91 CALCIUM PANTOTHENATE ASSAY

USP Reference Standards 11— USP Calcium Pantothenate RS

Standard Stock Solution of Calcium Pantothenate— Dissolve 50 mg of USP Calcium Pantothenate RS, previously dried and stored in the dark over phosphorus pentoxide and accurately weighed while protected from absorption of moisture during the weighing, in about 500 mL of water in a 1000-mL volumetric flask. Add 10 mL of 0.2 N acetic acid and 100 mL of sodium acetate solution (1 in 60), then dilute with water to volume. Each mL represents 50 µg of USP Calcium Pantothenate RS. Store under toluene in a refrigerator.

Standard Preparation— On the day of the assay, dilute a measured volume of Standard Stock Solution of Calcium Pantothenate with sufficient water so that it contains, in each mL, between 0.01 µg and 0.04 µg of calcium pantothenate, the exact concentration being such that the responses obtained as directed for Procedure, 2.0 and 4.0 mL of the Standard Preparation being used, are within the linear portion of the log-concentration response curve.

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

Basal Medium Stock Solution—

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 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 70 to 80, and add dilute hydrochloric acid (1 in 2) dropwise, with stirring, until the solids are dissolved. Cool, and add water to make 1000 mL. 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, and add water to make 200 mL. Store under toluene in a refrigerator.

Polysorbate 80 Solution— Dissolve 25 g of polysorbate 80 in alcohol to make 250 mL.

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, 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, and store under toluene.

Stock Culture of Lactobacillus plantarum— Dissolve 2.0 g of water-soluble yeast extract in 100 mL of water, add 500 mg of anhydrous dextrose, 500 mg of anhydrous sodium acetate, and 1.5 g of agar, and heat the mixture, with stirring, on a steam bath, until the agar dissolves. Add approximately 10-mL portions of the hot solution to test tubes, suitably close or cover the tubes, sterilize at 121, and allow the tubes to cool in an upright position. Prepare stab cultures in 3 or more of the tubes, using a pure culture of Lactobacillus plantarum,* incubating for 16 to 24 hours at any selected temperature between 30 and 37 but held constant to within ± 0.5, and finally store in a refrigerator. Prepare a fresh stab of the stock culture every week, and do not use for inoculum if the culture is more than 1 week old.

Culture Medium— To each of a series of test tubes containing 5.0 mL of Basal Medium Stock Solution add 5.0 mL of water containing 0.2 µg of calcium pantothenate. Plug the tubes with cotton, sterilize in an autoclave at 121, and cool.

Inoculum— Make a transfer of cells from the stock culture of Lactobacillus plantarum to a sterile tube containing 10 mL of culture medium. Incubate this culture for 16 to 24 hours at any selected temperature between 30 and 37 but held constant to within ± 0.5. The cell suspension so obtained is the inoculum.

Procedure— To similar test tubes add, in duplicate, 1.0 and/or 1.5, 2.0, 3.0, 4.0, and 5.0 mL, respectively, of the Standard Preparation. To each tube and to 4 similar tubes containing no Standard Preparation add 5.0 mL of Basal Medium Stock Solution and sufficient water to make 10 mL.

Calculation— Prepare a standard concentration-response curve as follows. For each level of the standard, calculate the response from the sum of the duplicate values of the transmittance as the difference, y = 2.00 S (of transmittance). Plot this response on the ordinate of cross-section paper against the logarithm of the mL of Standard Preparation per tube on the abscissa, using for the ordinate either an arithmetic or a logarithmic scale, whichever gives the better approximation to a straight line. Draw the straight line or smooth curve that best fits the plotted points.

Replication— Repeat the entire determination at least once, using separately prepared Assay Preparations. If the difference between the two log-potencies M is not greater than 0.08, their mean, bar(M), is the assayed log-potency of the test material (see The Confidence Interval and Limits of Potency 111). If the two determinations differ by more than 0.08, conduct one or more additional determinations. From the mean of two or more values of M that do not differ by more than 0.15, compute the mean potency of the preparation under assay.

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*  American Type Culture Collection No. 8014 is suitable. This strain formerly was known as Lactobacillus arabinosus 17-5.

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