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
Dissolve the anhydrous dextrose and sodium acetate in the solutions previously mixed, and adjust with 1 N sodium hydroxide to a pH of 6.8. Finally, dilute with water to 250 mL, and mix.
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.
CystineTryptophane 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.
AdenineGuanineUracil 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.
RiboflavinThiamine HydrochlorideBiotin 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 AcidNiacinPyridoxine 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.
To similar test tubes add, in duplicate, volumes of the Assay Preparation corresponding to 3 or more of the levels listed above for the Standard Preparation, including the levels of 2.0, 3.0, and 4.0 mL. To each tube add 5.0 mL of the Basal Medium Stock Solution and sufficient water to make 10 mL. Place one complete set of Standard and Assay tubes together in one tube rack and the duplicate set in a second rack or section of a rack, preferably in random order.
Cover the tubes of both series suitably to prevent contamination, and heat in an autoclave at 121 for 5 minutes. Cool, add 1 drop of inoculum to each tube, except 2 of the 4 tubes containing no Standard Preparation (to serve as the uninoculated blanks), and mix. Incubate the tubes at a temperature between 30 and 37, held constant to within ± 0.5 until, following 16 to 24 hours of incubation, there has been no substantial increase in turbidity in the tubes containing the highest level of standard during a 2-hour period.
Determine the transmittance of the tubes in the following manner: Mix the contents of each tube, and transfer to an optical container if necessary. Place the container in a spectrophotometer that has been set at a specific wavelength between 540 nm and 660 nm, and read the transmittance when a steady state is reached. This steady state is observed a few seconds after agitation when the galvanometer reading remains constant for 30 seconds or more. Allow approximately the same time interval for the reading on each tube.
With the transmittance set at 1.00 for the uninoculated blank, read the transmittance of the inoculated blank. With the transmittance set at 1.00 for the inoculated blank, read the transmittance for each of the remaining tubes. If there is evidence of contamination with a foreign microorganism, disregard the result of the assay.
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.
Calculate the response, y, adding together the two transmittances for each level of the Assay Preparation. Read from the standard curve the logarithm of the volume of the Standard Preparation corresponding to each of those values of y that fall within the range of the lowest and highest points plotted for the standard. Subtract from each logarithm so obtained the logarithm of the volume, in mL, of the Assay Preparation to obtain the difference, x, for each dosage level. Average the values of x for each of three or more dosage levels to obtain x = M¢, the log-relative potency of the Assay Preparation. Determine the quantity, in mg, of USP Calcium Pantothenate RS corresponding to the calcium pantothenate in the portion of material taken for assay as antilog:
M = antilog (M¢ + log R)
in which R is the number of mg of calcium pantothenate that was assumed to be present in each mg (or capsule or tablet) of the material taken for assay.
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, 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.
* American Type Culture Collection No. 8014 is suitable. This strain formerly was known as Lactobacillus arabinosus 17-5.
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