Specific thiol determination by micellar electrokinetic chromatography and on-column detection reaction with 2,2'-dipyridyldisulfide

A new method for specific determination of glutathione using micellar electrokinetic chromatography and on-column reaction with 2,2'-dipyridyldisulfide is described. 2,2'-Dipyridyldisulfide and a sample of glutathione are injected consecutively into the capillary as two discrete plugs separated with a short plug of background electrolyte. Due to the differences in the mobilities of the 2,2'-dipyridyldisulfide and glutathione, on-column mixing and reaction occur. Glutathione is in this reaction quantitatively transformed into a mixed disulfide concomitantly with formation of an equimolar amount of the 2-thiopyridone which is further separated by micellar electrokinetic chromatography and determined spectrophotometrically at 343 nm. The concentration of glutathione is thus estimated indirectly from the result of 2-thiopyridone determination.