6/10/2020 USP-NF 〈481〉 Riboflavin Assay

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〈481〉 RIBOFLAVIN ASSAY

ASSAY
• CHEMICAL METHODS, PROCEDURE 1
The following procedure is suitable for preparations in which ribo avin is a constituent of a mixture of several ingredients. In
using the procedure, keep the pH of solutions below 7.0, and protect the solutions from direct sunlight at all stages.
Standard ribo avin stock solution: To 50.0 mg of USP Ribo avin RS, previously dried and stored protected from light in a desiccator
over phosphorus pentoxide, add about 300 mL of 0.02 N acetic acid, and heat the mixture on a steam bath with frequent agitation
until the ribo avin has dissolved, then cool. To this solution add 0.02 N acetic acid to make 500 mL; then mix. Store the solution
under toluene in a refrigerator.
Dilute an accurately measured portion of this solution by using 0.02 N acetic acid to a concentration of 10.0 µg/mL of the dried
USP Ribo avin RS to obtain the Standard ribo avin stock solution. Store the solution under toluene in a refrigerator.
Standard solution: Dilute with water 10.0 mL of Standard ribo avin stock solution in a 100-mL volumetric ask to volume, and mix.
Each mL represents 1.0 µg of USP Ribo avin RS. Prepare a fresh Standard solution for each assay.
Sample solution: Place an amount of the material to be assayed in a ask of suitable size, and add a volume of 0.1 N hydrochloric
acid equal in mL to NLT 10 times the dry weight of the material in grams, but the resulting solution will contain NMT 100 µg/mL of
ribo avin. If the material is not readily soluble, comminute the material so that it may be evenly dispersed in the liquid. Agitate

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vigorously, and wash down the sides of the ask with 0.1 N hydrochloric acid.
Heat the mixture in an autoclave at 121°–123° for 30 min, and cool. If clumping occurs, agitate the mixture until the particles
are evenly dispersed. Adjust the mixture, with vigorous agitation, with sodium hydroxide solution1 to a pH of 6.0–6.5, then add
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hydrochloric acid solution1 immediately until no further precipitation occurs (usually at a pH of approximately 4.5, which is the
isoelectric point of many of the proteins present). Dilute the mixture with water to make a measured volume that contains about
0.11 µg of ribo avin in each mL, and lter through paper known not to adsorb ribo avin. To an aliquot of the ltrate add, with
vigorous agitation, sodium hydroxide solution1 to produce a pH of 6.6–6.8, dilute the solution with water to make a nal
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measured volume that contains approximately 0.1 µg of ribo avin in each mL, and if cloudiness occurs, lter again.
Instrumental conditions
(See Fluorescence Spectroscopy 〈853〉.)
Mode: Fluorescence
Excitation wavelength: 444 nm
FF

Emission wavelength: 530 nm

Analysis
Samples: Standard solution, Sample solution, and Blank
To each of four or more tubes (or reaction vessels) add 10.0 mL of the Sample solution. To each of two or more of these
tubes add 1.0 mL of the Standard solution, and mix; to each of two or more of the remaining tubes add 1.0 mL of water, and
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mix. To each tube add 1.0 mL of glacial acetic acid, mix, then add, with mixing, 0.50 mL of potassium permanganate solution
(1 in 25), and allow to stand for 2 min. To each tube add, with mixing, 0.50 mL of hydrogen peroxide solution, whereupon the
permanganate color is destroyed within 10 s. Shake the tubes vigorously until excess oxygen is expelled. Remove any gas
bubbles remaining on the sides of the tubes after foaming has ceased by tipping the tubes so that the solution ows slowly
from end to end.
Measure the uorescence of all tubes, designating the average reading from the tubes containing only the Sample solution
as IU, and designating the average from the tubes containing both the Sample solution and the Standard solution as IS. Then to
each of one or more tubes of each kind add, with mixing, 20 mg of sodium hydrosul te, and within 5 s again measure the
uorescence, designating the average reading as IB.
Calculate the quantity, in mg, of ribo avin (C17H20N4O6) in each mL of the Sample solution taken:

Result = [0.0001 × (IU − IB)]/(IS − IU)

IU = average reading of the tubes containing only the Sample solution

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6/10/2020 USP-NF 〈481〉 Riboflavin Assay
IB = average reading of uorescence of the tubes after adding 20 mg of sodium hydrosul te

IS = average reading of the tubes containing both the Sample solution and Standard solution

Calculate the quantity, in mg, of ribo avin (C17H20N4O6) in each capsule or tablet.

