Mode: LC

Detector: UV 265 nm

Column: 4.6-mm × 50-cm (prepared from two concatenated 4.6-mm × 25-cm columns); packing L1

Column temperature: 40

Flow rate: 1.5 mL/min

Injection size: 20 µL

System suitability

Sample: Standard solution

Suitability requirements

Relative standard deviation: NMT 5.0%

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of the labeled amount of vitamin A as retinol (C20H30O) in the portion of Oral Solution taken:

Result = (rU/rS) × (CS/CU) × F × 100

rU	=	= peak area of retinol or retinyl ester from the Sample solution
rS	=	= peak area of retinyl acetate from the Standard solution
CS	=	= concentration of retinyl acetate (C22H32O2) in the Standard solution (µg/mL)
CU	=	= nominal concentration of vitamin A, as retinol (C20H30O), in the Sample solution (µg/mL)
F	=	= factor used to convert retinyl acetate, the ester form present in USP Vitamin A RS, to retinol, 0.872

Acceptance criteria: 90.0%–250.0% of the labeled amount of vitamin A as retinol (C20H30O)

• Cholecalciferol or Ergocalciferol (Vitamin D)

[Note—Where vitamin D (cholecalciferol or ergocalciferol) is specified in the following procedure, use the chemical form present in the formulation and the relevant USP Reference Standard. Use low-actinic glassware throughout this procedure. ]

Diluent and Mobile phase: Prepare as directed for Vitamin A.

Standard solution: 5 µg/mL of USP Cholecalciferol RS or USP Ergocalciferol RS in Diluent

Sample solution: Transfer an accurately measured volume of Oral Solution, equivalent to 50 µg of cholecalciferol or ergocalciferol, to a 500-mL separatory funnel containing 10 mL of water and 20 mL of dehydrated alcohol. Add 150 mL of solvent hexane, insert the stopper, and shake for 1 min. Add another 150 mL of solvent hexane, insert the stopper, shake, and allow the layers to separate. Discard the aqueous layer. Drain the solvent hexane extract through anhydrous sodium sulfate into a 500-mL, round-bottom flask. Evaporate the solution to dryness with the aid of a rotary evaporator over a water bath maintained at about 65

. Immediately add 10.0 mL of Diluent, swirl to dissolve the residue, and filter.

Chromatographic system: Prepare as directed in Vitamin A.

System suitability

Sample: Standard solution

Suitability requirements

Relative standard deviation: NMT 5.0%

Analysis

Samples: Standard solution and Sample solution

Measure the peak areas for vitamin D. Calculate the percentage of the labeled amount of cholecalciferol (C27H44O) or ergocalciferol (C28H44O) in the portion of Oral Solution taken:

Result = (rU/rS) × (CS/CU) × F × 100

rU	=	= peak areas of cholecalciferol or ergocalciferol from the Sample solution
rS	=	= peak areas of cholecalciferol or ergocalciferol from the Standard solution
CS	=	= concentration of USP Cholecalciferol RS or USP Ergocalciferol RS in the Standard solution (µg/mL)
CU	=	= nominal concentration of cholecalciferol or ergocalciferol in the Sample solution (µg/mL)
F	=	= correction factor to account for the average amount of previtamin D present in the formulation, 1.09

Acceptance criteria: 90.0%–250.0% of the labeled amount of vitamin D as cholecalciferol (C27H44O) or ergocalciferol (C28H44O)

• Vitamin E

[Note—Use low-actinic glassware throughout this procedure. ]

Diluent: Acetonitrile and ethyl acetate (1:1)

Potassium hydroxide solution: Transfer 90 g of potassium hydroxide pellets to a 100-mL volumetric flask containing 60 mL of water. Mix to dissolve, cool, and dilute with water to volume.

