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Water-Soluble Vitamins Preparation
DEFINITION
Water-Soluble Vitamins Preparation is a mixture of two or
more of the following water-soluble vitamins: vitamin C as
Ascorbic Acid, Sodium Ascorbate, or Calcium Ascorbate;
vitamin B1 as Thiamine Hydrochloride or Thiamine
Mononitrate; vitamin B2 as Riboflavin; vitamin B3 as Niacin
or Niacinamide; vitamin B6 as Pyridoxine Hydrochloride;
vitamin B12 as Cyanocobalamin; Biotin; Folic Acid; and
pantothenic acid as Calcium Pantothenate or Racemic
Calcium Pantothenate with one or more inert substances and
suitable antioxidants. It contains NLT 90.0% and NMT
125.0% of the labeled amounts of vitamin C as ascorbic acid
(C6H8O6), vitamin B3 as niacin (C6H5NO2) or niacinamide
(C6H6N2O), vitamin B6 as pyridoxine (C8H11NO3), folic acid
(C19H19N7O6), pantothenic acid (C9H17NO5), vitamin B2 as
riboflavin (C17H20N4O6), thiamine as thiamine ion
(C12H17N4OS+), vitamin B12 as cyanocobalamin
(C63H88CoN14O14P), and biotin (C10H16N2O3S).
IDENTIFICATION
• A.
Analysis: Proceed as directed in Composition.
Acceptance criteria: The retention times of the main peaks
of the Sample solution correspond to those of the vitamins
claimed in each respective Standard solution.
COMPOSITION
[NOTE—In the following assays, where more than one assay
method is given for an individual ingredient, the
ffi
requirements may be met by following any one of the
specified methods, with the method used being stated in
the labeling only if Method 1 is not used.]
• CONTENT OF VITAMINS B1, B2, B3, AND B6, FOLIC ACID,
AND PANTOTHENIC ACID, Method 1
[NOTE—Where niacin or niacinamide, thiamine,
O
pyridoxine, riboflavin, calcium pantothenate, and folic
acid are specified in the following procedure, use the
vitamins present in the formulation and the relevant
USP Reference Standards. Use low-actinic glassware.]
Solution A: 10 mg/mL of ascorbic acid in water. Prepare
fresh at the time of use.
Solution B: Phosphoric acid and water (50:50)
Solution C: 1% sodium bicarbonate in water
Solution D: 0.1 M edetate disodium in water
Mobile phases: For gradient, see Table 1.
Mobile phase A: 0.1% trifluoroacetic acid in water
Mobile phase B: 0.1% trifluoroacetic acid in a mixture of
water and acetonitrile (88:12)
Table 1
Time Flow Rate Mobile Phase A Mobile Phase B
(min) (mL/min) (%)
0 1 100
8 1 100
20 1 0
26 1.5 0 100
40 1.5 0 100
41 1.5 100
50 1.5 100
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[NOTE—For preparation of the following Standard stock
solutions, dissolve the related USP Reference Standard in
water first. The solution can be swirled or placed on a steam
bath for about 5–10 min with frequent swirling to assist in
dissolution. When the vitamin has completely dissolved,
cool the solution to room temperature, add Solution B, and
dilute stepwise with water to final volume. Solution B should
be added in an amount resulting in a concentration of 0.5%
Solution B in the final volume of stock solution.]
Thiamine standard stock solution: 150 µg/mL of thiamine
from USP Thiamine Hydrochloride RS in water containing
0.5% Solution B
Riboflavin standard stock solution: 120 µg/mL of USP
Riboflavin RS in water containing 0.5% Solution B
Niacin (or Niacinamide) standard stock solution: 1.5 mg/
mL of USP Niacin RS or USP Niacinamide RS in water
containing 0.5% Solution B
Pyridoxine standard stock solution: 150 µg/mL of
pyridoxine from USP Pyridoxine Hydrochloride RS in water
containing 0.5% Solution B
Folic acid standard stock solution: 55 µg/mL of USP Folic
Acid RS in Solution C
Standard solution: Accurately transfer calculated volumes
al
of Thiamine standard stock solution, Riboflavin standard stock
solution, Niacin standard stock solution (or Niacinamide
standard stock solution), Pyridoxine standard stock solution,
and Folic acid standard stock solution into a 200-mL
ci volumetric flask to obtain a solution with final vitamin
concentrations similar to the nominal concentrations in the
Sample solution for the corresponding vitamins.
