| 17975887 |
Van Liempd SM, Kool J, Meerman JH, Irth H, Vermeulen NP: Metabolic profiling of endocrine-disrupting compounds by on-line cytochrome p450 bioreaction coupled to on-line receptor affinity screening. Chem Res Toxicol. 2007 Dec;20(12):1825-32. Epub 2007 Nov 1.
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 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. |
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