A Reliable Semiautomated Method for the Determination of Total Thiamine in Whole Blood by the Thiochrome Method with High-Performance Liquid Chromatography

Abstract

A sensitive and reliable method for the determination of total thiamine (vitamin B1) in whole blood has been developed which is suited for routine analysis. After extraction, and enzymatic hydrolysis of thiamine phosphate esters, thiamine is separated from interfering compounds by a fully automated high-performance liquid chromatographic (HPLC) system. Thiamine is detected fluorometrically as thiochrome. By calculating the concentration of thiamine on-line with the aid of a computer, it is possible to complete one analysis within four hours. Routine thiamine determinations can be carried out in a series of 120 samples within 48 hours. The within-assay and between-assay coefficients of variation of the analysis of total thiamine in whole blood were 4·2 and 4·4%, respectively. The between-assay analytical recovery of thiamine diphosphate added to blood samples was 99·9 ± 11·7% (mean ± SD). The HPLC method described has been applied also to the analysis of thiamine in plasma and erythrocytes. In agreement with other reports, it was found that about 80% of total thiamine of whole blood is present in the erythrocytes. Reference values of thiamine in whole blood of the human were found in the range 95–155 nmol/l.