• CHEMICAL METHODS, PROCEDURE 2

This procedure is suitable for the determination of ribo avin as a dietary ingredient or active pharmaceutical ingredient. [ NOTE—
Conduct the entire Analysis without exposure to direct sunlight.]
Standard solution: Transfer 50 mg of USP Ribo avin RS to a 1000-mL volumetric ask containing 50 mL of water. Add 5 mL of
acetic acid and su cient water to make 800 mL. Heat on a steam bath, protected from light, with frequent agitation until dissolved.
Cool to 25°, and dilute with water to volume. Dilute this solution with water to bring it within the operating sensitivity of the
uorometer used.
Sample solution: Transfer 50 mg of Ribo avin to a 1000-mL volumetric ask containing 50 mL of water. Add 5 mL of acetic acid and
su cient water to make 800 mL. Heat on a steam bath, protected from light, with frequent agitation until dissolved. Cool to 25°,
and dilute with water to volume. Dilute this solution with water to bring it to the same concentration as that of the Standard
solution.
Blank: Prepare as directed for the Sample solution, except omit the test specimen.
Instrumental conditions
(See Fluorescence Spectroscopy 〈853〉.)
Mode: Fluorescence
Excitation wavelength: 444 nm
Emission wavelength: 530 nm
Analysis
Samples: Standard solution, Sample solution, and Blank

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Measure the uorescence intensity of the Standard solution. Immediately after the reading, add to the solution 10 mg of
sodium hydrosul te, stirring with a glass rod until dissolved, and at once measure the uorescence again. [ NOTE— Depending
on the nal concentration of ribo avin in the solution, it may be necessary to increase the amount of sodium hydrosul te to
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suppress the uorescence activity completely.] The difference between the two readings represents the uorescence intensity
(IS) due to the Standard solution. Similarly, measure the uorescence intensity (IU) due to the Sample solution. Perform the blank
determination, and make any necessary correction.
Calculate the percentage of ribo avin (C17H20N4O6) in the portion of Ribo avin taken:
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Result = (IU/IS) × (CS/CU) × 100

IU = uorescence of the Sample solution

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IS = uorescence of the Standard solution

CS = concentration of USP Ribo avin RS in the Standard solution (µg/mL)

CU = concentration of ribo avin in the Sample solution (µg/mL)

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The following liquid chromatographic procedures are provided for the determination of ribo avin as an active pharmaceutical
ingredient, a dietary supplement ingredient, or a component in the dietary supplements or pharmaceutical dosage forms. Use the
appropriate USP Reference Standards.
Throughout these procedures, protect solutions containing and derived from the test specimen and the Reference Standards
from the atmosphere and light, preferably by the use of low-actinic glassware.
Chromatographic Methods, Procedure 1
This procedure can be used to determine ribo avin in:
Oil- and Water-Soluble Vitamins Capsules
Oil- and Water-Soluble Vitamins Tablets
Oil- and Water-Soluble Vitamins with Minerals Capsules
Oil- and Water-Soluble Vitamins with Minerals Tablets
Water-Soluble Vitamins Capsules
Water-Soluble Vitamins Tablets
Water-Soluble Vitamins with Minerals Capsules
Water-Soluble Vitamins with Minerals Tablets