Mobile phase: Methanol, acetonitrile, and n-hexane (46.5:46.5:7.0)

Standard solution: 0.3 mg/mL of USP Alpha Tocopherol RS in Diluent

Sample solution: Transfer an amount of Oral Solution equivalent to 1.5 mg of alpha tocopherol to a 125-mL conical flask fitted with a ground-glass joint, and add 25.0 mL of dehydrated alcohol. Attach a reflux condenser, and reflux in a boiling water bath for 1 min. Cautiously add 3 mL of Potassium hydroxide solution through the condenser, and continue to reflux for 30 min. Remove the flask from the bath, and rinse the condenser with about 15 mL of water. Cool, and transfer with a minimum volume of water to a 250-mL separatory funnel. Rinse the flask with 50 mL of n-hexane, and add the rinsings to the separatory funnel. Insert the stopper, shake vigorously for 1 min, and allow the layers to separate. Drain the aqueous layer into a second 250-mL separatory funnel, and repeat the extraction with 50 mL of n-hexane. Discard the aqueous layer, and combine the hexane extracts. Wash the combined extracts with 25 mL of water, allow the layers to separate, and discard the aqueous layer. Add 3 drops of glacial acetic acid, and repeat the washing procedure two more times. Filter the washed hexane layer through anhydrous sodium sulfate into a 250-mL round-bottom flask. Rinse the funnel and sodium sulfate with n-hexane, and add the rinsing to the hexane solution in the flask. Evaporate the hexane solution to dryness with the aid of a rotary evaporator over a water bath maintained at about 50

. Immediately add 5.0 mL of Diluent, and swirl to dissolve the residue.

Chromatographic system

Mode: LC

Detector: UV 291 nm

Column: 4.6-mm × 25-cm; packing L1

Column temperature: 40

Flow rate: 3.0 mL/min

Injection size: 20 µL

System suitability

Sample: Standard solution

Suitability requirements

Relative standard deviation: NMT 5.0%

Analysis

Samples: Standard solution and Sample solution

Measure the peak areas. Calculate the percentage of the labeled amount of vitamin E as alpha tocopherol (C29H50O2) in the portion of Oral Solution taken:

Result = (rU/rS) × (CS/CU) × 100

rU	=	= peak area of alpha tocopherol from the Sample solution
rS	=	= peak area of alpha tocopherol from the Standard solution
CS	=	= concentration of alpha tocopherol in the Standard solution (mg/mL)
CU	=	= nominal concentration of vitamin E, as alpha tocopherol, in the Sample solution (mg/mL)

Acceptance criteria: 90.0%–250.0% of the labeled amount of vitamin E.

• Ascorbic Acid, Method 1: Proceed as directed for Automated Methods of Analysis

, Assay for Ascorbic Acid.

Acceptance criteria: 90.0%–250.0% of the labeled amount of ascorbic acid (C6H8O6)

• Ascorbic Acid, Method 2

Sample solution: Transfer an accurately measured volume of Oral Solution, equivalent to 80 mg of ascorbic acid, to a conical flask. Add 50 mL of water, 100 mL of 0.1 N sulfuric acid VS, and 15.0 mL of 0.1 N iodine VS. Stir the contents for 30 s, and add 5 mL of starch TS. Immediately titrate with 0.1 N sodium thiosulfate VS to the disappearance of the color. Each mL of 0.1 N iodine is equivalent to 8.806 mg of ascorbic acid (C6H8O6).

Acceptance criteria: 90.0%–250.0% of the labeled amount of ascorbic acid (C6H8O6)

• Calcium Ascorbate, Method 1: Proceed as directed in Automated Methods of Analysis

, Assay for Ascorbic Acid.

Acceptance criteria: 90.0%–250.0% of the labeled amount of calcium ascorbate (C12H14CaO12·2H2O)

• Calcium Ascorbate, Method 2: Proceed as directed for Ascorbic Acid, Method 2. Each mL of 0.1 N iodine is equivalent to 10.66 mg of C12H14CaO12·2H2O.

Acceptance criteria: 90.0%–250.0% of the labeled amount of calcium ascorbate (C12H14CaO12·2H2O)

• Sodium Ascorbate, Method 1: Proceed as directed for Automated Methods of Analysis

, Assay for Ascorbic Acid.

Acceptance criteria: 90.0%–250.0% of the labeled amount of sodium ascorbate (C6H7NaO6)

• Sodium Ascorbate, Method 2: Proceed as directed for Ascorbic Acid, Method 2. Each mL of 0.1 N iodine is equivalent to 9.905 mg of C6H7NaO6.

Acceptance criteria: 90.0%–250.0% of the labeled amount of sodium ascorbate (C6H7NaO6)

• Cyanocobalamin

[Note—Use low-actinic glassware throughout this procedure. ]

Standard stock solution: 1.0 µg/mL of cyanocobalamin from USP Cyanocobalamin RS in 25% alcohol. Store in a refrigerator.

