Mode: LC

Detector: UV 200 nm

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

Flow rate: 1.2 mL/min

Injection volume: 100 µL

System suitability

Sample: Standard solution

Suitability requirements

Relative standard deviation: NMT 3.0%

Analysis

Samples: Standard solution and Sample solution

Measure the responses for the biotin peaks. Calculate the percentage of the labeled amount of biotin (C10H16N2O3S) in the portion of Tablets taken:

Result = (rU/rS) × (CS/CU) × 100

rU	=	= peak response from the Sample solution
rS	=	= peak response 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: On the day of the assay, dilute the Standard stock solution with water to a concentration of 0.1 ng/mL of USP Biotin RS.

Sample solution: Finely powder NLT 30 Tablets. Transfer a portion of the powder, equivalent to a nominal amount of 100 µg of biotin, to a 200-mL volumetric flask. Add 3 mL of 50% alcohol, and swirl to wet the contents. Heat the flask in a water bath at 60

–70

for 5 min. Sonicate for 5 min, dilute with diluted alcohol to volume, and filter. Dilute a volume of the filtrate quantitatively, and stepwise if necessary, with water to obtain a solution with 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 dilute with water to 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 NLT 10

. Filter the solution if a precipitate forms during storage.

Cystine–tryptophan solution: Suspend 4.0 g of l-cystine in 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 dilute with water to 1000 mL. Store under toluene in a cool place at a temperature NLT 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 dilute with water to 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: 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, and mix. Store under toluene.

Basal medium stock solution: Dissolve the 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

, 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

, 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. ] Make a transfer of cells from the Stock culture of Lactobacillus plantarum to a sterile tube containing 10 mL of Culture medium. Incubate this culture for 16–24 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 of 540–660 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 (S) as the difference, y = 2.00

S. 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, adding together the two transmittances 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 USP Biotin RS corresponding to the biotin in each mg of the portion of Tablets taken:

antilog M = antilog (M¢ + log R)

= number of µg of biotin assumed to be present in each mg in the portion of Tablets 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

, 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)

• Biotin, Method 3

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

Solution A: Transfer 800 mL of water and 100 mL of triethylamine to a 1000-mL volumetric flask. Add 80 mL of 85% phosphoric acid, and dilute with water to volume.

Mobile phase: Transfer 80 mL of acetonitrile and 10 mL of Solution A to a 1000-mL volumetric flask. Dilute with water to volume.

Standard solution: 0.6 µg/mL of USP Biotin RS in water

[Note—A portion of the Standard solution will be used to determine the percent recovery of biotin from the Solid-phase extraction procedure. ]

Sample solution: Finely powder NLT 20 Tablets. Transfer an amount of powdered Tablets to a volumetric flask to obtain a nominal concentration of 0.6 µg/mL of biotin. Add water up to 80% of the flask capacity, and sonicate for 30–40 min, with occasional mixing, to dissolve. Dilute with water to volume, and filter. Adjust the pH of the solution with either diluted acetic acid or 0.1 N sodium hydroxide to 6.0–7.0.

Solid-phase extraction: [Note—Condition the extraction column specified in this procedure in the following manner. Wash the column with a 2-mL portion of methanol. Equilibrate with a 2-mL portion of water. ] Separately pipet 5.0 mL of the Sample solution and Standard solution into freshly conditioned solid-phase extraction columns consisting of a mixed-mode packing with a sorbent mass of 60 mg. [Note—The mixed-mode packing consists of anion-exchange and reversed-phase sorbents. The reverse-phase component is a copolymer N-vinylpyrrolidone and divinylbenzene. The anion exchange moiety is a trialkylamino group.2 ]

Wash the column with 10 mL of 30% (v/v) methanol in water. Apply an appropriate volume (4.9 mL) of 30% (v/v) methanol in 0.1 N hydrochloric acid to the column. Collect the eluate in a 5-mL volumetric flask, containing 100 µL of 40% (w/v) sodium acetate in water, and dilute with 30% (v/v) methanol in 0.1 N hydrochloric acid to volume.

Chromatographic system

Mode: LC

Detector: UV 200 nm

Column: 4.6-mm × 25-cm; packing L1

Flow rate: 2 mL/min

Injection volume: 100 µL

System suitability

Samples: Standard solution and a portion of Standard solution that has undergone Solid-phase extraction

Suitability requirements

Tailing factor: NMT 1.5, Standard solution

Relative standard deviation: NMT 2.0%, Standard solution and Standard solution that has undergone Solid-phase extraction

Recovery: 95%–100%, Standard solution that has undergone Solid-phase extraction

Analysis

Samples: Standard solution and Sample solution that have both undergone Solid-phase extraction

Measure the responses for the biotin peak. Calculate the percentage of the labeled amount of biotin (C10H16N2O3S) in the portion of Tablets taken:

Result = (rU/rS) × (CS/CU) × 100

rU	=	= peak response from the Sample solution
rS	=	= peak response 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)

• 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: Finely powder NLT 30 Tablets. Transfer a portion of the powder, equivalent to a nominal amount of 100 µg of cyanocobalamin, to a 250-mL flask. Quantitatively add 100.0 mL of water, and carefully extract for 2 min. Filter 10 mL of the extract, and use the 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 volume: 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 responses for cyanocobalamin. Calculate the percentage of the labeled amount of cyanocobalamin (C63H88CoN14O14P) in the portion of Tablets taken:

Result = (rU/rS) × (CS/CU) × 100

rU	=	= peak response of cyanocobalamin from the Sample solution
rS	=	= peak response 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 cyanocobalamin 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 cyanocobalamin stock solution with water to a measured volume such that after the incubation period as directed 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 Standard solution. Prepare this solution fresh for each assay.

Sample solution: Finely powder NLT 20 Tablets. Transfer a portion of the powdered Tablets, equivalent to a nominal amount of 1.0 µg of cyanocobalamin, to an appropriate vessel containing, for each g of powdered Tablets 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 equivalent to the nominal activity 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 dilute with water to 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: Dissolve 20 g of polysorbate 80 in alcohol to make 200 mL. Store in a refrigerator.

