NOTE—In the following Assays,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— Proceed as directed for Assay preparationin the Assay for biotin,Method 2from the beginning through calculation of the net weight per Capsule.Transfer an accurately weighed portion of the 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— Accurately weigh not fewer than 20Capsules in a tared weighing bottle.Using a sharp blade,carefully cut open the Capsules,without loss of shell material,and transfer the contents as completely as possible to a beaker.Remove any contents adhering to the 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.Accurately 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 100µg of biotin,to a 200-mLvolumetric flask.Add 3mLof 50percent alcohol,and swirl to mix.Place 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 this solution quantitatively,and stepwise if necessary,with water to obtain a solution having a concentration of about 0.1ng per mL.

Assay preparation— Proceed as directed in the Assay for biotin,Method 2from the beginning through calculation of the net weight per Capsule.Transfer an accurately weighed portion of the Capsule contents,equivalent to 100µg of cyanocobalamin,to a 250-mLflask.Quantitatively add 100.0mLof water,and carefully extract for 2minutes.Filter about 10mLof this solution,and use the clear filtrate.

Assay preparation— Proceed as directed for the Assay preparationunder Assay for biotin,Method 2from the beginning through calculation of the net weight per Capsule.Transfer an accurately weighed portion of the Capsule contents to an appropriate vessel containing,for each g or mLof 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 test solution containing vitamin B12activity approximately equivalent to that of the Standard preparation.

Assay preparation— Proceed as directed in the Assay preparationin the Assay for biotin,Method 2from the beginning through calculation of the net weight per Capsule.Transfer an accurately weighed portion of the Capsule contents,equivalent to about 0.4mg of folic acid,to a 50-mLamber-colored centrifuge tube.Add 25.0mLof Internal standard preparation,insert the 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 in the Assay for biotin,Method 2from the beginning through calculation of the average net weight per Capsule.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.

Standard stock solution— Dissolve an accurately weighed quantity of USP Dexpanthenol RSin water,and dilute with water to obtain a solution having a known concentration of about 800µg per mL.Store in a refrigerator,protected from light,and use within 30days.

Standard preparation— On the day of the assay,dilute an accurately measured volume of Standard stock solutionwith water to obtain a solution having a known concentration of about 1.2µg of dexpanthenol per mL.

Assay preparation— Accurately weigh not fewer than 30Capsules in a tared weighing bottle.Using a sharp blade,carefully cut open the Capsules,without loss of shell material,and transfer the contents as completely as possible to a beaker.Remove any contents adhering to the empty Capsule 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 average net weight per Capsule.Dissolve an accurately weighed portion of the Capsule contents,equivalent to about 1.2mg of dexpanthenol or 2.4mg of panthenol,in 100.0mLof water.Quantitatively dilute a portion of this solution with water to obtain a solution having a concentration of about 1.2µg of dexpanthenol or 2.4µg of panthenol per mL.

Acid-hydrolyzed casein solution— Mix 100g of vitamin-free casein with 500mLof 6Nhydrochloric acid,and reflux the mixture for 8to 12hours.Remove the hydrochloric acid from the mixture by distillation under reduced pressure until a thick paste remains.Redissolve the resulting paste in about 500mLof water,adjust the solution with 1Nsodium hydroxide to a pHof 3.5±0.1,and add water to make 1000mL.Add 20g of activated charcoal,stir for 1hour,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-tryptophane solution— Suspend 4.0g of L-cystine and 1.0g of L-tryptophane (or 2.0g of D,L-tryptophane)in 700to 800mLof water,heat to 75±5,and add hydrochloric acid solution (1in 2)dropwise,with stirring,until the solids are dissolved.Cool,add water to make 1000mL,and mix.Store under toluene in a refrigerator at a temperature not below 10.

Adenine-guanine-uracil solution— Dissolve 200mg each of adenine sulfate,guanine hydrochloride,and uracil,with the aid of heat,in 10mLof 4Nhydrochloric acid,cool,add water to make 200mL,and mix.Store under toluene in a refrigerator.

Polysorbate 80solution— Dissolve 25g of polysorbate 80in alcohol to make 250mL,and mix.

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

p-Aminobenzoic acid-niacin-pyridoxine hydrochloride solution— Prepare a solution in neutral 25percent alcohol containing 10µg of p-aminobenzoic acid,50µg of niacin,and 40µg of pyridoxine hydrochloride per mL.Store in a refrigerator.