[NOTE—When calcium pantothenate presents in the
formulation, accurately weigh a calculated amount of USP
Calcium Pantothenate RS into a 200-mL volumetric flask to
obtain a solution with a final vitamin concentration similar
to that in the Sample solution. Add 30 mL of water, vortex
until dissolved, and then transfer the calculated volumes of
the other vitamin Standard stock solutions.] Add 1.0 mL of
Solution B and 1.0 mL of Solution A, and dilute with water
almost to volume. Add 2–3 drops of polysorbate 80 and
add water to final volume. Mix well by completely inverting
the flask at least 20 times. [NOTE—After inverting the flask
10–12 times, carefully remove the stopper to release the
built-up pressure.]
Sample solution: Transfer a calculated amount of
accurately weighed Water-Soluble Vitamins Preparation
into a suitable volumetric flask to obtain a solution
containing known nominal concentrations of vitamins in
the range of 2–130 µg/mL for thiamine, 1.5–110 µg/mL for
riboflavin, 10–800 µg/mL for niacin, 10–500 µg/mL for
niacinamide, 2–120 µg/mL for pyridoxine, 0.5–2.0 µg/mL
for folic acid, and 10-250 µg/mL for pantothenic acid. Add
about half volume of water and an aliquot of Solution D
equivalent to 2% of the volume of the flask, and swirl to
mix thoroughly. Place the flask on a steam bath for 2–3 min,
swirl frequently, and avoid overheating. Cool the flask to
(%) room temperature and add an aliquot of Solution C
equivalent to 10% of the volume of the flask, mix well,
0 sonicate for 1 min. Cool to room temperature, add an
0 aliquot of Solution A equivalent to 0.5% of the volume of
the flask and an aliquot of Solution B equivalent to 0.3% of
100 the volume of the flask, swirl to mix, and dilute with water
almost to volume. Add 2–3 drops of polysorbate 80 and
add water to final volume. [NOTE—If more than one dilution
is necessary to obtain the required concentrations of
0 vitamins, make the dilutions with water first and add
Solution A, Solution B, and polysorbate 80 to the last
0
dilution.] Mix well by completely inverting the flask at least
20 times with caution to release the built-up pressure. Pass a
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portion of the solution through a suitable filter of 0.45-µm
pore size, and discard the first milliliter of the filtrate.
Chromatographic system
(See Chromatography á621ñ, System Suitability.)
Mode: LC
Detector
Channel 1: UV 254 nm for 15 min, switch to 280 nm for
33 min, then return to 254 nm
Channel 2: UV 210 nm
Column: 4.6-mm × 25-cm; 5-µm packing L1
Flow rate: See Table 1.
Injection volume: 20 µL
System suitability
Sample: Standard solution
[NOTE—The relative retention times for ascorbic acid,
niacinamide, thiamine, pyridoxine, pantothenic acid,
folic acid, and riboflavin are 0.8, 1.0, 1.8, 3.3, 4.0,
5.0, and 6.2, respectively.]
Suitability requirements
Resolution: NLT 1.5 between the ascorbic acid and
niacinamide peaks
Relative standard deviation: NMT 2.0% for each
individual peak of niacin or niacinamide, thiamine,
pyridoxine, riboflavin, folic acid, and pantothenic acid
Analysis
Samples: Standard solution and Sample solution
Calculate separately the percentages of the labeled amount
of vitamin B1 as thiamine ion (C12H17N4OS+), vitamin B2 as
ci
riboflavin (C17H20N4O6), vitamin B3 as niacin (C6H5NO2) or
niacinamide (C6H6N2O), vitamin B6 as pyridoxine
(C8H11NO3), folic acid (C19H19N7O6), and pantothenic acid
(C9H17NO5) in the portion of Water-Soluble Vitamins
Preparation taken:
ffi
Result = (rU/rS) × (CS/CU) × 100
rU = peak area of the relevant vitamin from the Sample
solution
rS = peak area of the relevant vitamin from the
O
Standard solution
CS = concentration of the relevant vitamin Reference
Standard in the Standard solution (µg/mL)
CU = nominal concentration of the relevant vitamin in
the Sample solution (µg/mL)
Analysis: 90.0%–125.0% of the labeled amount of each
individual vitamin
• CONTENT OF VITAMINS B1, B2, B3, AND B6, AND
PANTOTHENIC ACID, Method 2
[NOTE—Where niacin or niacinamide, thiamine,
pyridoxine, riboflavin, and calcium pantothenate are
specified in the following procedure, use the vitamins
present in the formulation and the relevant USP
Reference Standards. Use low-actinic glassware.]
