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dimethyl sulfoxide, and swirl to wet the contents. Place the

Water-Soluble Vitamins Tablets flask in a water bath at 60°–70° for 5 min. Sonicate for
DEFINITION 5 min, dilute with water to volume, and filter.
Chromatographic system
Change to read: (See Chromatography á621ñ, System Suitability.)
Mode: LC
Water-Soluble Vitamins Tablets contain two or more of the Detector: UV 200 nm
following water-soluble vitamins: Ascorbic Acid or its Column: 4.6-mm × 15-cm; 3-µm packing L7
equivalent as Calcium Ascorbate or Sodium Ascorbate, Flow rate: 1.2 mL/min
Biotin, Cyanocobalamin, Folic Acid, Niacin or Niacinamide, Injection volume: 100 µL
pantothenic acid (as Calcium Pantothenate or Racemic System suitability
Calcium Pantothenate), Pyridoxine Hydrochloride, Sample: Standard solution
Riboflavin, and Thiamine Hydrochloride or Thiamine Suitability requirements
Mononitrate. Tablets contain NLT 90.0% and NMT 150.0% Relative standard deviation: NMT 3.0%
of the labeled amount of ascorbic acid (C6H8O6), Analysis
▲ (USP 1-May-2020) biotin (C10H16N2O3S), calcium
▲
Samples: Standard solution and Sample solution
pantothenate (C18H32CaN2O10), cyanocobalamin Measure the peak areas of biotin. Calculate the percentage
(C63H88CoN14O14P), folic acid (C19H19N7O6), niacin of the labeled amount of biotin (C10H16N2O3S) in the
(C6H5NO2) or niacinamide (C6H6N2O), pyridoxine portion of Tablets taken:
▲ (USP 1-May-2020) (C8H11NO3 · ▲ (USP 1-May-2020)), riboflavin
▲ ▲
Result = (rU/rS) × (CS/CU) × 100
(C17H20N4O6), and thiamine ▲as thiamine ion (C12H17N4OS
+)▲ (USP 1-May-2020) rU = peak area of biotin from the Sample solution

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They do not contain any form of Beta Carotene or Vitamin A, rS = peak area of biotin from the Standard solution
D, E, or K. They do not contain any minerals for which
CS = concentration of USP Biotin RS in the Standard
nutritional value is claimed. They may contain other labeled
solution (µg/mL)
added substances in quantities that are unobjectionable.
CU = nominal concentration of biotin in the Sample
IDENTIFICATION solution (µg/mL)
Add the following:
▲
• A. The retention times of the vitamin peaks of the Sample
ci Acceptance criteria: 90.0%–150.0% of the labeled amount
of biotin (C10H16N2O3S)
• BIOTIN, Method 2
solutions correspond to those of the corresponding vitamin
[NOTE—Use low-actinic glassware throughout this
ffi
peaks of the Standard solutions as obtained in the tests for
procedure.]
Strength.▲ (USP 1-May-2020)
Dehydrated mixtures yielding formulations similar to the
STRENGTH media described herein may be used provided that, when
[NOTE—In the following assays, where more than one constituted as directed, they have growth-promoting
assay method is given for an individual ingredient, the properties equal to or superior to those obtained with the
requirements may be met by following any one of the media prepared as described.
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specified methods, with the method used being stated Standard stock solution: 50 µg/mL of USP Biotin RS in 50%
in the labeling only if Method 1 is not used.] alcohol. Store in a refrigerator.
Standard solution: 0.1 ng/mL of USP Biotin RS in water,
Change to read: prepared by diluting the Standard stock solution with water
on the day of the assay
• ASCORBIC ACID, CALCIUM ASCORBATE, and SODIUM Sample solution: Finely powder NLT 30 Tablets. Transfer a
ASCORBATE portion of the powder, equivalent to 100 µg of biotin, to a
(See Vitamin C Assay á580ñ.) 200-mL volumetric flask. Add 3 mL of 50% alcohol, and
[NOTE—For labeling purposes, consider á580ñ, Method I swirl to wet the contents. Heat the flask in a water bath at
—Titrimetric Method as Method 1; ▲ á580ñ, Method II— 60°–70° for 5 min. Sonicate for 5 min, dilute with 50%
Chromatographic Method, Procedure 1 as Method 2 and alcohol to volume, and filter. Dilute a volume of the filtrate
á580ñ, Method II—Chromatographic Method, Procedure with water quantitatively, and stepwise if necessary, to
2 as Method 3.▲ (USP 1-May-2020)] obtain a solution with a concentration of 0.1 ng/mL.
Acceptance criteria: 90.0%–150.0% of the labeled amount Acid-hydrolyzed casein solution: Mix 100 g of vitamin-free
of ascorbic acid (C6H8O6), or its equivalent as calcium casein with 500 mL of 6 N hydrochloric acid, and reflux the
ascorbate (C12H14CaO12 · 2H2O), or sodium ascorbate mixture for 8–12 h. Remove the hydrochloric acid from the
(C6H7NaO6) mixture by distillation under reduced pressure until a thick
• BIOTIN, Method 1 paste remains. Redissolve the resulting paste in water,
[NOTE—Use low-actinic glassware throughout this adjust the solution with 1 N sodium hydroxide to a pH of
procedure.] 3.5 ± 0.1, and dilute with water to 1000 mL. Add 20 g of
Mobile phase: Mix 85 mL of acetonitrile, 1 g of sodium activated charcoal, stir for 1 h, and filter. Repeat the
perchlorate, and 1 mL of phosphoric acid, and dilute with treatment with activated charcoal. Store under toluene in a
water to 1000 mL. cool place at a temperature NLT 10°. Filter the solution if a
Standard stock solution: 0.333 mg/mL of USP Biotin RS in precipitate forms during storage.
dimethyl sulfoxide Cystine–tryptophan solution: Suspend 4.0 g of L-cystine
Standard solution: 5 µg/mL of USP Biotin RS prepared by in a solution of 1.0 g of L-tryptophan (or 2.0 g of
diluting the Standard stock solution in water D,L-tryptophan) in 700–800 mL of water. Heat to 70°–80°,
Sample solution: Finely powder NLT 20 Tablets. Transfer a and add dilute hydrochloric acid (1 in 2) dropwise, with
portion of the powder, equivalent to a nominal amount of stirring, until the solids are dissolved. Cool, and dilute with
1 mg of biotin, to a 200-mL volumetric flask. Add 3 mL of

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water to 1000 mL. Store under toluene in a cool place at a Culture medium: To each of a series of test tubes containing
temperature NLT 10°. 5.0 mL of Basal medium stock solution add 5.0 mL of water
Adenine–guanine–uracil solution: Dissolve 200 mg each containing 0.5 ng of biotin. Plug the tubes with cotton,
of adenine sulfate, guanine hydrochloride, and uracil, with sterilize in an autoclave at 121° for 15 min, and cool.
the aid of heat, in 10 mL of 4 N hydrochloric acid. Cool, Inoculum: [NOTE—A frozen suspension of Lactobacillus
and dilute with water to 200 mL. Store under toluene in a plantarum may be used as the stock culture, provided it
refrigerator. yields an Inoculum comparable to a fresh culture.] Transfer
Polysorbate 80 solution: 100 mg/mL of polysorbate 80 in cells from the Stock culture of Lactobacillus plantarum to a
alcohol sterile tube containing 10 mL of Culture medium. Incubate
Calcium pantothenate solution: 10 µg/mL of calcium this culture for 16–24 h at a temperature between 30° and
pantothenate in 50% alcohol. Store in a refrigerator. 37° held constant to within ±0.5°. The cell suspension so
Riboflavin–thiamine hydrochloride solution: 20 µg/mL of obtained is the Inoculum.
riboflavin and 10 µg/mL of thiamine hydrochloride in Analysis
0.02 N acetic acid. Store under toluene, protected from Samples: Standard solution and Sample solution
light, in a refrigerator. To similar separate test tubes add, in duplicate, 1.0 and/or
p-Aminobenzoic acid–niacin–pyridoxine hydrochloride 1.5, 2.0, 3.0, 4.0, and 5.0 mL of the Standard solution. To
solution: 10 µg/mL of p-aminobenzoic acid, 50 µg/mL of each tube and to four similar but empty tubes add
niacin, and 40 µg/mL of pyridoxine hydrochloride in a 5.0 mL of Basal medium stock solution and sufficient water
mixture of neutralized alcohol and water (1:3). Store in a to make 10 mL.
refrigerator. To similar test tubes add, in duplicate, volumes of the
Salt solution A: Dissolve 25 g of monobasic potassium Sample solution corresponding to 3 or more of the levels
phosphate and 25 g of dibasic potassium phosphate in 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

