Riboavin 5′-Phosphate Sodium

This article is compiled based on the United States Pharmacopeia (USP) – 2025 Edition

Issued and maintained by the United States Pharmacopeial Convention (USP)

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1 DEFINITION

Riboflavin 5′-Phosphate Sodium contains NLT the equivalent of 73.0% and NMT the equivalent of 79.0% of riboflavin (C₁₇H₂₀N₄O₆), calculated on the dried basis.

2 IDENTIFICATION

A. Color and Fluorescence of Solution

Sample solution: 0.01 mg/mL in water

Analysis: Alternately expose to transmitted light and long-wavelength UV light.

Acceptance criteria: The solution is pale greenish yellow by transmitted light, and it exhibits an intense yellowish green fluorescence by reflected light under long-wavelength UV light that disappears upon the addition of mineral acids or alkalies.

B. Identification Tests—General, Sodium 〈191〉 and Phosphate 〈191〉

Sample solution: To 0.5 g add 10 mL of nitric acid. Evaporate the mixture on a water bath to dryness, and ignite the residue until the carbon is removed. Dissolve the residue in 5 mL of water, and filter.

Acceptance criteria: The Sample solution meets the requirements.

3 ASSAY

Procedure

[Note—Conduct the assay so that all solutions are protected from actinic light at all stages, preferably by using low-actinic glassware.]

Standard solution: 0.35 µg/mL prepared as follows. Transfer 35 mg of USP Riboflavin RS to a 250-mL conical flask. Add 20 mL of pyridine and 75 mL of water, and dissolve the riboflavin by frequent shaking. Transfer the solution to a 1000-mL volumetric flask, and dilute with water to volume. Transfer 10.0 mL of this solution to a second 1000-mL volumetric flask, add sufficient 0.1 N sulfuric acid (about 4 mL) so that the final pH of the solution is between 5.9 and 6.1, and dilute with water to volume.

Sample solution: Transfer 50 mg of Riboflavin 5′-Phosphate Sodium to a 250-mL conical flask. Add 20 mL of pyridine and 75 mL of water, and dissolve the riboflavin by frequent shaking. Transfer the solution to a 1000-mL volumetric flask, and dilute with water to volume. Transfer 10.0 mL of this solution to a second 1000-mL volumetric flask, add sufficient 0.1 N sulfuric acid (about 4 mL) so that the final pH of the solution is between 5.9 and 6.1, and dilute with water to volume.

Blank: Proceed as directed in the Analysis, omitting the test specimen.

Instrumental conditions

(See Fluorescence Spectroscopy 〈853〉.)

Mode: Fluorescence

Excitation wavelength: 440 nm

Emission wavelength: 530 nm

Analysis

Samples: Standard solution, Sample solution, and Blank

Determine the maximum fluorescence intensities of the solutions against the Blank.

Calculate the percentage of riboflavin (C₁₇H₂₀N₄O₆) in the portion of Riboflavin 5′-Phosphate Sodium taken:

Result = (Iᵤ / Iₛ) × (Cₛ / Cᵤ) × 100

Iᵤ = fluorescence intensity from the Sample solution

Iₛ = fluorescence intensity from the Standard solution

Cₛ = concentration of USP Riboflavin RS in the Standard solution (µg/mL)

Cᵤ = concentration of Riboflavin 5′-Phosphate Sodium in the Sample solution (µg/mL)

Acceptance criteria: 73.0%–79.0% on the dried basis

4 IMPURITIES

Free Phosphate

Acid molybdate solution: Prepare a 70-mg/mL solution of ammonium molybdate in water. Dilute 25 mL of this solution with water to 200 mL. To this dilution slowly add 25 mL of 7.5 N sulfuric acid.

Ferrous sulfate solution: 100 mg/mL of ferrous sulfate in 0.15 N sulfuric acid, prepared just before use

Standard solution: 44.0 µg/mL of monobasic potassium phosphate in water

Sample solution: 3 mg/mL of Riboflavin 5′-Phosphate Sodium in water

Blank: Water

Instrumental conditions

(See Ultraviolet-Visible Spectroscopy 〈857〉.)

