Metabolic profiling of endocrine-disrupting compounds by on-line cytochrome p450 bioreaction coupled to on-line receptor affinity screening

Sebastiaan M Van Liempd, Jeroen Kool, John H Meerman, Hubertus Irth, Nico P Vermeulen

Research output: Contribution to JournalArticleAcademicpeer-review


We present a fully automated and hyphenated bioanalytical method for metabolic profiling of potentially harmful xenoestrogens. The system consists of an on-line cytochrome P450 bioreactor coupled to a reversed-phase, gradient high-performance liquid chromatograph. A C18 solid-phase extraction (SPE) unit is used as an interface between the P450 bioreactor and the HPLC column. The HPLC column is linked on-line to a high-resolution screening (HRS)-estrogen receptor alpha affinity detection (ERAD) assay. In effect, the P450 bioreactor produces metabolites that are subsequently trapped on-line by SPE and separated by HPLC. The separated metabolites are then screened on-line, at the moment of elution, for affinity toward estrogen receptor alpha (ERalpha) using the HRS-ERAD assay. The SPE method was optimized with methoxychlor (MXC) and its metabolites mono- and bis-OH-MXC. After optimization, the P450-bioreactor-SPE-HPLC system was made generally applicable to the biocatalysis and trapping of polar to highly apolar compounds. The precision of the P450-bioreactor-SPE-HPLC system is high (relative standard deviation<or=15%), and the HRS-ERAD assay is also very sensitive (having lower limits of detection of 250 ng for bis-OH-MXC and 240 ng for mono-OH-MXC). Finally, bioactivation of 2-hydroxy-4-methoxybenzophenone (benzophenone-3) into ERalpha-binding metabolites by P450 was studied using the validated P450-bioreactor-SPE-HPLC-ERAD system in combination with atmospheric pressure chemical ionization MS. This resulted in the detection of three ERalpha-binding metabolites, of which at least one, a hydroxylated metabolite initially detected only by ERalpha affinity, had not been described previously. The hyphenated P450-bioreactor-SPE-HPLC-HRS-ERAD methodology presented here will be of great interest in on-line research of metabolic activation of endocrine-disrupting compounds.

Original languageEnglish
Pages (from-to)1825-32
Number of pages8
JournalChemical Research in Toxicology
Issue number12
Publication statusPublished - Dec 2007


  • Animals
  • Automation
  • Biosensing Techniques
  • Biotransformation
  • Chromatography, High Pressure Liquid
  • Cytochrome P-450 Enzyme System
  • Endocrine Disruptors
  • Estrogen Receptor alpha
  • Microsomes, Liver
  • Protein Binding
  • Rats
  • Reproducibility of Results
  • Sensitivity and Specificity
  • Tandem Mass Spectrometry
  • Journal Article
  • Research Support, Non-U.S. Gov't


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