Analytical and Bioanalytical Chemistry

, Volume 408, Issue 15, pp 4021–4033 | Cite as

Stability issues in the determination of 19 urinary (free and conjugated) monohydroxy polycyclic aromatic hydrocarbons

  • Éric Gaudreau
  • René Bérubé
  • Jean-François Bienvenu
  • Normand Fleury
Research Paper


Data on the stability of monohydroxy polycyclic aromatic hydrocarbons (OH-PAHs; metabolites of PAHs) in urine are needed in order to effectively study the effects of PAHs in the body, but the relevant data are not available in the literature. Therefore, in this work, we investigated the stability of OH-PAHs in urine. For each OH-PAH studied, the free form (as opposed to the conjugated form) comprised <10 % of the total OH-PAH in urine samples obtained from a normal population, except for 9-OH-phenanthrene (where the free form represented 22.2 % of the total 9-OH-phenanthrene). 1-Naphthol and 9-OH-phenanthrene were found to be less stable in their free forms in urine than in their conjugated forms when the urine samples were stored at 4 °C or room temperature. Free 3-OH-fluoranthene was also very unstable at 4 °C or room temperature. The conjugated forms of the OH-PAHs were more stable than their corresponding free forms. However, the free and conjugated forms of all the OH-PAHs were stable in urine at −20 °C and −80 °C. A freeze and thaw assay also revealed that freezing and thawing had minimal impact on the stability of the OH-PAHs in urine. For the derivatized extracts, storing the samples under an argon atmosphere at 4 °C was found to maintain sample integrity. In order to measure the stabilities of 19 hydroxylated metabolites of PAHs in urine, we developed a method with sensitivity in the low pg/mL range using nine labeled internal standards. This method combined enzymatic deconjugation with liquid–liquid extraction, derivatization with N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA), and gas chromatography/tandem mass spectrometry (GC-MS/MS).

Graphical abstract

Stability of the conjugated forms of the OH-PAHs versus free forms (e.g. 1-naphthol)


Stability in urine Free and conjugated metabolites Freeze and thaw Monohydroxy polycyclic aromatic hydrocarbons Gas chromatography Tandem mass spectrometry 



The authors wish to acknowledge the contributions of the laboratory technicians Éric Daigle and Simon Chouinard, who respectively collaborated with us to develop and improve this analytical method. We would also like to thank Dr. Pierre Ayotte for his judicious advice about the publication.

Compliance with ethical standards

This study involves the use of human urine samples collected from multiple volunteer donors, including laboratory staff and members of their family. We obtained the consent of all participants to use the data obtained from those samples for research purposes. The urine samples were exclusively used for method development and validation. The samples were coded to maintain the anonymity of the participants and to disconnect the identity of each participant from the data generated from their urine sample. Several urine pools were also prepared to preserve anonymity. Moreover, no inter-individual (between-samples) comparisons were made.

Conflict of interest

The authors declare that there is no conflict of interest associated with this work.

Supplementary material

216_2016_9491_MOESM1_ESM.pdf (436 kb)
ESM 1 (PDF 436 kb)


