Mode: LC

Detector: UV 200 nm

Column: 4.6-mm × 15-cm; 3-µm packing L7

Flow rate: 1.2 mL/min

Injection size: 100 µL

System suitability

Sample: Standard solution

Suitability requirements

Relative standard deviation: NMT 3.0%

Analysis

Samples: Standard solution and Sample solution

Measure the peak areas of biotin. Calculate the percentage of the labeled amount of biotin (C10H16N2O3S) in the portion of Capsules taken:

Result = (rU/rS) × (CS/CU) × 100

rU	=	= peak area of biotin from the Sample solution
rS	=	= peak area of biotin from the Standard solution
CS	=	= concentration of USP Biotin RS in the Standard solution (µg/mL)
CU	=	= nominal concentration of biotin in the Sample solution (µg/mL)

Acceptance criteria: 90.0%–150.0% of the labeled amount of biotin (C10H16N2O3S)

• Biotin, Method 2

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

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: 50 µg/mL of USP Biotin RS in 50% alcohol. Store this solution in a refrigerator.

Standard solution: 0.1 ng/mL of USP Biotin RS in water, prepared by dilution of the Standard stock solution with water on the day of the assay.

–70

for 5 min. Sonicate for 5 min, dilute with 50% alcohol to volume, and filter. Dilute a volume of the filtrate quantitatively, and stepwise if necessary, with water to obtain a solution having a concentration of 0.1 ng/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 8–12 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.

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

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: 100 mg/mL of polysorbate 80 in alcohol

Calcium pantothenate solution: 10 µg/mL of calcium pantothenate in 50% alcohol. Store in a refrigerator.

Riboflavin–thiamine hydrochloride solution: 20 µg/mL of riboflavin and 10 µg/mL of thiamine hydrochloride 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: 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: 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.

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

Table 1

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
Calcium pantothenate solution	5 mL
Riboflavin–thiamine hydrochloride 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 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,1 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 0.5 ng of biotin. 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

Samples: Standard solution and Sample solution

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 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 a spectrophotometer cell. Place the cell in a spectrophotometer that has been set at a specific wavelength from 540 to 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 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

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 fall within the range of 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 biotin (C10H16N2O3S) in the portion of Capsules taken:

antilog M = antilog (M¢ + log R)

= number of µg of biotin assumed to be present in the portion of Capsules taken

Calculate the percentage of the labeled amount of biotin in the portion of Capsules taken:

Result = [(antilog M)/N] × 100

= nominal amount of biotin (C10H16N2O3S) in the portion of Capsules taken (µg)

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

, The 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 biotin (C10H16N2O3S)

• Cyanocobalamin, Method 1

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

Mobile phase: Methanol and water (7:13)

Standard stock solution: 10 µg/mL of USP Cyanocobalamin RS in water. [Note—Store this stock solution in a dark place, and discard after 1 week. ]

Standard solution: 1 µg/mL of USP Cyanocobalamin RS from the Standard stock solution diluted with water

Sample solution: Weigh NLT 30 Capsules in a tared weighing bottle. Open the Capsules, without the loss of shell material, and transfer the contents to a 100-mL beaker. Remove any contents adhering to the empty shells by washing, if necessary, 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. Transfer a portion of the Capsule contents, equivalent to 100 µg of cyanocobalamin, to a 250-mL flask. Add 100.0 mL of water, and carefully extract for 2 min. Filter 10 mL of the extract, and use the clear filtrate.

Chromatographic system

Mode: LC

Detector: 550 nm

Column: 4.6-mm × 15-cm; 5-µm packing L1

Flow rate: 0.5 mL/min

Injection size: 200 µL

System suitability

Sample: Standard solution

Suitability requirements

Relative standard deviation: NMT 3.0%

Analysis

Samples: Standard solution and Sample solution

Measure the peak areas of cyanocobalamin. Calculate the percentage of the labeled amount of cyanocobalamin (C63H88CoN14O14P) in the portion of Capsules taken:

Result = (rU/rS) × (CS/CU) × 100

rU	=	= peak area of cyanocobalamin from the Sample solution
rS	=	= peak area of cyanocobalamin from the Standard solution
CS	=	= concentration of USP Cyanocobalamin RS in the Standard solution (µg/mL)
CU	=	= nominal concentration of cyanocobalamin in the Sample solution (µg/mL)

Acceptance criteria: 90.0%–150.0% of the labeled amount of cyanocobalamin (C63H88CoN14O14P)

• Cyanocobalamin, Method 2

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

Standard stock solution: 1.0 µg/mL of 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 in the 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 the Standard solution. Prepare this solution fresh for each assay.

