Measurement of N7-(2'-hydroxyethyl)guanine in human DNA by gas chromatography electron capture mass spectrometry.
An improved method is presented, based on gas chromatography-electron capture mass spectrometry (GC-EC-MS), for measuring N7-(2'-hydroxyethyl)guanine (N7-HEG) in DNA from an in vivo sample. The method was used to detect this adduct in amounts of human DNA ranging from 0.07 to 11.5 microg isolated from granulocytes. In this method, the DNA is spiked with a stable isotope internal standard (N7-HEG-d4) and heated in water to release the adduct in a nucleobase form. After the adduct is extracted into 1-butanol, it is purified by reverse phase HPLC and derivatized with HONO, pentafluorobenzyl bromide, and pivalic anhydride. Further purification by silica solid phase extraction and reverse phase HPLC is done prior to injection into a GC-EC-MS. Relatively clean GC-EC-MS chromatograms result, contributing to the high sensitivity that is observed. In the samples tested, from 1.6 to 240 N7-HEG adducts in 10(7) nucleotides were observed, a 150-fold range.
Kao
Chi-Yu
CY
Department of Pharmaceutical Sciences in the Bouve College of Health Sciences, Barnett Institute, Northeastern University, Boston, Massachusetts 02115, USA.
Giese
Roger W
RW
D002851:Chromatography, High Pressure Liquid / Q000379:methods; D018736:DNA Adducts / Q000032:analysis*; D008401:Gas Chromatography-Mass Spectrometry / Q000295:instrumentation / Q000379:methods*; D006098:Granulocytes / Q000737:chemistry; D006147:Guanine / Q000031:analogs & derivatives* / Q000032:analysis*; D006801:Humans; D012680:Sensitivity and Specificity
D018736:DNA Adducts; C035018:N(7)-hydroxyethylguanine; D006147:Guanine