Monitoring of dithiothreitol clearance by means of micellar electrokinetic chromatography.
A new method for specific determination of dithiothreitol (DTT) using micellar electrokinetic chromatography and on-column reaction with reactive disulfide-2,2'-dipyridyldisulfide is described. DTT in this reaction is quantitatively transformed into a mixed disulfide concomitantly with formation of equimolar amount of the 2-thiopyridone that is further separated by micellar electrokinetic chromatography and determined spectrophotometrically at 343 nm. The concentration of DTT is thus estimated indirectly from the result of 2-thiopyridone determination. The linear detection range for concentration versus peak area for the assay is from 0.05 to 2.5 mM (correlation coefficient 0.993) with a detection limit of 2.5 microM. The inter-day reproducibility of the peak area was 1.35% and the inter-day reproducibility of the migration time 0.56%. The method can be applied for DTT monitoring both in chemical and biological systems.
Glatz
Zdenek
Z
Department of Biochemistry, Faculty of Science, Masaryk University, Brno, Czech Republic. glatz@chemi.muni.cz
Sevcíková
Petra
P
D000818:Animals; D020374:Chromatography, Micellar Electrokinetic Capillary / Q000379:methods*; D004229:Dithiothreitol / Q000032:analysis*; D008099:Liver / Q000737:chemistry; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence; D013552:Swine
D004229:Dithiothreitol