*3.7. Method Validation*

The method was validated using spiked samples, under the optimal conditions, in terms of linearity, sensitivity, selectivity, and precision (repeatability and between-day precision), decision limit (CCa), decision capability (CCb), and stability according to the European Decision 657/2002/EC [40].

Linearity was studied by triplicate analysis of working standard solutions at concentration levels between 0.5 ng μL−<sup>1</sup> to 10 ng μL−1. In milk, linearity was examined by triplicate analysis of spiked samples within the range of 50 μg kg−1–10,000 μg kg−<sup>1</sup> and calibration curves were calculated. Limits of detection (LOD) and quantification (LOQ) were considered as the concentration giving a signal to noise ratio of 3 and 10, respectively. The selectivity of the method was proved by the absence of interference of endogenous compounds in the analysis of blank milk samples.

Precision and accuracy were calculated by analyzing spiked samples at the concentration levels of 50 μg kg−1, 100 μg kg−<sup>1</sup> and 150 μg kg−1, which correspond to the 12 MRL, MRL, and 1 12 MRL of sulfonamides [6]. Within-day repeatability was examined by 4 measurements at the above concentration levels. Between-day precision was assessed by performing triplicate analysis at the same concentration levels in three days. The relative recovery was calculated using the formula of the percentage of the ratio of the analyte mass that was found in the spiked sample, to the spiked mass.

Decision limit (CCa) was calculated using the equation CCa = MRL + 1.64 × SD, where SD is the standard deviation of the duplicate measurements of twenty milk samples spiked at MRL concentrations of each analyte. Decision capability (CCb) was calculated using the equation CCb = CCa + 1.64 × SD, with the SD being the standard deviation of the duplicate measurements of twenty milk samples spiked at CCa concentrations of each sulfonamide.
