Two-dimensional high performance liquid chromatography for determination of homocysteine, methionine and cysteine enantiomers in human serum

A two-dimensional HPLC system with electrochemical detection was used for determination of homocysteine, methionine and cysteine enantiomers in biological samples. The amino acid separations were not possible only by using a chiral column. The compounds were separated from each other on an achiral column (Purospher RP-18 endcapped 250-4 mm, 5 μm) and their enantiomers were separated on Chirobiotic TAG (250-4.6 mm, 5 μm) column in an on-line system. The mobile phase composition and a choice of electrode potentials for detection were investigated. The l-enantiomers always eluted before the d-enantiomers. The proposed method was applied to the analysis of human serum of healthy volunteers and patients with multiple sclerosis. The limit of detection (LOD) and quantitation (LOQ) were defined as the concentration that produced a signal-to-noise ratio (S/N) of 3 and 10. The method LOD values were found to be between 0.05 and 0.50 μg mL−1. The range of LOQ values were between 0.17 and 1.67 μg mL−1, respectively.