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This is the procedure that involves the extraction of ribo avin from the formulation by the Diluent, heat, and mechanical shaking.
Unless speci ed in the individual monographs, the Standard solution, Sample solutions, and reagent solutions are prepared as
follows.
Diluent: Acetonitrile, glacial acetic acid, and water (5:1:94)
Mobile phase: A mixture of methanol, glacial acetic acid, and water (27:1:73) containing 140 mg of sodium 1-hexanesulfonate per
100 mL
Standard solution: Transfer 20 mg of USP Ribo avin RS to a 200-mL volumetric ask, and add 180 mL of Diluent. Immerse the ask
in a hot water bath maintained at 65°–70° for 10 min with regular shaking, or using a vortex mixer, until all of the solid materials are
dissolved. Chill rapidly in a cold water bath for 10 min to room temperature, and dilute with Diluent to volume.
Sample solution for capsules: Weigh NLT 20 capsules in a tared weighing bottle. Open the capsules, without loss of shell material,
and transfer the contents to a 100-mL beaker. Remove any contents adhering to the shells by washing with several portions of
ether. Discard the washings, and dry the capsule shells with the aid of a current of dry air until the odor of ether is no longer
perceptible. Weigh the empty capsule shells in the tared weighing bottle, and calculate the average net weight per capsule. Transfer
a portion of the capsule contents, equivalent to 2.5 mg of ribo avin, to a 50-mL centrifuge tube. Add 25.0 mL of Diluent, and mix
using a vortex mixer for 30 s to completely suspend the powder. Immerse the centrifuge tube in a hot water bath maintained at
65°–70°, heat for 5 min, and mix on a vortex mixer for 30 s. Return the tube to the hot water bath, heat for another 5 min, and mix
on a vortex mixer for 30 s. Filter a portion of the solution, cool to room temperature, and use the clear ltrate. [ NOTE— Use the
ltrate within 3 h of ltration.]
Sample solution for tablets: Finely powder NLT 30 tablets. Transfer a portion of the powder, equivalent to 2.5 mg of ribo avin, to a
50-mL centrifuge tube. Add 25.0 mL of Diluent, and mix using a vortex mixer for 30 s to completely suspend the powder. Immerse
the centrifuge tube in a hot water bath maintained at 65°–70°, heat for 5 min, and mix on a vortex mixer for 30 s. Return the tube to
the hot water bath, heat for another 5 min, and mix on a vortex mixer for 30 s. Filter a portion of the solution, cool to room
temperature, and use the clear ltrate. [ NOTE— Use the ltrate within 3 h of ltration.]
Chromatographic system

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(See Chromatography 〈621〉, System Suitability.)
Mode: LC
Detector: UV 280 nm
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Column: 4.6-mm × 25-cm; packing L1
Flow rate: 1 mL/min
Injection volume: 10 µL
System suitability
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Sample: Standard solution

Suitability requirements
Relative standard deviation: NMT 3.0%
Analysis
Samples: Standard solution and appropriate Sample solution
FF

Calculate the percentage of the labeled amount of ribo avin (C17H20N4O6) in the portion of sample taken:

Result = (rU/rS) × (CS/CU) × 100

rU = peak response of ribo avin from the appropriate Sample solution

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rS = peak response of ribo avin from the Standard solution

CS = concentration of USP Ribo avin RS in the Standard solution (mg/mL)

CU = nominal concentration of ribo avin in the appropriate Sample solution (mg/mL)

• CHROMATOGRAPHIC METHODS, PROCEDURE 2

This procedure can be used to determine ribo avin in:
Oil- and Water-Soluble Vitamins Capsules
Oil- and Water-Soluble Vitamins Tablets
Oil- and Water-Soluble Vitamins with Minerals Capsules
Oil- and Water-Soluble Vitamins with Minerals Tablets
Water-Soluble Vitamins Capsules
Water-Soluble Vitamins Tablets
Water-Soluble Vitamins with Minerals Capsules

