Quantitation of 7-ethylguanine in leukocyte DNA from smokers and nonsmokers by liquid chromatography-nanoelectrospray-high resolution tandem mass spectrometry - PubMed (original) (raw)

. 2011 Oct 17;24(10):1729-34.

doi: 10.1021/tx200262d. Epub 2011 Sep 14.

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Quantitation of 7-ethylguanine in leukocyte DNA from smokers and nonsmokers by liquid chromatography-nanoelectrospray-high resolution tandem mass spectrometry

Silvia Balbo et al. Chem Res Toxicol. 2011.

Abstract

There is considerable evidence for the exposure of humans to an unknown ethylating agent, and some studies indicate that cigarette smoking may be one source of this exposure. Therefore, we have developed a liquid chromatography-nanoelectrospray-high resolution tandem mass spectrometry-selected reaction monitoring (LC-NSI-HRMS/MS-SRM) method for the analysis of 7-ethyl-Gua in human leukocyte DNA, a readily available source of DNA. [(15)N(5)]7-Ethyl-Gua was used as the internal standard. Leukocyte DNA was isolated and treated by thermal neutral hydrolysis. The hydrolysate was partially purified by solid-phase extraction. The fraction containing 7-ethyl-Gua was analyzed by LC-NSI-HRMS/MS-SRM using the transition m/z 180 [M + H](+)→ m/z 152.05669 [Gua + H](+) for 7-ethyl-Gua and m/z 185 → m/z 157.04187 for the internal standard. The detection limit was approximately 10 amol on column, while the limit of quantitation was about 8 fmol/μmol Gua starting with 180 μg DNA (corresponding to 36 μg DNA on-column). Leukocyte DNA samples from 30 smokers and 30 nonsmokers were analyzed. Clear peaks for 7-ethyl-Gua and the internal standard were observed in most of the samples. The mean (±SD) level of 7-ethyl-Gua measured in leukocyte DNA from smokers was 49.6 ± 43.3 (range 14.6-181) fmol/μmol Gua, while that from nonsmokers was 41.3 ± 34.9 (range 9.64-157) fmol/μmol Gua. Although a significant difference between smokers and nonsmokers was not observed, the method described here is unique in the use of high resolution mass spectrometry and establishes for the first time the presence of 7-ethyl-Gua in human leukocyte DNA.

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Figures

Figure 1

Figure 1

Chromatograms obtained upon analysis of a standard mixture of 10 amol of 7-ethyl-Gua and 5 fmol [15N5]7-ethyl-Gua using LC-NSI-HRMS/MS-SRM. Panel (A) shows the results from the transition at m/z 180 [M + H]+ → m/z 152.05669 [Gua + H]+ for 7-ethyl-Gua. Panel (B) shows the corresponding transition m/z 185 [M + H]+ → m/z 157.04187 [Gua + H]+ for the internal standard. Results are shown with a 5 ppm mass tolerance.

Figure 2

Figure 2

Chromatograms obtained upon LC-NSI-HRMS/MS-SRM analysis of human leukocyte DNA (129 μg, 12.9 μg on column) containing 59.4 fmol/μmol Gua. The relatively higher amount of analyte in this sample allowed confirmation of its identity by additional monitoring of the accurate mass of the molecular ion of 7-ethyl-Gua and the internal standard. Panel (A) shows the result from monitoring of the accurate mass of 7-ethyl-Gua (m/z 180.08799). Panel (B) shows the result from the monitoring of the accurate mass of [15N5]7-ethyl-Gua (m/z 185.07317). Panel (C) shows the results from the transition at m/z 180 [M + H]+ → m/z 152.05669 [Gua + H]+ for 7-ethyl-Gua and panel (D) shows the corresponding transition m/z 185 [M + H]+ → m/z 157.04187 [Gua + H]+ for the internal standard. Results are shown with a 5 ppm mass tolerance.

Figure 3

Figure 3

Relationship of added to detected 7-ethyl-Gua. Various amounts of the DNA adduct were added to calf thymus DNA (0.3 mg, 30 μg on column) and analyzed by the method described in the text; R2 = 0.99. 7-Ethyl-Gua present in the calf thymus DNA was subtracted from each value

Figure 4

Figure 4

Chromatograms obtained upon LC-NSI-HRMS/MS-SRM analysis of human leukocyte DNA (14 μg, 1,4 μg on column) for 7-ethyl-Gua by the method described in the text. This sample contained 70.2 fmol/μmol Gua of 7-ethyl-Gua corresponding to 130 amol injected on column (1 μl injected out of a 10 μl sample). Panel (A), analyte; panel (B), internal standard. Results are shown with a 5 ppm mass tolerance.

Scheme 1

Scheme 1

Analytical scheme for determination of 7-ethyl-Gua in human leukocyte DNA

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