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water to make 500 mL. Add 5 drops of hydrochloric acid.
Store under toluene. medium stock solution and sufficient water to make 10 mL.
Salt solution B: Dissolve 10 g of magnesium sulfate, 0.5 g Place one complete set of Standard and sample tubes
of sodium chloride, 0.5 g of ferrous sulfate, and 0.5 g of together in one tube rack and the duplicate set in a second
manganese sulfate in water to make 500 mL. Add 5 drops rack or section of a rack, preferably in random order.
of hydrochloric acid, and mix. Store under toluene. Cover the tubes of both series to prevent contamination,
Basal medium stock solution: Dissolve anhydrous dextrose
and anhydrous sodium acetate in the solutions previously
mixed according to Table 1, and adjust with 1 N sodium
ci and sterilize in an autoclave at 121° for 5 min. Cool. Add
1 drop of Inoculum to each tube, except 2 of the 4 tubes
containing no Standard solution (the uninoculated
hydroxide to a pH of 6.8. Dilute with water to 250 mL. blanks). Incubate the tubes at a temperature between 30°
and 37° held constant to within ±0.5° until, following 16–
ffi
Table 1 24 h of incubation, there has been no substantial increase
Acid-hydrolyzed casein solution 25 mL in turbidity in the tubes containing the highest level of
Standard during a 2-h period.
Cystine–tryptophan solution 25 mL Determine the transmittance of the tubes in the following
Polysorbate 80 solution 0.25 mL manner. Mix the contents of each tube, and transfer to a
spectrophotometer cell. Place the cell in a
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Dextrose, anhydrous 10 g spectrophotometer that has been set at a specific

Sodium acetate, anhydrous 5g wavelength of 540–660 nm, and read the transmittance
when a steady state is reached. This steady state is
Adenine–guanine–uracil solution 5 mL observed a few seconds after agitation when the
Calcium pantothenate solution 5 mL galvanometer reading remains constant for 30 s or more.
Allow approximately the same time interval for the
Riboflavin–thiamine hydrochloride solution 5 mL reading on each tube.
p-Aminobenzoic acid–niacin–pyridoxine hydrochloride solu- With the transmittance set at 1.00 for the uninoculated
tion 5 mL blank, read the transmittance of the inoculated blank.
Salt solution A 5 mL
With the transmittance set at 1.00 for the inoculated
blank, read the transmittance for each of the remaining
Salt solution B 5 mL tubes. If there is evidence of contamination with a foreign
microorganism, disregard the result of the assay.
Stock culture of Lactobacillus plantarum: Dissolve 2.0 g of Calculation: Prepare a standard concentration-response
yeast extract in 100 mL of water. Add 500 mg of anhydrous curve as follows. For each level of the Standard, calculate
dextrose, 500 mg of anhydrous sodium acetate, and 1.5 g the response from the sum of the duplicate values of the
of agar, and heat the mixture on a steam bath, with stirring, transmittance (ΣS) as the difference, y = 2.00 − ΣS. Plot this
until the agar dissolves. Add 10-mL portions of the hot response on the ordinate of cross-section paper against the
solution to test tubes, close or cover the tubes, sterilize in logarithm of the milliliter of Standard solution per tube on
an autoclave at 121° for 15 min, and allow the tubes to cool the abscissa, using for the ordinate either an arithmetic or a
in an upright position. Prepare stab cultures in 3 or more of logarithmic scale, whichever gives the better
the tubes, using a pure culture of Lactobacillus plantarum, 1 approximation to a straight line. Draw the straight line or
incubating for 16–24 h at a temperature between 30° and smooth curve that best fits the plotted points.
37° held constant to within ±0.5°. Store in a refrigerator. Calculate the response, y = 2.00 − ΣU, adding together the
Prepare a fresh stab of the stock culture every week, and do two transmittances (ΣU) for each level of the Sample
not use for Inoculum if the culture is more than 1 week old. 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 lowest
1 ATCC
and highest points plotted for the Standard. Subtract from
#8014 is suitable. This strain was formerly known as Lactobacillus each logarithm so obtained the logarithm of the volume,
arabinosus 17-5.

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in mL, of the Sample solution to obtain the difference, X, anion-▲exchange▲ (ERR 1-May-2020) moiety is a trialkylamino
for each dosage level. Average the values of X for each of group.2 ]
three or more dosage levels to obtain X, which equals the Wash the column with 10 mL of 30% (v/v) methanol in
log-relative potency, M′, of the Sample solution. water. Apply an appropriate volume (4.9 mL) of 30%
Determine the quantity, in µg, of biotin (C10H16N2O3S) in (v/v) methanol in 0.1 N hydrochloric acid to the column.
the portion of Tablets taken: Collect the eluate in a 5-mL volumetric flask, containing
100 µL of 40% (w/v) sodium acetate in water, and dilute
antilog M = antilog (M′ + log R) with 30% (v/v) methanol in 0.1 N hydrochloric acid to
volume.
R = amount of biotin assumed to be present in the Chromatographic system
portion of Tablets taken (µg) (See Chromatography á621ñ, System Suitability.)
Mode: LC
Calculate the percentage of the labeled amount of biotin in Detector: UV 200 nm
the portion of tablets taken: (C10H16N2O3S) in the portion Column: 4.6-mm × 25-cm; packing L1
of Tablets taken: Flow rate: 2 mL/min
Injection volume: 100 µL
Result = [(antilog M)/N] × 100 System suitability
Samples: Standard solution and a portion of Standard
N = nominal amount of biotin in the portion of
solution that has undergone Solid-phase extraction
Tablets taken (µg)
Suitability requirements
Replication: Repeat the entire determination at least once, Tailing factor: NMT 1.5, Standard solution
using separately prepared Sample solutions. If the difference Relative standard deviation: NMT 2.0%, Standard

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between the two log-potencies M is NMT 0.08, their mean, solution and Standard solution that has undergone
M, is the assayed log-potency of the test material (see Solid-phase extraction
Design and Analysis of Biological Assays á111ñ, The Confidence Recovery: 95%–100%, Standard solution that has
Interval and Limits of Potency). If the two determinations undergone Solid-phase extraction
differ by more than 0.08, conduct one or more additional Analysis
Samples: Standard solution and Sample solution that have
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
ci both undergone Solid-phase extraction
Measure the responses for the biotin peak. Calculate the
percentage of the labeled amount of biotin (C10H16N2O3S)
of biotin (C10H16N2O3S) in the portion of Tablets taken:
ffi
Result = (rU/rS) × (CS/CU) × 100
Change to read:
• BIOTIN, Method 3 rU = peak response from the Sample solution
[NOTE—Use low-actinic glassware throughout this rS = peak response from the Standard solution
procedure.] CS = concentration of USP Biotin RS in the Standard
Solution A: Transfer 800 mL of water and 100 mL of solution (µg/mL)
O

