Conditions for Thiamin Assay by Cyanogen Bromide Oxidation

Abstract

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 0.25M 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 (1.8M) was introduced in place of the conventional BrCN (0.11M), obtaining 300% or more intensity of fluores-cence. For the oxidation of thiamin diphosphate, 0.15-0.2M of laboratory-prepared BrCN gave the highest intensity of fluorescence instead of the 0.25M for free thiamin. For simultaneous oxidation of free thiamin and thiamin diphosphate, therefore, 0.23-0.24M 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 0.04M for CNBr and about 0.16% for NaOH. When the sample contained an inhibitor of oxidation, such as ascorbic acid, the percentage of inhibition decreased inversely pro-portional 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.