Simultaneous determination of thiamine and pyrithiamine

Abstract

1. (1) Alkaline potassium ferricyanide oxidation of thiamine and pyrithiamine yields two different products as judged by excitation and emission characteristics. The former oxidation produces thiochrome and the latter pyrichrome. 2. (2) The differences in spectral characteristics of thiochrome and pyrichrome have been utilized to determine quantitatively thiamine and pyrithiamine concentration in a mixture of the two. 3. (3) Two generalized equations were derived to determine the concentration of thiamine and pyrithiamine in the presence of biological material: T = d ad − bc E 435 385 − b ad − bc E 480 385 = mνmoles thiamine/asay P = a ad − bc E 480 385 − c ad − bc E 480 385 = mνmoles pyrithiamine/asay where E = emission, P = pyrithiamine, T = thiamine, the numerator of the subscripts the emission wavelength, and the denominator the excitation wavelength. Constants a and b represent the relative molar fluorescence of thiochrome and pyrichrome when excited at 385 mμ and emitting at 435 mμ. Constants c and d represent the relative molar fluorescence of the same two respective compounds with the emission measured at 480 mμ and excitation at 385 mμ. 4. (4) The limit of detection for both thiamine and pyrithiamine is determined both by their absolute amounts and by the molar ratio of the two. When the molar ratio of pyrithiamine to thiamine was between 40 and 8, 12 mμmoles of pyrithiamine could be detected with recoveries ranging from 90 to 115%. With a similar range of molar ratios, 1.5 mμmoles of thiamine per assay could be detected with recoveries ranging from 90 to 100%. The limitations of this approach have been considered.