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

Vitamin solution B: Dissolve 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 according to Table 2, 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 the dextrose, sodium acetate, and ascorbic acid. Filter, if necessary. Add the Polysorbate 80 solution, adjust with 1 N sodium hydroxide to a pH of between 5.5 and 6.0, and dilute with Purified Water to 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 dilute with water to 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 into test tubes, cover the tubes, sterilize at 121

for 15 min in an autoclave (exhaust line temperature), 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.3 [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 each containing 10 mL of the Culture medium. 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 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 and 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 contents, place the container in a spectrophotometer that has been set at a wavelength of 530 nm, and read the transmittance 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 (S) as the difference, y = 2.00

Calculate the response, y, adding together the two transmittances 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 USP Cyanocobalamin RS corresponding to the cyanocobalamin in each mg of the portion of Tablets taken:

antilog M = antilog (M¢ + log R)

= number of µg of cyanocobalamin assumed to be present in each mg in the portion of Tablets taken

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: Finely powder NLT 30 Tablets. Transfer a portion of the powder, equivalent to a nominal amount of 0.4 mg of folic acid, to a 50-mL amber-colored centrifuge tube. Add 25.0 mL of Internal standard solution, 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 volume: 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 Tablets 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. ]

Reagent: Dissolve 7.5 g of edetate disodium, with stirring, in 500 mL of water containing 10 mL of ammonium hydroxide.

Diluent: 60 µg/mL of ammonium hydroxide

Mobile phase: Transfer 0.4 mL of triethylamine, 15 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.

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 methanol and 35.0 mL of Reagent, shake for 15 min in a water bath maintained at 60

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

Sample solution: Transfer a portion of finely powdered Tablets, equivalent to a nominal amount of 0.3 mg of folic acid, to a 125-mL stoppered flask. Add 10.0 mL of methanol and 35.0 mL of Reagent. 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 volume: 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 Tablets 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)

• 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: Finely powder NLT 30 Tablets. Transfer a portion of the powder, equivalent to a nominal amount of 15 mg of calcium pantothenate, to a centrifuge tube. Add 25.0 mL of the Internal standard solution, and shake vigorously for 10 min. Centrifuge, filter, and use the clear filtrate.

Chromatographic system

Mode: LC

Detector: UV 210 nm

Column: 3.9-mm × 15-cm; packing L1

Flow rate: 1.5 mL/min

Injection volume: 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 responses for calcium pantothenate and the internal standard. Calculate the percentage of the labeled amount of calcium pantothenate (C18H32CaN2O10) in the portion of Tablets taken:

Result = (RU/RS) × (CS/CU) × 100

RU	=	= peak response ratio of calcium pantothenate to p-hydroxybenzoic acid from the Sample solution
RS	=	= peak response 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 while protected from absorption of moisture during the 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 Analysis, 2.0–4.0 mL of the Standard solution being used, are within the linear portion of the log-concentration response curve.

Sample solution: Finely powder NLT 30 Tablets. Transfer a portion of the powder, 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 (16.66 mg/mL), dilute with water to volume, and filter. Dilute a volume of this solution to obtain a solution with 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 and 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

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.

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.

Basal medium stock solution: Dissolve the anhydrous Dextrose and anhydrous 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

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

, 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: To each of a series of test tubes containing 5.0 mL of Basal medium stock solution add 5.0 mL of water containing 0.2 µg of calcium pantothenate. Plug the tubes with cotton, sterilize in an autoclave at 121

, and cool.

Inoculum: [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, 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. Place the container in a spectrophotometer that has been set at a specific wavelength between 540 and 660 nm, and read the transmittance when a steady state is reached. This steady state is observed a few seconds after agitation when the galvanometer reading remains constant for 30 s or more. Allow approximately the same time interval for the reading on each tube.

Calculate the response, y, adding together the two transmittances 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 in each mg of the portion of Tablets taken:

antilog M = antilog (M¢ + log R)

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

111

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

• Calcium Pantothenate, Method 3

Buffer solution: 5.0 mg/mL of monobasic potassium phosphate in water. 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 Standard stock solution diluted with water

Sample solution: Finely powder NLT 20 Tablets. Transfer a portion of the powder, 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. 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 volume: 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 for calcium pantothenate. Calculate the percentage of the labeled amount of calcium pantothenate (C18H32CaN2O10) in the portion of Tablets taken:

Result = (rU/rS) × (CS/CU) × 100

rU	=	= peak area from the Sample solution
rS	=	= peak area 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. ]

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

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

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 using 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: Finely powder NLT 30 Tablets. Transfer a portion of the powder, equivalent to a nominal amount of 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 using 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 volume: 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 Tablets 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 Tablets 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 Tablets 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), 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, and 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. ] Finely powder NLT 20 Tablets. Transfer a portion of the powder, 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 of the powder equivalent to a nominal amount of 2 mg of pyridoxine. If pyridoxine is not present in the formulation, use a portion of the powder 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 volume: 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 for niacin. Calculate the percentage of the labeled amount of niacin (C6H5NO2) in the portion of Tablets 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 in Niacin, Method 2.

Analysis

Samples: Standard solution and Sample solution

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

Result = (rU/rS) × (CS/CU) × 100

rU	=	= peak area of niaciniamide from the Sample solution
rS	=	= peak area of niaciniamide 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 niaciniamide (C6H6N2O)

• Pyridoxine Hydrochloride, Method 2

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

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

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

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

Chromatographic system

Mode: LC

Detector: UV 254 nm

Column: 4.6-mm × 25-cm; packing L1

Flow rate: 1 mL/min

Injection volume: 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 for pyridoxine. Calculate the percentage of the labeled amount of pyridoxine hydrochloride (C8H11NO3·HCl) in the portion of Tablets 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: Prepare as directed in Niacin, Method 2.

Solution A: 6.8 g/L of sodium acetate in water

Mobile phase: Prepare a mixture of Solution A 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.

Sample solution: Prepare as directed in Niacin, Method 2.

Chromatographic system

Mode: LC

Detector: UV 254 nm

Column: 4.6-mm × 25-cm; packing L1

Flow rate: 1 mL/min

Injection volume: 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 for riboflavin. Calculate the percentage of the labeled amount of riboflavin (C17H20N4O6) in the portion of Tablets 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 Standard stock solution diluted with 0.2 N hydrochloric acid

Sample solution: Weigh and finely powder NLT 20 Tablets. Mix a portion of the powdered Tablets 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 volume: 20 µL

System suitability

Sample: Standard solution

Suitability requirements

Relative standard deviation: NMT 3.0%

Analysis

Samples: Standard solution and Sample solution

Measure the areas for the major peaks. For products containing thiamine hydrochloride, calculate the percentage of the labeled amount of thiamine hydrochloride (C12H17ClN4OS·HCl) in the portion of Tablets 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 Tablets 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 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: Weigh and finely powder NLT 20 Tablets. Transfer a portion of the powder, equivalent to a nominal amount of 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

Mode: LC

Detector: UV 270 nm

Column: 4.6-mm × 25-cm; packing L7

Column temperature: 50

Flow rate: 2 mL/min

Injection volume: 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 peaks. Calculate the percentage of the labeled amount of niacin (C6H5NO2) or niacinamide (C6H6N2O) in the portion of Tablets taken:

