Quantitative Determination of Thiamin and Its Phosphate Esters in Human Whole Blood by High-Performance Liquid Chromatography and Clinical Application

A sensitive high-performance liquid chromatography (HPLC) method was used for the determination of thiamin and its phosphate esters (thiamin triphosphate, TTP; thiamin diphosphate, TDP; and thiamin monophosphate, TMP) in normal human whole blood. The blood specimens deproteinized with cold ca. 7% trichloroacetic acid, were applied to a reversed-phase HPLC column (Shim-pack CLC-ODS) equipped with a spectrofluorometric system. Picogram levels of free thiamin and its phosphate esters were determined after conversion to the corresponding thiochrome derivatives in a post-column reaction with alkaline ferricyanide. The contents of thiamin and its phosphate esters were 24.8 (±4.2), 55.5 (±7.6), 1.6 (±0.8) ng/ml (N=8), and a trace amount for TTP, TDP, TMP, and thiamin, respectively. The most suitable method of sample preparation for HPLC determination of blood thiamin and its phosphate esters was found to be immediate deproteinization of the blood with trichloroacetic acid, followed by centrifugation and storage of the supernatant at -30°C until analysis could be performed.Although the total thiamin contents in human blood determined in the present study were similar to those reported previously using different procedures, the percentage of thiamin triphosphate (≅30%) was found to be high, which might be due to the existence of the enzyme which catalyses the synthesis of TTP from protein-bound TDP and ATP in the presence of Mg2+ and serum cofactor in human whole blood.