Determination of homocysteine in human plasma by micellar electrokinetic chromatography and in-capillary detection reaction with 2,2'-dipyridyl disulfide.

We present a new method for homocysteine quantitation in human plasma based on in-capillary reaction of homocysteine with 2,2'-dipyridyl disulfide. Homocysteine is in this so-called thiol-exchange reaction quantitatively transformed in mixed disulfide concomitantly with formation of an equimolar amount of 2-thiopyridone that is further separated by micellar electrokinetic chromatography and determined specifically at 343 nm. The concentration of homocysteine is thus estimated indirectly from the result of 2-thiopyridone determination. The linear detection range for concentration versus peak area for the assay was from 0.03-3 mM (correlation coefficient 0.994) with a detection limit of 6 microM and a limit of quantitation 20 microM. The inter-day reproducibility of the peak area and the migration time were 1.37% and 0.05%, respectively. The method is simple, relatively rapid and can be easily automated. Moreover the common capillary electrophoresis apparatus with a UV detector can be used to distinguish between normal and pathological hyperhomocysteinemia plasma samples.