triethylamine to a 1000-mL volumetric flask. Add 80 mL of CU = nominal concentration of biotin in the Sample
85% phosphoric acid, and dilute with water to volume. solution (µg/mL)
Mobile phase: Transfer 80 mL of acetonitrile and 10 mL of
Solution A to a 1000-mL volumetric flask. Dilute with water Acceptance criteria: 90.0%–150.0% of the labeled amount
to volume. of biotin (C10H16N2O3S)
Standard solution: 0.6 µg/mL of USP Biotin RS in water. • CYANOCOBALAMIN, Method 1
[NOTE—A portion of the Standard solution will be used to [NOTE—Use low-actinic glassware throughout this
determine the percent recovery of biotin from the procedure.]
Solid-phase extraction procedure.] Mobile phase: Methanol and water (7:13)
Sample solution: Finely powder NLT 20 Tablets. Transfer an Standard stock solution: 10 µg/mL of USP
amount of powdered Tablets to a volumetric flask to Cyanocobalamin RS in water. [NOTE—Store in a dark place,
obtain a nominal concentration of 0.6 µg/mL of biotin. Add and discard after 1 week.]
water up to 80% of the flask capacity, and sonicate for 30– Standard solution: 1 µg/mL of USP Cyanocobalamin RS
40 min, with occasional mixing, to dissolve. Dilute with from Standard stock solution diluted with water
water to volume, and filter. Adjust the solution with either Sample solution: Finely powder NLT 30 Tablets. Transfer a
diluted acetic acid or 0.1 N sodium hydroxide to a pH of portion of the powder, equivalent to 100 µg of
6.0–7.0. cyanocobalamin, to a 250-mL flask. Quantitatively add
Solid-phase extraction: [NOTE—Condition the extraction 100.0 mL of water, and carefully extract for 2 min. Filter
column specified in this procedure in the following manner. 10 mL of the extract, and use the filtrate.
Wash the column with a 2-mL portion of methanol. Chromatographic system
Equilibrate with a 2-mL portion of water.] (See Chromatography á621ñ, System Suitability.)
Separately pipet 5.0 mL of the Sample solution and Standard Mode: LC
solution into freshly conditioned solid-phase extraction Detector: 550 nm
columns consisting of a mixed-mode packing with a Column: 4.6-mm × 15-cm; 5-µm packing L1
sorbent mass of 60 mg. [NOTE—The mixed-mode packing Flow rate: 0.5 mL/min
consists of anion-exchange and reversed-phase sorbents. Injection volume: 200 µL
The reverse-phase component is a copolymer of
N-vinylpyrrolidone and divinylbenzene. The
2 A suitable cartridge is the Waters Oasis MAX Vac RC, particle size 30 µm,
part #86000371.

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System suitability Salt solution B: Dissolve 4.0 g of magnesium sulfate, 0.20 g

Sample: Standard solution of sodium chloride, 0.20 g of ferrous sulfate, and 0.20 g of
Suitability requirements manganese sulfate in water to make 200 mL. Add 2 drops
Relative standard deviation: NMT 3.0% of hydrochloric acid. Store this solution under toluene.
Analysis Polysorbate 80 solution: Dissolve 20 g of polysorbate 80 in
Samples: Standard solution and Sample solution alcohol to make 200 mL. Store in a refrigerator.
Measure the peak responses for cyanocobalamin. Calculate Vitamin solution A: Dissolve 10 mg of riboflavin, 10 mg of
the percentage of the labeled amount of cyanocobalamin thiamine hydrochloride, 100 µg of biotin, and 20 mg of
(C63H88CoN14O14P) in the portion of Tablets taken: niacin in 0.02 N acetic acid to make 400 mL. Store under
toluene, protected from light, in a refrigerator.
Result = (rU/rS) × (CS/CU) × 100 Vitamin solution B: Dissolve 20 mg of p-aminobenzoic
acid, 10 mg of calcium pantothenate, 40 mg of pyridoxine
rU = peak area of cyanocobalamin from the Sample hydrochloride, 40 mg of pyridoxal hydrochloride, 8 mg of
solution pyridoxamine dihydrochloride, and 2 mg of folic acid in a
rS = peak area of cyanocobalamin from the Standard mixture of water and neutralized alcohol (3:1) to make
solution 400 mL. Store, protected from light, in a refrigerator.
CS = concentration of USP Cyanocobalamin RS in the Basal medium stock solution: Prepare the medium
Standard solution (µg/mL) according to the following formula and directions. A
CU = nominal concentration of cyanocobalamin in the dehydrated mixture containing the same ingredients may
Sample solution (µg/mL) be used provided that, when constituted as directed in the
labeling, it yields a medium comparable to that obtained
Acceptance criteria: 90.0%–150.0% of the labeled amount from the formula given herein.
of cyanocobalamin (C63H88CoN14O14P) Add the ingredients in the order listed in Table 2, carefully

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dissolving cystine and tryptophan in the hydrochloric acid
Change to read: before adding the next eight solutions to the resulting
solution. Add 100 mL of water, and dissolve dextrose,
• CYANOCOBALAMIN, Method 2 sodium acetate, and ascorbic acid. Filter, if necessary.
[NOTE—Use low-actinic glassware throughout this Add the Polysorbate 80 solution, adjust with 1 N sodium
procedure.]
Standard stock solution: 1.0 µg/mL of USP
Cyanocobalamin RS in 25% alcohol. Store in a refrigerator.
ci hydroxide to a pH of 5.5–6.0, and dilute with Purified
Water to 250 mL.
Standard solution: Dilute a suitable volume of Standard Table 2
stock solution with water to a measured volume such that
L-Cystine 0.1 g
ffi
after the incubation period as described in the Analysis, the
difference in transmittance between the inoculated blank L-Tryptophan 0.05 g
and the 5.0-mL level of the Standard solution is NLT that 1 N Hydrochloric acid 10 mL
which corresponds to a difference of 1.25 mg in dried cell
weight. This concentration usually falls between 0.01 and Adenine–guanine–uracil solution 5 mL
0.04 ng/mL of Standard solution. Prepare this solution fresh Xanthine solution 5 mL
O

for each assay.

Sample solution: Finely powder NLT 20 Tablets. Transfer a Vitamin solution A 10 mL
portion of the powdered Tablets, equivalent to 1.0 µg of Vitamin solution B 10 mL
cyanocobalamin, to an appropriate vessel containing, for
each gram of powdered Tablets taken, 25 mL of an aqueous Salt solution A 5 mL
extracting solution prepared just before use to contain, in Salt solution B 5 mL
each 100 mL, 1.29 g of dibasic sodium phosphate, 1.1 g of
anhydrous citric acid, and 1.0 g of sodium metabisulfite. Asparagine solution 5 mL
Autoclave the mixture at 121° for 10 min. Allow any Acid-hydrolyzed casein solution 25 mL
undissolved particles of the extract to settle, and filter or
centrifuge if necessary. Dilute an aliquot of the clear Dextrose, anhydrous 10 g
solution with water to obtain a final solution containing Sodium acetate, anhydrous 5g
vitamin B12 activity approximately equivalent to that of the
Ascorbic acid 1g
Standard solution.
Acid-hydrolyzed casein solution: Prepare as directed in Polysorbate 80 solution 5 mL
Biotin, Method 2.
Asparagine solution: Dissolve 2.0 g of L-asparagine in water Tomato juice preparation: Centrifuge commercially
to make 200 mL. Store under toluene in a refrigerator. canned tomato juice so that most of the pulp is removed.
Adenine–guanine–uracil solution: Prepare as directed in Suspend 5 g/L of analytical filter aid in the supernatant, and
Biotin, Method 2. pass, with the aid of reduced pressure, through a layer of
Xanthine solution: Suspend 0.20 g of xanthine in 30– the filter aid. Repeat, if necessary, until a clear,
40 mL of water, heat to 70°, add 6.0 mL of 6 N ammonium straw-colored filtrate is obtained. Store under toluene in a
hydroxide, and stir until the solid is dissolved. Cool, and refrigerator.
dilute with water to 200 mL. Store under toluene in a Culture medium: [NOTE—A dehydrated mixture containing
refrigerator. the same ingredients may be used provided that, when
Salt solution A: Dissolve 10 g of monobasic potassium constituted as directed in the labeling, it yields a medium
phosphate and 10 g of dibasic potassium phosphate in equivalent to that obtained from the formula given
water to make 200 mL, and add 2 drops of hydrochloric herein.] Dissolve 0.75 g of yeast extract, 0.75 g of dried
acid. Store this solution under toluene. peptone, 1.0 g of anhydrous dextrose, and 0.20 g of
monobasic potassium phosphate in 60–70 mL of water.

