Automated high-performance liquid chromatographic method for the determination of homocysteine in plasma samples

Plasma homocysteine determination is essential for the diagnosis of inborn errors of metabolism of sulfur amino acids and is achieving considerable importance as a possible risk marker in vascular occlusive pathology. The aim of this study was therefore to develop a fast and sensitive method to assay total and free homocysteine and total and free cysteine in plasma samples, using an automated precolumn sample pretreatment including reduction with 2-mercaptoethanol, carboxymethylation of free thiols and derivation with o-phthalaldehyde. The chromatographic separation was accomplished in 7 min, the within-run and between-run R.S.D.s were all less than 4.3%, the response was linear in the range 0.4-150 microM for homocysteine and 4-1000 microM for cysteine and the mean recoveries were higher than 96%. Moreover, with minimal modification, the method allowed the evaluation of methionine, another important marker of transsulfuration and remethylation defects. The method was applied to the diagnosis of inborn errors involving sulfur amino acids metabolism and to detect mild hyperhomocysteinemia.