Riboflavin 5¢-Phosphate Sodium
(rye' boe flay vin fos' fate soe' dee um).
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C17H20N4NaO9P·2H2O 514.36
C17H20N4NaO9P 478.33
Riboflavin 5¢-(dihydrogen phosphate), monosodium salt, dihydrate;    
Riboflavine 5¢-(sodium hydrogen phosphate), dihydrate    
Anhydrous     [130-40-5].
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.
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.
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 
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
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 
Mode:  UV-Vis
Analytical wavelength:  700 nm
Cell:  1 cm
Analysis 
Samples:  Standard solution, Sample solution, and Blank
Transfer 10.0 mL each of the Standard solution, Sample solution, and Blank to separate 50-mL conical flasks. Add 10.0 mL of Acid molybdate solution and 5.0 mL of Ferrous sulfate solution to each flask, and mix. Determine the absorbances of the solutions against that of 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—Conduct this test so that all solutions are protected from actinic light at all stages, preferably by using low-actinic glassware. ]
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 this solution with Mobile phase to 160 µg/mL.
Standard solution:  Transfer 60 mg of USP Riboflavin RS to a 250-mL volumetric flask. Dissolve carefully in 1 mL of hydrochloric acid, and dilute with water to volume. Dilute with Mobile phase to 9.6 µg/mL.
Sample solution:  Prepare a 2-mg/mL solution of Riboflavin 5¢-Phosphate Sodium in water. Dilute this solution with Mobile phase to 160 µg/mL.
Chromatographic system 
Mode:  LC
Detector:  Fluorometer
Excitation wavelength:  440 nm
Emission wavelength:  530 nm
Column:  3.9-mm × 30-cm; packing L1
Flow rate:  2 mL/min
Injection size:  100 µL
System suitability 
Sample:  System suitability solution
The retention time of riboflavin 5¢-monophosphate is 20–25 min. The approximate relative retention times for the components are listed in Table 1.
Table 1
Name Relative
Retention
Time
Riboflavin 3¢4¢-diphosphate 0.23
Riboflavin 3¢5¢-diphosphate 0.39
Riboflavin 4¢5¢-diphosphate 0.58
Riboflavin 3¢-monophosphate 0.70
Riboflavin 4¢-monophosphate 0.87
Riboflavin 5¢-monophosphate 1.00
Riboflavin 1.63
Suitability requirements 
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
Measure the peak areas of the solutions. Identify the peaks to be measured in the Sample solution by comparison with the chromatogram obtained from the System suitability solution.
Calculate the percentage of free riboflavin in the portion of Riboflavin 5¢-Phosphate Sodium taken:
Result = (rU/rS) × (CS/CU) × 100
rU== peak area of free riboflavin from the Sample solution
rS== peak area of riboflavin 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)
Calculate the percentage of riboflavin diphosphates in the portion of Riboflavin 5¢-Phosphate Sodium taken:
Result = (rU/rS) × (CS/CU) × 100
rU== sum of the peak areas for any of the 3 riboflavin diphosphates from the Sample solution
rS== peak area of riboflavin 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 
Free riboflavin:  NMT 6.0% on the dried basis
Riboflavin diphosphates, as riboflavin:   NMT 6.0% on the dried basis
•  Limit of Lumiflavin
Alcohol-free chloroform:  Shake 20 mL of chloroform gently but thoroughly with 20 mL of water for 3 min, draw off the chloroform layer, and wash twice more with 20-mL portions of water. Finally, pass the chloroform through a dry filter paper, shake it for 5 min with 5 g of powdered anhydrous sodium sulfate, allow the mixture to stand for 2 h, and decant or filter the clear chloroform.
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 
Analytical wavelength:  440 nm
Cell:  1 cm
Analysis 
Samples:  Sample solution and Blank
Measure the absorbances of the Sample solution and Blank. Correct the absorbance of the Sample solution with the Blank.
Acceptance criteria:  The absorbance is NMT 0.025.
SPECIFIC TESTS
•  Residue on Ignition 281: NMT 25.0%
•  Optical Rotation, Specific Rotation 781S
Sample solution:  15 mg/mL in 5 N hydrochloric acid. Use the solution within 15 min.
Acceptance criteria:  +37.0 to +42.0
•  pH 791
Sample solution:  10 mg/mL solution
Acceptance criteria:  5.0–6.5
•  Loss on Drying 731: Dry a sample under vacuum over phosphorus pentoxide at 100 for 5 h: it loses NMT 7.5% of its weight.
ADDITIONAL REQUIREMENTS
•  Packaging and Storage: Preserve in tight, light-resistant containers.
•  USP Reference Standards 11
USP Phosphated Riboflavin RS
USP Riboflavin RS Click to View Structure
Auxiliary Information— Please check for your question in the FAQs before contacting USP.
Topic/Question Contact Expert Committee
Monograph Huy T. Dinh, M.S.
Scientific Liaison
1-301-816-8594
(DS2010) Monographs - Dietary Supplements
Reference Standards RS Technical Services
1-301-816-8129
rstech@usp.org
USP35–NF30 Page 4548