Different approaches for the separation of a set of nucleosides and nucleobases using capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) are described. Several electrolyte compositions have been tested for this purpose. The addition of appropriate amounts of borate to the carrier electrolyte allowed manipulating migration orders in CZE and MEKC by selective complexation of the nucleosides. For detection either UV or two different modes of mass spectrometric (MS) detection were employed. The latter approach included a comparison of two ion sources namely electrospray ionization (ESI) and atmospheric pressure photoionization (APPI) with respect to their potential in the detection of the selected compounds. Thereby it could be demonstrated that, in particular when it comes to the analysis of real samples, APPI-MS is the better choice if MS detection of purines and pyrimidines after separation by CZE is required.