Determination of free and total homocysteine in human plasma by high-performance liquid chromatography with fluorescence detection.

A simple, sensitive and precise method for the determination of both free and total homocysteine in human plasma is presented. The total homocysteine in plasma consists of free homocysteine (i.e. reduced plus oxidized homocysteine in the non-protein fraction of plasma) and protein-bound homocysteine. The thiol compounds in plasma, which are reduced or liberated from plasma proteins with tri-n-butylphosphine, are derivatized with a thiol-specific fluorogenic reagent, ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate. The derivatives are separated by reversed-phase high-performance liquid chromatography. The concentrations (mean +/- S.D.) of free and total homocysteine in plasma from 35 normal subjects were 1.94 +/- 0.46 and 6.18 +/- 1.19 nmol/ml, respectively.