Solution A: 100 mM potassium dihydrogen phosphate,
adjusted with 10 N phosphoric acid to a pH of 3.0
Solution B: 5% edetate disodium in water. Heat the solution
while stirring for complete dissolution.
Extraction solution: Solution A and Solution B (4:1)
Mobile phases: For gradient, see Table 2.
Mobile phase A: 45 mM hexane sulphonic acid
sodium salt, 75 mM potassium dihydrogen phosphate,
2.5% acetonitrile in water, adjusted with 10 N
phosphoric acid to a pH of 3.5
Mobile phase B: Mobile phase A and acetonitrile (2:1)
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Table 2
Time Mobile Phase A Mobile Phase B
(min) (%) (%)
0.0 100 0
6.0 100 0
15.0 72 28
19.0 72 28
20.0 30 70
23.0 20 80
23.2 0 100
24.8 100 0
32.0 100 0
Niacin (or Niacinamide) standard stock solution: 1.5 mg/
mL of USP Niacin RS or USP Niacinamide RS in Solution A
Pyridoxine standard stock solution: 350 μg/mL of
pyridoxine from USP Pyridoxine Hydrochloride RS in
Solution A
al
Thiamine standard stock solution: 275 µg/mL of thiamine
from USP Thiamine Hydrochloride RS in Solution A
Riboflavin standard stock solution: 120 µg/mL of USP
Riboflavin RS in Solution A. Heat the solution while stirring
for complete dissolution.
Calcium pantothenate standard stock solution: 825
µg/mL of pantothenic acid from USP Calcium
Pantothenate RS in water
Standard solution: Accurately transfer calculated volumes
of Thiamine standard stock solution, Riboflavin standard stock
solution, Niacin standard stock solution (or Niacinamide
standard stock solution), Pyridoxine standard stock solution,
and Calcium pantothenate standard stock solution into a
200-mL volumetric flask to obtain a solution with final
vitamin concentrations similar to the nominal
concentrations in the Sample solution for the corresponding
vitamins. Dilute with Solution A to volume, and mix well.
Sample solution: Transfer a calculated amount of
accurately weighed Water-Soluble Vitamins Preparation
into a suitable volumetric flask to obtain a solution
containing known nominal concentrations of vitamins in
the range of 9.5–18 µg/mL for thiamine, 7–18 µg/mL for
riboflavin, 84–290 µg/mL for niacin, 54–140 µg/mL for
niacinamide, 9–17 µg/mL for pyridoxine, and 27–140
µg/mL for pantothenic acid. Prepare as follows.
For Water-Soluble Vitamins Preparation containing
NMT 1% of vitamin B2: Add Extraction solution,
preheated to 90°, to 50% of the final volume. Vortex for
20 s, sonicate for 5 min, cool to room temperature, dilute
with water to final volume, and mix well. Pass a portion
of the solution through a 0.45-µm nylon filter, and discard
the first 2 mL of the filtrate.
For Water-Soluble Vitamins Preparation containing NLT
1% of vitamin B2: Add Extraction solution, preheated to
90°, to 50% of the final volume. Heat in boiling water bath
until the vitamin B2 particles are dissolved, vortex for 20 s,
sonicate for 5 min, cool to room temperature, dilute with
water to final volume, and mix well. Pass a portion of the
solution through a 0.45-µm nylon filter, and discard the
first 2 mL of the filtrate.
For Water-Soluble Vitamins Preparation containing
coated vitamins: Add Extraction solution, preheated to
90°, to 50% of the final volume. Add 3 mL of isopropanol.