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Add 10 mL of Tomato juice preparation and 1 mL of tubes and to 4 similar but empty tubes add 5.0 mL of Basal
Polysorbate 80 solution. Adjust with 1 N sodium hydroxide medium stock solution and sufficient water to make 10 mL.
to a pH of 6.8, and dilute with water to 100 mL. Place 10-mL To similar separate test tubes add, in duplicate, 1.0, 1.5,
portions of the solution in test tubes, and plug with cotton. 2.0, 3.0, and 4.0 mL of the Sample solution. To each tube
Sterilize the tubes and contents in an autoclave at 121° for add 5.0 mL of Basal medium stock solution and sufficient
15 min. Cool as rapidly as possible to avoid color formation water to make 10 mL. Place one complete set of Standard
resulting from overheating the medium. and sample tubes together in one tube rack and the
Suspension medium: Dilute a measured volume of Basal duplicate set in a second rack or section of a rack,
medium stock solution with an equal volume of water. Place preferably in random order.
10-mL portions of the diluted medium in test tubes. Cover the tubes to prevent bacterial contamination, and
Sterilize, and cool as directed for Culture medium. sterilize in an autoclave at 121° for 5 min, arranging to
Stock culture of Lactobacillus leichmannii: To 100 mL of reach this temperature in NMT 10 min by preheating the
Culture medium add 1.0–1.5 g of agar, and heat the mixture autoclave if necessary. Cool as rapidly as possible to avoid
on a steam bath, with stirring, until the agar dissolves. Place color formation resulting from overheating the medium.
10-mL portions of the hot solution in test tubes, cover the Take precautions to maintain uniformity of sterilizing and
tubes, sterilize at 121° for 15 min in an autoclave, and allow cooling conditions throughout the assay, because packing
the tubes to cool in an upright position. Inoculate three or the tubes too closely in the autoclave or overloading it
more of the tubes by stab transfer of a pure culture of may cause variation in the heating rate.
Lactobacillus leichmannii.3 [NOTE—Before first using a fresh Aseptically add 0.5 mL of Inoculum to each tube so
culture in this assay, make NLT 10 successive transfers of prepared, except 2 of the 4 containing no Standard
the culture in a 2-week period.] Incubate for 16–24 h at a solution (the uninoculated blanks). Incubate the tubes at a
temperature between 30° and 40° held constant to within temperature between 30° and 40°, held constant to
within ±0.5°, for 16–24 h.

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±0.5°. Store in a refrigerator.
Prepare fresh stab cultures at least 3 times each week, and Terminate growth by heating to a temperature NLT 80° for
do not use them for preparing the Inoculum if more than 5 min. Cool to room temperature. After agitating
4 days old. The activity of the microorganism can be contents, read the transmittance at 530 nm when a steady
increased by daily or twice-daily transfer of the stab state is reached. This steady state is observed a few
culture, to the point where definite turbidity in the liquid seconds after agitation when the reading remains
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.
ci 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
Inoculum: [NOTE—A frozen suspension of Lactobacillus blank, read the transmittance of the inoculated blank. If
leichmannii may be used as the stock culture, provided it the difference is greater than 5%, or if there is evidence of
ffi
yields an Inoculum comparable to a fresh culture.] Transfer contamination with a foreign microorganism, disregard
cells from the Stock culture of Lactobacillus leichmannii to two the results of the assay.
sterile tubes containing 10 mL each of the Culture medium. With the transmittance set at 100% for the uninoculated
Incubate these cultures for 16–24 h at a temperature blank, read the transmittance of each of the remaining
between 30° and 40° held constant to within ±0.5°. Under tubes. Disregard the results of the assay if the slope of the
aseptic conditions centrifuge the cultures, and decant the standard curve indicates a problem with sensitivity.
O

supernatant. Suspend the cells from the culture in 5 mL of Calculation: Prepare a standard concentration-response
Suspension medium, and combine. Using sterile Suspension curve by the following procedure. Test for and replace any
medium, adjust the volume so that a 1-in-20 dilution in aberrant individual transmittances. For each level of the
saline TS produces 70% transmittance when read on a Standard, calculate the response from the sum of the
suitable spectrophotometer that has been set at a duplicate values of the transmittances (ΣS) as the
wavelength of 530 nm, equipped with a 10-mm cell, and difference, y = 2.00 − ΣS. Plot this response on the ordinate
read against saline TS set at 100% transmittance. Prepare a of cross-section paper against the logarithm of the
1-in-400 dilution of the adjusted suspension using sterile milliliter of Standard solution per tube on the abscissa,
Basal medium stock solution. [NOTE—This dilution may be using for the ordinate either an arithmetic or a logarithmic
altered, when necessary, to obtain the desired test scale, whichever gives the better approximation to a
response.] The cell suspension is the Inoculum. straight line. Draw the straight line or smooth curve that
Calibration of spectrophotometer: Check the wavelength best fits the plotted points.
of the spectrophotometer periodically, using a standard Calculate the response, y = 2.00 − ΣU, adding together the
wavelength cell or other suitable device. Before reading any two transmittances (ΣU) for each level of the Sample
tests, calibrate the spectrophotometer for 0% and 100% solution. Read from the standard curve the logarithm of
transmittance, using water, the wavelength set at 530 nm. the volume of the Standard solution corresponding to
Analysis each of those values of y that falls within the range of the
Samples: Standard solution and Sample solution lowest and highest points plotted for the Standard.
Because of the high sensitivity of the test organism to Subtract from each logarithm so obtained the logarithm
minute amounts of vitamin B12 activity and to traces of of the volume, in mL, of the Sample solution to obtain
many cleansing agents, cleanse meticulously by suitable the difference, X, for each dosage level. Average the
means, followed preferably by heating at 250° for 2 h, values of X for each of three or more dosage levels to
using hard-glass 20-mm × 150-mm test tubes and other obtain X, which equals the log-relative potency, M′, of
necessary glassware. the Sample solution.
To separate test tubes add, in duplicate, 1.0, 1.5, 2.0, 3.0, Determine the quantity, in µg, of cyanocobalamin
4.0, and 5.0 mL of the Standard solution. To each of these (C63H88CoN14O14P) in the portion of Tablets taken:

antilog M = antilog (M′ + log R)

3 Pure cultures of Lactobacillus leichmannii (listed as Lactobacillus delbrueckii)
may be obtained as #7830 from ATCC, 10801 University Blvd., Manassas,
VA 20110-2209 (www.atcc.org).

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R = amount of cyanocobalamin assumed to be Analysis

present in the portion of Tablets taken (µg) Samples: Standard solution and Sample solution
Measure the peak areas for calcium pantothenate and the
Calculate the percentage of the labeled amount of internal standard.
cyanocobalamin (C63H88CoN14O14P) in the portion of Calculate the percentage of the labeled amount of calcium
Tablets taken: pantothenate (C18H32CaN2O10) in the portion of Tablets
taken:
Result = [(antilog M)/N] × 100
Result = (RU/RS) × (CS/CU) × 100
N = nominal amount of cyanocobalamin in the
portion of Tablets taken (µg) RU = peak area ratio of calcium pantothenate to
p-hydroxybenzoic acid from the Sample solution
Replication: Repeat the entire determination at least once, RS = peak area ratio of calcium pantothenate to
using separately prepared Sample solutions. If the p-hydroxybenzoic acid from the Standard
difference between the two log-potencies M is NMT 0.08, solution
their mean, M, is the assayed log-potency of the test CS = concentration of USP Calcium Pantothenate RS in
material (see ▲▲ (USP 1-May-2020) Design and Analysis of the Standard solution (mg/mL)
Biological Assays á111ñ, The Confidence Interval and Limits CU = nominal concentration of calcium pantothenate
of Potency). If the two determinations differ by more than in the Sample solution (mg/mL)
0.08, conduct one or more additional determinations.
From the mean of two or more values of M that do not Acceptance criteria: 90.0%–150.0% of the labeled amount
differ by more than 0.15, compute the mean potency of of calcium pantothenate (C18H32CaN2O10)
the preparation under assay.

