Determination of homocysteine in plasma by liquid chromatography with fluorescence detection

A liquid chromatographic (LC) method to assay homocysteine in plasma at micro-molar levels is described. The procedure consists of protein precipitation with trichloroacetic acid and a centrifugation step. The supernatant is then derivatized with a thiol-specific fluorochromophore and LC assayed with fluorescence detection at 385 nm excitation and 515 nm detection. LC analysis is performed by isocratic elution using an acetate buffer (0.05 M, pH 4) as a mobile phase and an alphaBond column. Recoveries based on a five-point standard curve calculation ranged from 96 to 112%. The limits of detection and quantification reached were 0.8 and 1.57 micromol/L, respectively.