High performance liquid chromatography with fluorimetric detection for the determination of total homocysteine in human plasma: method and clinical applications

Homocysteine (Hcy), an amino acid derived from methionine is an independent risk factor for coronary artery disease. We have developed a convenient high performance liquid chromatography assay with fluorimetric detection for the accurate determination of Hcy in human plasma. This assay involves direct addition of human plasma to methanolic monobromobimane for simultaneous protein precipitation and thiol derivatization. The assay was validated by adding authentic homocysteine and homocystine to plasma samples with recovery rates between 95.5 and 102.4%. The relative standard deviations for intraday (n=7) and interday (n=6, 10 consecutive days) analysis were 0.7¯3.9 and 0.7¯4.0%, respectively. Reference values of plasma total homocysteine (tHcy) have been determined for men and women, showing a significant difference (P<0.05) between genders. Preliminary results for occluded coronary artery disease and for maintenance hemodialysis are presented.