Physiochemical properties and quantitative determination of thiamine monophosphate disulfide.

Abstract

1. The ultraviolet absorption spectrum of thiamine monophosphate disulfide (TMPDS) changed with pH like thiamine, the isosbestic points being at 239 and 277mμ.2. pKa1, pKa2, and pKb were obtained from the titration curve. The pKa value obtained from the change in ultraviolet absorption spectrum with pH was almost the same as that obtained from titration, and the isoelectric point was 3.96. This value well corresponded to the properties of solubility and stability.3. It was hardly soluble in organic solvents. The solubility in water at 30° was 4.94% (w/v) at pH 7.0 and 4.50% (w/v) at pH 3.86.4. It could be separated from the related compounds by thin layer chromatography using pyridine-acetic acid-water as a solvent system.5. The anhydrate of TMPDS was hygroscopic, forming a stable powder-like material with 4-8 molecules of water in 24 hours.6. The stability of TMPDS solution (1-10mg/ml) after heating at various pH levels for 1-2 hours was greatest at pH 4 below 70°, but it was hydrolyzed at above 80°. It was unstable to light.7. The ultraviolet absorption spectrum and fluorescence spectrum of the thiochrome phosphate obtained by cysteine reduction and BrCN oxidation at pH 13.0 were the same as those of thiochrome obtained from thiamine. The recovery was more than 98%.8. The total thiamine value can be determined by measuring the ultraviolet absorbance and fluorometry in alkaline solution obtained by BrCN oxidation after cysteine reduction. Thiamine disulfide and free thiamine can be determined by fluorometry of the isobutanol extract of this solution. Thiamine monophosphate and free thiamine can be determined by ultraviolet absorbance and the fluorometry of the alkaline solution obtained by BrCN oxidation of the solution without cysteine reduction. Free thiamine can be determined by fluorometry of the isobutanol extract of this solution. TMPDS can be determined from each value thus obtained.9. TMPDS in the blood can be determined with 95% recovery by combined use of cysteine reduction and Takadiastase hydrolysis.