  1. 1.
    International Agency for Research on Cancer (IARC). IARC monographs on the evaluation of carcinogenic risks to humans, vol 92. Lyon: IARC; 2010. p. 1–853.Google Scholar
  2. 2.
    Ramesh A, Walker SA, Hood DB, Guillen MD, Schneider K, Weyand EH. Int J Toxicol. 2004;23:301–33.CrossRefGoogle Scholar
  3. 3.
    Agency for Toxic Substances and Disease Registry (ATSDR). Toxicological profile for polycyclic aromatic hydrocarbons. 1995. p. 1-487.
  4. 4.
    Romanoff LC, Li Z, Young KJ, Blakely III NC, Patterson Jr DG, Sandau CD. J Chromatogr B Analyt Technol Biomed Life Sci. 2006;835:47–54.CrossRefGoogle Scholar
  5. 5.
    Onyemauwa F, Rappaport SM, Sobus JR, Gajdosova D, Wu R, Waidyanatha S. J Chromatogr B Analyt Technol Biomed Life Sci. 2009;877:1117–25.CrossRefGoogle Scholar
  6. 6.
    Campo L, Rossella F, Fustinoni S. J Chromatogr B Analyt Technol Biomed Life Sci. 2008;875:531–40.CrossRefGoogle Scholar
  7. 7.
    Holm A, Molander P, Lundanes E, Ovrebo S, Greibrokk T. J Chromatogr B Analyt Technol Biomed Life Sci. 2003;794:175–83.CrossRefGoogle Scholar
  8. 8.
    Leroyer A, Jeandel F, Maitre A, Howsam M, Deplanque D, Mazzuca M, et al. Sci Total Environ. 2010;408:1166–73.CrossRefGoogle Scholar
  9. 9.
    Adetona O, Sjodin A, Zheng L, Romanoff LC, guilar-Villalobos M, Needham LL, et al. J Occup Environ Hyg. 2012;9:217–29.CrossRefGoogle Scholar
  10. 10.
    Benowitz NL, Jacob III P, Bernert JT, Wilson M, Wang L, Allen F, et al. Cancer Epidemiol Biomarkers Prev. 2005;14:1376–83.CrossRefGoogle Scholar
  11. 11.
    Bouchard M, Pinsonneault L, Tremblay C, Weber JP. Int Arch Occup Environ Health. 2001;74:505–13.CrossRefGoogle Scholar
  12. 12.
    Bouchard M, Normandin L, Gagnon F, Viau C, Dumas P, Gaudreau E, et al. J Toxicol Environ Health A. 2009;72:1534–49.CrossRefGoogle Scholar
  13. 13.
    Hagedorn HW, Scherer G, Engl J, Riedel K, Cheung F, Errington G, et al. J Anal Toxicol. 2009;33:301–9.CrossRefGoogle Scholar
  14. 14.
    Meeker JD, Barr DB, Serdar B, Rappaport SM, Hauser R. J Expo Sci Environ Epidemiol. 2007;17:314–20.CrossRefGoogle Scholar
  15. 15.
    Rossella F, Campo L, Pavanello S, Kapka L, Siwinska E, Fustinoni S. Occup Environ Med. 2009;66:509–16.CrossRefGoogle Scholar
  16. 16.
    Shealy DB, Barr JR, Ashley DL, Patterson Jr DG, Camann DE, Bond AE. Environ Health Perspect. 1997;105:510–3.CrossRefGoogle Scholar
  17. 17.
    van Wijnen JH, Slob R, Jongmans-Liedekerken G, van de Weerdt RH, Woudenberg F. Environ Health Perspect. 1996;104:530–4.CrossRefGoogle Scholar
  18. 18.
    Wang JJ, Frazer DG, Stone S, Goldsmith T, Law B, Moseley A, et al. Anal Chem. 2003;75:5953–60.CrossRefGoogle Scholar
  19. 19.
    Grimmer G, Jacob J, Dettbarn G, Naujack KW. Int Arch Occup Environ Health. 1997;69:231–9.CrossRefGoogle Scholar
  20. 20.
    Jacob III P, Wilson M, Benowitz NL. Anal Chem. 2007;79:587–98.CrossRefGoogle Scholar
  21. 21.
    Simon P, Morele Y, Delsaut P, Nicot T. J Chromatogr B Biomed Sci Appl. 1999;732:91–101.CrossRefGoogle Scholar
  22. 22.
    Jongeneelen FJ, Anzion RB, Leijdekkers CM, Bos RP, Henderson PT. Int Arch Occup Environ Health. 1985;57:47–55.CrossRefGoogle Scholar
  23. 23.
    Li Y, Li AC, Shi H, Zhou S, Shou WZ, Jiang X, et al. Rapid Commun Mass Spectrom. 2005;19:3331–8.CrossRefGoogle Scholar
  24. 24.
    Xu L, Spink DC. J Chromatogr B Analyt Technol Biomed Life Sci. 2007;855:159–65.CrossRefGoogle Scholar
