Analytical and Bioanalytical Chemistry

, Volume 385, Issue 5, pp 875–887 | Cite as

SPE-HPLC purification of endocrine-disrupting compounds from human serum for assessment of xenoestrogenic activity

  • Philip Sebastian Hjelmborg
  • Mandana Ghisari
  • Eva Cecilie Bonefeld-Jorgensen
Original Paper


Assessment of xenoestrogenic activity in human serum samples requires the removal of endogenous sex hormones to assure that the activity measured originates from xenobiotic compounds only. Serum samples representing high, medium and lower accumulation of persistent organic pollutants (POPs) were extracted using solid-phase extraction (SPE) followed by normal-phase high-performance liquid chromatography (NP-HPLC) for separation of POPs from endogenous hormones. The recovery of polychlorinated biphenyl (PCB) congeners in spiked serum samples was up to 86 %, making the extraction method suitable for the study. MVLN cells, stably transfected with an estrogen receptor (ER) luciferase reporter vector (estrogen response element chemically activated luciferase expression, ERE-CALUX), were exposed to the reconstituted SPE-HPLC extracts for determination of the integrated estrogenic activity. The effects of PCBs were analyzed by direct in vitro exposure of PCBs (nos. 138, 153, 180) and by ex vivo analysis of SPE-HPLC extracts from serum spiked with the PCBs. Similar effects on ER transactivation were observed for the direct in vitro and the ex vivo analysis experiments. The ER transactivation responses determined for actual serum samples were in the linear range of the dose-response curve. 17β-Estradiol titrations showed that the xenoestrogenic effects were mediated via ER. Moreover, our SPE-HPLC-ERE-CALUX assay was demonstrated to elicit high interlaboratory correlation. In the present study the combination of SPE-HPLC purification and the ex vivo estrogenic responses measured by ERE-CALUX was validated and considered to be a valuable tool to assess the combined ER effect of lipophilic serum POPs where additive/synergistic and agonistic/antagonistic effects are integrated giving an overall estimate of exposure and bioactivity.


Solid-phase extraction–high-performance liquid chromatography Estrogen response element chemically activated luciferase expression MVLN Endocrine disruption Xenoestrogens 



Bisphenol A


Bisphenol A dimethacrylate


Chemically activated luciferase expression


Coefficient of variation


Dextran-treated fetal calf serum


Dichlorodiphenyl dichloroethylene


Dichlorodiphenyl trichloroethane


Dulbecco’s modified Eagle’s medium


Dimethyl sulfoxide






Concentration exerting 40 % of the effect of the maximal effective concentration


Estrogen receptor


Estrogen response element


Gas chromatography


High-performance liquid chromatography


Female serum control


Male serum control


Logarithm of the octanol–water partitioning coefficient


Laboratoire de la Toxicologie Humaine


Mass spectrometry




Polychlorinated biphenyl


Persistent organic pollutant


Solid-phase extraction


Retention time


Eluotropic strength



We thank our colleagues from the Unit of Cellular and Molecular Toxicology: Manhai Long and Tanja Krüger for scientific support, and Anne Keblovszki and Birgitte Sloth Andersen for excellent technical assistance. The study was supported by grants from the European Commission: INUENDO (, grant no. QLK4-CT-2001-00202) and the Board of Danish Environmental Protection Agency.


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Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  • Philip Sebastian Hjelmborg
    • 1
  • Mandana Ghisari
    • 1
  • Eva Cecilie Bonefeld-Jorgensen
    • 1
  1. 1.Unit of Cellular and Molecular Toxicology, Institute of Public Health, Department of Environmental and Occupational MedicineUniversity of AarhusAarhus CDenmark

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