al
Acceptance criteria: 90.0%–150.0% of the labeled amount Change to read:
of cyanocobalamin (C63H88CoN14O14P)
• CALCIUM PANTOTHENATE, Method 2
Change to read: Standard stock solution: Dissolve 50 mg of USP Calcium
Pantothenate RS ▲▲ (USP 1-May-2020) in 500 mL of water in a
• FOLIC ACID, Method 1
▲
Proceed as directed in Folic Acid Assay á411ñ, Assay,
Procedure 1.▲ (USP 1-May-2020)
ci 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
Acceptance criteria: 90.0%–150.0% of the labeled amount µg/mL of USP Calcium Pantothenate RS. Store under
of folic acid (C19H19N7O6) toluene in a refrigerator.
ffi
Standard solution: On the day of the assay, dilute a volume
Change to read: of Standard stock solution with water to obtain a
concentration of 0.01–0.04 µg/mL of calcium
• FOLIC ACID, Method 2 pantothenate, the exact concentration being such that the
▲
Proceed as directed in Folic Acid Assay á411ñ, Assay, responses obtained as directed in the Analysis, 2.0 and
Procedure 2.▲ (USP 1-May-2020) 4.0 mL of the Standard solution being used, are within the
O

Acceptance criteria: 90.0%–150.0% of the labeled amount linear portion of the log-concentration response curve.
of folic acid (C19H19N7O6) Sample solution: Finely powder NLT 30 Tablets. Transfer a
• CALCIUM PANTOTHENATE, Method 1 portion of the powder, equivalent to a nominal amount of
Mobile phase: Phosphoric acid and water (1:1000) 50 mg of calcium pantothenate, to a 1000-mL volumetric
Internal standard solution: 80 mg of p-hydroxybenzoic flask containing 500 mL of water. Add 10 mL of 0.2 N acetic
acid in 3 mL of alcohol. Add 50 mL of water and 7.1 g of acid and 100 mL of sodium acetate solution (1 in 60), dilute
dibasic sodium phosphate, and dilute with water to with water to volume, and filter. Dilute a volume of this
1000 mL. Adjust with phosphoric acid to a pH of 6.7. solution to obtain a solution with approximately the same
Standard solution: 0.6 mg/mL of USP Calcium concentration as that of the Standard solution.
Pantothenate RS in Internal standard solution Acid-hydrolyzed casein solution: Mix 100 g of vitamin-free
Sample solution: Finely powder NLT 30 Tablets. Transfer a casein with 500 mL of 6 N hydrochloric acid, and reflux the
portion of the powder, equivalent to 15 mg of calcium mixture for 8–12 h. Remove the hydrochloric acid from the
pantothenate, to a centrifuge tube. Add 25.0 mL of the mixture by distillation under reduced pressure until a thick
Internal standard solution, and shake vigorously for 10 min. paste remains. Redissolve the resulting paste in water,
Centrifuge, filter, and use the clear filtrate. adjust the solution with 1 N sodium hydroxide to a pH of
Chromatographic system 3.5 ± 0.1, and dilute with water to 1000 mL. Add 20 g of
(See Chromatography á621ñ, System Suitability.) activated charcoal, stir for 1 h, and filter. Repeat the
Mode: LC treatment with activated charcoal. Store under toluene in a
Detector: UV 210 nm cool place at a temperature NLT 10°. Filter the solution if a
Column: 3.9-mm × 15-cm; packing L1 precipitate forms during storage.
Flow rate: 1.5 mL/min Cystine–tryptophan solution: Suspend 4.0 g of L-cystine
Injection volume: 10 µL in a solution of 1.0 g of L-tryptophan (or 2.0 g of
System suitability D,L-tryptophan) in 700–800 mL of water, heat to 70°–80°,
Sample: Standard solution and add dilute hydrochloric acid (1 in 2) dropwise, with
[NOTE—The relative retention times for calcium stirring, until the solids are dissolved. Cool, and dilute with
pantothenate and p-hydroxybenzoic acid are about water to 1000 mL. Store under toluene in a cool place at a
0.5 and 1.0, respectively.] temperature NLT 10°.
Suitability requirements Adenine–guanine–uracil solution: Dissolve 200 mg each
Relative standard deviation: NMT 3.0% of adenine sulfate, guanine hydrochloride, and uracil, with
the aid of heat, in 10 mL of 4 N hydrochloric acid. Cool,

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and dilute with water to 200 mL. Store under toluene in a 37° held constant to within ±0.5°. The cell suspension is the
refrigerator. Inoculum.
Polysorbate 80 solution: 100 mg/mL of polysorbate 80 in Analysis
alcohol Samples: Standard solution and Sample solution
Riboflavin–thiamine hydrochloride–biotin solution: 20 To similar separate test tubes add, in duplicate, 1.0 and/or
µg/mL of riboflavin, 10 µg/mL of thiamine hydrochloride, 1.5, 2.0, 3.0, 4.0, and 5.0 mL of the Standard solution. To
and 0.04 µg/mL of biotin in 0.02 N acetic acid. Store under each tube and to 4 similar but empty tubes add 5.0 mL of
toluene, protected from light, in a refrigerator. Basal medium stock solution and sufficient water to make
p-Aminobenzoic acid–niacin–pyridoxine hydrochloride 10 mL.
solution: 10 µg/mL of p-aminobenzoic acid, 50 µg/mL of To similar separate test tubes add, in duplicate, volumes
niacin, and 40 µg/mL of pyridoxine hydrochloride in a of the Sample solution corresponding to three or more of
mixture of neutralized alcohol and water (1:3). Store in a the levels specified for the Standard solution, including the
refrigerator. levels of 2.0, 3.0, and 4.0 mL. To each tube add 5.0 mL
Salt solution A: Dissolve 25 g of monobasic potassium of the Basal medium stock solution and sufficient water to
phosphate and 25 g of dibasic potassium phosphate in make 10 mL. Place one complete set of Standard and
water to make 500 mL. Add 5 drops of hydrochloric acid. sample tubes together in one tube rack and the duplicate
Store under toluene. set in a second rack or section of a rack, preferably in
Salt solution B: Dissolve 10 g of magnesium sulfate, 0.5 g random order.
of sodium chloride, 0.5 g of ferrous sulfate, and 0.5 g of Cover the tubes of both series to prevent contamination,
manganese sulfate in water to make 500 mL. Add 5 drops and sterilize in an autoclave at 121° for 5 min. Cool, and
of hydrochloric acid. Store under toluene. add 1 drop of Inoculum to each tube, except 2 of the 4
Basal medium stock solution: Dissolve the anhydrous tubes containing no Standard solution (the uninoculated
blanks). Incubate the tubes at a temperature between 30°

al
dextrose and anhydrous sodium acetate in the solutions
previously mixed according to Table 3, and adjust with 1 N and 37°, held constant to within ±0.5° until, following 16–
sodium hydroxide to a pH of 6.8. Dilute with water to 24 h of incubation, there has been no substantial increase
250 mL. in turbidity in the tubes containing the highest level of
Standard during a 2-h period.
Table 3 Determine the transmittance of the tubes in the following
Acid-hydrolyzed casein solution

