Advertisement

Analytical and Bioanalytical Chemistry

, Volume 406, Issue 22, pp 5293–5302 | Cite as

New validated LC-MS/MS method for the determination of three alkylated adenines in human urine and its application to the monitoring of alkylating agents in cigarette smoke

  • Yongfeng Tian
  • Hongwei HouEmail author
  • Xiaotao Zhang
  • An WangEmail author
  • Yong Liu
  • Qingyuan HuEmail author
Research Paper

Abstract

A highly specific liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed for simultaneous determination of urinary N 3-methyladenine (N 3-MeA), N 3-ethyladenine (N 3-EtA), and N 3-(2-hydroxyethyl)adenine (N 3-HOEtA). Chromatographic separation was achieved on a hydrophilic interaction liquid chromatography column, with a mobile phase gradient prepared from aqueous 10 mM ammonium formate–acetonitrile (5:95 v/v, pH 4.0). Quantification of the analytes was done by multiple reaction monitoring using a triple-quadrupole mass spectrometer in positive-ionization mode. The limits of quantification were 0.13, 0.02, and 0.03 ng/mL for N 3-MeA, N 3-EtA, and N 3-HOEtA, respectively. Intraday and interday variations (relative standard deviations) ranged from 0.6 to 1.3 % and from 3.7 to 7.5 %. The recovery ranges of N 3-MeA, N 3-EtA, and N 3-HOEtA in urine were 80.1–97.3 %, 83.3–90.0 %, and 100.0–110.0 %, respectively. The proposed method was successfully applied to urine samples from 251 volunteers including 193 regular smokers and 58 nonsmokers. The results showed that the levels of urinary N 3-MeA, N 3-EtA, and N 3-HOEtA in smokers were significantly higher than those in nonsmokers. Furthermore, the level of urinary N 3-MeA in smokers was found to be positively correlated with the level of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (r = 0.48, P < 0.001, N = 192). This method is appropriate for routine analysis and accurate quantification of N 3-MeA, N 3-EtA, and N 3-HOEtA. It is also a useful tool for the surveillance of alkylating agent exposure.

Figure

A new validated LC-MS/MS method couple with Oasis MCX column for simultaneous determination of three alkylated DNA adducts and evaluation of correlation with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol in smokers’ urine

Keywords

N3-Methyladenine N3-Ethyladenine N3-(2-Hydroxyethyl)adenine Urine Liquid chromatography–tandem mass spectrometry 

Notes

Acknowledgments

This work was supported by the National Natural Science Foundation of China (No. 21277174, 21347002). The authors thank Gao Na from the Institute of Clinical Pharmacology (School of Medicine, Zhengzhou University, China) for providing 24-h urine samples of smokers and blank urine samples of nonsmokers.