Result = (rU/rS) × (CS/CU) × 100

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

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 Tablets 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 niacin (C6H5NO2) or niacinamide (C6H6N2O), pyridoxine hydrochloride (C8H11NO3·HCl), riboflavin (C17H20N4O6), and thiamine as thiamine hydrochloride (C12H17ClN4OS·HCl) or thiamine mononitrate (C12H17N5O4S)

[Note—Commercially available atomic absorption standard solutions for the minerals, where applicable, may be used where preparation of a Standard stock solution is described in the following assays. Use deionized water where water is specified. Where atomic absorption spectrophotometry is specified in the assay, the Standard solutions and the Sample solution may be diluted quantitatively with the solvent specified, if necessary, to yield solutions of suitable concentrations adaptable to the linear or working range of the instrument. ]

• Calcium, Method 1

Lanthanum chloride solution: 267 mg/mL of lanthanum chloride heptahydrate in 0.125 N hydrochloric acid

Calcium standard solution: 400 µg/mL of calcium. Dissolve 1.001 g of calcium carbonate, previously dried at 300

for 3 h and cooled in a desiccator for 2 h, in 25 mL of 1 N hydrochloric acid. Boil to expel carbon dioxide, and dilute with water to 1000 mL.

Standard stock solution: 100 µg/mL of calcium from Calcium standard solution diluted with 0.125 N hydrochloric acid

Standard solutions: Into separate 100-mL volumetric flasks pipet 1.0, 1.5, 2.0, 2.5, and 3.0 mL of the Standard stock solution. To each flask add 1.0 mL of Lanthanum chloride solution, and dilute with water to volume to obtain concentrations of 1.0, 1.5, 2.0, 2.5, and 3.0 µg/mL of calcium.

Sample solution: Finely powder NLT 20 Tablets. Transfer a portion of the powder, equivalent to 5 Tablets, to a porcelain crucible. Heat the crucible in a muffle furnace maintained at 550

for 6–12 h, and cool. Add 60 mL of hydrochloric acid, and boil gently on a hot plate or steam bath for 30 min, intermittently rinsing the inner surface of the crucible with 6 N hydrochloric acid. Cool, and quantitatively transfer the contents of the crucible to a 100-mL volumetric flask. Rinse the crucible with small portions of 6 N hydrochloric acid, and add the rinsings to the flask. Dilute with water to volume, and filter, discarding the first 5 mL of the filtrate. Dilute this solution, quantitatively, with 0.125 N hydrochloric acid to obtain a nominal concentration of 2 µg/mL of calcium, adding 1 mL of Lanthanum chloride solution per 100 mL of the final volume.

Instrumental conditions

Mode: Atomic absorption spectrophotometry

Lamp: Calcium hollow-cathode

Flame: Nitrous oxide–acetylene

Analytical wavelength: Calcium emission line at 422.7 nm

Blank: 0.125 N hydrochloric acid containing 1 mL of Lanthanum chloride solution per 100 mL

Analysis

Samples: Standard solutions and Sample solution

Determine the absorbances of the solutions against the Blank. Plot the absorbances of the Standard solutions versus the concentration, in µg/mL, of calcium, and draw the straight line best fitting the five plotted points. From the graph so obtained, determine the concentration, C, in µg/mL of calcium in the Sample solution.

Calculate the percentage of the labeled amount of calcium (Ca) in the portion of Tablets taken:

Result = (C/CU) × 100

C	=	= measured concentration of calcium in the Sample solution (µg/mL)
CU	=	= nominal concentration of calcium in the Sample solution (µg/mL)

Acceptance criteria: 90.0%–125.0% of the labeled amount of calcium (Ca)

• Chromium, Method 1

Chromium standard solution: 1000 µg/mL of chromium from potassium dichromate, previously dried at 120

for 4 h, in water. Store in a polyethylene bottle.

Standard stock solution: 10 µg/mL of chromium from Chromium standard solution diluted with 6 N hydrochloric acid and water (1 in 20)

Standard solutions: Transfer 10.0 and 20.0 mL of the Standard stock solution to separate 100-mL volumetric flasks, and transfer 15.0 and 20.0 mL of the Standard stock solution to separate 50-mL volumetric flasks. Dilute the contents of each of the four flasks with 0.125 N hydrochloric acid to volume to obtain concentrations of 1.0, 2.0, 3.0, and 4.0 µg/mL of chromium.

Sample solution: Proceed as directed in Calcium, Method 1, except prepare the Sample solution to contain 1 µg/mL of chromium and omit the use of the Lanthanum chloride solution.

Instrumental conditions

Mode: Atomic absorption spectrophotometry

Lamp: Chromium hollow-cathode

Flame: Air–acetylene

Analytical wavelength: Chromium emission line at 357.9 nm

Blank: 0.125 N hydrochloric acid

Analysis

Samples: Standard solutions and Sample solution

Determine the absorbances of the solutions against the Blank. Plot the absorbances of the Standard solutions versus the concentration, in µg/mL, of chromium, and draw the straight line best fitting the four plotted points. From the graph so obtained, determine the concentration, C, in µg/mL of chromium in the Sample solution.

Calculate the percentage of the labeled amount of chromium (Cr) in the portion of Tablets taken:

Result = (C/CU) × 100

C	=	= measured concentration of chromium in the Sample solution (µg/mL)
CU	=	= nominal concentration of chromium in the Sample solution (µg/mL)

Acceptance criteria: 90.0%–160.0% of the labeled amount of chromium (Cr)

• Copper, Method 1

Copper standard solution: Dissolve 1.00 g of copper foil in a minimum volume of a 50% (v/v) solution of nitric acid, and dilute with a 1% (v/v) solution of nitric acid to 1000 mL. This solution contains 1000 µg/mL of copper.

Standard stock solution: 100 µg/mL of copper from Copper standard solution diluted with 0.125 N hydrochloric acid

Standard solutions: To separate 200-mL volumetric flasks transfer 1.0, 2.0, 4.0, 6.0, and 8.0 mL of the Standard stock solution. Dilute with water to volume to obtain concentrations of 0.5, 1.0, 2.0, 3.0, and 4.0 µg/mL of copper.

Sample solution: Proceed as directed in Calcium, Method 1, except prepare the Sample solution to contain 2 µg/mL of copper and omit the use of the Lanthanum chloride solution.

