Metabolic Profiling of Endocrine-Disrupting Compounds by On-Line Cytochrome P450 Bioreaction Coupled to On-Line Receptor Affinity Screening

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<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.