Improved detection limit for a direct determination of 8-hydroxy-2'-deoxyguanosine in untreated urine samples by capillary electrophoresis with optical detection.
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 nucleosides/nucleotides 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 electro-osmotic 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 microM for 8OHdG (LOD = 42 nM) and 0.2-5.0 microM 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.
Tůma
Petr
P
Charles University, 3rd Faculty of Medicine, Charles University in Prague, Centre of Biomedical Sciences, Ruská 87, 10000 Prague 10, Czech Republic.
Samcová
Eva
E
Kvasnicová
Vladimíra
V
D000080242:8-Hydroxy-2'-Deoxyguanosine; D000368:Aged; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000652:urine*; D019075:Electrophoresis, Capillary / Q000379:methods*; D006801:Humans; D008297:Male; D008875:Middle Aged; D012680:Sensitivity and Specificity
D000080242:8-Hydroxy-2'-Deoxyguanosine; D003849:Deoxyguanosine