Sample solution: Proceed as directed in Ascorbic Acid, Method 1, through “calculate the average net weight per Capsule.” Transfer a portion of the Capsule contents, equivalent to 1.0 µg of cyanocobalamin, to an appropriate vessel containing, for each g of Capsule contents taken, 25 mL of an aqueous extracting solution prepared just before use to contain 12.9 mg/mL of dibasic sodium phosphate, 11.0 mg/mL of anhydrous citric acid, and 10 mg/mL 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: Prepare as directed in Calcium Pantothenate, Method 2.

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: Prepare as directed in Calcium Pantothenate, Method 2.

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. Add 2 drops of hydrochloric acid. Store this solution under toluene.

Polysorbate 80 solution: 20 g of polysorbate 80 in alcohol to make 200 mL. 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 2, carefully dissolving cystine and tryptophan in the hydrochloric acid before adding the next eight solutions to the resulting solution. Add 100 mL of water, and dissolve the dextrose, sodium acetate, and ascorbic acid. Filter, if necessary. Add the Polysorbate 80 solution, adjust with 1 N sodium hydroxide to a pH between 5.5 and 6.0, and add Purified Water to make 250 mL.

Table 2

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 pass, 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.2 [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. ] Make a transfer of 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. [Note—This dilution may be altered, when necessary, to obtain the desired test response. ] The cell suspension so obtained is the Inoculum.

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, with the wavelength set at 530 nm.

Analysis

Samples: Standard solution and Sample solution

Because of the high sensitivity of the test organism to minute 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 its 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

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 in the portion of Capsules taken:

antilog M = antilog (M¢ + log R)

= number of µg of cyanocobalamin that was assumed to be present in the portion of Capsules taken

Calculate the percentage of the labeled amount of cyanocobalamin in the portion of Capsules taken

Result = [(antilog M)/N] × 100

= nominal amount of cyanocobalamin in the portion of Capsules taken (µg)

111

Acceptance criteria: 90.0%–150.0% of the labeled amount of cyanocobalamin (C63H88CoN14O14P)

• Folic Acid, Method 1

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

Reagent A: 25% solution of tetrabutylammonium hydroxide in methanol

Reagent B: Transfer 5.0 g of pentetic acid to a 50-mL volumetric flask. Using sonication if necessary, dissolve in and dilute with 1 N sodium hydroxide to volume.

Mobile phase: 2 g of monobasic potassium phosphate in 650 mL of water. Add 12.0 mL of Reagent A, 7.0 mL of 3 N phosphoric acid, and 240 mL of methanol. Cool to room temperature, adjust with phosphoric acid or ammonia TS to a pH of 7.0, dilute with water to 1000 mL, and filter. Recheck the pH before use by adding water or methanol to the prepared Mobile phase to obtain baseline separation of folic acid and the internal standard. The pH may be increased up to 7.15 to obtain better separation. [Note—The methanol and water content may be varied (between 1% and 3%). ]

Internal standard solution: Transfer 40 mg of methylparaben to a 1000-mL volumetric flask, and add 220 mL of methanol to dissolve. Dissolve 2.0 g of monobasic potassium phosphate in 300 mL of water in a separate beaker, quantitatively transfer this solution to the flask containing the methylparaben solution, and add an additional 300 mL of water. Add 19 mL of Reagent A, 7 mL of 3 N phosphoric acid, and 30 mL of Reagent B. Adjust with ammonia TS to a pH of 9.8, bubble nitrogen through the solution for 30 min, dilute with water to volume, and mix.