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This Water-Soluble Vitamins

is the procedure with Minerals
that involves Tablets of ribo avin from the formulation by the Extraction solvent, heat, and
the extraction
mechanical shaking.
Unless speci ed in the individual monographs, the Standard solution, Sample solutions, and reagent solutions are prepared as
follows.
Extraction solvent: Transfer 1 mL of glacial acetic acid and 2.5 g of edetate disodium to a 100-mL volumetric ask. Dissolve in and
dilute with water to volume. Mix the resulting solution with methanol (3:1).
Solution A: 6.8 g of sodium acetate per 1000 mL of water
Mobile phase: Prepare a mixture of Solution A and methanol (13:7). Add 2 mL of triethylamine per L of the mixture, and adjust with
glacial acetic acid to a pH of 5.2.
Standard stock solution: Transfer 20 mg of USP Ribo avin RS to a 200-mL volumetric ask, and add 180 mL of Extraction solvent.
Immerse the ask for 5 min in a water bath maintained at 65°–75°. Mix well, and repeat if necessary until dissolved. Chill rapidly in
a cold water bath to room temperature, and dilute with Extraction solvent to volume.
Standard solution: Dilute 5.0 mL of the Standard stock solution with Extraction solvent to 25.0 mL.
Sample solution for capsules: Weigh NLT 20 capsules in a tared weighing bottle. Open the capsules, without loss of shell material,
and transfer the contents to a beaker. Remove any contents adhering to the shells by washing with several portions of ether.
Discard the washings, and dry the capsule shells with the aid of a current of dry air. Weigh the empty capsule shells in the tared
weighing bottle, and calculate the net weight of the capsule contents. Transfer a portion of the capsule contents, equivalent to 2
mg of ribo avin, to a 200-mL volumetric ask. Add 100.0 mL of Extraction solvent, and mix for 20 min using a wrist-action shaker.
Immerse the ask in a water bath maintained at 70°–75°, and heat for 20 min. Mix on a vortex mixer for 30 s, cool to room
temperature, and lter. Use the clear ltrate.
Sample solution for tablets: Finely powder NLT 20 tablets. Transfer a portion of the powder, equivalent to 2 mg of ribo avin, to a
200-mL volumetric ask. Add 100.0 mL of Extraction solvent, and mix for 20 min using a wrist-action shaker. Immerse the ask in a
water bath maintained at 70°–75°, and heat for 20 min. Mix on a vortex mixer for 30 s, cool to room temperature, and lter. Use the
clear ltrate.

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Chromatographic system
(See Chromatography 〈621〉, System Suitability.)
Mode: LC
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Detector: UV 254 nm
Column: 4.6-mm × 25-cm; packing L1
Flow rate: 1 mL/min
Injection volume: 20 µL
IC

System suitability
Sample: Standard solution
Suitability requirements
Relative standard deviation: NMT 3.0%
Analysis
FF

Samples: Standard solution and appropriate Sample solution

Calculate the percentage of the labeled amount of ribo avin (C17H20N4O6) in the portion of sample taken:

Result = (rU/rS) × (CS/CU) × 100

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rU = peak response of ribo avin from the appropriate Sample solution

rS = peak response of ribo avin from the Standard solution

CS = concentration of USP Ribo avin RS in the Standard solution (mg/mL)

CU = nominal concentration of ribo avin in the appropriate Sample solution (mg/mL)

• CHROMATOGRAPHIC METHODS, PROCEDURE 3

This procedure can be used to determine ribo avin in:
Oil- and Water-Soluble Vitamins Capsules
Oil- and Water-Soluble Vitamins Tablets
Oil- and Water-Soluble Vitamins with Minerals Capsules
Oil- and Water-Soluble Vitamins with Minerals Tablets
Water-Soluble Vitamins Capsules
Water-Soluble Vitamins Tablets

