Analysis of Dideoxyadenosine Triphosphate by Capillary Electrophoresis with Fluorescence Detection. Derivatization Through the Adenine Group

Abstract

The adenine base of 2′,3′-dideoxyadenosine-5′-triphosphate (ddATP) was chosen as the target for precapillary derivatization for its analysis by CE with fluorescence detection. Several fluorophores, such as chloroacetaldehyde, 9-fluorenylmethyloxycarbonylchloride (FMOC), fluorescamine (Fluram), o-phthaldialdehyde (OPA) and complexation with terbium (Tb), gadolinium (Gd), and phenanthroline (phen) were tried. Precapillary derivatization of ddATP with chloroacetaldehyde showed poor peak shape for the ε-ddATP adduct and degradation peaks of ε-adenine, possibly due to the low sample pH of 4.0. Precapillary derivatization of ddATP with OPA/β-mercaptoethanol, Fluram, and FMOC showed no fluorescent derivatization products. Complexation of ddATP with Tb-Gd-phen produced a sharp fluorescent peak. The sensitivity of the Tb-Gd-ddATP-phen complex was found to be 20 times higher than UV detection of ddATP.

Keywords:

• 2′,3′-Dideoxyadenosine-5′-triphosphate
• CE
• Didanosine
• Fluorescence detection
• Human immunodeficiency virus
• Precapillary derivatization

ACKNOWLEDGMENTS

Funding of this work by the Flemish Fund for Scientific Research (Research Project G.0355.06) is gratefully acknowledged. S.M thanks the onderzoeksfonds/K. U. Leuven for financial support.