M.J.C. Rozemeijer

Procedures for analysing phenolic metabolites of polychlorinated dibenzofurans, -dibenzo-p-dioxins and -biphenyls extracted from a microsomal assay: optimising solid-phase adsorption clean-up and derivatisation methods

Abstract A microsomal assay with the enzyme cytochrome P450 offers a good experimental model to study the principles of the metabolism of polychlorinated dibenzofurans, -dibenzo- p -dioxins and -biphenyls (PCDFs, PCDDs, PCBs). To study the phenolic metabolites on a gas chromatograph with mass spectrometric or electron-capture detection, some clean-up is necessary to remove coextracted phospholipids and other interferences. In this study, two solid-phase clean-up columns were studied: Florisil and alumina B, as well as a destructive column: an acidic:basic impregnated silica column (ABSC). The elution profile and recovery of a methoxytetrachlorodibenzofuran (methoxy-TCDF) and a dimethoxytetrachlorobiphenyl (dimethoxy-TCB) were determined using pentane or dichloromethane as eluent. For comparison, a TCB and a TCDD were assessed as well. Both Florisil and alumina B yielded satisfactory results for all compounds. The ABSC showed low recoveries for the dimethoxy-TCB. Next both solid-phases were applied on the extract from microsomal assays with PCDFs and PCDDs. A successive combination of Florisil and alumina B resulted in samples sufficiently purified to allow for low concentration analysis. In addition attention was focused on several derivatisation methods. Methylation with methyl iodide and diazomethane and acetylation in an aqueous phase and an organic phase were studied. For the purpose of the present study the method with methyl iodide suited best.

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.