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 0.1 ng/mL for N3-methyladenine and 0.2 ng/mL for both N7-methylguanine and O(6)-methylguanine (S/N=3). The limit of quantitation (LOQ) was found to be 0.5 ng/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
Jiri
J
Department of Molecular Biology of Cancer, Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Videnska 1083, Prague 4, Czech Republic.
Sykora
David
D
Nilsson
Robert
R
Vodicka
Pavel
P
D000225:Adenine / Q000031:analogs & derivatives* / Q000737:chemistry; D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004247:DNA / Q000187:drug effects; D018736:DNA Adducts / Q000737:chemistry*; D006147:Guanine / Q000031:analogs & derivatives* / Q000737:chemistry; D008297:Male; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization; D013463:Sulfuric Acid Esters / Q000737:chemistry / Q000494:pharmacology*; D013737:Testis / Q000378:metabolism
D018736:DNA Adducts; D013463:Sulfuric Acid Esters; C025946:3-methyladenine; D006147:Guanine; C008450:7-methylguanine; D004247:DNA; C008449:O-(6)-methylguanine; D000225:Adenine; C007482:dimethyl sulfate