Monitoring of dimethyl sulphate-induced N3-methyladenine, N7-methylguanine and O(6)-methylguanine DN
March 29th, 2008 | by admin |Monitoring of dimethyl sulphate-induced N3-methyladenine, N7-methylguanine and O(6)-methylguanine DNA adducts using reversed-phase high performance liquid chromatography and mass spectrometry.
This work describes the determination of N3-methyladenine, N7-methylguanine and O(6)-methylguanine adducts in dimethyl sulphate-treated salmon-testes DNA employing reversed-phase high performance liquid chromatography (RP-HPLC) with UV-vis detection, followed by mass-spectrometric verification using electrospray ionisation in positive mode ESI(+). Within validation parameters, accuracy, precision, calibration parameters, limit of detection (LOD) and quantitation (LOQ) as well as stability of standard stock solutions were tested and presented for UV/vis detection. The limit of detection (LOD) was found to be 0.1ng/mL for N3-methyladenine and 0.2ng/mL for both N7-methylguanine and O(6)-methylguanine (S/N=3). The limit of quantitation (LOQ) was found to be 0.5ng/mL for all measured compounds, (S/N=10). Quantitative results were obtained for each substance based on eight-point calibration. Intra- and inter-day precisions were within 1.73-6.96 and 2.26-7.58%, respectively, and correlation coefficients of calibration curves (R(2)) ranged from 0.9992 to 0.9997. Relative proportion of N7-methylguanine was accounted for 61.53+/-2.97% (R.S.D.=4.8), N3-methyladenine for 38.19+/-2.99% (R.S.D.=9.6) and O(6)-methylguanine for 0.29+/-0.02% (R.S.D.=5.1), respectively. The application of the above-mentioned techniques provides a valuable contribution for simultaneous determination of methylated DNA adducts, and may represent a suitable approach for similar monitoring/screening studies.
Chadt J, Sykora D, Nilsson R, Vodicka P.
Department of Molecular Biology of Cancer, Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Videnska 1083, Prague 4, Czech Republic; Department of Biochemistry, Faculty of Science, Charles University, Albertov 2030, Prague 2, Czech Republic.