[NOTE—If there is no coated vitamin B2 in the preparation
do not add isopropanol.] Vortex, boil for 5 min, vortex for
20 s, sonicate for 5 min, cool to room temperature, dilute
with water to final volume, and mix well. Pass a portion
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3

of the solution through a 0.45 µm nylon filter, and discard Table 3 (continued)
the first 2 mL of the filtrate. Time Buffer Acetonitrile
For Water-Soluble Vitamins Preparation containing (min) (%) (%)
starch, flour, and cellulose: Add Extraction solution, 5.0 82.0 18.0
preheated to 90°, to 50% of the final volume. Boil for
5 min, vortex for 20 s, sonicate for 5 min, cool to room 5.5 75.0 25.0
temperature, and add acetonitrile to 15% of the final 6.5 75.0 25.0
volume. [NOTE—If there is no coated vitamin B2 in the
preparation do not add acetonitrile.] Vortex, dilute with 7.0 99.0 1.0
water to final volume, and mix well. Pass a portion of the 15.0 99.0 1.0
solution through a 0.45-µm nylon filter, and discard the
first 2 mL of the filtrate.
Chromatographic system Standard solution: 1 µg/mL of USP Cyanocobalamin
(See Chromatography á621ñ, System Suitability.) (Crystalline) RS in water
Mode: LC Riboflavin solution: 80 µg/mL of USP Riboflavin RS in water.
Detector: UV 254 nm for vitamins B1, B3, and B6; UV [NOTE—The solution can be swirled or placed on a steam
280 nm for vitamin B2; UV 210 nm for pantothenic acid bath for about 5–10 min with frequent swirling to assist in
Column: 4.6-mm × 15-cm; 5-µm packing L1 dissolution.]
Flow rate: 1 mL/min System suitability solution: Mix equal volumes of Standard
Injection volume: 10 µL solution and Riboflavin solution.
System suitability Sample solution: Transfer a calculated amount of
Sample: Standard solution accurately weighed Water-Soluble Vitamins Preparation,
nominally equivalent to 25 µg of cyanocobalamin, to a

al
Suitability requirements
Relative standard deviation: NMT 2.0% for each flask. Add 25 mL of water accurately measured. Record the
individual peak of niacin or niacinamide, thiamine, weight of the flask, sonicate for 5 min, and shake vigorously
pyridoxine, riboflavin, and pantothenic acid for 2 min. Add water to the previously recorded weight to
Analysis compensate for any solvent loss. Pass a portion of the
Samples: Standard solution and Sample solution solution through a polysulfone membrane filter of 0.45-µm
Calculate separately the percentages of the labeled amount
of vitamin B1 as thiamine ion (C12H17N4OS+), vitamin B2 as
riboflavin (C17H20N4O6), vitamin B3 as niacin (C6H5NO2) or
ci pore size, and discard the first 1 mL of the filtrate.
Chromatographic system
(See Chromatography á621ñ, System Suitability.)
Mode: LC
niacinamide (C6H6N2O), vitamin B6 as pyridoxine
Detector: UV 361 nm
(C8H11NO3), and pantothenic acid (C9H17NO5) in the
ffi
Column: 2.1-mm × 10-cm; 1.7-µm packing L1
portion of Water-Soluble Vitamins Preparation taken: Column temperature: 35°
Flow rate: 0.5 mL/min
Result = (rU/rS) × (CS/CU) × 100 Injection volume: 15 µL
System suitability
rU = peak area of the relevant vitamin from the Sample
Samples: Standard solution and System suitability solution
solution
O

[NOTE—The relative retention times for

rS = peak area of the relevant vitamin from the
cyanocobalamin and riboflavin are 1.0 and 1.04,
Standard solution
respectively.]
CS = concentration of the relevant vitamin Reference
Suitability requirements
Standard in the Standard solution (µg/mL)
Resolution: NLT 1.5 between cyanocobalamin and
CU = nominal concentration of the relevant vitamin in
riboflavin, System suitability solution
the Sample solution (µg/mL)
Relative standard deviation: NMT 2.0%, Standard
Acceptance criteria: 90.0%–125.0% of the labeled amount solution
of each individual vitamin Analysis
• CONTENT OF VITAMIN B12 Samples: Standard solution and Sample solution
[NOTE—Use low-actinic glassware throughout this Calculate the percentage of the labeled amount of vitamin
procedure. Inject samples within 30 min.] B12 as cyanocobalamin (C63H88CoN14O14P) in the portion
Buffer: Dissolve 470.5 mg of hexane sulphonic acid of Water-Soluble Vitamins Preparation taken:
sodium salt in water, add 1 mL of phosphoric acid, dilute
with water to 1000 mL, and mix. Adjust with 50% Result = (rU/rS) × (CS/CU) × 100
potassium hydroxide to a pH of 3.5.
