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.
Sevcíková
Petra
P
Department of Biochemistry, Faculty of Science, Masaryk University, Kotlárská 2, 611 37 Brno, Czech Republic.
Glatz
Zdenek
Z
Tomandl
Josef
J
D015082:2,2'-Dipyridyl / Q000031:analogs & derivatives* / Q000737:chemistry*; D020374:Chromatography, Micellar Electrokinetic Capillary / Q000379:methods*; D004220:Disulfides / Q000737:chemistry*; D006710:Homocysteine / Q000097:blood*; D006801:Humans; D015203:Reproducibility of Results
D004220:Disulfides; D006710:Homocysteine; C001105:2,2'-dipyridyl disulfide; D015082:2,2'-Dipyridyl