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Water-Soluble
Water-Soluble Vitamins
Vitamins with
with Minerals
Minerals Capsules
Tablets
This is the procedure that involves the extraction of ribo avin from the formulation by mixtures of organic solvents, heat, and
mechanical shaking.
Unless speci ed in the individual monographs, the Standard solutions, Sample solutions, and reagent solutions are prepared as
follows.
Diluent: 25 mg/mL of edetate disodium in water
Mobile phase: Transfer 0.4 mL of triethylamine, 15.0 mL of glacial acetic acid, and 350 mL of methanol to a 2000-mL volumetric
ask. Dilute with 0.008 M sodium 1-hexanesulfonate to volume.
Standard stock solution: 0.08 mg/mL of USP Ribo avin RS in Diluent, with heating if necessary
Standard solution for capsules/tablets: Transfer 5.0 mL of Standard stock solution to a stoppered 125-mL ask. Add 10.0 mL of a
mixture of methanol and glacial acetic acid (9:1) and 30.0 mL of a mixture of methanol and ethylene glycol (1:1). Insert the stopper,
shake for 15 min in a water bath maintained at 60°, and cool. Filter, discarding the rst few mL of the ltrate.
Standard solution for oral solution: 8 µg/mL of USP Ribo avin RS in Diluent, diluted from the Standard stock solution
Sample solution for capsules: Weigh NLT 20 capsules in a tared weighing bottle. Open the capsules, without the loss of shell
material, and transfer the contents to a 100-mL beaker. Remove any contents adhering to the empty shells by washing, if necessary,
with several portions of ether. Discard the washings, and dry the capsule shells with the aid of a current of dry air until the odor of
ether is no longer perceptible. Weigh the empty capsule shells in the tared weighing bottle, and calculate the average net weight per
capsule. Transfer a portion of the capsule contents, equivalent to 0.4 mg of ribo avin, to a stoppered 125-mL ask. Add 10.0 mL of
a mixture of methanol and glacial acetic acid (9:1) and 30.0 mL of a mixture of methanol and ethylene glycol (1:1). Insert the
stopper, shake for 15 min in a water bath maintained at 60°, and cool. Filter, discarding the rst few mL of the ltrate.
Sample solution for oral solution: Equivalent to 8 µg/mL of ribo avin from oral solution in the Diluent
Sample solution for tablets: Weigh and nely powder NLT 20 tablets. Transfer a portion of the powder, equivalent to 0.4 mg of
ribo avin, to a stoppered 125-mL ask. Add 10.0 mL of a mixture of methanol and glacial acetic acid (9:1) and 30.0 mL of a mixture
of methanol and ethylene glycol (1:1). Insert the stopper, shake for 15 min in a water bath maintained at 60°, and cool. Filter,

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discarding the rst few mL of the ltrate.
Chromatographic system
(See Chromatography 〈621〉, System Suitability.)
IA
Mode: LC
Detector: UV 270 nm
Column: 4.6-mm × 25-cm; packing L7
Column temperature: 50°
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Flow rate: 2 mL/min

Injection volume: 5 µL
System suitability
Sample: Standard solution
Suitability requirements
FF

Relative standard deviation: NMT 2.0%

Analysis
Samples: Appropriate Standard solution and appropriate Sample solution
For capsules and tablets, calculate the percentage of the labeled amount of ribo avin (C17H20N4O6) in the portion of sample
taken:
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Result = (rU/rS) × (CS/CU) × 100

rU = peak response of ribo avin from the appropriate Sample solution

rS = peak response of ribo avin from the Standard solution for capsules/tablets

CS = concentration of USP Ribo avin RS in the Standard solution for capsules/tablets (mg/mL)

CU = nominal concentration of ribo avin in the appropriate Sample solution (mg/mL)

For oral solution, calculate the percentage of the labeled amount of ribo avin (C17H20N4O6) in the portion of sample taken:

Result = (rU/rS) × (CS/CU) × 100

rU = peak response of ribo avin from the Sample solution for oral solution

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rS = peak response of ribo avin from the Standard solution for oral solution

CS = concentration of USP Ribo avin RS in the Standard solution for oral solution (mg/mL)

CU = nominal concentration of ribo avin in the Sample solution for oral solution (mg/mL)

• CHROMATOGRAPHIC METHODS, PROCEDURE 4

This procedure can be used to determine ribo avin in:
Oil- and Water-Soluble Vitamins Capsules
Oil- and Water-Soluble Vitamins Tablets
Oil- and Water-Soluble Vitamins with Minerals Capsules
Oil- and Water-Soluble Vitamins with Minerals Tablets
Water-Soluble Vitamins Capsules
Water-Soluble Vitamins Tablets
Water-Soluble Vitamins with Minerals Capsules
Water-Soluble Vitamins with Minerals Tablets
This is a newly added procedure as part of the USP monograph modernization efforts. The procedure uses hydrophilic
interaction liquid chromatography (HILIC), and the sample preparation involves the extraction of ribo avin from the formulation by
the Diluent, heat, and mechanical shaking.
Unless speci ed in the individual monographs, the Standard solution, Sample solutions, and reagent solutions are prepared as
follows.
Diluent: Methanol, glacial acetic acid, and water (50:1:49)
Solution A: 50 mM ammonium formate; adjust with ammonium hydroxide to a pH of 9.0.
Solution B: Acetonitrile
Mobile phase: Gradient elution. See Table 1.

