STUDIES ON THIAMINE PHOSPHATES : (III) QUANTITATIVE DETERMINATION OF S-BENZOYLTHIAMINE O-MONOPHOSPHATE

Abstract

Optimum conditions for the quantitative determination of S-benzoylthiamine O-monophosphate (BTMP) were defined based on the results of fundamental investigations on the conditions of hydrolysis of BTMP to free thiamine with Taka-diastase B and of debenzoylation to thiamine monophosphate (TMP) with cysteine. When BTMP was hydrolyzed with Taka-diastase B of the final concentration of about 1%, at pH 5-6,37℃ for 5-15 hours, free thiamine was obtained quantitatively, and consequently, it was determined by the fluorometric procedure (Taka-diastase method). When BTMP was heated at 50℃, pH 5 for 30 minutes with cysteine of 1000-10000 parts in weight to BTMP, TMP was produced quantitatively, and then free thiamine was obtained by the hydrolysis of the solution with Taka-diastase B, at pH 4.5,37℃, for about 2 hours (cysteine・Taka-diastase method). For the quantitative determination of BTMP in aqueous solution, Taka-diastase method and cysteine・Taka-diastase method were both useful. When BTMP, free thiamine and esterified thiamine were contained together in blood, cysteine・Taka-diastase method and suitable combination of both methods were seemed to be effective for the determination of total thiamine and of BTMP respectively.