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

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

Pyridoxal-calcium pantothenate solution— Dissolve 40mg of pyridoxal hydrochloride and 375µg of calcium pantothenate in 10percent alcohol to make 200mL,and mix.Store in a refrigerator,and use within 30days.

Polysorbate 40-oleic acid solution— Dissolve 25g of polysorbate 40and 0.25g of oleic acid in 20percent alcohol to make 500mL,and mix.Store in a refrigerator,and use within 30days.

Modified pantothenate medium—

Double-strength modified pantothenate medium— Prepare as directed under Modified pantothenate medium,but make the final dilution to 125mLinstead of 250mL.Prepare fresh.

Stock culture of Pediococcus acidilactici— Dissolve in about 800mLof water,with the aid of heat,6.0g of peptone,4.0g of pancreatic digest of casein,3.0g of yeast extract,1.5g of beef extract,1.0g of dextrose,and 15.0g of agar.Adjust with 0.1Nsodium hydroxide or 0.1Nhydrochloric acid to a pHof between 6.5and 6.6,dilute with water to 1000mL,and mix.Add 10-mLportions of the solution to culture tubes,place caps on the tubes,and sterilize in an autoclave at 121for 15minutes.Cool on a slant,and store in a refrigerator.Prepare a stock culture of Pediococcus acidilactici*on a slant of this medium.Incubate at 35for 20to 24hours,and store in a refrigerator.Maintain the stock culture by monthly transfer onto fresh slants.

Inoculum— Inoculate three 250-mLportions of Modified pantothenate mediumfrom a stock culture slant,and incubate at 35for 20to 24hours.Centrifuge the suspension from the combined portions,and wash the cells with Modified pantothenate medium.Resuspend the cells in sufficient Modified pantothenate mediumso that a 1in 50dilution,when tested in a 13-mm diameter test tube,gives 80%light transmission at 530nm.Transfer 1.2-mLportions of this stock suspension to glass ampuls,seal,freeze in liquid nitrogen,and store in a freezer.On the day of the assay,allow the ampuls to reach room temperature,mix the contents,and dilute 1mLof thawed culture with sterile saline TSto 150mL.[NOTE—This dilution may be altered when necessary to obtain the desired test response.]

Procedure— Prepare in triplicate a series of eight culture tubes by adding the following quantities of water to the tubes within a set:5.0,4.5,4.0,3.5,3.0,2.0,1.0,and 0.0mL.To these same tubes,and in the same order,add 0.0,0.5,1.0,1.5,2.0,3.0,4.0,and 5.0mLof the Standard preparation.

Calculation— Draw a dose-response curve on arithmetic graph paper by plotting the average response,in percent transmittance,for each set of tubes of the standard curve against the standard level concentrations.The curve is drawn by connecting each adjacent pair of points with a straight line.From this standard curve,determine by interpolation the potency,in terms of dexpanthenol,of each tube containing portions of the Assay preparation.Divide the potency of each tube by the amount of the Assay preparationadded to it,to obtain the individual responses.Calculate the mean response by averaging the individual responses that vary from their mean by not more than 15%,using not less than half the total number of tubes.Calculate the potency of the portion of the material taken for assay,in terms of dexpanthenol,by multiplying the mean response by the appropriate dilution factor.

Assay preparation— Proceed as directed in the Assay for biotin,Method 2from the beginning through calculation of the net weight per Capsule.Transfer an accurately weighed portion of the 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,filter,and use the clear filtrate.

Assay preparation— Proceed as directed in the Assay for biotin,Method 2from the beginning through calculation of the net weight per Capsule.Transfer a portion of the 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 2from the beginning through calculation of the average net weight per Capsule.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 Capsules contents.Dilute with water to volume,mix,and filter.

Assay preparation— Proceed as directed in the Assay for biotin,Method 2from the beginning through calculation of the average net weight of the Capsule.Transfer an accurately weighed portion of the Capsule contents,equivalent to 10mg of niacinamide and 2.5mg each of pyridoxine hydrochloride,riboflavin,and thiamine hydrochloride,to a 50-mLcentrifuge tube,and add 25.0mLof Diluting solution.Mix,using a vortex mixer,for 30seconds to completely suspend the specimen.Immerse the centrifuge tube in a hot water bath maintained at 65to 70,heat for 5minutes,and mix,using a vortex mixer,for 30seconds.Return the tube to the hot water bath,heat for another 5minutes,and mix,using a vortex mixer,for 30seconds.Filter a portion of the solution,cool to room temperature,and use the clear filtrate.[NOTE—Use the filtrate within 3hours after filtration.]

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

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

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