Discontinuous electrophoretic stacking system for cholate-based electrokinetic chromatographic separation of 8-hydroxy-2'-deoxyguanosine from unmodified deoxynucleosides.

Abstract

The stacking and baseline-resolved separation of the oxidative damage marker, 8-hydroxy-2'-deoxyguanosine (8-OHdG), from unmodified deoxynucleosides in under 4 min is reported. Separations of 8-OHdG from 2'-deoxyadenosine, 2'-deoxycytosine, 2'-deoxyguanosine, and thymidine are accomplished using micellar electrokinetic capillary chromatography with sodium cholate. Importantly, the use of sulfate, intentionally added to the sample matrix, results in effective stacking of 8-OHdG and other analytes. This work extends electrokinetic stacking injection of neutral analytes to include deoxynucleosides. The procedure works well with either electrokinetic or hydrodynamic injection. The separation buffer and sample matrix composition were optimized to effect stacking conditions with an uncoated 50 microm fused-silica capillary. The lower limit of detection for the analytes is in the nanomolar range, and is more than an order of magnitude lower than without stacking. With 30 s (5.7 cm) electrokinetic injections, stacking and baseline separation of 8-hydroxy-2'-deoxyguanosine from the unmodified nucleosides is accomplished, even in the presence of a 400-fold excess of unmodified deoxynucleosides.