Quenching effect of electrolytes on the fluorescence intensity of riboflavin and thiochrome.

After the discovery of riboflavin, and its identification as vitaminr B,, assay methods based on the measurement of its fluorescence intensity were described (v. Euler & Adler, 1934; Cohen, 1934; Josephy, 1934). Similar methods of assay were developed for vitamin B1 after Jansen (1936) had shown that thiochrome, the fluorescent oxidation product of thiamin, could be used as the basis for the fluorimetric estimation of this vitamin. These methods have been developed by numerous workers for use in the determination of the vitamin content of foodstuffs, tissues and body fluids. In all such assays the fluorescent intensity of riboflavin is measured in aqueous or butanol solution; thiamin is extracted, oxidized to thiochrome and measured fluorimetrically in isobutanol solution. The methods used for both vitamins (cf. papers quoted by Najjar, 1941, and the Report of the Vitamin B1 SubCommittee (Appendix A), Medical Research Council, 1943), do not prevent the appearance in the purified extract of a number of substances present in the original material, particularly since a considerable amount of water is soluble in butanol. Such substances might significantly influence the fluorescence intensity ofriboflavin or thiochrome, and thus interfere with the accuracy ofthe assay. An investigation has therefore been made on the effect of a number of substances on the fluorescence intensity of the two fluorescent pigments. The quenching effect of oxygen, which was stressed by Weil-Malherbe & Weiss (1942), was not taken into account because all measurements were made in equilibrium with air and the standards used were equally affected.