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

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.