Standard solution: Dilute a suitable volume of Standard stock solution with water to a measured volume such that after the incubation period as described for Analysis, the difference in transmittance between the inoculated blank and the 5.0-mL level of the Standard solution is NLT that which corresponds to a difference of 1.25 mg in dried cell weight. This concentration usually falls between 0.01 and 0.04 ng/mL of Standard solution. Prepare this solution fresh for each assay.

Sample solution: Transfer an accurately measured volume of Oral Solution, assumed to contain 1.0 µg of cyanocobalamin, to an appropriate vessel containing, for each mL of the Oral Solution taken, 25 mL of an aqueous extracting solution prepared just before 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 min. 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 solution containing vitamin B12 activity approximately equivalent to that of the Standard 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 8–12 h. 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 h, and filter. Repeat the treatment with activated charcoal. Store under toluene in a cool place at a temperature not below 10

. Filter the solution if a precipitate forms during storage.

Asparagine solution: Dissolve 2.0 g of l-asparagine in water to make 200 mL. Store under toluene in a refrigerator.

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.

Xanthine solution: Suspend 0.20 g of xanthine in 30–40 mL of water, heat to 70

, add 6.0 mL of 6 N ammonium hydroxide, and stir until the solid is dissolved. Cool, and add water to make 200 mL. Store under toluene in a refrigerator.

Salt solution A: Dissolve 10 g of monobasic potassium phosphate and 10 g of dibasic potassium phosphate in water to make 200 mL, and add 2 drops of hydrochloric acid. Store this solution under toluene.

Salt solution B: Dissolve 4.0 g of magnesium sulfate, 0.20 g of sodium chloride, 0.20 g of ferrous sulfate, and 0.20 g of manganese sulfate in water to make 200 mL, and add 2 drops of hydrochloric acid. Store this solution under toluene.

Polysorbate 80 solution: 100 mg/mL of polysorbate 80 in alcohol. Store in a refrigerator.

Vitamin solution A: 10 mg of riboflavin, 10 mg of thiamine hydrochloride, 100 µg of biotin, and 20 mg of niacin in 0.02 N acetic acid to make 400 mL. Store under toluene, protected from light, in a refrigerator.

Vitamin solution B: 20 mg of p-aminobenzoic acid, 10 mg of calcium pantothenate, 40 mg of pyridoxine hydrochloride, 40 mg of pyridoxal hydrochloride, 8 mg of pyridoxamine dihydrochloride, and 2 mg of folic acid in a mixture of water and neutralized alcohol (3:1) to make 400 mL. Store, protected from light, in a refrigerator.

Basal medium stock solution: Prepare the medium according to the following formula and directions. A dehydrated mixture containing the same ingredients may be used provided that, when constituted as directed in the labeling, it yields a medium comparable to that obtained from the formula given herein.

Add the ingredients in the order listed in Table 1, carefully dissolving cystine and tryptophan in the hydrochloric acid before adding the next eight solutions in the resulting solution. Add 100 mL of water, and dissolve dextrose, sodium acetate, and ascorbic acid. Filter, if necessary, add the Polysorbate 80 solution, adjust with 1 N sodium hydroxide to a pH of 5.5–6.0, and add Purified Water to make 250 mL.

Table 1

l-Cystine	0.1 g
l-Tryptophan	0.05 g
1 N Hydrochloric acid	10 mL
Adenine–guanine–uracil solution	5 mL
Xanthine solution	5 mL
Vitamin solution A	10 mL
Vitamin solution B	10 mL
Salt solution A	5 mL
Salt solution B	5 mL
Asparagine solution	5 mL
Acid-hydrolyzed casein solution	25 mL
Dextrose, anhydrous	10 g
Sodium acetate, anhydrous	5 g
Ascorbic acid	1 g
Polysorbate 80 solution	5 mL

Tomato juice preparation: Centrifuge commercially canned tomato juice so that most of the pulp is removed. Suspend 5 g/L of analytical filter-aid in the supernatant, and filter, with the aid of reduced pressure, through a layer of the filter-aid. Repeat, if necessary, until a clear, straw-colored filtrate is obtained. Store under toluene in a refrigerator.