Cystine–tryptophan solution
ci
25 mL

25 mL
manner. Mix the contents of each tube, and transfer to an
optical container if necessary. Read the transmittance
between 540 and 660 nm when a steady state is reached.
Polysorbate 80 solution 0.25 mL This steady state is observed a few seconds after agitation
when the galvanometer reading remains constant for 30 s
ffi
Dextrose, anhydrous 10 g or more. Allow approximately the same time interval for
Sodium acetate, anhydrous 5g the reading on each tube.
With the transmittance set at 1.00 for the uninoculated
Adenine–guanine–uracil solution 5 mL blank, read the transmittance of the inoculated blank.
Riboflavin–thiamine hydrochloride–biotin solution 5 mL With the transmittance set at 1.00 for the inoculated
blank, read the transmittance for each of the remaining
O

p-Aminobenzoic acid–niacin–pyridoxine hydrochloride solu- tubes. If there is evidence of contamination with a foreign
tion 5 mL
microorganism, disregard the result of the assay.
Salt solution A 5 mL Calculation: Prepare a standard concentration-response
Salt solution B 5 mL
curve as follows. For each level of the Standard, calculate
the response from the sum of the duplicate values of the
transmittance (ΣS) as the difference, y = 2.00 − ΣS. Plot this
Stock culture of Lactobacillus plantarum: Dissolve 2.0 g of response on the ordinate of cross-section paper against
yeast extract in 100 mL of water. Add 500 mg of anhydrous the logarithm of the milliliter of Standard solution per tube
dextrose, 500 mg of anhydrous sodium acetate, and 1.5 g on the abscissa, using for the ordinate either an arithmetic
of agar, and heat the mixture on a steam bath, with stirring, or a logarithmic scale, whichever gives the better
until the agar dissolves. Add 10-mL portions of the hot approximation to a straight line. Draw the straight line or
solution to the test tubes, close or cover the tubes, sterilize smooth curve that best fits the plotted points.
in an autoclave at 121° for 15 min, and allow the tubes to Calculate the response, y = 2.00 − ΣU, adding together the
cool in an upright position. Prepare stab cultures in three two transmittances (ΣU) for each level of the Sample
or more of the tubes, using a pure culture of Lactobacillus
solution. Read from the standard curve the logarithm of
plantarum1 incubating for 16–24 h at a temperature
the volume of the Standard solution corresponding to
between 30° and 37° held constant to within ±0.5°. Store
each of those values of y that fall within the range of the
in a refrigerator. Prepare a fresh stab of the stock culture
lowest and highest points plotted for the Standard.
every week, and do not use for Inoculum if the culture is
Subtract from each logarithm so obtained the logarithm
more than 1 week old.
of the volume, in mL, of the Sample solution to obtain
Culture medium: To each of a series of test tubes containing
the difference, X, for each dosage level. Average the
5.0 mL of Basal medium stock solution add 5.0 mL of water
values of X for each of three or more dosage levels to
containing 0.2 µg of calcium pantothenate. Plug the tubes
obtain X, which equals the log-relative potency, M′, of
with cotton, sterilize in an autoclave at 121° for 15 min,
the Sample solution.
and cool.
Determine the quantity, in mg, of calcium pantothenate
Inoculum: [NOTE—A frozen suspension of Lactobacillus
(C18H32CaN2O10) in the portion of Tablets taken:
plantarum may be used as the stock culture, provided it
yields an Inoculum comparable to a fresh culture.] Transfer
antilog M = antilog (M′ + log R)
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

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R = amount of calcium pantothenate assumed to be Acceptance criteria: 90.0%–150.0% of the labeled amount
present in the portion of Tablets taken (mg) of calcium pantothenate (C18H32CaN2O10)

Calculate the percentage of the labeled amount of Change to read:

calcium pantothenate (C18H32CaN2O10) in the portion of
Tablets taken: • NIACIN OR NIACINAMIDE, PYRIDOXINE HYDROCHLORIDE,
RIBOFLAVIN, and THIAMINE, Method 1
Result = [(antilog M)/N] × 100 [NOTE—Use low-actinic glassware throughout this
procedure.]
N = nominal amount of calcium pantothenate in the Diluent: Acetonitrile, glacial acetic acid, and water
portion of Tablets taken (mg) (5:1:94)
Mobile phase: A mixture of methanol, glacial acetic acid,
Replication: Repeat the entire determination at least once, and water (27:1:73) containing 140 mg of sodium
using separately prepared Sample solutions. If the 1-hexanesulfonate per 100 mL
difference between the two log-potencies M is NMT 0.08, Standard solution: [NOTE—Use USP Niacin RS in place of
their mean, M, is the assayed log-potency of the test USP Niacinamide RS for formulations containing niacin.]
material (see Design and Analysis of Biological Assays á111ñ, Transfer 80 mg of USP Niacinamide RS, 20 mg of USP
The Confidence Interval and Limits of Potency). If the two Pyridoxine Hydrochloride RS, 20 mg of USP Riboflavin RS,
determinations differ by more than 0.08, conduct one or and 20 mg of USP Thiamine Hydrochloride RS to a 200-mL
more additional determinations. From the mean of two or volumetric flask, and add 180 mL of Diluent. Immerse the
more values of M that do not differ by more than 0.15, flask in a hot water bath maintained at 65°–70° for 10 min
compute the mean potency of the preparation under with regular shaking or using a vortex mixer, until all the
assay. solid materials are dissolved. Chill rapidly in a cold water

al
Acceptance criteria: 90.0%–150.0% of the labeled amount bath for 10 min to room temperature, and dilute with
of calcium pantothenate (C18H32CaN2O10) Diluent to volume.
• CALCIUM PANTOTHENATE, Method 3 Sample solution: Finely powder NLT 30 Tablets. Transfer a
Buffer solution: Dissolve 10.0 g of monobasic potassium portion of the powder, equivalent to 10 mg of niacinamide
phosphate in 2000 mL of water, and adjust with phosphoric and 2.5 mg each of pyridoxine hydrochloride, riboflavin,
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
ci 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
Pantothenate RS in water. Prepare fresh every 4 weeks. centrifuge tube in a hot water bath maintained at 65°–70°,
Store in a refrigerator. heat for 5 min, and mix using a vortex mixer for 30 s. Return
ffi
Standard solution: 40 µg/mL of USP Calcium the tube to the hot water bath, heat for another 5 min, and
Pantothenate RS from Standard stock solution diluted with mix on a vortex mixer for 30 s. Filter a portion of the
water solution, cool to room temperature, and use the clear
Sample solution: Finely powder NLT 20 Tablets. Transfer a filtrate. [NOTE—Use the filtrate within 3 h of filtration.]
portion of the powder, equivalent to a nominal amount of Chromatographic system
10 mg of calcium pantothenate, to a 250-mL volumetric (See Chromatography á621ñ, System Suitability.)
O

flask. Add 10 mL of methanol, and swirl the flask to disperse. Mode: LC

Dilute with water to volume, mix, and filter. Detector: UV 280 nm
Chromatographic system Column: 3.9-mm × 30-cm; packing L1
(See Chromatography á621ñ, System Suitability.) Flow rate: 1 mL/min
Mode: LC Injection volume: 10 µL
Detector: UV 205 nm System suitability
Column: 3.9-mm × 30-cm; 5-µm packing L1 Sample: Standard solution
Column temperature: 50° ▲
▲ (USP 1-May-2020)
Flow rate: 2 mL/min Suitability requirements
Injection volume: 25 µL Relative standard deviation: NMT 3.0%
System suitability Analysis
Sample: Standard solution Samples: Standard solution and Sample solution
Suitability requirements ▲
Calculate the percentage of the labeled amount of
Relative standard deviation: NMT 3.0% vitamin B1 as thiamine ion (C12H17N4OS+), vitamin B2 as
Analysis riboflavin (C17H20N4O6), vitamin B3 as niacin (C6H5NO2) or
Samples: Standard solution and Sample solution niacinamide (C6H6N2O), vitamin B6 as pyridoxine
Measure the peak areas for calcium pantothenate. Calculate
(C8H11NO3) ▲▲ (ERR 1-May-2020) in the portion of Tablets
the percentage of the labeled amount of calcium
pantothenate (C18H32CaN2O10) in the portion of Tablets taken:
taken: Result = (rU/rS) × (CS/CU) × 100
Result = (rU/rS) × (CS/CU) × 100 rU = peak area of the relevant vitamin from the
corresponding Sample solution
rU = peak area of calcium pantothenate from the
rS = peak area of the relevant vitamin from the
Sample solution
corresponding Standard solution
rS = peak area of calcium pantothenate from the
CS = concentration of the relevant vitamin Reference
Standard solution
Standard in the corresponding Standard solution
CS = concentration of USP Calcium Pantothenate RS in
(µg/mL)
the Standard solution (mg/mL)
CU = nominal concentration of calcium pantothenate
in the Sample solution (mg/mL)