Instrumental conditions

Mode: Atomic absorption spectrophotometry

Lamp: Copper hollow-cathode

Flame: Air–acetylene

Analytical wavelength: Copper emission line at 324.7 nm

Blank: 0.125 N hydrochloric acid

Analysis

Samples: Standard solutions and Sample solution

Determine the absorbances of the solutions against the Blank. Plot the absorbances of the Standard solutions versus the concentration, in µg/mL, of copper, and draw the straight line best fitting the five plotted points. From the graph so obtained, determine the concentration, C, in µg/mL of copper in the Sample solution.

Calculate the percentage of the labeled amount of copper (Cu) in the portion of Tablets taken:

Result = (C/CU) × 100

C	=	= measured concentration of copper in the Sample solution (µg/mL)
CU	=	= nominal concentration of copper in the Sample solution (µg/mL)

Acceptance criteria: 90.0%–125.0% of the labeled amount of copper (Cu)

• Fluoride, Method 1

[Note—Store all solutions in plastic containers. ]

3 M sodium acetate solution: Dissolve 408 g of sodium acetate in 600 mL of water in a 1000-mL volumetric flask. Allow the solution to equilibrate to room temperature, and dilute with water to volume. Adjust with a few drops of acetic acid to a pH of 7.0.

Sodium citrate solution: Dissolve 222 g of sodium citrate in 250 mL of water in a 1000-mL volumetric flask. Add 28 mL of perchloric acid, and dilute with water to volume.

Fluoride standard stock solution: 500 µg/mL of fluoride from a quantity of sodium fluoride, previously dried at 100

for 4 h and cooled in a desiccator in water

Intermediate stock solution A: 100 µg/mL of fluoride from Fluoride standard stock solution diluted with water

Intermediate stock solution B: 10 µg/mL of fluoride from Fluoride standard stock solution diluted with water

Standard solutions: To five separate 100-mL volumetric flasks transfer 3.0, 5.0, and 10.0 mL of Intermediate stock solution B and 5.0 and 10.0 mL of Intermediate stock solution A. To each flask add 10.0 mL of 1 N hydrochloric acid, 25 mL of 3 M sodium acetate solution, and 25.0 mL of Sodium citrate solution. Dilute the contents of each flask with water to volume to obtain concentrations of 0.3, 0.5, 1.0, 5.0, and 10.0 µg/mL of fluoride.

Sample solution: Transfer a quantity of the finely powdered Tablets, equivalent to a nominal amount of 200 µg of fluoride, to a 100-mL volumetric flask. Add 10.0 mL of 1 N hydrochloric acid, 25.0 mL of 3 M sodium acetate solution, and 25.0 mL of Sodium citrate solution, and dilute with water to 100 mL.

Analysis

Samples: Standard solutions and Sample solution

To separate plastic beakers, each containing a plastic-coated stirring bar, transfer 50.0 mL each of the Standard solutions and the Sample solution. Measure the potentials (see pH

791

), in mV, of the Standard solutions and the Sample solution with a pH meter capable of a minimum reproducibility of ±0.2 mV and equipped with a fluoride-specific ion-indicating electrode and a calomel reference electrode. [Note—When taking measurements, immerse the electrodes in the solution, stir on a magnetic stirrer with an insulated top until equilibrium is attained (1–2 min), and record the potential. Rinse and dry the electrodes between measurements, taking care to avoid damaging the crystal of the specific-ion electrode. ]

Plot the logarithms of fluoride concentrations, in µg/mL, of the Standard solutions versus the potential, in mV. From the standard response curve and the measured potential of the Sample solution, determine the concentration, C, in µg/mL, of fluoride in the Sample solution.

Calculate the percentage of the labeled amount of fluorine (F) in the portion of Tablets taken:

Result = (C/CU) × 100

C	=	= measured concentration of fluoride in the Sample solution (µg/mL)
CU	=	= nominal concentration of fluorine in the Sample solution (µg/mL)

Acceptance criteria: 90.0%–160.0% of the labeled amount of fluorine (F)

• Fluoride, Method 2

[Note—Use plastic containers and deionized water throughout this procedure. ]

pH 10.0 buffer: Add 214 mL of 0.1 N sodium hydroxide to 1000 mL of 0.05 M sodium bicarbonate.

Mobile phase: Alcohol, 0.1 N sulfuric acid, and water (20:5:175)

Standard stock solution: 220 µg/mL of USP Sodium Fluoride RS in water. This solution contains 100 µg/mL of fluoride.

Standard solution: [Note—Condition the solid-phase extraction column specified for use in the Standard solution and the Sample solution in the following manner. Using a vacuum at a pressure not exceeding 5 mm of mercury, wash the column with 1 column volume of methanol followed by 1 column volume of pH 10.0 buffer. Do not allow the column top to dry. If the top of the column becomes dry, recondition the column. ] Transfer 10.0 mL of the Standard stock solution to a 100-mL volumetric flask. Add 75 mL of water, and adjust with 0.1 N sodium hydroxide to a pH of 10.4 ± 0.1. Dilute with water to volume. Filter, discarding the first 15 mL of the filtrate. Transfer 25.0 mL of the filtrate to a 50-mL volumetric flask, add 15.0 mL of water, and adjust with 0.1 N sodium hydroxide to a pH of 10.0. Dilute with pH 10.0 buffer to volume. Elute a portion of this solution through a 3-mL solid-phase extraction column containing L1 packing that is connected through an adaptor to a second solid-phase extraction column containing sulfonylpropyl strong cation-exchange packing. Discard the first 3 mL of the eluate, and collect the rest of the eluate in a suitable flask for injection into the chromatograph.

Sample solution: Finely powder NLT 20 Tablets. Transfer a portion of powdered Tablets, equivalent to a nominal amount of 1 mg of fluorine, to 15 mL of water, and shake vigorously. Rinse the sides of the flask with 15 mL of water to a 100-mL volumetric flask. Add 15 mL of water, and allow to stand for 10 min. Dilute with water to 85 mL, adjust with 1 N sodium hydroxide to a pH of 10.4 ± 0.1, and dilute with water to 100 mL. Proceed as directed for the Standard solution, beginning with “Filter, discarding the first 15 mL of the filtrate.”

Chromatographic system

Mode: LC

Detector: Conductivity

Columns

Guard: 4.6-mm × 3-cm; packing L17

Analytical: 7.8-mm × 30-cm; packing L17

Flow rate: 0.5 mL/min

Injection volume: 100 µL

System suitability

Sample: Standard solution

Suitability requirements

Relative standard deviation: NMT 2.0%

Analysis

Samples: Standard solution and Sample solution

Measure the peak areas for fluoride. Calculate the percentage of the labeled amount of fluorine (F) in the portion of Tablets taken:

Result = (rU/rS) × (CS/CU) × 100

rU	=	= peak area of fluorine from the Sample solution
rS	=	= peak area of fluorine from the Standard solution
CS	=	= concentration of fluoride in the Standard solution (µg/mL)
CU	=	= nominal concentration of fluorine in the Sample solution (µg/mL)

Acceptance criteria: 90.0%–160.0% of the labeled amount of fluorine (F)

• Iodide

Bromine water: To 20 mL of bromine in a glass-stoppered bottle add 100 mL of water. Insert the stopper into the bottle, and shake. Allow to stand for 30 min, and use the supernatant.