Culture medium

[Note—A dehydrated mixture containing the same ingredients may be used provided that, when constituted as directed in the labeling, it yields a medium equivalent to that obtained from the formula given herein. ]

Dissolve 0.75 g of yeast extract, 0.75 g of dried peptone, 1.0 g of anhydrous dextrose, and 0.20 g of monobasic potassium phosphate in 60–70 mL of water. Add 10 mL of Tomato juice preparation and 1 mL of Polysorbate 80 solution. Adjust with 1 N sodium hydroxide to a pH of 6.8, and add water to make 100 mL. Place 10-mL portions of the solution in test tubes, and plug with cotton. Sterilize the tubes and contents in an autoclave at 121

for 15 min. Cool as rapidly as possible to avoid color formation resulting from overheating the medium.

Suspension medium: Dilute a measured volume of Basal medium stock solution with an equal volume of water. Place 10-mL portions of the diluted medium in test tubes. Sterilize, and cool as directed for Culture medium.

Stock culture of Lactobacillus leichmannii: To 100 mL of Culture medium add 1.0–1.5 g of agar, and heat the mixture on a steam bath, with stirring, until the agar dissolves. Place 10-mL portions of the hot solution in test tubes, cover the tubes, sterilize at 121

for 15 min in an autoclave, and allow the tubes to cool in an upright position. Inoculate three or more of the tubes by stab transfer of a pure culture of Lactobacillus leichmannii. [Note—Before first using a fresh culture in this assay, make NLT 10 successive transfers of the culture in a 2-week period. ]

Incubate for 16–24 h at a temperature between 30

and 40

held constant to within ±0.5

. Store in a refrigerator.

Prepare fresh stab cultures at least three times each week, and do not use them for preparing the Inoculum if more than 4 days old. The activity of the microorganism can be increased by daily or twice-daily transfer of the stab culture to the point where definite turbidity in the liquid Inoculum can be observed 2–4 h after inoculation. A slow-growing culture seldom gives a suitable response curve and may lead to erratic results.

Inoculum: [Note—A frozen suspension of Lactobacillus leichmannii may be used as the stock culture, provided it yields an Inoculum comparable to a fresh culture. ]

Transfer cells from the Stock culture of Lactobacillus leichmannii to two sterile tubes containing 10 mL of the Culture medium each. Incubate these cultures for 16–24 h at a temperature between 30

and 40

held constant to within ± 0.5

. Under aseptic conditions centrifuge the cultures, and decant the supernatant. Suspend the cells from the culture in 5 mL of sterile Suspension medium, and combine. Using sterile Suspension medium, adjust the volume so that a 1-in-20 dilution in saline TS produces 70% transmittance when read on a suitable spectrophotometer that has been set at a wavelength of 530 nm, equipped with a 10-mm cell, and read against saline TS set at 100% transmittance. Prepare a 1-in-400 dilution of the adjusted suspension using sterile Basal medium stock solution. The cell suspension so obtained is the Inoculum. [Note—This dilution may be altered, when necessary, to obtain the desired test response. ]

Calibration of spectrophotometer: Check the wavelength of the spectrophotometer periodically, using a standard wavelength cell or other suitable device. Before reading any tests, calibrate the spectrophotometer for 0% and 100% transmittance, using water and with the wavelength set at 530 nm.

Analysis: Because of the high sensitivity of the test organism to minimum amounts of vitamin B12 activity and to traces of many cleansing agents, cleanse meticulously by suitable means, followed preferably by heating at 250

for 2 h, using hard-glass 20-mm × 150-mm test tubes, and other necessary glassware.

To separate test tubes add, in duplicate, 1.0, 1.5, 2.0, 3.0, 4.0, and 5.0 mL of the Standard solution. To each of these tubes and to four similar empty tubes, add 5.0 mL of Basal medium stock solution and sufficient water to make 10 mL.

To similar separate test tubes add, in duplicate, 1.0, 1.5, 2.0, 3.0, and 4.0 mL of the Sample solution. To each tube add 5.0 mL of Basal medium stock solution and sufficient water to make 10 mL. Place one complete set of standard and sample 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 to prevent bacterial contamination, and sterilize in an autoclave at 121

for 5 min, arranging to reach this temperature in NMT 10 min by preheating the autoclave if necessary. Cool as rapidly as possible to avoid color formation resulting from overheating the medium. Take precautions to maintain uniformity of sterilizing and cooling conditions throughout the assay, because packing the tubes too closely in the autoclave or overloading it may cause variation in the heating rate.

Aseptically add 0.5 mL of Inoculum to each tube so prepared, except two of the four containing no Standard solution (the uninoculated blanks). Incubate the tubes at a temperature between 30

and 40

, held constant to within ±0.5

, for 16–24 h.