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CU = nominal concentration of the relevant vitamin in Change to read:

the corresponding Sample solution (µg/
mL)▲ (USP 1-May-2020) • RIBOFLAVIN, Method 2
▲
Proceed as directed in Riboflavin Assay á481ñ, Assay,
Acceptance criteria: 90.0%–150.0% ▲of the labeled Chromatographic Methods, Procedure 2.▲ (USP 1-May-2020)
amount of each individual vitamin▲ (USP 1-May-2020) Acceptance criteria: 90.0%–150.0% of the labeled amount
of riboflavin (C17H20N4O6)
Change to read:
Change to read:
• NIACIN, Method 2
▲
Proceed as directed in Niacin or Niacinamide Assay á441ñ, • THIAMINE, Method 2
Assay, Chromatographic Methods, Procedure ▲
Proceed as directed in Thiamine Assay á531ñ, Assay,
2.▲ (USP 1-May-2020) Chromatographic Methods, Procedure 2.▲ (USP 1-May-2020)
Acceptance criteria: 90.0%–150.0% of the labeled amount Acceptance criteria: 90.0%–150.0% of the labeled amount
of niacin (C6H5NO2) of thiamine ▲as thiamine ion (C12H17N4OS+)▲ (USP 1-May-2020)

Change to read: Change to read:

• NIACINAMIDE, Method 2 • NIACIN OR NIACINAMIDE, PYRIDOXINE HYDROCHLORIDE,
▲
Proceed as directed in Niacin or Niacinamide Assay á441ñ, RIBOFLAVIN, and THIAMINE, Method 3
Assay, Chromatographic Methods, Procedure [NOTE—Use low-actinic glassware throughout this
2.▲ (USP 1-May-2020) procedure.]
Acceptance criteria: 90.0%–150.0% of the labeled amount

al
Reagent: 25 mg/mL of edetate disodium in water
of niacinamide (C6H6N2O) Mobile phase: Transfer 0.4 mL of triethylamine, 15.0 mL of
• PYRIDOXINE HYDROCHLORIDE, Method 2 glacial acetic acid, and 350 mL of methanol to a 2000-mL
[NOTE—Use low-actinic glassware throughout this volumetric flask. Dilute with 0.008 M sodium
procedure.] 1-hexanesulfonate to volume.
Extraction solvent, Mobile phase, and Sample solution: Standard stock solution: 1.5 mg/mL of USP Niacin RS or
Prepare as directed in Niacin, Method 2.
Standard stock solution: 0.1 mg/mL of USP Pyridoxine
Hydrochloride RS in Extraction solvent
ci USP Niacinamide RS, 0.24 mg/mL of USP Pyridoxine
Hydrochloride RS, 0.08 mg/mL of USP Riboflavin RS, and
0.24 mg/mL of USP Thiamine Hydrochloride RS in the
Standard solution: Transfer 5.0 mL of Standard stock Reagent, with heating if necessary
solution to a 25-mL volumetric flask, and dilute with Standard solution: Transfer 5.0 mL of Standard stock
ffi
Extraction solvent to volume. solution to a stoppered 125-mL flask. Add 10.0 mL of a
Chromatographic system mixture of methanol and glacial acetic acid (9:1), and
(See Chromatography á621ñ, System Suitability.) 30.0 mL of a mixture of methanol and ethylene glycol (1:1).
Mode: LC Insert the stopper, shake for 15 min in a water bath
Detector: UV 254 nm maintained at 60°, and cool. Filter, discarding the first few
Column: 4.6-mm × 25-cm; packing L1 milliliters of the filtrate.
O

Flow rate: 1 mL/min Sample solution: Weigh and finely powder NLT 20 Tablets.
Injection volume: 20 µL Transfer a portion of the powder, equivalent to 7.5 mg of
System suitability niacin or niacinamide, 1.2 mg of pyridoxine hydrochloride,
Sample: Standard solution 0.4 mg of riboflavin, and 1.2 mg of thiamine hydrochloride,
Suitability requirements to a stoppered 125-mL flask. Add 10.0 mL of a mixture of
Relative standard deviation: NMT 3.0% methanol and glacial acetic acid (9:1) and 30.0 mL of a
Analysis mixture of methanol and ethylene glycol (1:1). Insert the
Samples: Standard solution and Sample solution stopper, shake for 15 min in a water bath maintained at 60°,
Measure the peak areas for pyridoxine. and cool. Filter, discarding the first few milliliters of the
Calculate the percentage of the labeled amount of filtrate.
pyridoxine hydrochloride (C8H11NO3 · HCl) in the portion Chromatographic system
of Tablets taken: (See Chromatography á621ñ, System Suitability.)
Mode: LC
Result = (rU/rS) × (CS/CU) × 100 Detector: UV 270 nm
Column: 4.6-mm × 25-cm; packing L7
rU = peak area of pyridoxine from the Sample solution Column temperature: 50°
rS = peak area of pyridoxine from the Standard solution Flow rate: 2.0 mL/min
CS = concentration of USP Pyridoxine Injection volume: 5 µL
Hydrochloride RS in the Standard solution System suitability
(mg/mL) Sample: Standard solution
CU = nominal concentration of pyridoxine Suitability requirements
hydrochloride in the Sample solution (mg/mL) Relative standard deviation: NMT 2.0%
Analysis
Acceptance criteria: 90.0%–150.0% of the labeled amount Samples: Standard solution and Sample solution
of pyridoxine hydrochloride (C8H11NO3 · HCl) ▲
Calculate the percentage of the labeled amount of
vitamin B1 as thiamine ion (C12H17N4OS+), vitamin B2 as
riboflavin (C17H20N4O6), vitamin B3 as niacin (C6H5NO2) or
niacinamide (C6H6N2O), vitamin B6 as pyridoxine
(C8H11NO3) ▲▲ (ERR 1-May-2020) in the portion of Tablets
taken:

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Result = (rU/rS) × (CS/CU) × 100 Folic acid standard stock solution: 0.2 mg/mL of folic
acid from USP Folic Acid RS in Solution C
rU = peak area of the relevant vitamin from the Calcium Pantothenate standard stock solution:
corresponding Sample solution 1.0 mg/mL of calcium pantothenate from USP Calcium
rS = peak area of the relevant vitamin from the Pantothenate RS in Mobile phase A
corresponding Standard solution System suitability solution: Transfer 0.5 mL of Ascorbic acid
CS = concentration of the relevant vitamin Reference standard stock solution, 2.5 mL of Thiamine standard stock
Standard in the corresponding Standard solution solution, and 0.5 mL of Niacin or niacinamide standard stock
(µg/mL) solution into 10-mL volumetric flask and dilute with Mobile
CU = nominal concentration of the relevant vitamin in phase A to volume.
the corresponding Sample solution (µg/ Sample solution 1: Weigh and finely powder NLT 20
mL)▲ (USP 1-May-2020) Tablets. Transfer a calculated amount of accurately
weighed powder into a 100-mL volumetric flask to obtain a
Acceptance criteria: 90.0%–150.0% ▲of the labeled solution containing known nominal concentrations of
amount of each individual vitamin▲ (USP 1-May-2020) thiamine, riboflavin, and calcium pantothenate in the
ranges of 5–30 µg/mL for thiamine, 10–60 µg/mL for
Change to read: riboflavin, and 25–150 µg/mL for calcium pantothenate.
Add Solution B to about half the volume and mix on a vortex
▲
• FOLIC ACID, Method 3; ASCORBIC ACID, NIACIN OR mixer for 5 min. Heat the flask at 65°–70° for 5 min, swirling
NIACINAMIDE, PYRIDOXINE HYDROCHLORIDE, CALCIUM frequently, and sonicate additionally for 5 min. Cool to
PANTOTHENATE, RIBOFLAVIN, and THIAMINE, Method 4 room temperature, dilute with Mobile phase A to volume,
[NOTE—Use low-actinic glassware.] and mix well. Pass a portion of the solution through a
Solution A: 0.56 g of edetate disodium and 2.04 g of suitable filter of 0.45-µm pore size and discard the first

