The fluorimetric determination of riboflavin in urine

lumichrome (6:7-dimethyl-isoalloxazine) is pro- duced by the irradiation ofriboflavin, while Warburg & Christian (1933) showed that lumiflavin (6:7:9- trimethyl-isoalloxazine) is formed in alkaline solution.Both these compounds exhibit a blue fluorescence.Karrer et al. (1934) showed that the nature of the solvent had a considerable influence on the yield of the decomposition products, but did not investigate the products of decomposition in butanol-pyridine-acetic acid solution.The decom- position in this solution is so much more rapid than in aqueous solution that it is quite possible that the decomposition takes a different course.We found that the pH of the butanol-pyridine-acetic acid solution, diluted fourfold with water and ethanol, was 4-6.The method is sufficiently sensitive for most purposes.The smallest amount of riboflavin which can be measured with an accuracy of 10 % is about 0-1 Lg.The limit of sensitivity of the method, i.e. the smallest amount which can be regarded as significantly greater than zero, is aboui 0-01 Zg.The smallest concentration which can be measured depends upon the size of the aliquot taken, which in turn depends upon the amount of interfering substances present.SUMMARY 1.A modification of the fluorimetric method of determining riboflavin in biological materials, based on the method of Najjar (1941) but differing in several important respects, is described.' 2. The method differs from those previously described by including a test for specificity.3. The photochemical destruction of riboflavin is not a simple exponential function of time, but is dependent upon the concentration of the ,decomposition products, i.e. a reversible reaction is involved.4. High concentrations of KMnO4 rapidly destroy riboflavin. The introduction of an additional purification step involving adsorption on Florisil did not increase the specificity of the method.6.In general, figures obtained by the proposed method agreed closely with those obtained by a microbiological method.7. The accuracy of the method is about 10 % and the limit of sensitivity about 0 01 ,ug.We wish to thank Miss C. E. Francis for some of the analyses in Table 9 and Dr C. White for the analyses made by the microbiological method in Table 8.