Terminate growth by heating to a temperature NLT 80

for 5 min. Cool to room temperature. After agitating the contents, read the transmittance at 530 nm when a steady state is reached. This steady state is observed a few seconds after agitation when the reading remains constant for 30 s or more. Allow approximately the same time interval for the reading on each tube.

With the transmittance set at 100% for the uninoculated blank, read the transmittance of the inoculated blank. If the difference is greater than 5% or if there is evidence of contamination with a foreign microorganism, disregard the results of the assay.

With the transmittance set at 100% for the uninoculated blank, read the transmittance of each of the remaining tubes. Disregard the results of the assay if the slope of the standard curve indicates a problem with sensitivity.

Calculation: Prepare a standard concentration-response curve by the following procedure. Test for and replace any aberrant individual transmittances. For each level of the Standard, calculate the response from the sum of the duplicate values of the transmittances (SS) as the difference, y = 2.00

SS. Plot this response on the ordinate of cross-section paper against the logarithm of the mL of Standard solution 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 = 2.00

SU, adding together the two transmittances (SU) for each level of the Sample solution. Read from the standard curve the logarithm of the volume of the Standard solution corresponding to each of those values of y that falls 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 Sample solution 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, which equals the log-relative potency, M¢, of the Sample solution. Determine the quantity, in µg, of cyanocobalamin (C63H88CoN14O14P), in the portion of Oral Solution taken:

antilog M = antilog (M¢ + log R)

= number of µg of cyanocobalamin assumed to be present in the portion of Oral Solution taken

Calculate the percentage of the labeled amount of cyanocobalamin in the portion of the Oral Solution taken:

Result = [(antilog M)/N] x 100

= nominal amount of cyanocobalamin in the portion of Oral Solution taken

Replication: Repeat the entire determination at least once, using separately prepared Sample solutions. If the difference between the two log-potencies M is NMT 0.08, their mean, M, is the assayed log-potency of the test material (see Design and Analysis of Biological Assays

111

, Vitamin B12 Activity). 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.

Acceptance criteria: 90.0%–450.0% of the labeled amount of cyanocobalamin (C63H88CoN14O14P)

• Calcium Pantothenate, Method 1

Mobile phase: 0.2 M monobasic sodium phosphate and methanol (97:3). Adjust with 1.7 M phosphoric acid to a pH of 3.2 ± 0.1.

Standard solution: 80 µg/mL of USP Calcium Pantothenate RS in Mobile phase

System suitability solution: 80 µg/mL of USP Racemic Panthenol RS in Mobile phase. Mix the resulting solution and Standard solution (1:1).

Sample solution: Equivalent to 80 µg/mL of calcium pantothenate from Oral Solution, in Mobile phase

Chromatographic system

Mode: LC

Detector: UV 210 nm

Column: 4.0-mm × 10-cm; packing L1

Flow rate: 1 mL/min

Injection size: 20 µL

System suitability

Samples: Standard solution and System suitability solution

Suitability requirements

Resolution: NLT 1.5 between panthenol and calcium pantothenate, System suitability solution

Tailing factor: NMT 2.0 for both the calcium pantothenate and the panthenol peaks, Standard solution

Relative standard deviation: NMT 2.0%

Analysis

Samples: Standard solution and Sample solution

Measure the peak areas for calcium pantothenate. Calculate the percentage of the labeled amount of calcium pantothenate (C18H32CaN2O10) in the portion of the Oral Solution taken:

Result = (rU/rS) × (CS/CU) × 100

rU	=	= peak area of calcium pantothenate from the Sample solution
rS	=	= peak area of calcium pantothenate from the Standard solution
CS	=	= concentration of USP Calcium Pantothenate RS in the Standard solution (mg/mL)
CU	=	= nominal concentration of calcium pantothenate in the Sample solution (mg/mL)

Acceptance criteria: 90.0%–150.0% of the labeled amount of calcium pantothenate (C18H32CaN2O10)

• Calcium Pantothenate, Method 2

Standard stock solution: Dissolve 50 mg of USP Calcium Pantothenate RS, previously dried and stored in the dark over phosphorus pentoxide and protected from absorption of moisture while weighing, in 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), and dilute with water to volume to obtain a concentration of 50 µg/mL of USP Calcium Pantothenate RS. Store under toluene in a refrigerator.