Standard solution: 0.016 mg/mL of USP Folic Acid RS in Internal standard solution

Sample solution: Proceed as directed in Ascorbic Acid, Method 1, through “calculate the average net weight per Capsule.” Transfer an amount of Capsule contents to a suitable centrifuge tube, and add a volume of Internal standard solution to obtain a nominal concentration of 0.016 mg/mL of folic acid. Shake by mechanical means for 10 min, and centrifuge. Filter a portion of the clear supernatant, and use the filtrate.

Chromatographic system

Mode: LC

Detector: UV 280 nm

Column: 3.9-mm × 30-cm; packing L1

Flow rate: 1 mL/min

Injection size: 15 µL

System suitability

Sample: Standard solution

[Note—The relative retention times for folic acid and methylparaben are about 0.8 and 1.0, respectively. ]

Suitability requirements

Relative standard deviation: NMT 3.0%

Analysis

Samples: Standard solution and Sample solution

Measure the peak areas for folic acid and methylparaben. Calculate the percentage of the labeled amount of folic acid (C19H19N7O6) in the portion of Capsules taken:

Result = (RU/RS) × (CS/CU) × 100

RU	=	= peak area ratio of folic acid to methylparaben from the Sample solution
RS	=	= peak area ratio of folic acid to methylparaben from the Standard solution
CS	=	= concentration of USP Folic Acid RS in the Standard solution (µg/mL)
CU	=	= nominal concentration of folic acid in the Sample solution (µg/mL)

Acceptance criteria: 90.0%–150.0% of the labeled amount of folic acid (C19H19N7O6)

• Folic Acid, Method 2

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

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

Diluent: 60 µg/mL of ammonium hydroxide

Standard stock solution: 60 µg/mL of USP Folic Acid RS in Diluent. Prepare this solution fresh daily.

Standard solution: Mix 5.0 mL of Standard stock solution with 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). Shake for 15 min in a water bath maintained at 60

, and cool. Filter, discarding the first few mL of the filtrate.

Sample solution: Proceed as directed for the Sample solution in Ascorbic Acid, Method 1, through “calculate the net average weight per Capsule.” Transfer a portion of the Capsule contents, equivalent to 0.3 mg of folic acid, to a 125-mL stoppered 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). Shake for 15 min in a water bath maintained at 60

, and cool. Filter, discarding the first few mL of the filtrate.

Chromatographic system

Mode: LC

Detector: UV 270 nm

Column: 4.6-mm × 25-cm; packing L7

Column temperature: 50

Flow rate: 2 mL/min

Injection size: 5 µL

System suitability

Sample: Standard solution

Suitability requirements

Relative standard deviation: NMT 2.0%

Analysis

Samples: Standard solution and Sample solution

Measure the areas of the major peaks. Calculate the percentage of the labeled amount of folic acid (C19H19N7O6) in the portion of Capsules taken:

Result = (rU/rS) × (CS/CU) × 100

rU	=	= peak area of folic acid from the Sample solution
rS	=	= peak area of folic acid from the Standard solution
CS	=	= concentration of USP Folic Acid RS in the Standard solution (µg/mL)
CU	=	= nominal concentration of folic acid in the Sample solution (µg/mL)

Acceptance criteria: 90.0%–150.0% of the labeled amount of folic acid (C19H19N7O6)

• Dexpanthenol or Panthenol

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

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.

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

Sample solution: Weigh NLT 30 Capsules in a tared weighing bottle. 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 a portion of the Capsule contents, equivalent to 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 concentration of 1.2 µg/mL of dexpanthenol or 2.4 µg/mL of panthenol.

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 700–800 mL of water, heat to 75 ± 5

, and add hydrochloric acid solution (1 in 2) dropwise, with stirring, until the solids are dissolved. Cool, 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 neutral 25% alcohol. Store in a refrigerator.

Salt solution A: 50 mg/mL of monobasic potassium phosphate and 50 mg/mL of dibasic potassium phosphate in water. Add 10 drops of hydrochloric acid per L of solution. Store under toluene.

Salt solution B: 20 mg/mL of magnesium sulfate, 1 mg/mL of sodium chloride, 1 mg/mL of ferrous sulfate, and 1 mg/mL of manganese sulfate in water. Add 10 drops of hydrochloric acid per L of the solution. Store under toluene.

