Total homocysteine levels in plasma: high-performance liquid chromatographic determination with electrochemical detection and glassy carbon electrode.

An isocratic high-performance liquid chromatography with electrochemical detection (HPLC-ED) method for the determination of total plasma homocysteine [H(e)] has been developed. The electrochemical detection is performed using a glassy-carbon electrode that is not specific for thiol groups. We have tried to solve the problem of specificity focusing our work on chromatographic resolution and have obtained good results without coelution of other thiol compounds or any substances mentioned as common interferences for carbon electrode methods: uric acid, ascorbic acid and salicylates. Thirty samples a day can be assayed for total homocysteine with a lower limit of detection of 2 pmol, and a limit of quantification of 1.0 micromol/l, with a coefficient of variation (C.V.) <20%. For a concentration of total plasma homocysteine of 9.36 micromol/l, the intra- and inter-assay C.V.s were of 3.86% and 5.55% respectively. The analytical recovery achieved in the preparation of the samples ranged from 85.0% to 98.3% and the electrochemical response was linear up to 100 micromol/l.