The Fluorometric Determination of Riboflavin in Urine and Other Biological Fluids

The increasing frequency with which riboflavin deficiency is being discovered in man has given an impetus to the study of this substance in biological fluids wi’ith a view to providing a quick and accurate diagnost,ic method. For this purpose chemical methods of determination are distinctly to be preferred to the more laborious and time-consuming biological and microbiological methods. Several procedures have been developed for the estimation of riboflavin in urine, either directly or after some method of concent’ration. The flavin has been measured quantitatively by its color, by use of a step-photometer (1, 2), by its fluorescence in aqueous solutions (3-5) by converting it into lumiflavin which may be measured calorimetrically in chloroform solutions (6, 7), or by its potential as measured with the polarograph (8). The fluorometric method appealed to us because of its simplicity and sensitivity and because a convenient apparatus for measuring fluorescence photometrically is now available.’ Although fluorometric methods have been criticized, justly so we believe, by a number of workers in this field, we have been able to modify the procedures used in such a way as to avoid recognized difficulties and to obtain accurate reproducible results. Our early experiments convinced us that t)wo sources of error inherent in all methods in which fluorescence was measured in aqueous solutions were the turbidity of the solution and the formation of gaseous emulsions. The addition of potassium permanganate followed by hydrogen peroxide, a procedure used to oxidize interfering fluorescent substances, results in the formation of