al
potassium phosphate monobasic per 1000 mL of water. milliliter of the filtrate.
Adjust with phosphoric acid to a pH of 3.0. Sample solution 2: Transfer a portion of finely powdered
Solution B: Acetonitrile; acetic acid, glacial; and water Tablets into a 100-mL volumetric flask to obtain a solution
(5:1:94) containing known nominal concentrations of ascorbic acid
Solution C: Aqueous solution containing 2 mL of and niacin or niacinamide in the ranges of 50–300 µg/mL
ammonium hydroxide and 1 g of sodium perchlorate per
100 mL
Solution D: Aqueous solution containing 2 mL of
ci for ascorbic acid and 12–65 µg/mL for niacin or
niacinamide, add Solution A to about half the volume, and
mix on a vortex mixer for 5 min. Sonicate for 10 min with
ammonium hydroxide, 1 g of sodium perchlorate, and 2 g occasional shaking. Cool to room temperature, dilute with
of sodium ascorbate per 100 mL Solution A to volume, and mix well. Pass a portion of the
ffi
Mobile phase A: 0.02% trifluoroacetic acid in water solution through a suitable filter of 0.45-µm pore size, and
Mobile phase B: Acetonitrile discard the first milliliter of the filtrate.
Mobile phase: See Table 4. Sample solution 3: Transfer a portion of finely powdered
Tablets into a 100-mL volumetric flask to obtain a solution
Table 4 containing known nominal concentrations of pyridoxine
Time Mobile Phase A Mobile Phase B hydrochloride and folic acid in the ranges of 5–30 µg/mL
O

(min) (%) (%) for pyridoxine hydrochloride and 2–12 µg/mL for folic
0 100 0 acid, add Solution D to about half the volume, heat in a
water bath at 40° for 5 min, and sonicate for 10 min with
1.0 100 0 occasional shaking. Cool to room temperature, dilute with
6.0 95 5 Solution D to volume, and mix well. Pass a portion of the
solution through a suitable filter of 0.45-µm pore size, and
15.0 83 17 discard the first milliliter of the filtrate.
17.0 80 20 Standard solution 1: Transfer calculated volumes of
Thiamine standard stock solution, Riboflavin standard stock
19.0 80 20 solution, and Calcium pantothenate standard stock solution
19.1 100 0 into a suitable volumetric flask to obtain a solution with final
vitamin concentrations similar to the nominal
25.0 100 0 concentrations in Sample solution 1 for the corresponding
vitamins. Dilute with Mobile phase A to volume, and
Ascorbic acid standard stock solution: 1.0 mg/mL of mix well.
ascorbic acid from USP Ascorbic Acid RS in Solution A Standard solution 2: Transfer calculated volumes of
Thiamine standard stock solution: 0.2 mg/mL of Ascorbic acid standard stock solution and Niacin or
thiamine from USP Thiamine Hydrochloride RS in Solution niacinamide standard stock solution to a suitable volumetric
B. The solution can be placed on a steam bath for about 5– flask to obtain a solution with final vitamin concentrations
10 min with frequent swirling to assist in dissolution. similar to the nominal concentrations in Sample solution 2
Riboflavin standard stock solution: 0.2 mg/mL of USP for the corresponding vitamins. Dilute with Mobile phase A
Riboflavin RS in Solution B. Heat the solution to 65°–70° for to volume, and mix well.
10 min to assist in dissolution. Standard solution 3: Transfer calculated volumes of
Niacin or niacinamide standard stock solution: Pyridoxine standard stock solution and Folic acid standard
1.0 mg/mL of USP Niacin RS or USP Niacinamide RS in stock solution into a suitable volumetric flask to obtain a
Solution A solution with final vitamin concentrations similar to the
Pyridoxine standard stock solution: 0.2 mg/mL of nominal concentrations in Sample solution 3 for the
pyridoxine from USP Pyridoxine Hydrochloride RS in corresponding vitamins. Dilute with Solution C to volume,
Solution C and mix well.

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Chromatographic system CU = nominal concentration of the relevant vitamin in

(See Chromatography á621ñ, System Suitability.) the corresponding Sample solution (µg/mL)
Mode: LC
Detectors Acceptance criteria: 90.0%–150.0% of the labeled amount
For calcium pantothenate: UV 210 nm of each individual vitamin▲ (USP 1-May-2020)
For thiamine, ascorbic acid, niacin or niacinamide, and
riboflavin: UV 254 nm PERFORMANCE TESTS
For pyridoxine hydrochloride and folic acid: UV • DISINTEGRATION AND DISSOLUTION á2040ñ, Dissolution:
280 nm Meet the requirements
Column: 4.6-mm × 15-cm; 5-µm packing L1 • WEIGHT VARIATION á2091ñ: Meet the requirements
Column temperature: 25° CONTAMINANTS
Flow rate: 1.0 mL/min • MICROBIAL ENUMERATION TESTS á2021ñ: The total aerobic
Injection volume: 20 µL microbial count does not exceed 3 × 103 cfu/g, and the
System suitability combined molds and yeasts count does not exceed 3 ×
Samples: System suitability solution, Standard solution 1, 102 cfu/g.
Standard solution 2, and Standard solution 3 • ABSENCE OF SPECIFIED MICROORGANISMS á2022ñ, Test
Suitability requirements Procedures, Test for Absence of Salmonella Species and Test
Resolution: NLT 3.5 between the thiamine and ascorbic for Absence of Escherichia coli: Meet the requirements
acid peaks; NLT 4.0 between the ascorbic acid and niacin
or niacinamide peaks, System suitability solution ADDITIONAL REQUIREMENTS
Relative standard deviation: NMT 2.0% for each • PACKAGING AND STORAGE: Preserve in tight, light-resistant
individual peak, Standard solution 1, Standard solution containers.
• LABELING: The label states that the product is Water-Soluble

al
2, and Standard solution 3
Analysis Vitamins Tablets. The label also states the quantity of each
Samples: Sample solutions 1–3 and Standard solutions 1–3 vitamin in terms of metric units per dosage unit, and where
Calculate the percentage of the labeled amount of necessary, the salt form in which it is present. Where more
vitamin B1 as thiamine ion (C12H17N4OS+), vitamin B2 as than one assay method is given for a particular vitamin, the
riboflavin (C17H20N4O6), vitamin B3 as niacin (C6H5NO2) or labeling states which assay method is used only if Method
niacinamide (C6H6N2O), vitamin B6 as pyridoxine
(C8H11NO3), calcium pantothenate (C18H32CaN2O10),
▲
ascorbic acid (C6H8O6),▲ (ERR 1-May-2020) and folic acid
ci 1 is not used.

Change to read:
(C19H19N7O6), in the portion of Tablets taken: • USP REFERENCE STANDARDS á11ñ
ffi
▲
USP Ascorbic Acid RS▲ (USP 1-May-2020)
Result = (rU/rS) × (CS/CU) × 100 USP Biotin RS
USP Calcium Pantothenate RS
rU = peak area of the relevant vitamin from the USP Cyanocobalamin RS
corresponding Sample solution USP Folic Acid RS
rS = peak area of the relevant vitamin from the USP Niacin RS
corresponding Standard solution
O

USP Niacinamide RS
CS = concentration of the relevant vitamin Reference USP Pyridoxine Hydrochloride RS
Standard in the corresponding Standard solution USP Riboflavin RS
(µg/mL) USP Thiamine Hydrochloride RS

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