Conditions for thiamin assay by cyanogen bromide oxidation

Optimum conditions for the assay of thiamin were studied using a cyanogen bromide (BrCN) oxidation method. The adopted procedure included neither pre-purification of samples through an ion exchanger nor extraction of the thiochrome into an organic solvent. The 0.25 M BrCN (the concentration before the addition of alkali) and the final NaOH concentration of approx. 1% gave the highest yield of thiochrome by a laboratory-prepared BrCN. To obtain the highest intensity of fluorescence, a concentrated BrCN (1.8 M) was introduced in place of the conventional BrCN (0.11 M), obtaining 300% or more intensity of fluorescence. For the oxidation of thiamin diphosphate, 0.15-0.2 M of laboratory-prepared BrCN gave the highest intensity of fluorescence instead of the 0.25 M for free thiamin. For simultaneous oxidation of free thiamin and thiamin diphosphate, therefore, 0.23-0.24 M of laboratory-prepared BrCN was deduced to give the best yield of fluorescence. With a solution of commercially obtained solid CNBr, optimum concentrations for the oxidation of thiamin were about 0.04 M for CNBr and about 0.16% for NaOH. When the sample contained in inhibitor of oxidation, such as ascorbic acid, the percentage of inhibition decreased inversely proportional to the concentration of the sample in a rough approximation. The degree of inhibition was not reduced by the increased amount of BrCN reagent. Thus the possibility was indicated that thiamin in an ascorbic acid-contaminated sample could be determined accurately by extrapolating values for serially diluted samples.