rU = peak area of cyanocobalamin from the Sample
Mobile phase: Acetonitrile and Buffer. For gradient, see
solution
Table 3.
rS = peak area of cyanocobalamin from the Standard
solution
Table 3
CS = concentration of cyanocobalamin from USP
Time Buffer Acetonitrile Cyanocobalamin (Crystalline) RS in the Standard
(min) (%) (%)
solution (µg/mL)
0 99.0 1.0 CU = nominal concentration of cyanocobalamin in the
Sample solution (µg/mL)
0.5 99.0 1.0

1.2 97.7 2.3 Acceptance criteria: 90.0%–125.0%

• CONTENT OF VITAMIN C
1.4 95.0 5.0
[NOTE—Protect samples from air, light, and heat.]
2.5 93.0 7.0 Buffer: 2.04 g/L of monobasic potassium phosphate in
water. Adjust with phosphoric acid to a pH of 3.0.

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Mobile phase: Buffer
Diluent: 0.56 g of edetate disodium and 2.04 g of
monobasic potassium phosphate per 1000 mL of water.
Adjust with phosphoric acid to a pH of 3.0.
Standard solution: 0.25 mg/mL of USP Ascorbic Acid RS in
Diluent
Sample solution: Transfer a calculated amount of
accurately weighed Water-Soluble Vitamins Preparation,
nominally equivalent to 25 mg of ascorbic acid, into a
100-mL volumetric flask. Add 60 mL of Diluent, shake
mechanically for 15 min, dilute with Diluent to volume, mix
well, and pass through a membrane filter of 0.45-µm pore
size, discarding the first 4 mL of the filtrate.
Chromatographic system
(See Chromatography á621ñ, System Suitability.)
Mode: LC
Detector: UV 245 nm
Column: 4.6-mm × 25-cm; 5-µm packing L1
Flow rate: 1.0 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
Calculate the percentage of the labeled amount of
vitamin C as ascorbic acid (C6H8O6) in the portion of
Water-Soluble Vitamins Preparation taken:
Result = (rU/rS) × (CS/CU) × 100
ci
rU = peak area of ascorbic acid from the Sample
ffi
solution
rS = peak area of ascorbic acid from the Standard
solution
CS = concentration of ascorbic acid from USP Ascorbic
Acid RS in the Standard solution (mg/mL)
CU = nominal concentration of ascorbic acid in the
O
Sample solution (mg/mL)
Acceptance criteria: 90.0%–125.0%
• CONTENT OF BIOTIN, Method 11
Dehydrated mixtures yielding formulations similar to the
media described herein may be used provided that, when
constituted as directed, they have growth-promoting
properties equal to or superior to those obtained with the
media prepared as described herein.
Standard stock solution: 50 µg/mL of USP Biotin RS in 50%
alcohol. Store this solution in a refrigerator.
Standard solution: 0.1 ng/mL of USP Biotin RS in water,
prepared by dilution of the Standard stock solution with
water on the day of the assay
Sample solution: Transfer a portion of the Water-Soluble
Vitamins Preparation, equivalent to 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 50%
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
1 AOAC-certified
VitaFast Microbiological Microtiter Plate Test to
Quantitate Biotin, Art. No. P1003, is available.
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3.5 ± 0.1, and dilute with water to make 1000 mL. Add 20 g
of activated charcoal, stir for 1 h, and filter. Repeat the
treatment with activated charcoal. Store under toluene in a
cool place at a temperature not below 10°. Filter the
solution if a precipitate forms during storage.
Cystine–tryptophan solution: Suspend 4.0 g of l-cystine
in a solution of 1.0 g of l-tryptophan (or 2.0 g of
d,l-tryptophan) in 700–800 mL of water. Heat to 70°–80°,
and add dilute hydrochloric acid (1:2) dropwise, with
stirring, until the solids are dissolved. Cool, and add water
to make 1000 mL. Store under toluene in a cool place at a
temperature 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 add water to make 200 mL. Store under toluene in a
refrigerator.
