Metabolic profiling of endocrine-disrupting compounds by on-line cytochrome p450 bioreaction coupled to on-line receptor affinity screening |
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Authors: | Van Liempd Sebastiaan M Kool Jeroen Meerman John H Irth Hubertus Vermeulen Nico P |
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Institution: | LACDR-Division of Molecular Toxicology, Department of Chemistry and Pharmaceutical Sciences, Vrije Universiteit, Amsterdam, The Netherlands. |
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Abstract: | 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
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