á481ñRIBOFLAVIN ASSAY
The following procedure is suitable for preparations in which riboflavin is a constituent of a mixture of several ingredients.In employing it,keep the pHof solutions below 7.0,and protect the solutions from direct sunlight at all stages.
Standard Riboflavin Stock Solution— To 50.0mg of USP Riboflavin RS,previously dried and stored protected from light in a desiccator over phosphorus pentoxide,add about 300mLof 0.02Nacetic acid,and heat the mixture on a steam bath,with frequent agitation,until the riboflavin has dissolved.Then cool,add 0.02Nacetic acid to make 500mL,and mix.Store under toluene in a refrigerator.
Dilute an accurately measured portion of this solution,using 0.02Nacetic acid,to a concentration of 10.0µg of the dried USP Riboflavin RSper mL,to obtain the Standard Riboflavin Stock Solution.Store under toluene in a refrigerator.
Standard Preparation— Dilute 10.0mLof Standard Riboflavin Stock Solutionwith water in a 100-mLvolumetric flask to volume,and mix.Each mLrepresents 1.0µg of USP Riboflavin RS.Prepare fresh Standard Preparationfor each assay.
Assay Preparation— Place an amount of the material to be assayed in a flask of suitable size,and add a volume of 0.1Nhydrochloric acid equal in mLto not less than 10times the dry weight of the material in g,but the resulting solution shall contain not more than 100µg of riboflavin per mL.If the material is not readily soluble,comminute it so that it may be evenly dispersed in the liquid.Then agitate vigorously,and wash down the sides of the flask with 0.1Nhydrochloric acid.
Heat the mixture in an autoclave at 121
to 123for 30minutes,and cool.If clumping occurs,agitate the mixture until the particles are evenly dispersed.Adjust the mixture,with vigorous agitation,to a pHof 6.0to 6.5with sodium hydroxide solution,*then add hydrochloric acid solution*immediately until no further precipitation occurs (usually at a pHof approximately 4.5,the isoelectric point of many of the proteins present).Dilute the mixture with water to make a measured volume that contains about 0.11µg of riboflavin in each mL,and filter through paper known not to adsorb riboflavin.To an aliquot of the filtrate add,with vigorous agitation,sodium hydroxide solution*to produce a pHof 6.6to 6.8,dilute the solution with water to make a final measured volume that contains approximately 0.1µg of riboflavin in each mL,and if cloudiness occurs,filter again.
to 123for 30minutes,and cool.If clumping occurs,agitate the mixture until the particles are evenly dispersed.Adjust the mixture,with vigorous agitation,to a pHof 6.0to 6.5with sodium hydroxide solution,*then add hydrochloric acid solution*immediately until no further precipitation occurs (usually at a pHof approximately 4.5,the isoelectric point of many of the proteins present).Dilute the mixture with water to make a measured volume that contains about 0.11µg of riboflavin in each mL,and filter through paper known not to adsorb riboflavin.To an aliquot of the filtrate add,with vigorous agitation,sodium hydroxide solution*to produce a pHof 6.6to 6.8,dilute the solution with water to make a final measured volume that contains approximately 0.1µg of riboflavin in each mL,and if cloudiness occurs,filter again.Procedure— To each of four or more tubes (or reaction vessels)add 10.0mLof the Assay Preparation.To each of two or more of these tubes add 1.0mLof the Standard Preparation,and mix,and to each of two or more of the remaining tubes add 1.0mLof water,and mix.To each tube add 1.0mLof glacial acetic acid,mix,then add,with mixing,0.50mLof potassium permanganate solution (1in 25),and allow to stand for 2minutes.To each tube add,with mixing,0.50mLof hydrogen peroxide solution,whereupon the permanganate color is destroyed within 10seconds.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 flows slowly from end to end.
In a suitable fluorophotometer,having an input filter of narrow transmittance range with a maximum at about 440nm and an output filter of narrow transmittance range with a maximum at about 530nm,measure the fluorescence of all tubes,designating the average reading from the tubes containing only the Assay Preparationas IUand the average from the tubes containing both the Assay Preparationand the Standard Preparationas IS.Then to each of one or more tubes of each kind add,with mixing,20mg of sodium hydrosulfite,and within 5seconds again measure the fluorescence,designating the average reading as IB.
Calculation— Calculate the quantity,in mg,of C17H20N4O6in each mLof the Assay Preparationtaken by the formula:
0.0001(IU–IB)/(IS–IU).
Calculate the quantity,in mg,of C17H20N4O6in each capsule or tablet.
*
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.