NOTE—In the following Assays, where more than one Assay method 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 1 is not used.

(Official January 1, 2007)

Assay preparation— Proceed as directed for Assay preparation in the Assay for biotin, Method 2 from the beginning through calculation of the net weight per Capsule. Transfer an accurately weighed portion of the 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— Accurately weigh not fewer than 20 Capsules 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 100 µg of biotin, to a 200-mL volumetric flask. Add 3 mL of 50 percent alcohol, and swirl to mix. Place 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 this solution quantitatively, and stepwise if necessary, with water to obtain a solution having a concentration of about 0.1 ng per mL.

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

Assay preparation— Proceed as directed for the Assay preparation under Assay for biotin, Method 2 from 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 mL 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 test solution containing vitamin B12 activity approximately equivalent to that of the Standard preparation.

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

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

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

Assay preparation— Accurately weigh not fewer than 30 Capsules 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 1.2 mg of dexpanthenol or 2.4 mg of panthenol, in 100.0 mL of water. Quantitatively dilute a portion of this solution with water to obtain a solution having a concentration of about 1.2 µg of dexpanthenol or 2.4 µg of panthenol per mL.

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

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

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

Riboflavin-thiamine hydrochloride-biotin solution— Prepare a solution of riboflavin, thiamine hydrochloride, and biotin in 0.02 N acetic acid containing 20 µg of riboflavin, 10 µg of thiamine hydrochloride, and 0.04 µg 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 25 percent alcohol containing 10 µg of p-aminobenzoic acid, 50 µg of niacin, and 40 µg of pyridoxine hydrochloride per mL. Store in a refrigerator.

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

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

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

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

Modified pantothenate medium—

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

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

Inoculum— Inoculate three 250-mL portions of Modified pantothenate medium from a stock culture slant, and incubate at 35 for 20 to 24 hours. Centrifuge the suspension from the combined portions, and wash the cells with Modified pantothenate medium. Resuspend the cells in sufficient Modified pantothenate medium so that a 1 in 50 dilution, when tested in a 13-mm diameter test tube, gives 80% light transmission at 530 nm. Transfer 1.2-mL portions 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 1 mL of thawed culture with sterile saline TS to 150 mL. [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 0.0 mL. 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 5.0 mL of 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 preparation added 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 2 from the beginning through calculation of the net weight per Capsule. Transfer an accurately weighed portion of the 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, filter, and use the clear filtrate.

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

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

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

Assay preparation— Prepare as directed for Assay preparation in 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 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 niacin, Method 2, through “calculate the net weight of the Capsule contents.” Transfer an accurately weighed portion of the Capsule contents, equivalent to about 7.5 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.