Improved detection limit for a direct determination of 8-hydroxy-2 '-deoxyguanosine in untreated urine samples by capillary electrophoresis with optical detection

摘要

Method for a direct determination of 8-hydroxy-2-deoxyguanosine (8OHdG) in untreated urine samples by capillary electrophoresis with optical detection was developed. Optimisation of conditions resulted in a significant lowering of the limit of detection (LOD) by a factor of 400 as compared to our previous study. Optimum separation of 8OHdG from other urine components was achieved using the separation electrolyte containing 80 mM 2-(cyclohexylamino)ethanesulfonic acid, 9 mM LiOH (pH 8.6), and 0.1 mM[ cetyltrimethylammonium bromide ensuring the electro-osmotic flow inversion. In the model aqueous samples, these conditions allow separating 8OHdG and 2'-deoxyguanosine (dG) from other nucleosidesucleotides including 2-deoxycitidine 5'-monophosphate (dCMP), thymidine 5'-monophosphate (TMP), adenosine (A), and thymidine (T). On the other hand, 2'-deoxyadenosine 5-monophosphate (dAMP) and 2'-deoxyguanosine 5'-monophosphate (dGMP) migrate together, and guanosine (G), 2-deoxyadenosine (dA), 2'-deoxycytidine (dC) are transported as neutral species with the electroosmotic flow. In the spiked urine samples, 8OHdG and dG are well separated from each other and from other urine components and exhibit a linear calibration over the concentration range of 0.1-2.0 muM for 8OHdG (LOD = 42 nM) and 0.2-5.0 muM for dG (LOD = 86 nM), but urine metabolites interfere with the determination of dCMP, TMP, A and T. Method is applicable to untreated urine samples with slightly enhanced levels of 8OHdG compared to that found in healthy individuals. (C) 2004 Elsevier B.V. All rights reserved.