Pyridoxal–calcium pantothenate solution: 200 µg/mL of pyridoxal hydrochloride and 1.875 µg/mL of calcium pantothenate in 10% alcohol. Store in a refrigerator, and use within 30 days.

Polysorbate 40–oleic acid solution: 50 mg/mL of polysorbate 40 and 0.5 mg/mL of oleic acid in 20% alcohol. 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, and adjust with 1 N sodium hydroxide to a pH of 6.8. Dilute with water to 250 mL.

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 in Modified pantothenate medium, but make the final dilution to 125 mL instead of 250 mL. Prepare fresh.

Stock culture of Pediococcus acidilactici: Dissolve in 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 6.5–6.6, and dilute with water to 1000 mL. 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 acidilactici3 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 sterile 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 sterile Modified pantothenate medium. Resuspend the cells in enough sterile Modified pantothenate medium 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 sterile 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. 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 NLT 80

, such as by steaming at atmospheric pressure in a sterilizer for 5–10 min. Cool, and determine the percentage transmittance of the suspensions, in cells of equal path length, 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 percentage of 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. To obtain the individual responses, divide the potency of each tube by the amount of the Sample solution added to it. 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 Capsules taken:

Result = (P/N) × 100

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

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

• Calcium Pantothenate, Method 1

Mobile phase: Phosphoric acid and water (1:1000)

Internal standard solution: 80 mg of p-hydroxybenzoic acid in 3 mL of alcohol. Add 50 mL of water and 7.1 g of dibasic sodium phosphate, and dilute with water to 1000 mL. Adjust with phosphoric acid to a pH of 6.7.

Standard solution: 0.6 mg/mL of USP Calcium Pantothenate RS in Internal standard solution

Sample solution: Proceed as directed in Ascorbic Acid, Method 1, through “calculate the average net weight per Capsule.” To a centrifuge tube, transfer an amount of mixed Capsule contents and a volume of Internal standard solution to obtain a nominal concentration of 0.6 mg/mL in the Sample solution.

Chromatographic system

Mode: LC

Detector: UV 210 nm

Column: 3.9-mm × 15-cm; packing L1

Flow rate: 1.5 mL/min

Injection size: 10 µL

System suitability

Sample: Standard solution

[Note—The relative retention times for calcium pantothenate and p-hydroxybenzoic acid are about 0.5 and 1.0, respectively. ]

Suitability requirements

Relative standard deviation: NMT 3.0%

Analysis

Samples: Standard solution and Sample solution

Measure the peak areas of calcium pantothenate and the internal standard. Calculate the percentage of the labeled amount of calcium pantothenate (C18H32CaN2O10) in the portion of Capsules taken:

Result = (RU/RS) × (CS/CU) × 100

RU	=	= peak area ratio of calcium pantothenate to p-hydroxybenzoic acid from the Sample solution
RS	=	= peak area ratio of calcium pantothenate to p-hydroxybenzoic acid 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 in the 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: Proceed as directed in Ascorbic Acid, Method 1, through “calculate the average net weight per Capsule.” Transfer a portion of the Capsule contents, equivalent to a nominal amount of 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 volume of this solution to obtain a solution having approximately 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

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

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 anhydrous dextrose and anhydrous sodium acetate in the solutions previously mixed according to Table 4, and adjust with 1 N sodium hydroxide to a pH of 6.8. Dilute with water to 250 mL.

Table 4

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 plantarum1 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: Add 5.0 mL of water containing 0.2 µg of calcium pantothenate to each of a series of test tubes containing 5.0 mL of Basal medium stock solution. 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 of 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

Samples: Standard solution and Sample solution

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, and 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.

Calculate the response y = 2.00

SU, adding together the two transmittances for each level of the Sample solution (SU). 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 calcium pantothenate (C18H32CaN2O) in the portion of Capsules taken:

antilog M = antilog (M¢ + log R)

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

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

Result = [(antilog M)/N] × 100

= nominal amount of calcium pantothenate in the portion of Capsules taken (mg)

111

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

• Calcium Pantothenate, Method 3

Buffer solution: Dissolve 10.0 g of monobasic potassium phosphate in 2000 mL of water, and adjust with phosphoric acid to a pH of 3.5.