Standard solution: On the day of the assay, dilute a volume of Standard stock solution with water to obtain a concentration of 0.01–0.04 µg/mL of calcium pantothenate, the exact concentration being such that the responses obtained as directed for Analysis, 2.0 and 4.0 mL of the Standard solution being used, are within the linear portion of the log-concentration response curve.

Sample solution: Transfer an accurately measured volume of Oral Solution, equivalent to 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 a measured volume of this solution quantitatively, and stepwise if necessary, with water to obtain a solution having about the same concentration as that of the Standard solution.

. Filter the solution if a precipitate forms during storage.

Cystine–tryptophan solution: Suspend 4.0 g of l-cystine in a solution of 1.0 g of l-tryptophan (or 2.0 g of d,l-tryptophan) in 700–800 mL of water, heat to 70

–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 cool place at a temperature not below 10

Polysorbate 80 solution: 100 mg/mL of polysorbate 80 in alcohol

Riboflavin–thiamine hydrochloride–biotin solution: 20 µg/mL of riboflavin, 10 µg/mL of thiamine hydrochloride, and 0.04 µg/mL of biotin in 0.02 N acetic acid. Store under toluene, protected from light, in a refrigerator.

p-Aminobenzoic acid–niacin–pyridoxine hydrochloride solution: 10 µg/mL of p-aminobenzoic acid, 50 µg/mL of niacin, and 40 µg/mL of pyridoxine hydrochloride in a mixture of neutralized alcohol and water (1:3). 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. 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. Store under toluene.

Basal medium stock solution: Dissolve the anhydrous dextrose and anhydrous sodium acetate in the solutions previously mixed according to Table 2, and adjust with 1 N sodium hydroxide to a pH of 6.8. Dilute with water to 250 mL.

Table 2

Acid-hydrolyzed casein solution	25 mL
Cystine–tryptophan solution	25 mL
Polysorbate 80 solution	0.25 mL
Dextrose, anhydrous	10 g
Sodium acetate, anhydrous	5 g
Adenine–guanine–uracil solution	5 mL
Riboflavin–thiamine hydrochloride–biotin solution	5 mL
p-Aminobenzoic acid–niacin–pyridoxine hydrochloride solution	5 mL
Salt solution A	5 mL
Salt solution B	5 mL

Stock culture of Lactobacillus plantarum: Dissolve 2.0 g of 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 on a steam bath, with stirring, until the agar dissolves. Add 10-mL portions of the hot solution to the test tubes, close or cover the tubes, sterilize in an autoclave at 121

for 15 min, and allow the tubes to cool in an upright position. Prepare stab cultures in three or more of the tubes, using a pure culture of Lactobacillus plantarum, incubating for 16–24 h at a temperature between 30

and 37

held constant to within ±0.5

. 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

for 15 min, and cool.

Inoculum: [Note—A frozen suspension of Lactobacillus plantarum may be used as the stock culture, provided it yields an Inoculum comparable to a fresh culture. ]

Transfer cells from the Stock culture of Lactobacillus plantarum to a sterile tube containing 10 mL of Culture medium. Incubate this culture for 16–24 h at a temperature between 30

and 37

held constant to within ±0.5

. The cell suspension so obtained is the Inoculum.

Analysis: To similar separate test tubes add, in duplicate, 1.0 and/or 1.5, 2.0, 3.0, 4.0, and 5.0 mL of the Standard solution. To each tube and to four similar empty tubes, add 5.0 mL of Basal medium stock solution and sufficient water to make 10 mL.

To similar separate test tubes add, in duplicate, volumes of the Sample solution corresponding to three or more of the levels specified for the Standard solution, 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 sample 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 to prevent contamination, and sterilize in an autoclave at 121

for 5 min. Cool, add 1 drop of Inoculum to each tube, except two of the four tubes containing no Standard solution (the uninoculated blanks). Incubate the tubes at a temperature between 30

and 37

, held constant to within ±0.5

until, following 16–24 h of incubation, there has been no substantial increase in turbidity in the tubes containing the highest level of Standard during a 2-h 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. Read the transmittance between 540 and 660 nm when a steady state is reached. This steady state is observed a few seconds after agitation when the galvanometer reading remains constant for 30 s 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 (SS) as the difference, y = 2.00

Calculate the response, y = 2.00

SU, adding together the two transmittances (SU) for each level of the Sample solution. Read from the standard curve the logarithm of the volume of the Standard solution 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 Sample solution 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, which equals the log-relative potency, M¢, of the Sample solution. Determine the quantity, in mg, of USP Calcium Pantothenate RS corresponding to the calcium pantothenate (C18H32CaN2O10) in the portion of Oral Solution taken:

antilog M = antilog (M¢ + log R)

= number of mg of calcium pantothenate assumed to be present in the portion of Oral Solution taken

Calculate the percentage of the labeled amount of calcium pantothenate (C18H32CaN2O10) in the portion of Oral Solution taken:

Result = [(antilog M)/N] × 100

= nominal amount of calcium pantothenate in the portion of Oral Solution (mg)

111

, Confidence Interval and Limits of Potency). 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.