Polysorbate 80 solution: 100 mg/mL of polysorbate 80 in
alcohol
Calcium pantothenate solution: 10 µg/mL of calcium
pantothenate in 50% alcohol. Store in a refrigerator.
Riboflavin–thiamine hydrochloride solution: 20 µg/mL of
riboflavin and 10 µg/mL of thiamine hydrochloride in
al
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)
Salt solution A: 25 g of monobasic potassium phosphate
and 25 g of dibasic potassium phosphate in water to make
500 mL. Add 5 drops of hydrochloric acid. Store under
toluene.
Salt solution B: 10 g of magnesium sulfate, 0.5 g of sodium
chloride, 0.5 g of ferrous sulfate, and 0.5 g of manganese
sulfate in water to make 500 mL. Add 5 drops of
hydrochloric acid, and mix. Store under toluene.
Basal medium stock solution: Dissolve anhydrous dextrose
and anhydrous sodium acetate in the solutions previously
mixed according to Table 4, and adjust with 1 N sodium
hydroxide to a pH of 6.8. Dilute with water to 250 mL.
Table 4
Name Amount
Acid-hydrolyzed casein solution 25 mL
Cystine–tryptophan solution 25 mL
Polysorbate 80 solution 0.25 mL
Dextrose, anhydrous 10 g
Sodium acetate, anhydrous 5g
Adenine–guanine–uracil solution 5 mL
Calcium pantothenate solution 5 mL
Riboflavin–thiamine hydrochloride
solution 5 mL
p-Aminobenzoic acid–niacin–pyri-
doxine hydrochloride solution 5 mL
Salt solution A 5 mL
Salt solution B 5 mL
Lactobacillus plantarum stock culture: 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
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an autoclave at 121° for 15 min, and allow the tubes to cool
in an upright position. Prepare stab cultures in three or
more of the tubes, using a pure culture of Lactobacillus
plantarum, 2 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 the Inoculum if the culture
is more than 1 week old.
Culture medium: To each of a series of test tubes containing
5.0 mL of Basal medium stock solution add 5.0 mL of water
containing 0.5 ng of biotin. Plug the tubes with cotton,
sterilize in an autoclave at 121° for 15 min, and cool.
Inoculum: [NOTE—A frozen suspension of Lactobacillus
plantarum may be used as the stock culture, provided it
yields an inoculum comparable to a fresh culture.] Transfer
cells from the Lactobacillus plantarum stock culture 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
ci
of the Basal medium stock solution and sufficient water to
make 10 mL. Place one complete set of Standard solution
ffi
and Sample solution tubes together in one tube rack and
place 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
O
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 solution 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 solution,
calculate the response from the sum of the duplicate values
of the transmittance (ΣS) as the difference, y = 2.00 − ΣS.
Plot this response against the logarithm of the mL of the
2 ATCC #8014 is suitable. This strain was formerly known as Lactobacillus
arabinosus 17-5.
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5
Standard solution per tube on the abscissa, using for the
ordinate either an arithmetic or a logarithmic scale,
whichever gives the better approximation to a straight line.
Draw the straight line or smooth curve that best fits the
plotted points.
Calculate the response, y = 2.00 − Σu, adding together the
two transmittances (Σu) 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 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.
Calculate the amount, in µg, of biotin (C10H16N2O3S) in the
portion of Water-Soluble Vitamins Preparation taken:
antilog M = antilog (M′ + log R)
M′ = log-relative potency
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R = amount of biotin presumed to be present in the
portion of Water-Soluble Vitamins Preparation
taken (µg)
Calculate the percentage of the labeled amount of biotin
(C10H16N2O3S) in the portion of Water-Soluble Vitamins
Preparation taken:
Result = [(antilog M)/N] × 100
N = nominal amount of biotin in the portion of
Water-Soluble Vitamins Preparation taken (µg)
Replication: Repeat the entire determination at least once,
using separate preparations of Sample solution. 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 á111ñ,
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 Water-Soluble Vitamins
Preparation under assay.