Mobile phase: Methanol and Buffer solution (1:9)

Standard stock solution: 0.25 mg/mL of USP Calcium Pantothenate RS in water. Prepare fresh every 4 weeks. Store in a refrigerator.

Standard solution: 40 µg/mL of USP Calcium Pantothenate RS from the Standard stock solution diluted with water

Sample solution: Proceed as directed for the Sample solution in Ascorbic Acid, Method 1, through “calculate the average net weight per Capsule.” Transfer a portion of the Capsule contents, equivalent to a nominal amount of 10 mg of calcium pantothenate, to a 250-mL volumetric flask. Add 10 mL of methanol, and swirl the flask to disperse the Capsule contents. Dilute with water to volume, mix, and filter.

Chromatographic system

Mode: LC

Detector: UV 205 nm

Column: 3.9-mm × 30-cm; 5-µm packing L1

Column temperature: 50

Flow rate: 2 mL/min

Injection size: 25 µL

System suitability

Sample: Standard solution

Suitability requirements

Relative standard deviation: NMT 3.0%

Analysis

Samples: Standard solution and Sample solution

Measure the peak areas of calcium pantothenate. Calculate the percentage of the labeled amount of calcium pantothenate (C18H32CaN2O10) in the portion of Capsules 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)

• Niacin or Niacinamide, Pyridoxine Hydrochloride, Riboflavin, and Thiamine, Method 1

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

Mobile phase: A mixture of methanol, glacial acetic acid, and water (27:1:73) containing 140 mg of sodium 1-hexanesulfonate per 100 mL

Diluent: Acetonitrile, glacial acetic acid, and water (5:1:94)

Standard solution: [Note—Use USP Niacin RS in place of USP Niacinamide RS for formulations containing niacin. ] Transfer 80 mg of USP Niacinamide RS, 20 mg of USP Pyridoxine Hydrochloride RS, 20 mg of USP Riboflavin RS, and 20 mg of USP Thiamine Hydrochloride RS to a 200-mL volumetric flask, and add 180 mL of Diluent. Immerse the flask in a hot water bath maintained at 65

–70

for 10 min with regular shaking or on a vortex mixer, until all the solid materials are dissolved. Chill rapidly in a cold water bath for 10 min to room temperature, and dilute with Diluent to volume.

Sample solution: Proceed as directed in Ascorbic Acid, Method 1, through “calculate the average net weight per Capsule.” Transfer a 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. Add 25.0 mL of Diluent, and mix on a vortex mixer for 30 s to completely suspend the powder. Immerse the centrifuge tube in a hot water bath maintained at 65

–70

, heat for 5 min, and mix on a vortex mixer for 30 s. Return the tube to the hot water bath, heat for another 5 min, and mix on a vortex mixer for 30 s. Filter a portion of the solution, cool to room temperature, and use the clear filtrate. [Note—Use the filtrate within 3 h of filtration. ]

Chromatographic system

Mode: LC

Detector: UV 280 nm

Column: 3.9-mm × 30-cm; packing L1

Flow rate: 1 mL/min

Injection size: 10 µL

System suitability

Sample: Standard solution

[Note—The relative retention times for niacinamide, pyridoxine, riboflavin, and thiamine are about 0.3, 0.5, 0.8, and 1.0, respectively. ]

Suitability requirements

Relative standard deviation: NMT 3.0%

Analysis

Samples: Standard solution and Sample solution

Measure the peak areas for niacin or niacinamide, pyridoxine, riboflavin, and thiamine. Calculate the percentage of the labeled amount of niacinamide (C6H6N2O) in the portion of Capsules taken:

Result = (rU/rS) × (CS/CU) × 100

rU	=	= peak area of niacinamide from the Sample solution
rS	=	= peak area of niacinamide from the Standard solution
CS	=	= concentration of USP Niacinamide RS in the Standard solution (mg/mL)
CU	=	= nominal concentration of niacinamide in the Sample solution (mg/mL)

For formulations containing niacin:

Result = (rU/rS) × (CS/CU) × 100

rU	=	= peak area of niacin from the Sample solution
rS	=	= peak area of niacin from the Standard solution
CS	=	= concentration of USP Niacin RS in the Standard solution (mg/mL)
CU	=	= nominal concentration of niacin in the Sample solution (mg/mL)