Analysis

Sample: Tablets

Transfer a portion of finely powdered Tablets, equivalent to a nominal amount of 3 mg of iodide, to a nickel crucible. Add 5 g of sodium carbonate, 5 mL of 50% (w/v) sodium hydroxide solution, and 10 mL of alcohol, taking care that the entire specimen is moistened. Heat the crucible on a steam bath to evaporate the alcohol, then dry the crucible at 100

for 30 min to prevent spattering upon subsequent heating. Transfer the crucible with its contents to a furnace heated to 500

, and heat the crucible for 15 min. [Note—Heating at 500

is necessary to carbonize any organic matter present; a higher temperature may be used, if necessary, to ensure complete carbonization of all organic matter. ] Cool the crucible, add 25 mL of water, cover the crucible with a watchglass, and boil gently for 10 min. Filter the solution, and wash the crucible with boiling water, collecting the filtrate and washings in a beaker. Add phosphoric acid until the solution is neutral to methyl orange, then add 1 mL excess of phosphoric acid. Add excess of Bromine water, and boil the solution gently until colorless and then for 5 min longer. Add a few crystals of salicylic acid, and cool the solution to 20

. Add 1 mL of phosphoric acid and 0.5 g of potassium iodide, and titrate the liberated iodine with 0.005 N sodium thiosulfate VS, adding starch TS when the liberated iodine color has nearly disappeared.

Calculate the percentage of the labeled amount of iodine (I) in the portion of Tablets taken:

Result = V × NA × F × Ime × (Aw/W) × (100/L)

V	=	= volume of sodium thiosulfate consumed (mL)
NA	=	= actual normality of the sodium thiosulfate solution used (meq/mL)
F	=	= correction factor to convert mg to µg, 1000 µg/mg
Ime	=	= milliequivalent of I, 21 mg/meq
Aw	=	= average weight of the Tablets
W	=	= weight of the portion of Tablets taken
L	=	= labeled amount of iodine (µg/Tablet)

Acceptance criteria: 90.0%–160.0% of the labeled amount of iodine (I)

• Iron, Method 1

Iron standard stock solution: Transfer 100 mg of iron powder to a 1000-mL volumetric flask. Dissolve in 25 mL of 6 N hydrochloric acid, dilute with water to volume, and mix.

Standard solutions: To separate 100-mL volumetric flasks transfer 2.0, 4.0, 5.0, 6.0, and 8.0 mL of Iron standard stock solution. Dilute the contents of each flask with water to volume to obtain concentrations of 2.0, 4.0, 5.0, 6.0, and 8.0 µg/mL of iron.

Sample solution: Proceed as directed in Calcium, Method 1, except prepare the Sample solution to contain a nominal concentration of 5 µg/mL of iron and omit the use of the Lanthanum chloride solution.

Instrumental conditions

Mode: Atomic absorption spectrophotometry

Lamp: Iron hollow-cathode

Flame: Air–acetylene

Analytical wavelength: Iron emission line at 248.3 nm

Blank: 0.125 N hydrochloric acid

Analysis

Samples: Standard solutions and Sample solution

Determine the absorbances of the solutions against the Blank. Plot the absorbances of the Standard solutions versus the concentration, in µg/mL, of iron, and draw the straight line best fitting the five plotted points. From the graph so obtained, determine the concentration, C, in µg/mL, of iron in the Sample solution.

Calculate the percentage of the labeled amount of iron (Fe) in the portion of Tablets taken:

Result = (C/CU) × 100

C	=	= measured concentration of iron in the Sample solution (µg/mL)
CU	=	= nominal concentration of iron in the Sample solution (µg/mL)

Acceptance criteria: 90.0%–125.0% of the labeled amount of iron (Fe)

• Magnesium, Method 1

Lanthanum chloride solution: Prepare as directed in Calcium, Method 1.

Magnesium standard solution: Transfer 1.0 g of magnesium ribbon to a 1000-mL volumetric flask, dissolve in 50 mL of 6 N hydrochloric acid, dilute with water to volume, and mix to obtain a solution with a concentration of 1000 µg/mL of magnesium.

Standard stock solution: 20 µg/mL of magnesium from Magnesium standard solution diluted with 0.125 N hydrochloric acid

Standard solutions: To separate 100-mL volumetric flasks transfer 1.0, 1.5, 2.0, 2.5, and 3.0 mL of Standard stock solution. To each flask add 1.0 mL of Lanthanum chloride solution, and dilute with 0.125 N hydrochloric acid to volume to obtain concentrations of 0.2, 0.3, 0.4, 0.5, and 0.6 µg/mL of magnesium.

Sample solution: Proceed as directed in Calcium, Method 1, except prepare the Sample solution to contain a nominal concentration of 0.4 µg/mL of magnesium.

Instrumental conditions

Mode: Atomic absorption spectrophotometry

Lamp: Magnesium hollow-cathode

Flame: Air–acetylene

Analytical wavelength: Magnesium emission line at 285.2 nm

Blank: 0.125 N hydrochloric acid containing 1 mL of Lanthanum chloride solution per 100 mL

Analysis

Samples: Standard solutions and Sample solution

Determine the absorbances of the solutions against the Blank. Plot the absorbances of the Standard solutions versus the concentration, in µg/mL, of magnesium, and draw the straight line best fitting the five plotted points. From the graph so obtained, determine the concentration, C, in µg/mL, of magnesium in the Sample solution.

Calculate the percentage of the labeled amount of magnesium (Mg) in the portion of Tablets taken:

Result = (C/CU) × 100

C	=	= measured concentration of magnesium in the Sample solution (µg/mL)
CU	=	= nominal concentration of magnesium in the Sample solution (µg/mL)

Acceptance criteria: 90.0%–125.0% of the labeled amount of magnesium (Mg)

• Manganese, Method 1

Manganese standard stock solution: Transfer a weighed amount of 1.00 g of manganese to a 1000-mL volumetric flask. Dissolve in 20 mL of nitric acid, dilute with 6 N hydrochloric acid to volume, and mix to obtain a solution with a concentration of 1000 µg/mL of manganese.