References

  1. 1.
    Hu C-W, Liu H-H, Li Y-J, Chao M-R (2012) Direct analysis of 5-methylcytosine and 5-methyl-2′-deoxycytidine in human urine by isotope dilution LC-MS/MS: correlations with N-methylated purines and oxidized DNA lesions. Chem Res Toxicol 25:462–470CrossRefGoogle Scholar
  2. 2.
    Pfeifer GP, Denissenko MF, Olivier M, Tretyakova N, Hecht SS, Hainaut P (2002) Tobacco smoke carcinogens, DNA damage and p53 mutations in smoking-associated cancers. Oncogene 21:7435–7451CrossRefGoogle Scholar
  3. 3.
    Tompkins EM, McLuckie KIE, Jones DJL, Farmer PB, Brown K (2009) Mutagenicity of DNA adducts derived from ethylene oxide exposure in the pSP189 shuttle vector replicated in human Ad293 cells. Mutat Res 678:129–137CrossRefGoogle Scholar
  4. 4.
    Swenberg JA, Dyroff MC, Bedell MA, Popp JA, Huh N, Kristein U, Rajewsky MF (1984) O 4-Ethyldeoxythymidine, but not O 6-ethyldeoxyguanosine, accumulates in hepatocyte DNA of rats exposed continuously to diethylnitrosamine. Proc Natl Acad Sci U S A 81:1692–1695CrossRefGoogle Scholar
  5. 5.
    Scherer E, Timmer AP, Emmelot P (1980) Formation by diethylnitrosamine and persistence of O 4-ethylthymidine in rat liver DNA in vivo. Cancer Lett 10:1–6CrossRefGoogle Scholar
  6. 6.
    Prevost V, Shuker DEG (1996) Cigarette smoking and urinary 3-alkyladenine excretion in man. Chem Res Toxicol 9:439–444CrossRefGoogle Scholar
  7. 7.
    Chao M-R, Wang C-J, Chang L-W, Hu C-W (2005) Quantitative determination of urinary N7-ethylguanine in smokers and non-smokers using an isotope dilution liquid chromatography/tandem mass spectrometry with on-line analyte enrichment. Carcinogenesis 27:146–151CrossRefGoogle Scholar
  8. 8.
    Hu C-W, Lin B-H, Chao M-R (2011) Quantitative determination of urinary N3-methyladenine by isotope-dilution LC-MS/MS with automated solid-phase extraction. Int J Mass Spectrom 304:68–73CrossRefGoogle Scholar
  9. 9.
    Kopplin A, Eberle-Adamkiewicz G, Glüsenkamp K-H, Nehls P, Kirstein U (1995) Urinary excretion of 3-methyladenine and 3-ethyladenine after controlled exposure to tobacco smoke. Carcinogenesis 16:2637–2641CrossRefGoogle Scholar
  10. 10.
    Singer B (1976) All oxygens in nucleic acids react with carcinogenic ethylating agents. Nature 264:333–339CrossRefGoogle Scholar
  11. 11.
    Beranek DT (1990) Distribution of methyl and ethyl adducts following alkylation with monofunctional alkylating agents. Mutat Res 231:11–30CrossRefGoogle Scholar
  12. 12.
    Shuker DEG, Prevost V, Friesen MD, Li D, Ohshima H, Bartsch H (1993) Urinary markers for measuring exposure to endogenous and exogenous alkylating agents and precursors. Environ Health Perspect 99:33–37CrossRefGoogle Scholar
  13. 13.
    Xiong W, Hou H, Jiang X, Tang G, Hu Q (2010) Simultaneous determination of four tobacco-specific N-nitrosamines in mainstream smoke for Chinese Virginia cigarettes by liquid chromatography-tandem mass spectrometry and validation under ISO and “Canadian intense” machine smoking regimes. Anal Chim Acta 674:71–78CrossRefGoogle Scholar
  14. 14.
    Fay LB, Leaf CD, Gremaud E, Aeschlimann J-M, Steen C, Shuker DEG, Turesky RJ (1997) Urinary excretion of 3-methyladenine after consumption of fish containing high levels of dimethylamine. Carcinogenesis 18:1039–1044CrossRefGoogle Scholar
  15. 15.
    Hecht SS (1999) DNA adduct formation from tobacco-specific N-nitrosamines. Mutat Res 424:127–142CrossRefGoogle Scholar
  16. 16.
    Carmella SG, Akerkar SA, Richie JP, Hecht SS (1995) Intraindividual and interindividual differences in metabolites of the tobacco-specific lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in smokers’ urine. Cancer Epidemiol Biomarkers Prev 4:635–642Google Scholar
  17. 17.
    Hu C-W, Hsu Y-W, Chen J-L, Tam L-M, Chao M-R (2013) Direct analysis of tobacco-specific nitrosamine NNK and its metabolite NNAL in human urine by LC–MS/MS: evidence of linkage to methylated DNA lesions. Arch Toxicol 88:291–299CrossRefGoogle Scholar
  18. 18.
    Vineis P, Perera F (2000) DNA adducts as markers of exposure to carcinogens and risk of cancer. Int J Cancer 88:325–328CrossRefGoogle Scholar
  19. 19.
    Prevost V, Shuker DEG, Bartsch H, Pastorelli R, Stillwell WG, Trudel LJ, Tannenbaum SR (1990) The determination of urinary 3-methyladenine by immunoaffinity chromatography-monoclonal antibody-based ELISA: use in human biomonitoring studies. Carcinogenesis 11:1747–1751CrossRefGoogle Scholar
  20. 20.
    Prevost V, Shuker DEG, Friesen MD, Eberle G, Ajewsky MFR, Bartsch H (1993) Immunoaffinity purification and gas chromatography–mass spectrometric quantification of 3-alkyladenines in urine: metabolism studies and basal excretion levels in man. Carcinogenesis 14:199–204CrossRefGoogle Scholar
  21. 21.
    Shuker DEG, Bailey E, Parry A, Lamb J, Farmer PB (1987) The determination of urinary 3-methyladenine in humans as a potential monitor of exposure to methylating agents. Carcinogenesis 8:959–962CrossRefGoogle Scholar
  22. 22.
    Friesen MD, Garren L, Prevost V, Shuker DEG (1991) Isolation of urinary 3-methyladenine using immunoaffinity columns prior to determination by low-resolution gas chromatography–mass spectrometry. Chem Res Toxicol 4:102–106CrossRefGoogle Scholar
  23. 23.
    Feng S, Roethig HJ, Liang Q, Kinser R, Jin Y, Scherer G, Urban M, Riedel K (2006) Evaluation of urinary 1-hydroxypyrene, S-phenylmercapturic acid, trans, trans-muconic acid, 3-methyladenine, 3-ethyladenine, 8-hydroxy-2′-deoxyguanosine and thioethers as biomarkers of exposure to cigarette smoke. Biomarkers 11:28–52CrossRefGoogle Scholar
  24. 24.
    Hu C-W, Chen M-C, Ho H-H, Chao M-R (2012) Simultaneous quantification of methylated purines in DNA by isotope dilution LC-MS/MS coupled with automated solid-phase extraction. Anal Bioanal Chem 402:1199–1208CrossRefGoogle Scholar
  25. 25.
    Hu C-W, Chao M-R (2012) Direct-acting DNA alkylating agents present in aqueous extracts of areca nut and its products. Chem Res Toxicol 25:2386–2392CrossRefGoogle Scholar
  26. 26.
    Food and Drug Administration (2001) Guidance for industry - bioanalytical method validation. http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/UCM070107.Pdf. Accessed 31 Jan 2014
  27. 27.
    Hou H, Zhang X, Tian Y, Tang G, Liu Y, Hu Q (2012) Development of a method for the determination of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol in urine of nonsmokers and smokers using liquid chromatography/tandem mass spectrometry. J Pharm Biomed Anal 63:17–22CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  1. 1.China National Tobacco Quality Supervision and Test CenterZhengzhouChina
  2. 2.Anhui Institute of Optics and Fine MechanicsChinese Academy of ScienceHefeiChina

Personalised recommendations