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

Abstract

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.

DNA adducts in granulocytes of hospital workers exposed to ethylene oxide

“…Further purification by silica solid phase extraction and reverse phase HPLC was done prior to injection into a GC-EC-MS. This has contributed to the high sensitivity observed as shown by the relatively clean GC-EC-MS chromatograms [Kao and Giese, 2005]. The N7-HEG adducts were detected in all samples with levels of 11.1 and 15.2 adducts/10 7 nucleotides in the duplicate sample.…”

“…Furthermore, although not all samples were analyzed in duplicate, little variation in the adduct level was seen in a duplicate sample. In prior work on method development [Kao and Giese, 2005], it has been demonstrated that the agreement (coefficient of variation) between triplicate 10 pg standards subjected to the method was 3%, and values for duplicate DNA samples spiked with 11 pg of N7-HEG differed by only 2%.…”

“…The N7-HEG adducts were analyzed in 0.1-11.5 mg of DNA by a GC-EC-MS method, as described in detail elsewhere by Kao and Giese [2005]. With the exception of one sample, all coded samples were analyzed only once due to the small amount of available DNA.…”

“…Subjects with homozygous deletion of the glutathione S-transferase T1 (GSTT1) gene (null genotype) were also found to have a significantly higher mean level of HEV as compared to subjects with at least one copy of the gene (positive genotype) [Yong et al, 2001]. Recently, an improved chromatography-electron capturemass spectrometry (GC-EC-MS) method has been developed for the analysis of N7-HEG adducts in human tissues [Kao and Giese, 2005]. Here this technique is used to quantify N7-HEG adducts in the small quantity of granulocyte DNA remaining from earlier studies of this group of subjects.…”

The application of mass spectrometry in molecular dosimetry: Ethylene oxide as an example

“…N7-HEG in lymphocytes varies from 0.21 to 1.85 adducts/10 7 nucleotides with GC/ HRMS analysis (Wu et al, 1999b), and not detectable to 2.6 adducts/10 7 nucleotides with analysis by immunochemical assay (van Delft et al, 1994), and 0.37 to 0.66 adducts/10 7 nucleotides with 32 P-postlabeling assay Zhao & Hemminki, 2002). The levels in granulocytes ranged from 1.6 to 240 adducts/10 7 nucleotides with GC/ECNCI-MS analysis (Kao & Giese, 2005;Yong et al, 2007). These results are also consistent with the observation that a great variation exists in the N7-HEG concentrations in the urine of non-exposed humans (Huang et al, 2008).…”