Integrating gel permeation chromatography clean-up in the analysis of metabolites of polychlorinated biphenyls, dibenzo- p-dioxins and dibenzofurans extracted from a microsomal assay A comparison of different mobile phases

Abstract

The metabolism of polychlorinated biphenyls, dibenzo- p-dioxins and dibenzofurans (PCBs, PCDDs, PCDFs) can be studied well with a cytochrome P450 containing microsomal assay. In the present study, the residues of microsomal assays were extracted with organic solvents to determine the metabolites of the studied compounds with GC-ECD and GC-MS. Extracts of microsomal assays contained a matrix which interferes with these type of measurements. The matrix was hard to remove completely with solid phase adsorption chromatography. To overcome the problem of the interfering compounds, clean-up properties of a gel permeation chromatography system were studied for this specific matrix. The clean-up was firstly studied with model compounds and two different mobile phases. Thereupon the most appropriate mobile phase was applied to the extracts of a microsomal assay. Acetone and cyclohexane-dichloromethane (CH-DCM, 1:1, v/v) were compared as mobile phases. The elution profiles of several lipids and some organohalogen compounds (OHCs) were determined. The mixture CH-DCM yielded the best separation between lipids and the OHCs. The results were discussed with a theoretical evaluation on the basis of interactions between solute, solvent and stationary phase. Recoveries were assessed for the GPC procedure and appeared to be good (98–100% for the column itselt and 80–100% for the sample transfer from vial to column). The GPC was integrated in the clean-up of the extract of a microsomal PCDF metabolism assay yielding satisfactory results.