Standard stock solution: 50 µg/mL of manganese from Manganese standard stock solution diluted with 0.125 N hydrochloric acid

Standard solutions: To separate 100-mL volumetric flasks transfer 1.0, 1.5, 2.0, 3.0, and 4.0 mL of Standard stock solution. Dilute the contents of each flask with 0.125 N hydrochloric acid to volume to obtain solutions with concentrations of 0.5, 0.75, 1.0, 1.5, and 2.0 µg/mL of manganese.

Sample solution: Proceed as directed in Calcium, Method 1, except prepare the Sample solution to contain a nominal concentration of 1 µg/mL of manganese and omit the use of the Lanthanum chloride solution.

Instrumental conditions

Mode: Atomic absorption spectrophotometry

Lamp: Manganese hollow-cathode

Flame: Air–acetylene

Analytical wavelength: Manganese emission line at 279.5 nm

Blank: 0.125 N hydrochloric acid

Analysis

Samples: Standard solutions and Sample solution

Determine the absorbances of the solutions against the Blank. Plot the absorbances of the Standard solutions versus the concentration, in µg/mL, of manganese, and draw the straight line best fitting the five plotted points. From the graph so obtained, determine the concentration, C, in µg/mL, of manganese (Mn) in the Sample solution.

Calculate the percentage of the labeled amount of manganese (Mn) in the portion of Tablets taken:

Result = (C/CU) × 100

C	=	= measured concentration of manganese in the Sample solution (µg/mL)
CU	=	= nominal concentration of manganese in the Sample solution (µg/mL)

Acceptance criteria: 90.0%–125.0% of the labeled amount of manganese (Mn)

• Molybdenum, Method 1

Diluent: 20 mg/mL of ammonium chloride in water

Molybdenum standard solution: Transfer 1.0 g of molybdenum wire to a 1000-mL volumetric flask, and dissolve in 50 mL of nitric acid, warming if necessary. Dilute with water to volume, and mix to obtain a solution with a concentration of 1000 µg/mL of molybdenum.

Standard stock solution: 100 µg/mL of molybdenum from Molybdenum standard solution diluted with water

Standard solutions: To separate 100-mL volumetric flasks transfer 2.0, 10.0, and 25.0 mL of the Standard stock solution, and add 5.0 mL of perchloric acid to each flask. Gently boil the solution in each flask for 15 min, cool to room temperature, and dilute each with Diluent to volume to obtain concentrations of 5.0, 10.0, and 25.0 µg/mL of molybdenum.

Sample solution: Transfer a portion of the powder, equivalent to a nominal amount of 1000 µg of molybdenum, to a suitable flask, and add 12 mL of nitric acid. [Note—The volume of nitric acid may be varied to ensure that the powder is uniformly dispersed. ] Carefully swirl the flask to disperse the test specimen. Sonicate for 10 min, or until the test specimen is completely dissolved. Gently boil the solution for 15 min, and cool to room temperature. Carefully add 8 mL of perchloric acid, heat until perchloric acid fumes appear, and swirl the flask to dissipate the fumes. Repeat the heating and swirling until no fumes are present. Cool to room temperature. Quantitatively transfer the contents of the flask to a 100-mL volumetric flask with the aid of Diluent, and dilute with Diluent to volume.

Instrumental conditions

Mode: Atomic absorption spectrophotometry

Lamp: Molybdenum hollow-cathode

Flame: Nitrous oxide–acetylene

Analytical wavelength: Molybdenum emission line at 313.3 nm

Blank: Diluent and perchloric acid (20:1)

Analysis

Samples: Standard solutions and Sample solution

Determine the absorbances of the solutions against the Blank. Plot the absorbances of the Standard solutions versus the concentration, in µg/mL, of molybdenum, and draw the straight line best fitting the three plotted points. From the graph so obtained, determine the concentration, C, in µg/mL, of molybdenum in the Sample solution.

Calculate the percentage of the labeled amount of molybdenum (Mo) in the portion of Tablets taken:

Result = (C/CU) × 100

C	=	= measured concentration of molybdenum in the Sample solution (µg/mL)
CU	=	= nominal concentration of molybdenum in the Sample solution (µg/mL)

Acceptance criteria: 90.0%–160.0% of the labeled amount of molybdenum (Mo)

• Molybdenum, Method 2

Sodium fluoride solution: Add 200 mL of water to 10 g of sodium fluoride, stir until the solution is saturated, and filter. Store in a polyethylene bottle.

Ferrous sulfate solution: 4.98 mg/mL of ferrous sulfate in water

Potassium thiocyanate solution: 200 mg/mL of potassium thiocyanate in water

20% Stannous chloride solution: Transfer 40 g of stannous chloride to a beaker, add 20 mL 6.5 N hydrochloric acid, and heat the solution until the stannous chloride is dissolved. Cool, and dilute with water to 100 mL.

Diluted stannous chloride solution: 20% Stannous chloride solution diluted with water (1 in 25). Prepare this solution fresh at the time of use.

Standard solution: 20 µg/mL of molybdenum in water

Sample: A portion of finely powdered Tablets, equivalent to a nominal amount of 40 µg of molybdenum

Instrumental conditions

Mode: UV-Vis

Cell: 1 cm

Analytical wavelength: 465 nm

Blank: Amyl alcohol

Analysis

Samples: Standard solution and Sample

Transfer the Sample and 2.0 mL of the Standard solution to separate 200-mL beakers. Add 20 mL of nitric acid to each beaker. Cover each beaker with a watchglass, and boil slowly on a hot plate for 45 min. Cool to room temperature. Add 6 mL of perchloric acid, cover the beakers with a watchglass, and continue the heating until digestion is complete, as indicated when the liquid becomes colorless or pale yellow. Evaporate the solutions in the beakers to dryness. Rinse the sides of the beakers and the watchglasses with water, and add more water to make 50 mL in each beaker. Gently boil the water solution for a few min. Cool to room temperature. Add 2 drops of methyl orange TS, and neutralize with ammonium hydroxide. Add 8.2 mL of hydrochloric acid. Quantitatively transfer the contents of the beakers to separate 100-mL volumetric flasks, rinse the beakers with water, transfer the rinsings to the corresponding volumetric flasks, and dilute with water to volume. Transfer 50.0 mL of each solution to separatory funnels. To each separatory funnel add 1.0 mL of Sodium fluoride solution, 0.5 mL of Ferrous sulfate solution, 4.0 mL of Potassium thiocyanate solution, 1.5 mL of 20% Stannous chloride solution, and 15.0 mL of amyl alcohol, and shake the separatory funnels for 1 min. Allow the layers to separate, and discard the aqueous layers. Add 25 mL of Diluted stannous chloride solution to each separatory funnel, and shake gently for 15 s. Allow the layers to separate, and discard the aqueous layers. Transfer the organic layers from each separatory funnel to a centrifuge tube, and centrifuge at 2000 rpm for 10 min. Determine the absorbances of the organic phases from the Standard solution and the Sample, and correct with the Blank.

