An improved method for the determination of endogenous thiamine and its phosphate esters in biological material

Nonphosphorylated thiamine was extracted from tissue homogenates in saline or sucrose into ethylbutylketone-acetonitrile using counter-ion extraction and reextracted into hydrochloric acid. The dried samples were then analyzed by the fluorimetric thiochrome method. Phosphorylated thiamine was treated with Takadiastase to split off the phosphate groups and was thereafter extracted and measured. No column chromatography was needed for the separation of thiamine before the thiochrome assay. A higher accuracy and sensitivity for the thiamine assay was thus achieved.