RESUMO
A simple and inexpensive liquid chromatographic method for the determination of seven sulphonamides in animal tissues was validated. The measurement uncertainty of the method was estimated using two approaches: a 'top-down' approach based on in-house validation data, which used either repeatability data or intra-laboratory reproducibility; and a 'bottom-up' approach, which included repeatability data from spiking experiments. The decision limits (CCalpha) applied in the European Union were calculated for comparison. The bottom-up approach was used to identify critical steps in the analytical procedure, which comprised extraction, concentration, hexane-wash and HPLC-UV analysis. Six replicates of porcine kidney were fortified at the maximum residue limit (100 microg kg(-1)) at three different stages of the analytical procedure, extraction, evaporation, and final wash/HPLC analysis, to provide repeatability data for each step. The uncertainties of the gravimetric and volumetric measurements were estimated and integrated in the calculation of the total combined uncertainties by the bottom-up approach. Estimates for systematic error components were included in both approaches. Combined uncertainty estimates for the seven compounds using the 'top-down' approach ranged from 7.9 to 12.5% (using reproducibility) and from 5.4 to 9.5% (using repeatability data) and from 5.1 to 9.0% using the bottom-up approach. CCalpha values ranged from 105.6 to 108.5 microg kg(-1). The major contributor to the combined uncertainty for each analyte was identified as the extraction step. Since there was no statistical difference between the uncertainty values obtained by either approach, the analyst would be justified in applying the 'top-down' estimation using method validation data, rather than performing additional experiments to obtain uncertainty data.
