The HPLC determination of total thiamin (Vitamin B1) in foods

Abstract A high-performance liquid chromatography (HPLC) method for the routine determination of total thiamin in foods is described. After acid and enzymatic hydrolysis, thiamin was oxidized to thiochrome, and the oxidized fraction was purified and concentrated with solid-phase extraction (SPE). Several commercially available SPE columns were tested. Thiochrome was analyzed with a C 18 HPLC column, using a mixture of methanol and 50 m M phosphate buffer (pH 7.0) (30:70) as the mobile phase. Fluorescence detection (ex 366 nm, em 436 nm) was used. The method was verified using the 14 C technique. Labeled thiamin, [ thiazole -2- 14 C]thiamin hydrochloride, was added to four different food matrices (cheese, pork, potato, and wheat flour), and the distribution of 14 C activity was measured at several stages of the analytical procedure. On the average, 14% of the labeled thiamin was lost in the thiochrome oxidation and in the SPE procedure. About 70% of the residual activity was located in the major peak of the reversed-phase thiochrome chromatogram. The total recovery of the added 14 C activity was estimated at 60% in both samples and standards. The distribution of activity was found to be similar in the four food matrices and the standards.