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The crude extract of kohlrabi (Brassica oleracea gongylodes) was prepared as a rich source of peroxidase and its enzymatic and specific activities in the presence of cross-linked polyvinylpyrrolidone as a stabilizer were determined. This extract was used to catalyze the oxidation of thiamine in the presence of hydrogen peroxide to thiochrome at pH 8.0. Thiochrome shows strong fluorescence at 436 nm with excitation at 370 nm. Based on the obtained results, a sensitive and simple spectrofluorimetric method was developed for the determination of thiamine. In the optimum conditions, the calibration graph was linear from 2´10-7 to 1´10-4 mol L-1, with a detection limit of 6.2´10-8 mol L-1. The relative standard deviation (RSD) was 1.2% for 5´10-6 mol L-1 thiamine. The method was successfully applied to the determination of thiamine in vitamin B1 and vitamin B complex tablets and vitamin B complex syrup.