Mode: UV-Vis

Analytical wavelength: 700 nm

Cell: 1 cm

Analysis

Samples: Standard solution, Sample solution, and Blank

Transfer 10.0 mL each to separate 50-mL conical flasks. Add 10.0 mL of Acid molybdate solution and 5.0 mL of Ferrous sulfate solution. Mix and determine the absorbances against the Blank.

Acceptance criteria: NMT 1% as PO₄. The absorbance of the Sample solution is NMT that of the Standard solution.

Free Riboflavin and Riboflavin Diphosphates

[Note—Protect all solutions from actinic light.]

Mobile phase: Methanol and 0.054 M monobasic potassium phosphate (15:85)

System suitability solution: Prepare a 2-mg/mL solution of USP Phosphated Riboflavin RS in water. Dilute to 160 µg/mL with Mobile phase.

Standard solution: 60 mg USP Riboflavin RS in 250 mL. Dissolve in 1 mL hydrochloric acid, dilute with water to volume, then dilute to 9.6 µg/mL with Mobile phase.

Sample solution: 2-mg/mL solution of Riboflavin 5′-Phosphate Sodium in water. Dilute to 160 µg/mL with Mobile phase.

Chromatographic system

Mode: LC

Detector: Fluorometer

Excitation: 440 nm

Emission: 530 nm (monochromator) or 470 nm (filter)

Column: 3.9-mm × 30-cm; packing L1

Flow rate: 2 mL/min

Injection size: 100 µL

The retention time of riboflavin 5′-monophosphate is 20–25 min.

System suitability

Resolution: NLT 1.0 between riboflavin 4′-monophosphate and riboflavin 5′-monophosphate

Relative standard deviation: NMT 1.5% for riboflavin 5′-monophosphate

Analysis

Samples: Standard solution and Sample solution

Identify peaks by comparison with the System suitability solution.

Calculate free riboflavin:

Result = (rᵤ / rₛ) × (Cₛ / Cᵤ) × 100

rᵤ = peak area of free riboflavin from Sample

rₛ = peak area of riboflavin from Standard

Cₛ = concentration of USP Riboflavin RS (µg/mL)

Cᵤ = concentration of Riboflavin 5′-Phosphate Sodium (µg/mL)

Calculate riboflavin diphosphates:

Result = (rᵤ / rₛ) × (Cₛ / Cᵤ) × 100

rᵤ = sum of peak areas of riboflavin diphosphates

rₛ = peak area of riboflavin from Standard

Cₛ = concentration of USP Riboflavin RS (µg/mL)

Cᵤ = concentration of Riboflavin 5′-Phosphate Sodium (µg/mL)

Acceptance criteria:

Free riboflavin: NMT 6.0% (dried basis)

Riboflavin diphosphates: NMT 6.0% (dried basis)

Limit of Lumiflavin

Alcohol-free chloroform: Prepare by washing chloroform with water and drying over anhydrous sodium sulfate.

Sample solution: Shake 35 mg of Riboflavin 5′-Phosphate Sodium with 10 mL of Alcohol-free chloroform for 5 min, and filter.

Blank: Alcohol-free chloroform

Instrumental conditions

(See Ultraviolet-Visible Spectroscopy 〈857〉.)

Analytical wavelength: 440 nm

Cell: 1 cm

Analysis

Measure absorbance of Sample vs Blank.

Acceptance criteria: Absorbance NMT 0.025.

5 SPECIFIC TESTS

Residue on Ignition 〈281〉: NMT 25.0%

Optical Rotation, Specific Rotation 〈781S〉:

Sample: 15 mg/mL in 5 N HCl, used within 15 min

Acceptance criteria: +37.0° to +42.0°

pH 〈791〉: 10 mg/mL

Acceptance criteria: 5.0–6.5

Loss on Drying 〈731〉:

Dry under vacuum over phosphorus pentoxide at 100° for 5 h

Acceptance criteria: NMT 7.5%

6 ADDITIONAL REQUIREMENTS

Packaging and Storage: Preserve in tight, light-resistant containers.

USP Reference Standards 〈11〉

USP Phosphated Riboflavin RS

USP Riboflavin RS