Determination of thiamine and its phosphate esters by gradient-elution high-performance liquid chromatography.

A high-performance liquid chromatographic method for the determination of thiamine and its phosphate esters using gradient elution is presented. Chromatography was performed on an octadecyl C18 column with post-column derivatization using potassium ferricyanide: thiamine was oxidized to thiochrome, which was detected fluorimetrically. The gradient system avoids the use of a modifier and an ion-pairing reagent. The proposed method enables the separation of thiamine triphosphate, thiamine pyrophosphate, thiamine monophosphate and free thiamine, at concentrations of ca. 2 nM, within 10 min The within-assay precision for blood and serum total thiamine was less than 2 and 5%, respectively, and the between-day variation was less than 3 and 5%, respectively. The method is simple and rapid, and it can be used for screening of thiamine deficiency and for clinical study of various diseases related to vitamin B1 deficiency.