Workpackage 3 Optimisation screening techniques

Objectives

The following screening techniques will be optimised

• CALUX

• ASE-CALUX

• AH-PCR

• GCxGC-MS 

• GC-LRMS(/MS)

Their applicability for dioxin analysis in food and feed will be thoroughly tested in an interlaboratory test.

Period

February 2002 - October 2004

WP co-ordinator

Scientific Institute of Public Health

Results

The DIFFERENCE project focuses on the development, optimisation and validation of screening methods for dioxin analysis, including bio-analytical and chemical screening techniques (CALUX, GC-LRMS/MS, GCxGC-ECD) and on the optimisation and validation of new extraction and clean-up procedures. The different screening techniques are optimised in the first year of the project. Good results were obtained for GC-LRMS/MS analysis. For GCxGC-ECD, different column combinations have been tested to optimise the chromatography (in close relation to the DIAC project). GCxGC-ECD showed good separation results (see chromatogram). For the CALUX bioassay a preliminary interlaboratory study was organised to optimise and evaluate CALUX.

Figure. The two-dimensional separation of 90 PCBs (partly visible) and dioxins and furans by comprehensive GCxGC-EC.

The performance of these techniques is assessed in an international validation study and the results are compared with the reference technique GC-HRMS. Furthermore, the validation study is casrried out according to ISO standards. The following combinations are tested on their quantitative performance: GC-HRMS (reference), CALUX bio-assay, GC-LRMS/MS, GCxGC-ECD, ASE-CALUX, and ASE-GC-HRMS. A large number of samples has been prepared by RIVO for the test series. These samples include pork, milk, fish, fish oil, compound feed and standard solutions.

The first round of the validation study (consisting of 3 rounds) focussed on the goodness-of-fit of the calibration curve and on the accuracy of the methods.

In round 2 the detection capability and selectivity were assessed. In round 3, the accuracy and robustness of the methods were evaluated on several samples of different origin (pork tissue, chicken, compound
feed, sepiolithic clay, egg, chicken tissue and vegetable oil). The results of round 3 are summarized in Table 1. The CVW is below 30% for all the labs, which is the EU tolerance for screening method results. (no precision data are available for GCxGC-ECD). The uncorrected CALUX results underestimate the total TEQ concentration of the feed sample. The results of the CALUX labs (= lab A, D, E and H) are more than 50% lower than the median of the GC-HRMS results. The SSZ-scores of the CALUX labs are unsatisfactory (Figure 1). Applying recovery correction improves the total TEQ results of these labs, since the SSZ-scores for CALUX* are satisfactory. The results of the GC-LRMS/MS and GCxGC-ECD are all satisfactory, however only 1 result for GCxGC-ECD was reported. The resultsof the ASE + GC-HRMS show a larger variation than those of GC-HRMS with conventional extraction, but their SSZ-score is still satisfactory.

The results of validation round 1 and 2 have recently been published in:
Van Loco, J., S. P. J. Van Leeuwen, P. Roos, S. Carbonnelle, J. de Boer, L. Goeyens and H. Beernaert (2004). The international validation of bio- and chemical- analytical screening methods for dioxins and dioxin-like PCBs: the DIFFERENCE project rounds 1 and 2. Talanta 63(5): 1169-1182.