Separately calculate the percentage of the labeled amount of pyridoxine hydrochloride (C8H11NO3·HCl), riboflavin (C17H20N4O6), and thiamine hydrochloride (C12H17ClN4OS·HCl) in the portion of Capsules taken:

Result = (rU/rS) × (CS/CU) × 100

rU	=	= peak area of the corresponding vitamin from the Sample solution
rS	=	= peak area of the corresponding vitamin from the Standard solution
CS	=	= concentration of the relevant USP Reference Standard in the Standard solution (mg/mL)
CU	=	= nominal concentration of the corresponding vitamin in the Sample solution (mg/mL)

For products containing thiamine mononitrate, calculate the percentage of the labeled amount of thiamine mononitrate (C12H17N5O4S) in the portion of Capsules taken:

Result = (rU/rS) × (CS/CU) × (Mr1/Mr2) × 100

rU	=	= peak area of thiamine from the Sample solution
rS	=	= peak area of thiamine from the Standard solution
CS	=	= concentration of USP Thiamine Hydrochloride RS in the Standard solution (mg/mL)
CU	=	= nominal concentration of thiamine mononitrate in the Sample solution (mg/mL)
Mr1	=	= molecular weight of thiamine mononitrate, 327.36
Mr2	=	= molecular weight of thiamine hydrochloride, 337.27

Acceptance criteria: 90.0%–150.0% of the labeled amount of niacinamide (C6H6N2O) or niacin (C6H5NO2), pyridoxine hydrochloride (C8H11NO3·HCl), riboflavin (C17H20N4O6), and thiamine as thiamine hydrochloride (C12H17ClN4OS·HCl) or thiamine mononitrate (C12H17N5O4S)

• Niacin, Method 2

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

Solution A: Transfer 1 mL of glacial acetic acid and 2.5 g of edetate disodium to a 100-mL volumetric flask. Dissolve in and dilute with water to volume.

Extraction solvent: Solution A and methanol (3:1)

Mobile phase: 0.1 M sodium acetate solution (13.6 mg/mL of sodium acetate in water). Adjust with acetic acid to a pH of 5.4. [Note—A small amount of methanol (up to 1%) may be added to the Mobile phase to improve resolution. ]

Standard stock solution: 1 mg/mL of USP Niacin RS in Extraction solvent

Standard solution: Transfer 5.0 mL of Standard stock solution to a 25-mL volumetric flask. Dilute with Extraction solvent to volume.

Sample solution: [Note—This preparation is suitable for the determination of niacin or niacinamide, pyridoxine, and riboflavin, when present in the formulation. ] Weigh NLT 20 Capsules in a tared weighing bottle. 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 a portion of the Capsule contents, equivalent to a nominal amount of 2 mg of riboflavin, to a 200-mL volumetric flask. If riboflavin is not present in the formulation, use a portion equivalent to a nominal amount of 2 mg of pyridoxine. If pyridoxine is not present in the formulation, use a portion equivalent to a nominal amount of 20 mg of niacin or niacinamide. Add 100.0 mL of Extraction solvent, and mix for 20 min, using a wrist-action shaker. Immerse the flask in a water bath maintained at 70

–75

, and heat for 20 min. Mix on a vortex mixer for 30 s, cool to room temperature, and filter. Use the clear filtrate.

Chromatographic system

Mode: LC

Detector: UV 254 nm

Column: 4.6-mm × 25-cm; packing L1

Flow rate: 1 mL/min

Injection size: 20 µL

System suitability

Sample: Standard solution

Suitability requirements

Relative standard deviation: NMT 3.0%

[Note—If necessary, flush the column with methanol between injections. ]

Analysis

Samples: Standard solution and Sample solution

Measure the peak areas of niacin. Calculate the percentage of the labeled amount of niacin (C6H5NO2) in the portion of Capsules taken:

Result = (rU/rS) × (CS/CU) × 100

rU	=	= peak area of niacin from the Sample solution
rS	=	= peak area of niacin from the Standard solution
CS	=	= concentration of USP Niacin RS in the Standard solution (mg/mL)
CU	=	= nominal concentration of niacin in the Sample solution (mg/mL)

Acceptance criteria: 90.0%–150.0% of the labeled amount of niacin (C6H5NO2)

• Niacinamide, Method 2

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

Extraction solvent, Mobile phase, Standard stock solution, Standard solution, Sample solution, and Chromatographic system: Using USP Niacinamide RS in place of USP Niacin RS, proceed as directed for Niacin, Method 2.

