*2.5. Method Validation*

The method quantification limit (MQL) of 2 μg/kg was validated according to the Chinese Standard [39]. The calibration curves were prepared by the peak area ratios of ENR to the isotope standard plotted against the concentration. Recovery and accuracy were examined by comparing the measured concentrations from processed samples with different spiking levels. Intra- and inter-assay precision was assessed by relative standard deviation (RSD) of the measured positive samples. The RSD was calculated for all determinations in a spiking experiment with three replicates. The content of ENR in the samples was calculated according to the following equation:

$$X = \frac{C \times V \times 1000}{m \times 1000}$$

where *X* (μg/kg) is the content of ENR in the samples; *C* (ng/mL) indicates the concentration of ENR detected in the samples; *V* (mL) is the volume of the extraction reagent; and *m* (g) stands for the weight of the sample.

The decision limit (CCα) and the detection capability (CCβ) of the method were obtained according to Commission Decision 2002/657/EC. We performed the measurement of noise on 20 blank samples (grass carp), with ENR-*d5* as the surrogate, and then the decision limit was obtained; this was greater than 3 times the signal-to-noise ratio from the blank sample. Furthermore, we spiked ENR in 20 blank samples (grass carp) at the concentration of CCα, and calculated the detection capability with the sum of CCα + 1.64 times the SD of within-laboratory reproducibility (n = 20).
