115 DEXPANTHENOL ASSAY

USP Reference Standards 11— USP Dexpanthenol RS.

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

Standard Preparation— On the day of the assay, prepare a water dilution of the Standard Stock Solution of Dexpanthenol to contain 1.2 µg of dexpanthenol per mL.

Assay Preparation— Proceed as directed in the individual monograph for preparing a solution expected to contain approximately the equivalent of the dexpanthenol concentration in the Standard Preparation.

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.

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 mL to 800 mL of water, heat to 75 ± 5, and add dilute hydrochloric acid (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 containing, in each mL, 20 µg of riboflavin, 10 µg of thiamine hydrochloride, and 0.04 µg of biotin, by dissolving riboflavin, thiamine hydrochloride, and biotin in 0.02 N acetic acid. Store, protected from light, under toluene in a refrigerator.

Para-aminobenzoic Acid–Niacin–Pyridoxine Hydrochloride Solution— Prepare a solution in neutral 25 percent alcohol to contain 10 µg of para-aminobenzoic acid, 50 µg of niacin, and 40 µg of pyridoxine hydrochloride in each mL. Store in a refrigerator.

Salt Solution A— 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, mix, and store under toluene.

Salt Solution B— 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, mix, and 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 2000 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.

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 between 6.5 and 6.6, adjust the volume with water to 1000 mL, and mix. Add approximately 10-mL portions of the solution to culture tubes, place caps on the tubes, and sterilize 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 1:50 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 mL, 4.5 mL, 4.0 mL, 3.5 mL, 3.0 mL, 2.0 mL, 1.0 mL, and 0.0 mL. To these same tubes, and in the same order, add 0.0 mL, 0.5 mL, 1.0 mL, 1.5 mL, 2.0 mL, 3.0 mL, 4.0 mL, 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 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.

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*  American Type Culture Collection No. 8042 is suitable.

Auxiliary Information— Staff Liaison : Lawrence Evans, III, Ph.D., Scientist