Analysis

Samples: Standard solution and Sample solution

Measure the peak areas of niacinamide. Calculate the percentage of the labeled amount of niacinamide (C6H6N2O) in the portion of Capsules taken:

Result = (rU/rS) × (CS/CU) × 100

rU	=	= peak area of niacinamide from the Sample solution
rS	=	= peak area of niacinamide from the Standard solution
CS	=	= concentration of USP Niacinamide RS in the Standard solution (mg/mL)
CU	=	= nominal concentration of niacinamide in the Sample solution (mg/mL)

Acceptance criteria: 90.0%–150.0% of the labeled amount of niacinamide (C6H6N2O)

• Pyridoxine Hydrochloride, Method 2

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

Extraction solvent, Mobile phase, and Sample solution: Prepare as directed for Niacin, Method 2.

Standard stock solution: 0.1 mg/mL of USP Pyridoxine Hydrochloride RS in Extraction solvent

Standard solution: 20 µg/mL of USP Pyridoxine Hydrochloride RS from the Standard stock solution diluted with Extraction solvent

Chromatographic system

Mode: LC

Detector: UV 254 nm

Column: 4.6-mm × 25-cm; packing L1

Flow rate: 1 mL/min

Injection size: 20 µL

System suitability

Sample: Standard solution

Suitability requirements

Relative standard deviation: NMT 3.0%

Analysis

Samples: Standard solution and Sample solution

Measure the peak areas of pyridoxine. Calculate the percentage of the labeled amount of pyridoxine hydrochloride (C8H11NO3·HCl) in the portion of Capsules taken:

Result = (rU/rS) × (CS/CU) × 100

rU	=	= peak area of pyridoxine from the Sample solution
rS	=	= peak area of pyridoxine from the Standard solution
CS	=	= concentration of USP Pyridoxine Hydrochloride RS in the Standard solution (mg/mL)
CU	=	= nominal concentration of pyridoxine hydrochloride in the Sample solution (mg/mL)

Acceptance criteria: 90.0%–150.0% of the labeled amount of pyridoxine hydrochloride (C8H11NO3·HCl)

• Riboflavin, Method 2

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

Extraction solvent and Sample solution: Prepare as directed for Niacin, Method 2.

Solution B: 6.8 mg/mL of sodium acetate in water

Mobile phase: Prepare a mixture of Solution B and methanol (13:7). Add 2 mL of triethylamine per L of the mixture, and adjust with glacial acetic acid to a pH of 5.2.

Standard stock solution: Transfer 20 mg of USP Riboflavin RS to a 200-mL volumetric flask, and add 180 mL of Extraction solvent. Immerse the flask for 5 min in a water bath maintained at 65

–75

. Mix well, and repeat if necessary until dissolved. Chill rapidly in a cold water bath to room temperature, and dilute with Extraction solvent to volume.

Standard solution: Dilute 5.0 mL of Standard stock solution with Extraction solvent to 25.0 mL.

Chromatographic system

Mode: LC

Detector: UV 254 nm

Column: 4.6-mm × 25-cm; packing L1

Flow rate: 1 mL/min

Injection size: 20 µL

System suitability

Sample: Standard solution

Suitability requirements

Relative standard deviation: NMT 3.0%

Analysis

Samples: Standard solution and Sample solution

Measure the peak areas of riboflavin. Calculate the percentage of the labeled amount of riboflavin (C17H20N4O6) in the portion of Capsules taken:

Result = (rU/rS) × (CS/CU) × 100

rU	=	= peak area of riboflavin from the Sample solution
rS	=	= peak area of riboflavin from the Standard solution
CS	=	= concentration of USP Riboflavin RS in the Standard solution (mg/mL)
CU	=	= nominal concentration of riboflavin in the Sample solution (mg/mL)

