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Measurement of N7-(2‘-Hydroxyethyl)guanine in Human DNA by Gas Chromatography Electron Capture Mass Spectrometry

2004; American Chemical Society; Volume: 18; Issue: 1 Linguagem: Inglês

10.1021/tx049854d

1520-5010

Chi‐Yu Kao, Roger W. Giese,

DNA and Nucleic Acid Chemistry

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 μg 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 107 nucleotides were observed, a 150-fold range.