DEVELOPMENT OF A CAPILLARY ELECTROPHORESIS PRECONCENTRATION METHOD FOR THE ANALYSIS OF DIDEOXYADENOSINE TRIPHOSPHATE

Abstract

The compound 2′,3′-dideoxyadenosine-5′-triphosphate (ddATP) is the intracellular active metabolite of the antiretroviral drug didanosine. Since micellar electrokinetic capillary chromatography with fluorescence detection (MEKC-fluo) by means of precapillary derivatization allowed quantitation of ddATP only in pmol range, development of a more sensitive CE method was intended. CE preconcentration based on electrophoretic effects was tested for additional sensitivity enhancement. Large volume sample stacking (LVSS) using diethylenetriamine (DETA) as an EOF suppressant (ES) in combination with MEKC-fluo was found to be better, but the presence of excess unreacted reagent in the sample hindered the separation of the derivatized nucleotides. Some miscellaneous effects and precapillary derivatization of ddATP at low scale improved the sensitivity of the MEKC-fluo method by minimizing dilution of the sample, which allowed reaching the fmol level that can be found in cells (LOQ = 10 fmol/sample). However, cell studies showed a drastic decrease in fluorescence intensity of the derivatized ddATP peak in the presence of peripheral blood mononuclear cells. Therefore, LVSS-ES using DETA was combined with CZE-UV for the preconcentration of underivatized compounds, which showed promising results with the sensitivity of ddATP being 160 times higher compared to CZE-UV detection without stacking.