Acceptance criteria: 90.0%–150.0% of the labeled amount of riboflavin (C17H20N4O6)

• Thiamine, Method 2

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

Solution A: 1.88 mg/mL of sodium 1-hexanesulfonate in 0.1% phosphoric acid

Mobile phase: Solution A and acetonitrile (46:9)

Standard stock solution: 0.1 mg/mL of USP Thiamine Hydrochloride RS in 0.2 N hydrochloric acid

Standard solution: 0.02 mg/mL of USP Thiamine Hydrochloride RS from the Standard stock solution diluted with 0.2 N hydrochloric acid

Sample solution: Proceed as directed for the Sample solution in Ascorbic Acid, Method 1, through “calculate the average net weight per Capsule.” Mix a portion of the Capsule contents with a volume of 0.2 N hydrochloric acid to obtain a nominal concentration of 0.02 mg/mL of thiamine. Shake for 15 min with a wrist-action shaker, and heat to boiling for 30 min. Cool to room temperature, and filter. Use the clear filtrate.

Chromatographic system

Mode: LC

Detector: UV 254 nm

Column: 4.6-mm × 25-cm; packing L1

Flow rate: 2 mL/min

Injection size: 20 µL

System suitability

Sample: Standard solution

Suitability requirements

Relative standard deviation: NMT 3.0%

Analysis

Samples: Sample solution and Standard solution

Measure the peak areas of the major peaks. For products containing thiamine hydrochloride, calculate the percentage of the labeled amount of thiamine hydrochloride (C12H17ClN4OS·HCl) in the portion of Capsules taken:

Result = (rU/rS) × (CS/CU) × 100

rU	=	= peak area of thiamine from the Sample solution
rS	=	= peak area of thiamine from the Standard solution
CS	=	= concentration of USP Thiamine Hydrochloride RS in the Standard solution (mg/mL)
CU	=	= nominal concentration of thiamine hydrochloride in the Sample solution (mg/mL)

For products containing thiamine mononitrate, calculate the percentage of the labeled amount of thiamine mononitrate (C12H17N5O4S) in the portion of Capsules taken:

Result = (rU/rS) × (CS/CU) × (Mr1/Mr2) × 100

rU	=	= peak area of thiamine from the Sample solution
rS	=	= peak area of thiamine from the Standard solution
CS	=	= concentration of USP Thiamine Hydrochloride RS in the Standard solution (mg/mL)
CU	=	= nominal concentration of thiamine mononitrate in the Sample solution (mg/mL)
Mr1	=	= molecular weight of thiamine mononitrate, 327.36
Mr2	=	= molecular weight of thiamine hydrochloride, 337.27

Acceptance criteria: 90.0%–150.0% of the labeled amount of thiamine as thiamine hydrochloride (C12H17ClN4OS·HCl) or thiamine mononitrate (C12H17N5O4S)

• Niacin or Niacinamide, Pyridoxine Hydrochloride, Riboflavin, and Thiamine, Method 3

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

Reagent: 25 mg/mL of edetate disodium in water

Mobile phase: Transfer 0.4 mL of triethylamine, 15.0 mL of glacial acetic acid, and 350 mL of methanol to a 2000-mL volumetric flask. Dilute with 0.008 M sodium 1-hexanesulfonate to volume.

Standard stock solution: 1.5 mg/mL of USP Niacin RS or USP Niacinamide RS, 0.24 mg/mL of USP Pyridoxine Hydrochloride RS, 0.08 mg/mL of USP Riboflavin RS, and 0.24 mg/mL of USP Thiamine Hydrochloride RS in the Reagent, with heating if necessary

Standard solution: Transfer 5.0 mL of Standard stock solution 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 min in a water bath maintained at 60

, and cool. Filter, discarding the first few mL of the filtrate.

Sample solution: Proceed as directed for the Sample solution in Ascorbic Acid, Method 1, through “calculate the average net weight per Capsule.” Transfer a portion of the Capsule contents, equivalent to 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 min in a water bath maintained at 60

, and cool. Filter, discarding the first few mL of the filtrate.

Chromatographic system