A Method for the Simultaneous Determination of Pyrithiamine and Thiamine in Biological Materials

Abstract

A more precise and sensitive method for the separate determination of Pyrithiamine (PyT) and thiamine in biological materials was described. The method was based on the different behavior of these 2 compounds toward the oxidation with cyanogen bromide or potassium ferricyanide, and on the different characteristics of the fluorescence spectra of the oxidation products. Thiamine and PyT in biological materials were extracted by homogenizing and heating in an acidic medium, followed by deproteinization with metaphosphoric acid, adsorption on permutit and elution with hot KC1-HC1. For the determination of PyT, an aliquot of the eluate was oxidized with alkaline ferricyanide (1% in 10% NaOH) and the oxidation product, after extraction into iso-butanol, was determined fluorometrically using a spectrofluorometer with an excitation at 430 mu and emission at 460 mu. In the case of the thiamine determination, the same eluate was oxidized with cyanogen bromide and isobutanol extracted thiochrome was determined at 375 mu. exciting and 420 mu emitting wavelengths. The lowest limit of detection for PyT was less than 0.3 mu mole, and that for thiamine was less than 0.03 mumole per assay.