Acceptance criteria: 90.0%–150.0% of the labeled amount of calcium pantothenate (C18H32CaN2O10)

• Dexpanthenol or Panthenol, Method 1

Mobile phase and Chromatographic system: Proceed as directed in Calcium Pantothenate, Method 1.

Standard solution: 80 µg/mL of USP Dexpanthenol RS or USP Racemic Panthenol RS in Mobile phase. [Note—Use USP Dexpanthenol RS to analyze Oral Solution that contains dexpanthenol and USP Racemic Panthenol RS to analyze Oral Solution that contains panthenol. ]

System suitability solution: 80 µg/mL of USP Calcium Pantothenate RS in Mobile phase. Mix the resulting solution and Standard solution (1:1).

Sample solution: Equivalent to 80 µg/mL of dexpanthenol or panthenol from the Oral Solution in Mobile phase

Analysis

Samples: Standard solution and Sample solution

Measure the areas for panthenol. Calculate the percentage of the labeled amount of dexpanthenol or panthenol (C9H19NO4) in the portion of the Oral Solution taken:

Result = (rU/rS) × (CS/CU) × 100

rU	=	= peak area of dexpanthenol or panthenol from the Sample solution
rS	=	= peak area of dexpanthenol or panthenol from the Standard solution
CS	=	= concentration of USP Dexpanthenol RS or USP Racemic Panthenol RS in the Standard solution (mg/mL)
CU	=	= nominal concentration of dexpanthenol or panthenol in the Sample solution (mg/mL)

Acceptance criteria: 90.0%–150.0% of the labeled amount of dexpanthenol or panthenol (C9H19NO4)

• Dexpanthenol or Panthenol, Method 2

[Note—The following procedure is applicable also to the determination of the dextrorotatory component of racemic panthenol in preparations containing panthenol. ]

Dehydrated mixtures yielding formulations similar to the media described herein may be used provided that, when constituted as directed, they have growth-promoting properties equal to or superior to those obtained with the media prepared as described herein.

Standard stock solution: 800 µg/mL of USP Dexpanthenol RS or 1600 µg/mL of USP Racemic Panthenol RS in water. Store in a refrigerator, protected from light, and use within 30 days. [Note—Use USP Dexpanthenol RS to analyze Oral Solution that contains dexpanthenol and USP Racemic Panthenol RS to analyze Oral Solution that contains panthenol. ]

Standard solution: On the day of the assay, prepare 1.2 µg/mL of dexpanthenol or 2.4 µg/mL of racemic panthenol from the Standard stock solution with water.

Sample solution: 1.2 µg/mL of dexpanthenol or 2.4 µg/mL of panthenol from the Oral Solution in water

Acid-hydrolyzed casein solution: Mix 100 g of vitamin-free casein with 500 mL of 6 N hydrochloric acid, and reflux the mixture for 8–12 h. 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 h, and filter. Repeat the treatment with activated charcoal. Store under toluene in a cool place at a temperature not below 10

. Filter the solution if a precipitate forms during storage.

Cystine–tryptophan solution: Suspend 4.0 g of l-cystine in a solution of 1.0 g of l-tryptophan (or 2.0 g of d,l-tryptophan) in 700 to 800 mL of water, heat to 75 ± 5

, and add 6 M hydrochloric acid solution dropwise, with stirring, until the solids are dissolved. Cool, add water to make 1000 mL, and mix. Store under toluene in a cool place 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: 100 mg/mL of polysorbate 80 in alcohol

Riboflavin–thiamine hydrochloride–biotin solution: Prepare a solution of riboflavin, thiamine hydrochloride, and biotin in 0.02 N acetic acid containing 20 µg/mL of riboflavin, 10 µg/mL of thiamine hydrochloride, and 0.04 µg/mL of biotin. Store under toluene, protected from light, in a refrigerator.

p-Aminobenzoic acid–niacin–pyridoxine hydrochloride solution: Prepare a solution in neutral 25% alcohol containing 10 µg/mL of p-aminobenzoic acid, 50 µg/mL of niacin, and 40 µg/mL of pyridoxine hydrochloride. 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 mix. Store under toluene.

