A: Dissolve 1mg in 100mLof water:the solution is pale greenish yellow by transmitted light,and it exhibits an intense yellowish green fluorescence under long-wavelength UVlight that disappears upon the addition of mineral acids or alkalies.

B: To 0.5g add 10mLof nitric acid,evaporate the mixture on a water bath to dryness,ignite the residue until the carbon is removed,dissolve the residue in 5mLof water,and filter:the filtrate so obtained responds to the tests for Sodium á191ñand for Phosphate á191ñ.

Test solution: 15mg per mL,in 5Nhydrochloric acid.

Acid molybdate solution— Dilute 25mLof ammonium molybdate solution (7in 100)with water to 200mL.To this dilution add slowly 25mLof 7.5Nsulfuric acid,and mix.

Ferrous sulfate solution— Just prior to use,prepare a 1in 10solution of ferrous sulfate in 0.15Nsulfuric acid.

Standard preparation— Prepare a solution in water containing 44.0µg of monobasic potassium phosphate in each mL.

Test preparation— Transfer 300mg of Riboflavin 5¢-Phosphate Sodium to a 100-mLvolumetric flask,dissolve in and dilute with water to volume,and mix.

Procedure— Transfer 10.0mLeach of the Standard preparationand the Test preparationto separate 50-mLconical flasks,add 10.0mLof Acid molybdate solutionand 5.0mLof Ferrous sulfate solutionto each flask,and mix.Concomitantly determine the absorbances of the solutions,in 1-cm cells,at the wavelength of maximum absorbance at about 700nm,with a suitable spectrophotometer,using as the blank a mixture of 10.0mLof water,10.0mLof Acid molybdate solution,and 5.0mLof Ferrous sulfate solution:the absorbance of the solution from the Test preparationis not greater than that from the Standard preparation(1%as PO4).

Mobile phase— Mix 850mLof 0.054Mmonobasic potassium phosphate with 150mLof methanol,filter,and degas the solution.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard preparation— Transfer 60mg of USP Riboflavin RS,accurately weighed,to a 250-mLvolumetric flask,dissolve carefully in 1mLof hydrochloric acid,dilute with water to volume,and mix.Pipet a 4-mLaliquot into a 100-mLvolumetric flask,dilute with Mobile phaseto volume,and mix.

Test preparation— Transfer 100.0mg of Riboflavin 5¢-Phosphate Sodium to a 100-mLvolumetric flask,dissolve in 50mLof water,dilute with Mobile phaseto volume,and mix.Pipet 8mLof this solution into a 50-mLvolumetric flask,dilute with Mobile phaseto volume,and mix.

System suitability preparation— Dissolve USP Phosphated Riboflavin RSin water to obtain a solution containing 2mg per mL.Add an equal volume of Mobile phase,and mix.Dilute 8mLof this solution with Mobile phaseto 50mL,and mix.

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a fluorometric detector set at 440-nm excitation wavelength and provided with a 470-nm emission filter or set at about 530nm for a fluorescence detector that uses a monochromator for emission wavelength selection,and a 3.9-mm ×30-cm column that contains packing L1.The flow rate is about 2.0mLper minute.Chromatograph the System suitability preparation,and record the peak responses.The retention time for riboflavin 5¢-monophosphate is about 20to 25minutes,and the approximate relative retention times for the components are as follows:

Procedure— Separately inject equal volumes (about 100µL)of the Standard preparation,the Test preparation,and the System suitability preparationinto the chromatograph.Measure the peak responses obtained from the Standard preparationand the Test preparation,identifying the peaks to be measured in the chromatogram of the Test preparationby comparison of retention times with those of the peaks in the chromatogram of the System suitability preparation.Calculate the percentage of free riboflavin taken by the formula: and calculate the percentage of riboflavin in the form of riboflavin diphosphates taken by the formula: in which Cis the concentration,in mg per mL,of USP Riboflavin RSin the Standard preparation,rFis the riboflavin peak response,if any,obtained from the Test preparation,rDis the sum of the responses for any of the 3riboflavin diphosphate peaks obtained from the Test preparation,and rSis the riboflavin peak response obtained from the Standard preparation.Not more than 6.0%of free riboflavin and not more than 6.0%of riboflavin diphosphates,as riboflavin,calculated on the dried basis,are found.

Standard preparation— Transfer about 35mg of USP Riboflavin RS,accurately weighed,to a 250-mLconical flask,add 20mLof pyridine and 75mLof water,and dissolve the riboflavin by frequent shaking.Transfer the solution to a 1000-mLvolumetric flask,dilute with water to volume,and mix.Transfer 10.0mLof this solution to a second 1000-mLvolumetric flask,add sufficient 0.1Nsulfuric acid (about 4mL)so that the final pHof the solution is between 5.9and 6.1,dilute with water to volume,and mix.The Standard preparationso obtained contains about 0.35µg of riboflavin per mL.

Assay preparation— Transfer about 50mg of Riboflavin 5¢-Phosphate Sodium,accurately weighed,to a 250-mLconical flask,add 20mLof pyridine and 75mLof water,and dissolve by frequent shaking.Transfer the solution to a 1000-mLvolumetric flask,dilute with water to volume,and mix.Transfer 10.0mLof this solution to a second 1000-mLvolumetric flask,add sufficient 0.1Nsulfuric acid (about 4mL)so that the final pHof the solution is between 5.9and 6.1,dilute with water to volume,and mix.

Procedure— With a suitable fluorometer,determine the maximum fluorescence intensities,ISand IU,of the Standard preparationand the Assay preparation,respectively,at about 530nm,using an excitation wavelength of about 440nm.Calculate the quantity,in mg,of C17H20N4O6in the portion of Riboflavin 5¢-Phosphate Sodium taken by the formula: in which Cis the concentration,in µg per mL,of USP Riboflavin RSin the Standard preparation.