Calculate the percentage of the labeled amount of molybdenum (Mo) in the portion of Tablets taken:

Result = (AU/AS) × [(V × CS)/MU] × 100

AU	=	= absorbance of the solution from the Tablets
AS	=	= absorbance of the solution from the Standard solution
V	=	= volume of the Standard solution analyzed, 2.0 mL
CS	=	= concentration of molybdenum in the Standard solution (µg/mL)
MU	=	= nominal amount of molybdenum in the Sample (µg)

Acceptance criteria: 90.0%–160.0% of the labeled amount of molybdenum (Mo)

• Phosphorus, Method 1

Sulfuric acid solution: Cautiously add sulfuric acid to water (37.5: 100), and mix.

Ammonium molybdate solution: 50 mg/mL of ammonium molybdate in Sulfuric acid solution and water (2:3). [Note—Dissolve in water first, then dilute with Sulfuric acid solution to volume. ]

Hydroquinone solution: 5 mg/mL of hydroquinone in water. Add 1 drop of sulfuric acid per 100 mL of solution.

Sodium bisulfite solution: 200 mg/mL of sodium bisulfite in water

Phosphorus standard stock solution: Weigh 4.395 g of monobasic potassium phosphate, previously dried at 105

for 2 h and stored in a desiccator, and transfer to a 1000-mL volumetric flask. Dissolve in water, add 6 mL of sulfuric acid as a preservative, dilute with water to volume, and mix to obtain a solution with a concentration of 1000 µg/mL of phosphorus.

Standard solution: 20 µg/mL of phosphorus from Phosphorus standard stock solution diluted with water

Sample solution: [Note—Finely powder and weigh a counted number of Tablets. ] Transfer a portion of the powder, equivalent to a nominal amount of 100 mg of phosphorus, to 25 mL of nitric acid, and digest on a hot plate for 30 min. Add 15 mL of hydrochloric acid, and continue the digestion to the cessation of brown fumes. Cool, and transfer the contents of the flask to a 500-mL volumetric flask with the aid of small portions of water. Dilute with water to volume. Transfer 10.0 mL of this solution to a 100-mL volumetric flask, and dilute with water to volume.

Instrumental conditions

Mode: UV-Vis

Cell: 1 cm

Analytical wavelength: 650 nm

Analysis

Samples: Standard solution and Sample solution

To three separate 25-mL volumetric flasks transfer 5.0 mL each of the Standard solution, the Sample solution, and water to provide the blank. To each of the three flasks add 1.0 mL each of Ammonium molybdate solution, Hydroquinone solution, and Sodium bisulfite solution, and swirl to mix. Dilute the contents of each flask with water to volume, and allow the flasks to stand for 30 min. Determine the absorbances of the solutions against the blank.

Calculate the percentage of the labeled amount of phosphorus (P) in the portion of Tablets taken:

Result = (AU/AS) × (CS/CU) × 100

AU	=	= absorbance of the Sample solution
AS	=	= absorbance of the Standard solution
CS	=	= concentration of phosphorus in the Standard solution (µg/mL)
CU	=	= nominal concentration of phosphorus in the Sample solution (µg/mL)

Acceptance criteria: 90%–125% of the labeled amount of phosphorus (P)

• Potassium

Potassium standard solution: 100 µg/mL of potassium from potassium chloride, previously dried at 105

for 2 h, in water

Standard stock solution: 10 µg/mL of potassium from Potassium standard solution diluted with 0.125 N hydrochloric acid

Standard solutions: Transfer 5.0, 10.0, 15.0, 20.0, and 25.0 mL of the Standard stock solution to separate 100-mL volumetric flasks. Dilute the contents of each flask with 0.125 N hydrochloric acid to volume to obtain solutions containing 0.5, 1.0, 1.5, 2.0, and 2.5 µg/mL of potassium.

Sample solution: Proceed as directed in Calcium, Method 1, except prepare the Sample solution to contain a nominal amount of 1 µg/mL of potassium and omit the use of the Lanthanum chloride solution.

Instrumental conditions

Mode: Atomic absorption spectrophotometry

Lamp: Potassium hollow-cathode

Flame: Air–acetylene

Analytical wavelength: Potassium emission line at 766.5 nm

Blank: Water

Analysis

Samples: Standard solutions and Sample solution

Determine the absorbances of the solutions against the Blank. Plot the absorbances of the Standard solutions versus the concentration, in µg/mL, of potassium, and draw the straight line best fitting the five plotted points. From the graph so obtained, determine the concentration, C, in µg/mL, of potassium in the Sample solution.

Calculate the percentage of the labeled amount of potassium (K) in the portion of Tablets taken:

Result = (C/CU) × 100

C	=	= measured concentration of potassium in the Sample solution (µg/mL)
CU	=	= nominal concentration of potassium in the Sample solution (µg/mL)

Acceptance criteria: 90.0%–125.0% of the labeled amount of potassium (K)

• Selenium, Method 1

Diluent: Prepare as directed in Molybdenum, Method 1.

Selenium standard solution: [Caution—Selenium is toxic; handle it with care. ] Dissolve 1 g of metallic selenium in a minimum volume of nitric acid. Evaporate to dryness. Add 2 mL of water, and evaporate to dryness. Repeat the addition of water and the evaporation to dryness three times. Dissolve the residue in 3 N hydrochloric acid, transfer to a 1000-mL volumetric flask, and dilute with 3 N hydrochloric acid to volume to obtain a concentration of 1000 µg/mL of selenium.

Standard stock solution: 100 µg/mL of selenium from Selenium standard solution diluted with water

Standard solutions: To separate 100-mL volumetric flasks transfer 5.0, 10.0, and 25.0 mL of the Standard stock solution, and add 5.0 mL of perchloric acid to each flask. Gently boil the solutions for 15 min, cool to room temperature, and dilute each with Diluent to volume to obtain solutions with concentrations of 5.0, 10.0, and 25.0 µg/mL of selenium.

Sample solution: Transfer a portion of powder, equivalent to a nominal amount of 1000 µg of selenium, to a suitable flask, and add 12 mL of nitric acid. [Note—The volume of nitric acid may be varied to ensure that the powder is uniformly dispersed. ] Carefully swirl the flask to disperse the test specimen. Sonicate for 10 min or until the test specimen is completely dissolved. Gently boil the solution for 15 min, and cool to room temperature. Carefully add 8 mL of perchloric acid to the flask, heat the flask until perchloric acid fumes appear, and swirl the flask to dissipate the fumes. Repeat the heating and swirling until the fumes appear again. Cool to room temperature. Transfer the contents of the flask to a 50-mL volumetric flask with the aid of Diluent, and dilute with Diluent to volume.

