[8] Determination of thiamin and its phosphate esters in human blood, plasma, and urine

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This chapter focuses on the determination of thiamin and its phosphate esters in human blood, plasma, and urine. There are several functional tests for the estimation of triphosphate (TPP) concentration. Among them are the glucose tolerance test and the erythrocyte transketolase assay. The transketolase assay has become a standard test used to estimate thiamin deficiency. The thiochrome method introduces the oxidation of thiamin to the fluorescent thiochrome, which is then determined by fluorometry. The use of high-performance liquid chromatography (HPLC) allows separation of the thiamin compounds under analysis. The chapter proposes a new method, which combines the advantages of high sensitivity and determination of the four compounds in one chromatographic run. The conversion of thiamin compounds into fluorescent derivatives is obtained by alkaline oxidation, which converts thiamin into the fluorescent thiochrome. Cyanogen bromide is chosen as it is the most efficient oxidizing agent.

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      The method by determining erythrocyte transketolase activity only quantifies TDP content, and has poor inter-assay precision and sample storage stability because of rapid inactivation of the transketolase [26]. HPLC methods can be used for simultaneous determination of thiamine and its phosphate esters in human whole blood [17,25,27–32]. However, current HPLC methods required pre- or post-column fluorescence derivatization to improve sensitivity.

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