the Assay for vitamin A,Method 3under Oil-and Water-Soluble Vitamins with Minerals Tablets,beginning with “Add 5mLof water.”

Assay preparation— Transfer an accurately measured volume of the solution retained as specified in the directions for Assay preparationin the Assay for vitamin A,Method 1to a suitable container,and evaporate,if necessary,in vacuum at room temperature to obtain a solution having a concentration of about 2µg of cholecalciferol or ergocalciferol per mL.

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

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

Assay preparation— Transfer not less than 20mL,accurately measured,of the solution retained as specified in the directions for Assay preparationin the Assay for vitamin A,Method 1to 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 2mg of alpha tocopherol,alpha tocopheryl acetate,or alpha tocopheryl acid succinate per mL.

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

Assay preparation— Accurately weigh not fewer than 20Capsules 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-mLbeaker.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 55mg of vitamin E,to a container having a polytef-lined screw cap.Add about 0.5g of sodium bicarbonate,1.5mLof Lecithin solution,and 12.5mLof 2,2,4-trimethylpentane,and disperse on a vortex mixer.Add 6mLof 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 12minutes.Add 6mLof dimethyl sulfoxide,mix on a vortex mixer to form a suspension,and shake for 12minutes.Add 6mLof 3N Methanolic sulfuric acid solution,mix on a vortex mixer to form a suspension,and shake for 12minutes.Add 12.5mLof 2,2,4-trimethylpentane,mix on a vortex mixer to form a suspension,and shake for 10minutes.Centrifuge for about 10minutes 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 preparationis equivalent to that of the Standard preparation.Evaporate nearly to dryness,add several mLof methanol,and evaporate the remaining 2,2,4-trimethylpentane.Dilute with methanol to volume,and mix.

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

Assay preparation— Transfer not less than 20mL,accurately measured,of the solution retained as specified in the directions for Assay preparationin the Assay for vitamin A,Method 1to 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 20µg of phytonadione per mL.

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

Iodine solution— Transfer about 10mg of iodine crystals to a 100-mLvolumetric flask.Dissolve in and dilute with cyclohexane to volume,and mix.Dilute 10mLof this solution with cyclohexane to 100mL,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 2µg of beta carotene per mL.

Assay preparation 2— Accurately weigh not fewer than 20Capsules,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 2mg of beta carotene,to a 500-mLsaponification flask.Add 100mLof alcohol,6mLof Potassium hydroxide solution,and a magnetic stirring bar.Attach an air condenser to the flask,and heat under reflux for 45minutes with constant stirring.Cool to room temperature,add 170mLof solvent hexane,and stir for 30minutes.Quantitatively transfer the contents of the flask to a 500-mLseparatory funnel with portions of solvent hexane.Allow the layers to separate for 5to 10minutes,and transfer the upper organic layer to a 500-mLvolumetric flask.Transfer the lower aqueous layer into the saponification flask,add 170mLof solvent hexane,and stir for an additional 20minutes.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 10minutes.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 3g of anhydrous sodium sulfate,shake,and allow to settle.Quantitatively transfer a volume of this solution,equivalent to about 100µg of beta carotene,to a 50-mLvolumetric flask.Evaporate under a stream of nitrogen to dryness,and immediately add cyclohexane.Add 2mLof Iodine solution,and heat for 15minutes in a water bath maintained at 65.Cool rapidly,dilute with cyclohexane to volume,and mix.

Procedure— Determine the absorbance of Assay preparation 1or Assay preparation 2at the wavelength of maximum absorbance at 452nm,using cyclohexane as the blank.Calculate the quantity,in mg,of beta carotene (C40H56)in the Capsules taken by the formula: in which Lis the labeled amount,in mg,of beta carotene in each Capsule;Dis 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;AUis the absorbance of the Assay preparation;and 223is the absorptivity of beta carotene at 452nm.