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The following procedure is suitable for preparations in which riboflavin is a constituent of a mixture of several ingredients. In employing it, keep the pH of solutions below 7.0, and protect the solutions from direct sunlight at all stages.

Standard Riboflavin Stock Solution—
To 50.0 mg of USP Riboflavin 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 riboflavin has dissolved. Then cool, add 0.02 N acetic acid to make 500 mL, and mix. Store under toluene in a refrigerator.
Dilute an accurately measured portion of this solution, using 0.02 N acetic acid, to a concentration of 10.0 µg of the dried USP Riboflavin RS per mL, to obtain the Standard Riboflavin Stock Solution. Store under toluene in a refrigerator.

Standard Preparation—
Dilute 10.0 mL of Standard Riboflavin Stock Solution with water in a 100-mL volumetric flask to volume, and mix. Each mL represents 1.0 µg of USP Riboflavin RS. Prepare fresh Standard Preparation for each assay.

Assay Preparation—
Place an amount of the material to be assayed in a flask of suitable size, and add a volume of 0.1 N hydrochloric acid equal in mL to not less than 10 times the dry weight of the material in g, but the resulting solution shall contain not more than 100 µg 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 0.1 N hydrochloric acid.
Heat the mixture in an autoclave at 121 to 123 for 30 minutes, and cool. If clumping occurs, agitate the mixture until the particles are evenly dispersed. Adjust the mixture, with vigorous agitation, to a pH of 6.0 to 6.5 with sodium hydroxide solution,* then add hydrochloric acid solution* immediately until no further precipitation occurs (usually at a pH of 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 0.11 µg 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 pH of 6.6 to 6.8, dilute the solution with water to make a final measured volume that contains approximately 0.1 µg of riboflavin in each mL, and if cloudiness occurs, filter again.

To each of four or more tubes (or reaction vessels) add 10.0 mL of the Assay Preparation. To each of two or more of these tubes add 1.0 mL of the Standard Preparation, and mix, and to each of two or more of the remaining tubes add 1.0 mL of water, and 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 minutes. To each tube add, with mixing, 0.50 mL of hydrogen peroxide solution, whereupon the permanganate color is destroyed within 10 seconds. 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 440 nm and an output filter of narrow transmittance range with a maximum at about 530 nm, measure the fluorescence of all tubes, designating the average reading from the tubes containing only the Assay Preparation as IU and the average from the tubes containing both the Assay Preparation and the Standard Preparation as IS. Then to each of one or more tubes of each kind add, with mixing, 20 mg of sodium hydrosulfite, and within 5 seconds again measure the fluorescence, designating the average reading as IB.

Calculate the quantity, in mg, of C17H20N4O6 in each mL of the Assay Preparation taken by the formula:
0.0001(IU – IB) / (IS – IU).
Calculate the quantity, in mg, of C17H20N4O6 in 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.

Auxiliary Information—
Staff Liaison : Lawrence Evans, III, Ph.D., Scientist
Expert Committee : (DSN05) Dietary Supplements - Non-Botanicals
USP29–NF24 Page 2590
Phone Number : 1-301-816-8389