A new fluorometric determination of thiamine.

Several different types of assay procedure have been proposed for the quantitative determination of thiamine. In a discussion (1) of the difficulties encountered in some of the methods, it has been pointed out that calorimetric (2, 3) and fluorometric (4) methods often suffer from a lack of sufficient sensitivity or specificity. The procedure described in the present communication, while not so sensitive as the microbiological assay (l), is sensitive enough for materials of relatively low potency. Furthermore, since the determination, unlike all others, depends upon the cyanogen bromide reaction, and since the more commonly encountered pyridine derivatives which might react with the reagent have been found to cause little or no interference, the method possesses a rather high degree of specificity. Investigation of the cyanogen bromide reaction has shown (5) that thiamine, if present in relatively high concentration, produces a colored compound with this reagent. It was concluded that utilization of this reaction for the calorimetric determination of thiamine is not practicable because of the high concentration of the vitamin required. Further investigation has revealed, however, that under different and simpler experimental conditions thiamine produces with cyanogen bromide a highly fluorescent compound. This reaction has been made the basis for the following simple and rapid fluorometric determination of the vitamin. In addition to its simplicity and relatively high degree of sensitivity, the method has the added advantage of requiring the same reagents as are used in the determinations of nicotinic acid (6) and nicotinamide (7).