An improved high-performance liquid chromatography method for the determination of homocysteine in human plasma

Elevated plasma homocysteine is a known risk factor in arteriosclerotic vas cular disease. To measure homocysteine in a large number of samples we have developed a rapid, simple, robust and inexpensive reversed-phase HPLC meth od for routine analysis. Mercaptopropionylglycine was used as the internal standard and an external calibration in plasma was performed. Improvement w as achieved by the use of gradient elution (using a sodium acetate buffer a nd methanol) resulting in a higher number of samples analyzed per day. Plas ma samples were reduced with tributylphosphine and the proteins were precip itated with perchloric acid before addition of internal standard. The analy tes were derivatized by use of 7-fluorobenzofurazone-4-sulfonic acid ammoni um salt. For calibration human plasma was spiked with nine different concen trations of homocysteine (range 2-50 mu mol L-1). The inter-assay precision of replicate (n = 29) analysis of the concentration of homocysteine in a s ample of pooled plasma was 3.0%. The limit of detection, defined as three t imes the signal-to-noise ratio, was 0.25 mu mol L-1. The linearity of the a ssay was confirmed for a plasma concentration range of 2-2000 mu mol L-1. T he variation of duplicate analyses of 842 plasma samples was 2.6 +/- 1.7%.