Determination of thiamin and thiamin phosphate esters in blood by liquid chromatography with post-column derivatization.

We demonstrate a liquid-chromatographic method involving post-column derivatization for determining the concentration of thiamin and its phosphate esters in human blood. Blood, erythrocytes, or plasma 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 hydroxide solution to the column effluent with a proportioning pump converts thiamin phosphates into fluorophores, the intensities of which are measured with a spectrofluorophotometer. Thiamin, thiamin monophosphate, thiamin pyrophosphate, and thiamin triphosphate eluted as single peaks; no coeluting substances were detected. Thiamin pyrophosphate was the ester present in greatest concentration, followed by thiamin triphosphate; thiamin monophosphate and thiamin were present in slight amounts. This method allows easy determination of thiamin and its phosphate esters in 0.1 mL of blood.