  25. 25.
    Goen T, Schaller KH, Drexler H. Int J Hyg Environ Health. 2012;215:229–32.CrossRefGoogle Scholar
  26. 26.
    Jongeneelen FJ, Anzion RB, Henderson PT. J Chromatogr. 1987;413:227–32.CrossRefGoogle Scholar
  27. 27.
    Chetiyanukornkul T, Toriba A, Kameda T, Tang N, Hayakawa K. Anal Bioanal Chem. 2006;386:712–8.CrossRefGoogle Scholar
  28. 28.
    Marie C, Bouchard M, Heredia-Ortiz R, Viau C, Maitre A. J Appl Toxicol. 2010;30:402–10.Google Scholar
  29. 29.
    Ariese F, Verkaik M, Hoornweg GP, van de Nesse RJ, Jukema-Leenstra SR, Hofstraat JW, et al. J Anal Toxicol. 1994;18:195–204.CrossRefGoogle Scholar
  30. 30.
    Ruzgyte A, Bouchard M, Viau C. J Anal Toxicol. 2005;29:533–8.CrossRefGoogle Scholar
  31. 31.
    Galceran MT, Moyano E. J Chromatogr A. 1996;731:75–84.CrossRefGoogle Scholar
  32. 32.
    Pigini D, Cialdella AM, Faranda P, Tranfo G. Rapid Commun Mass Spectrom. 2006;20:1013–8.CrossRefGoogle Scholar
  33. 33.
    Xu X, Zhang J, Zhang L, Liu W, Weisel CP. Rapid Commun Mass Spectrom. 2004;18:2299–308.CrossRefGoogle Scholar
  34. 34.
    Mattarozzi M, Musci M, Careri M, Mangia A, Fustinoni S, Campo L, et al. J Chromatogr A. 2009;1216:5634–9.CrossRefGoogle Scholar
  35. 35.
    Li Z, Romanoff LC, Trinidad DA, Hussain N, Jones RS, Porter EN, et al. Anal Chem. 2006;78:5744–51.CrossRefGoogle Scholar
  36. 36.
    Li Z, Sandau CD, Romanoff LC, Caudill SP, Sjodin A, Needham LL, et al. Environ Res. 2008;107:320–31.CrossRefGoogle Scholar
  37. 37.
    Li Z, Romanoff LC, Trinidad DA, Pittman EN, Hilton D, Hubbard K, et al. Anal Bioanal Chem. 2014;406:3119–29.CrossRefGoogle Scholar
  38. 38.
    Gupta MK, Jain R, Singh P, Ch R, Mudiam MK. J Anal Toxicol. 2015;39:365–73.CrossRefGoogle Scholar
  39. 39.
    Gaudreau E, Bienvenu J-F, Bérubé R, Daigle E, Chouinard S. Using the Agilent 7000B triple quadrupole GC/MS for parts per trillion detection of PAH metabolites in human urine (Agilent application note). 2012.
  40. 40.
    Health Canada. Report on human biomonitoring of environmental chemicals in Canada. Results of the Canadian Health Measures Survey Cycle 2 (2009–2011). 2013.
  41. 41.
    Kovacs A, Kende A, Mortl M, Volk G, Rikker T, Torkos K. J Chromatogr A. 2008;1194:139–42.CrossRefGoogle Scholar
  42. 42.
    Poole CF. J Chromatogr A. 2013;1296:2–14.CrossRefGoogle Scholar
  43. 43.
    Centers for Disease Control and Prevention (CDC). Third national report on human exposure to environmental chemicals (years 2001–2002). 2005.
  44. 44.
    Centers for Disease Control and Prevention (CDC). Fourth national report on human exposure to environmental chemicals (years 2003–2004). 2009.
  45. 45.
    Matuszewski BK, Constanzer ML, Chavez-Eng CM. Anal Chem. 2003;75:3019–30.CrossRefGoogle Scholar
  46. 46.
    Matuszewski BK. J Chromatogr B Analyt Technol Biomed Life Sci. 2006;830:293–300.CrossRefGoogle Scholar
  47. 47.
    Schantz MM, Benner BA, Jr., Heckert NA, Sander LC, Sharpless KE, Vander Pol SS, et al. Anal Bioanal Chem. 2015;407:2945–54.Google Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Éric Gaudreau
    • 1
  • René Bérubé
    • 1
  • Jean-François Bienvenu
    • 1
  • Normand Fleury
    • 1
  1. 1.Laboratoire du Centre de Toxicologie (CTQ)Institut national de santé publique du Québec (INSPQ)QuébecCanada

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