Tritium isotope effect in reversed-phase high-performance liquid chromatographic analysis of dopamine and dihydroxyphenylacetic acid

Abstract

A tritium isotope effect has been demonstrated in the high-performance liquid chromatographic analysis of dopamine and its acidic metabolite dihydroxyphenylacetic acid. The chromatographic system consisted of tributyl- n-phosphate, bound to a ChromSpher C 8 column, as stationary phase, and a citrate buffer, containing the ion-pairing agent perchlorate, as the mobile phase. For detection we used continuous electrochemical monitoring (for the total amount of solutes) and discontinuous liquid scintillation counting (for radiolabelled molecules) of the column effluent. [ 3H] Dopamine and [ 3H] dihydroxyphenylacetic acid were biosynthesized by incubation of homogenates of striatal tissue from rat brains with 3H-labelled L-tyrosine. The tritium-labelled compounds were eluted before the corresponding unlabelled analogues. The capacity factor reduction increased with the number of tritium atoms incorporated in the molecules: for single, double and triple tritium-labelled dopamine the separation factors amounted to 1.015, 1.028 and 1.033, respectively. No isotope separation was observed for 7- 14C-labelled dopamine and dihydroxyphenylacetic acid. The isotope effect observed is ascribed to a decrease in lipophilicity following tritium substitution for hydrogen.