Automated sample clean-up and fractionation of chlorpyrifos, chlorpyrifos-methyl and metabolites in mussels using normal-phase liquid chromatography.

An automated method based on normal-phase LC has been developed for the sample clean-up of mussel extracts prior to gas chromatographic analysis of residues of chlorpyrifos, chlorpyrifos-methyl and their metabolites chlorpyrifos-methyl-oxon and 3,5,6-trichloro-2-pyridinol. Pesticides were extracted by means of a high speed blender using acetonitrile-acetone (90:10, v/v). The extract obtained was filtered and concentrated using rotavapor and the residue was dissolved in hexane. One ml of the hexanoic extract was injected on the silica-gel column, using hexane as mobile phase. Pesticides and metabolites were eluted in fat-free fractions with different mixtures of hexane-ethyl acetate. Diode array detection allowed monitoring on-line the elution of lipids. Purified extracts were analyzed by GC using nitrogen-phosphorus detection for quantitation and MS for confirmatory purposes. The method is fully automated from the injection of the extract to the collection of fractions, which are directly injected into the GC system. In this way, neither further clean-up nor solvent exchange were necessary prior to GC analysis. Recoveries obtained from fortified mussel samples at two concentration levels-100 and 20 ng g-1 for parent pesticides and 200 and 40 ng g-1 for metabolites-were higher than 90%. Limits of detection of the whole procedure of analysis were lower than 1 ng g-1 for parent pesticides and than 10 ng g-1 for metabolites. This method has been successfully applied to bioconcentration studies with mussels exposed to chlorpyrifos. Chlorpyrifos and its metabolic derivative 3,5,6-trichloro-2-pyridinol were detected and confirmed by MS in analyzed samples.