An improved method for the electrophoretic separation and fluorometric determination of thiamine and its phosphates in animal tissues.

An improved method for electrophoretic separation and fluorometric (or radiometric) determination of thiamine and thiamine mono-, di- and tri-phosphates in animal tissues (liver, small intestine, kidney, heart, brain) is described. The amount of total thiamine handled was 1.5-3.5 microgram. The procedure includes: acid excretion of the compounds from the tissue, deproteinization with TCA, purification of the extract on partially deactivated charcoal, elution with 10% n-propanol in 0.1 N formic acid, lyophilization, electrophoresis on gelatinized cellulose acetate strips, elution of the thiaminic bands with 50% ethanol and finally oxidation to thiochromes and reading of eluate fluorescence in a spectrofluorophotometer. The mean internal recovery was 83.3% and the external one 83.5%. Dephosphorylation of the T phosphates was rather scanty and each thiamine compound was comparably recovered (82.0-85.7%), apparently TTP being the worst and T the best recovered compound.