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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

Abstract

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)

Keywords

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

Notes

Acknowledgments

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)

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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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