Pyridoxal–calcium pantothenate solution: Dissolve 40 mg of pyridoxal hydrochloride and 375 µg of calcium pantothenate in 10% 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% alcohol to make 500 mL, and mix. Store in a refrigerator, and use within 30 days.

Modified pantothenate medium: Dissolve anhydrous dextrose and sodium acetate in the solutions previously mixed according to Table 3, adjust with 1 N sodium hydroxide to a pH of 6.8, dilute with water to 250 mL, and mix.

Table 3

Acid-hydrolyzed casein solution	25 mL
Cystine–tryptophan solution	25 mL
Polysorbate 80 solution	0.25 mL
Dextrose, anhydrous	10 g
Sodium acetate, anhydrous	5 g
Adenine–guanine–uracil solution	5 mL
Riboflavin–thiamine hydrochloride–biotin solution	5 mL
p-Aminobenzoic acid–niacin–pyridoxine hydrochloride solution	5 mL
Salt solution A	5 mL
Salt solution B	5 mL
Pyridoxal–calcium pantothenate solution	5 mL
Polysorbate 40–oleic acid solution	5 mL

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 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 in 800 mL of water, with the aid of heat. Adjust with 0.1 N sodium hydroxide or 0.1 N hydrochloric acid to a pH 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 min. Cool on a slant, and store in a refrigerator. Prepare a stock culture of Pediococcus acidilactici1 on a slant of this medium. Incubate at 35

for 20–24 h, 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 20–24 h. 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. ]

Analysis: 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 solution.

Prepare in duplicate a series of five culture tubes by adding the following quantities of water to the tubes within a set: 4.0, 3.5, 3.0, 2.0, and 1.0 mL. To these same tubes, and in the same order, add 1.0, 1.5, 2.0, 3.0, and 4.0 mL of the Sample solution.

Add 5.0 mL of Double-strength modified pantothenate medium to each tube, and mix. Cover the tubes with metal caps, and sterilize in an autoclave at 121

for 5 min. Cool to room temperature in a chilled water bath, and inoculate each tube with 0.5 mL of the Inoculum. Allow to incubate at 37

for 16 h. Terminate growth by heating to a temperature not below 80

, such as by steaming at atmospheric pressure in a sterilizer for 5 to 10 min. Cool, and concomitantly determine the percentage transmittance of the suspensions, in cells of equal pathlength, on a suitable spectrophotometer, at a wavelength of 530 nm.

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 of each tube containing portions of the Sample solution. Divide the potency of each tube by the amount of the Sample solution added to it, to obtain the individual responses. Calculate the mean response by averaging the individual responses that vary from their mean by NMT 15%, using NLT half the total number of tubes. Calculate the potency of the portion of the material taken for assay, by multiplying the mean response by the appropriate dilution factor. Calculate the percentage of the labeled amount of dexpanthenol or panthenol in the portion of Oral Solution taken:

Result = (P/N) × 100

P	=	= calculated potency of dexpanthenol or panthenol in the portion of Oral Solution taken (mg)
N	=	= nominal amount of dexpanthenol or panthenol in the portion of Oral Solution taken (mg)

Acceptance criteria: 90.0%–150.0% of the labeled amount of dexpanthenol or panthenol (C9H19NO4)

• Niacin or Niacinamide

[Note—Use low-actinic glassware throughout this procedure. ]

Diluent: 25 mg/mL of edetate disodium in water

Mobile phase: Mix 0.4 mL of triethylamine, 15.0 mL of glacial acetic acid, and 350 mL of methanol, and dilute with 0.008 M sodium 1-hexanesulfonate to 2000 mL.

Standard solution: 0.10 mg/mL of USP Niacin RS or USP Niacinamide RS in Diluent. [Note—Use USP Niacin RS for Oral Solution that contains niacin and USP Niacinamide RS for Oral Solution that contains niacinamide. ]

Sample solution: Dilute an accurately measured volume of Oral Solution with Diluent to obtain a solution with a concentration of 0.1 mg/mL of niacin or niacinamide.

Chromatographic system