NOTE—Where more than one Assaymethod is given for an individual ingredient,the requirements may be met by following any one of the specified methods,the method used being stated in the labeling only if Method 1is not used.

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 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 vitamin A.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 3,through “calculate the net weight of the Capsule contents.”Transfer an accurately weighed portion of the Capsule contents,equivalent to about 10µg of cholecalciferol or ergocalciferol,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 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 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— Proceed as directed for Assay preparationin 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 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 3,through “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.

Assay preparation— Accurately weigh not fewer than 20Capsules 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-mLbeaker.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 100mg of ascorbic acid,to a 200-mLvolumetric flask,and proceed as directed in the Assay for ascorbic acid,Method 1under Oil-and Water-Soluble Vitamins with Minerals Tablets,beginning with “and add about 75mLof metaphosphoric-acetic acids TS.”

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 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 1mg of biotin,to a 200-mLvolumetric flask.Add 3mLof dimethyl sulfoxide,and swirl to wet the contents.Place the flask in a water bath at 60to 70for 5minutes.Sonicate for 5minutes,dilute with water to volume,mix,and filter.

Assay preparation— Proceed as directed for Assay preparationin 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 100µg of biotin,to a 200-mLvolumetric flask,add 3mLof 50%alcohol,and swirl to wet the contents.Heat the flask in a water bath at 60to 70for 5minutes.Sonicate for 5minutes,dilute with 50percent 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 0.1ng per mL.

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

Assay preparation— Proceed as directed for Assay preparationin 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 1.0µg of cyanocobalamin,to an appropriate vessel containing,for each g of Capsule contents taken,25mLof an aqueous extracting solution prepared just prior to use to contain,in each 100mL,1.29g of dibasic sodium phosphate,1.1g of anhydrous citric acid,and 1.0g of sodium metabisulfite.Autoclave the mixture at 121for 10minutes.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 B12activity approximately equivalent to that of the Standard preparation.

Assay preparation— Proceed as directed for Assay preparationin 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 0.4mg of folic acid,to a 50-mLcentrifuge tube.Add 25.0mLof Internal standard preparation,insert a stopper,shake by mechanical means for 10minutes,and centrifuge.Filter a portion of the clear supernatant,and use the filtrate.

Assay preparation— Proceed as directed for Assay preparationin 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 0.3mg of folic acid,to a stoppered 125-mLflask.Add 10.0mLof a mixture of methanol and glacial acetic acid (9:1)and 30.0mLof a mixture of methanol and ethylene glycol (1:1).Insert the stopper,shake for 15minutes in a water bath maintained at 60,and cool.Filter,discarding the first few mLof 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 15mg of calcium pantothenate,to a centrifuge tube.Transfer 25.0mLof Internal standard preparationto the centrifuge tube,and shake vigorously for 10minutes.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 50mg of calcium pantothenate,to a 1000-mLvolumetric flask containing 500mLof water.Add 10mLof 0.2Nacetic acid and 100mLof sodium acetate solution (1in 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 preparationin 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 10mg of calcium pantothenate,to a 250-mLvolumetric flask,add 10mLof 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 10mg of niacinamide and 2.5mg each of pyridoxine hydrochloride,riboflavin,and thiamine hydrochloride,to a 50-mLcentrifuge tube.Proceed as directed for Assay preparationin the Assay for niacin or niacinamide,pyridoxine hydrochloride,riboflavin,and thiamine,Method 1under Oil-and Water-Soluble Vitamins with Minerals Tablets,beginning with “Add 25.0mLof 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 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 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 2mg of riboflavin,to a 250-mLvolumetric flask.If riboflavin is not present in the formulation,use a portion equivalent to about 2mg of pyridoxine.If pyridoxine is not present in the formulation,use a portion equivalent to about 20mg of niacin or niacinamide.Add 100.0mLof Extraction solvent,and mix for 20minutes,using a wrist-action shaker.Immerse the flask in a water bath maintained at 70to 75,and heat for 20minutes.Mix on a vortex mixer for 30seconds,cool to room temperature,and filter.Use the clear filtrate.

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

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

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

Assay preparation— Proceed as directed for Assay preparationin 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 2mg of thiamine,to a 250-mLvolumetric flask.Add 100.0mLof 0.2Nhydrochloric acid,shake for 15minutes with a wrist-action shaker,and heat to boiling for 30minutes.Cool to room temperature,and filter.Use the clear filtrate.

Assay preparation— Proceed as directed for Assay preparationin 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 7.5mg of niacin or niacinamide,1.2mg of pyridoxine hydrochloride,0.4mg of riboflavin,and 1.2mg of thiamine hydrochloride,to a stoppered 125-mLflask.Add 10.0mLof a mixture of methanol and glacial acetic acid (9:1)and 30.0mLof a mixture of methanol and ethylene glycol (1:1).Insert the stopper,shake for 15minutes in a water bath maintained at 60,and cool.Filter,discarding the first few mLof the filtrate.