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Table 1
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Time (min) Solution A (%) Solution B (%)

0 11 89
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8 17 83

15 23 77
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20 30 70

21 50 50

24 50 50
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25 11 89

30 11 89

Standard solution: Transfer 20 mg of USP Ribo avin RS to a 200-mL volumetric ask, and add 160 mL of Diluent. Immerse the ask
in a hot water bath maintained at 65°–70° for 10 min with regular shaking or using a vortex mixer, until all of the solid materials are
dissolved. Chill rapidly in a cold water bath for 10 min to room temperature, and dilute with Diluent to volume.
Sample solution for capsules: Weigh NLT 20 capsules in a tared weighing bottle. Open the capsules, without the loss of shell
material, and transfer the contents to a 100-mL beaker. Remove any contents adhering to the empty shells by washing, if necessary,
with several portions of ether. Discard the washings, and dry the capsule shells with the aid of a current of dry air until the odor of
ether is no longer perceptible. Weigh the empty capsule shells in the tared weighing bottle, and calculate the average net weight per
capsule. Transfer a portion of the capsule contents, equivalent to 2.5 mg of ribo avin, to a 50-mL centrifuge tube. Add 25.0 mL of
Diluent, and mix using a vortex mixer for 30 s to completely suspend the powder. Immerse the centrifuge tube in a hot water bath

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maintained at 68°, heat for 10 min, and mix on a vortex mixer for 30 s. Return the tube to the hot water bath, heat for another 10
min, and mix on a vortex mixer for 30 s. Filter a portion of the solution, cool to room temperature, and use the clear ltrate.
Sample solution for tablets: Finely powder NLT 30 tablets. Transfer a portion of the powder, equivalent to 2.5 mg of ribo avin, to a
50-mL centrifuge tube. Add 25.0 mL of Diluent, and mix using a vortex mixer for 30 s to completely suspend the powder. Immerse
the centrifuge tube in a hot water bath maintained at 65°–70°, heat for 10 min, and mix on a vortex mixer for 30 s. Return the tube
to the hot water bath, heat for another 10 min, and mix on a vortex mixer for 30 s. Filter a portion of the solution, cool to room
temperature, and use the clear ltrate. [ NOTE— Use the ltrate within 3 h of ltration.]
Chromatographic system
(See Chromatography 〈621〉, System Suitability.)
Mode: LC
Detector: UV 267 nm
Column: 4.6-mm × 15-cm; 3.5-µm packing L68
Flow rate: 1.2 mL/min
Injection volume: 10 µL
System suitability
Sample: Standard solution
Suitability requirements
Relative standard deviation: NMT 2.0%
Analysis
Samples: Standard solution and appropriate Sample solution
Calculate the percentage of the labeled amount of ribo avin (C17H20N4O6) in the portion of sample taken:

Result = (rU/rS) × (CS/CU) × 100

rU = peak response of ribo avin from the appropriate Sample solution

rS = peak response of ribo avin from the Standard solution

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CS = concentration of USP Ribo avin RS in the Standard solution (mg/mL)

CU = nominal concentration of ribo avin in the appropriate Sample solution (mg/mL)

ADDITIONAL REQUIREMENTS
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• USP REFERENCE STANDARDS 〈11〉

USP Ribo avin RS
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1 The concentrations of the hydrochloric acid and sodium hydroxide solutions used are not stated in each instance, because these
concentrations may be varied depending upon the amount of material taken for assay, volume of test solution, and buffering effect of
material.

Auxiliary Information- Please check for your question in the FAQs before contacting USP.
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Topic/Question Contact Expert Committee

<481> RIBOFLAVIN ASSAY Natalia Davydova NBDS2020 Non-botanical Dietary

Scienti c Liaison Supplements

Most Recently Appeared In:

Pharmacopeial Forum: Volume No. 41(6)

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