Acceptance criteria: 90.0%–125.0%
• CONTENT OF BIOTIN, Method 2
Solution A: 0.1% of phosphoric acid, 0.1% of sodium
perchlorate, and 10% of acetonitrile in water
Solution B: 0.1% of phosphoric acid, 0.1% of sodium
perchlorate, and 40% of acetonitrile in water
Mobile phase: See Table 5.
Table 5
Time Solution A Solution B
(min) (%) (%)
0 100 0
13.0 100 0
13.2 0 100
16.2 0 100
16.4 100 0
25.0 100 0
Standard stock solution: Transfer 34 mg of USP Biotin RS
into a 250-mL volumetric flask, add 15 mL ethanol, vortex
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(EST)
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6

for 30 s, and sonicate at 60° for 2 min. Add water preheated Result = (rU/rS) × (CS/CU) × 100
to 50°–80°, vortex, and sonicate at 80° for 10 min, followed
by 10-min sonication without heat. Cool to room rU = peak area of biotin from the Sample solution
temperature, dilute with water to volume, and mix well. rS = peak area of biotin from the Standard solution
Standard solution: 2 µg/mL of USP Biotin RS prepared by CS = concentration of USP Biotin RS in the Standard
diluting the Standard stock solution in water solution (µg/mL)
Sample solution: Transfer a calculated amount of CU = nominal concentration of biotin in the Sample
accurately weighed Water-Soluble Vitamins Preparation, solution (µg/mL)
nominally equivalent to 200 µg of biotin, into a 100-mL
volumetric flask. Prepare as follows. Acceptance criteria: 90.0%–125.0%
For Water-Soluble Vitamins Preparation containing
NMT 0.1% of biotin: Add water preheated to 50°–80°, CONTAMINANTS
vortex for 20 s, sonicate at 60° for 10 min, followed by • MICROBIAL ENUMERATION TESTS á2021ñ: The total aerobic
10-min sonication without heat, and cool to room microbial count does not exceed 103 cfu/g, and the total
temperature. Dilute with water to volume, and mix well. combined molds and yeasts count does not exceed 102 cfu/
Pass a portion of the solution through a 0.45-mm nylon g.
filter, and discard the first 2 mL of the filtrate. • ABSENCE OF SPECIFIED MICROORGANISMS á2022ñ, Test
For Water-Soluble Vitamins Preparation containing NLT Procedures, Test for Absence of Salmonella Species and Test
0.1% of biotin: Add 5 mL of ethanol, vortex, add water for Absence of Escherichia coli: Meets the requirements
preheated to 50°–80°, vortex for 20 s, sonicate at 60° for ADDITIONAL REQUIREMENTS
10 min, followed by 10-min sonication without heat, and • PACKAGING AND STORAGE: Preserve in tight, light- and
cool to room temperature. Dilute with water to volume, moisture-resistant containers.

al
and mix well. Pass a portion of the solution through a • LABELING: The label states the quantity of each vitamin in
0.45-mm nylon filter, and discard the first 2 mL of the terms of metric units and, where necessary, the chemical
filtrate. form in which a vitamin is present. The label also states the
Chromatographic system name and content of any carriers and antioxidants added.
(See Chromatography á621ñ, System Suitability.) Where more than one assay method is given for a particular
Mode: LC vitamin, the labeling states with which assay method the
Detector: UV 200 nm
Column: 4.6-mm × 15-cm; 3-µm packing L7
Column temperature: 30°
ci product complies only if Method 1 is not used.
• USP REFERENCE STANDARDS á11ñ
USP Ascorbic Acid RS
Flow rate: 1.0 mL/min USP Biotin RS
Injection volume: 10 µL USP Calcium Pantothenate RS
ffi
System suitability USP Cyanocobalamin (Crystalline) RS
Sample: Standard solution USP Folic Acid RS
Suitability requirements USP Niacin RS
Relative standard deviation: NMT 3.0% USP Niacinamide RS
Analysis USP Pyridoxine Hydrochloride RS
Samples: Standard solution and Sample solution USP Riboflavin RS
O

Calculate the percentage of the labeled amount of biotin USP Thiamine Hydrochloride RS▲ (USP 1-May-2021)
(C10H16N2O3S) in the portion of Water-Soluble Vitamins
Preparation taken:

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