Determination of thiamine and its phosphate esters in human and rat blood by high-performance liquid chromatography with post-column derivatization.

A method for the determination of thiamine and thiamine phosphate esters in human and rat blood by liquid chromatography with post-column derivatization, and distribution of thiamine and its phosphate esters in human and various animals is demonstrated. Blood is deproteinized and centrifuged. Aliquots of the samples are applied to a mu Bondapak C18 column attached to a high-performance liquid chromatograph. Addition of potassium ferricyanide sodium hydroxyide solution to the column effluent with a proportioning pump converts thiamine phosphate into fluorophores, the intensities of which are measured with a spectrofluorophotometer. In human blood, thiamine pyrophosphate was present in the greatest concentration, followed by thiamine triphosphate; thiamine monophosphate and thiamine were present in small amounts. In rat blood, thiamine pyrophosphate was found in the largest amount, followed by thiamine monophosphate. Thiamine triphosphate and thiamine were present in small amounts.