Determination of Total Homocysteine in Human Plasma by Isocratic High-Performance Liquid Chromatography

A simple, sensitive and precise isocratic HPLC method for the determination of total homocysteine in human plasma is described. The thiol compounds were liberated from plasma proteins by reduction with tri-n-butylphosphine and derivatized with a thiol-specific fluorogenic marker, 7-fluoro-benzo-2-oxa-1, 3-diazole-4-sulphonate. The derivatives were separated isocratically within 7 min by reversed-phase HPLC using a Superspher 100 RP-18 column as stationary phase. By using this approach more than 200 samples a day can be assayed for total homocysteine. The method was linear up to 100 μmol/l and proved to be sensitive with a detection limit of 0.1 μmol/l and the lowest limit of reliable quantification of 0.5 μmol/l for homocysteine in buffer. Intra- and inter-assay coefficients of variation were both <4% at a concentration of 10 umol/1 homocysteine. Similar results were obtained for homocysteine concentrations between 0.5 and 100 μmol/l. The analytical recovery for these concentrations ranged from 94.9 to 117.0%. As compared to other protocols published so far, this modified method is less complicated but equally sensitive and reproducible and allows a rapid determination of total homocysteine and cysteine in human plasma under routine conditions.