Instrumental conditions

Mode: Atomic absorption spectrophotometry

Lamp: Selenium hollow-cathode

Flame: Air–acetylene

Analytical wavelength: Selenium emission line at 196.0 nm

Blank: Diluent and perchloric acid (20:1)

Analysis

Samples: Standard solutions and Sample solution

Determine the absorbances of the solutions against the Blank. Plot the absorbances of the Standard solutions versus the concentration, in µg/mL, of selenium, and draw the straight line best fitting the three plotted points. From the graph so obtained, determine the concentration, C, in µg/mL, of selenium in the Sample solution.

Calculate the percentage of the labeled amount of selenium (Se) in the portion of Tablets taken:

Result = (C/CU) × 100

C	=	= measured concentration of selenium in the Sample solution (µg/mL)
CU	=	= nominal concentration of selenium in the Sample solution (µg/mL)

Acceptance criteria: 90.0%–160.0% of the labeled amount of selenium (Se)

• Selenium, Method 2

Hydrochloric acid solution: Hydrochloric acid diluted with water (1 in 10)

50% Ammonium hydroxide solution: Ammonium hydroxide diluted with water (1 in 2)

Reagent A: 9 mg/mL of edetate disodium and 25 mg/mL of hydroxylamine hydrochloride in water. [Note—Dissolve edetate disodium in a portion of water first, add hydroxylamine hydrochloride, then dilute with water to volume. ]

Reagent B: Transfer 200 mg of 2,3-diaminonaphthalene to a 250-mL separatory funnel, and add 200 mL of 0.1 N hydrochloric acid. Wash the solution with three 40-mL portions of cyclohexane, and discard the cyclohexane layer. Filter the solution into a brown bottle, and cover the solution with a 1-cm layer of cyclohexane. This solution is stable for 1 week if stored in a refrigerator.

Standard stock solution: [Caution—Selenium is toxic; handle it with care. ] Dissolve 1 g of metallic selenium in a minimum volume of nitric acid. Evaporate to dryness. Add 2 mL of water, and evaporate to dryness. Repeat the addition of water and evaporation to dryness three times. Dissolve the residue in 3 N hydrochoric acid, transfer to a 1000-mL volumetric flask, and dilute with 3 N hydrochloric acid to volume to obtain a solution with a concentration of 1000 µg/mL of selenium. Dilute a volume of the solution with 0.125 N hydrochloric acid to obtain a concentration of 2.0 µg/mL of selenium.

Standard solution: Transfer 10.0 mL of the Standard stock solution to a glass-stoppered flask. Add 1 mL of perchloric acid and 1 mL of Hydrochloric acid solution, and dilute with water to 20 mL.

Sample solution: Transfer a portion of finely powdered Tablets, equivalent to a nominal amount of 20 µg of selenium, to a suitable flask. Add 10 mL of nitric acid, and warm gently on a hot plate. Continue heating until the initial nitric acid reaction has subsided, then add 3 mL of perchloric acid. [Caution—Exercise care at this stage, because the perchloric acid reaction becomes vigorous. ] Continue heating on the hot plate until the appearance of white fumes of perchloric acid or until the digest begins to darken. Add 0.5 mL of nitric acid, and resume heating, adding additional amounts of nitric acid if further darkening occurs. Digest for 10 min after the first appearance of perchloric acid fumes or until the digest becomes colorless. Cool the flask. Add 2.5 mL of Hydrochloric acid solution, and return the flask to the hot plate to expel residual nitric acid. Heat the mixture for 3 min after it begins to boil. Cool the flask to room temperature, and dilute with water to 20 mL.

Instrumental conditions

Mode: UV

Cell: 1 cm

Analytical wavelength: 380 nm

Blank: 1 mL of perchloric acid and 1 mL of Hydrochloric acid solution diluted with water to 20 mL

Analysis

Samples: Standard solution and Sample solution

Treat the Sample solution, Standard solution, and Blank as follows. Add 5 mL of Reagent A to each flask, and swirl gently to mix. Adjust the solution in each flask with 50% Ammonium hydroxide solution to a pH of 1.1 ± 0.1. Add 5 mL of Reagent B to each flask, and swirl gently to mix. Place the flasks in a water bath maintained at 50

, and equilibrate for 30 min, taking care that the flasks are covered to protect them from light. Cool to room temperature, and transfer the contents of each flask to separate separatory funnels. Transfer 10.0 mL of cyclohexane to each separatory funnel, and extract vigorously for 1 min. Discard the aqueous layer. Transfer the cyclohexane layer to a centrifuge tube, and centrifuge at 1000 rpm for 1 min to remove any remaining water. Determine the absorbances of the solutions from the Samples against the solution from the Blank.

Calculate the percentage of the labeled amount of selenium (Se) in the portion of Tablets taken:

Result = (AU/AS) × [(V × CS)/MU] × 100

AU	=	= absorbance of the cyclohexane layer from the Sample solution
AS	=	= absorbance of the cyclohexane layer from the Standard solution
V	=	= volume of the Standard stock solution used to prepare the Standard solution, 10 mL
CS	=	= concentration of selenium in the Standard stock solution (µg/mL)
MU	=	= nominal amount of selenium in the Sample solution (µg)

Acceptance criteria: 90.0%–160.0% of the labeled amount of selenium (Se)

• Zinc, Method 1

Zinc standard stock solution: 1000 µg/mL of zinc from zinc oxide dissolved in 5 M hydrochloric acid (3.89 mg/mL) and diluted with water to final volume. [Note—Dissolve in 5 M hydrochloric acid by warming if necessary, cool, then dilute to final volume. ]

Standard stock solution: 50 µg/mL of zinc from Zinc standard stock solution diluted with 0.125 N hydrochloric acid

Standard solutions: Transfer 1.0, 2.0, 3.0, 4.0, and 5.0 mL of the Standard stock solution to separate 100-mL volumetric flasks. Dilute the contents of each flask with 0.125 N hydrochloric acid to volume to obtain concentrations of 0.5, 1.0, 1.5, 2.0, and 2.5 µg/mL of zinc.

Sample solution: Proceed as directed in Calcium, Method 1, except prepare the Sample solution to contain a nominal concentration of 2 µg/mL of zinc and omit the use of the Lanthanum chloride solution.

Instrumental conditions