*2.7. Method Validation*

#### 2.7.1. Screening Detection Limit

According to SANTE/11813/2017 [33], the screening detection limit (SDL) was examined with similar process, but less replicates, which has been applied in many reported works [34–36]. Fishery drug of Pure Chinese herb was fortified with mixed standard solutions at di fferent concentrations in six duplicates together with their non-spiked counterparts, which were used for the examination of the screening detection limit, and all compounds satisfied 100% detection criterion at their SDL. Simultaneously, an additional criterion, identity confirmation through the 13C/12C-ratio, was satisfied for each target compound at the corresponding theoretical SDL [34–36]. In our experiment, 1 mg/kg, 10 mg/kg, 50 mg/kg, 100 mg/kg, and 500 mg/kg of these mix target samples were prepared respectively. All these fortified samples were pretreated following the aforementioned method (2.4). Results showed that 54, 80, 85, 86, and 89 compounds were screened positive at 1 mg/kg, 10 mg/kg, 50 mg/kg, 100 mg/kg, and 500 mg/kg, respectively.

## 2.7.2. Accuracy and Repeatability

The accuracy and repeatability of the screening method were investigated under the fortified concentrations of 10 mg/kg and 100 mg/kg in fishery drug of pure Chinese herb. For compounds at the detection limit of 500 ng/mL on the mass spectrometer, fortified samples of 500 mg/kg were prepared independently. Under the fortified concentration of 10 mg/kg and 100 mg/kg, compounds with the instrument detection limit of 10 ng/mL and below can be readily detected. Over sixty compounds showed the recovery of 70%–110% at spiked 10 and 100mg/kg; fifteen compounds with 110%–120% at 10 mg/kg; twelve compounds with 110%–120% at 100 mg/kg; and three compounds including chlorpyrifos, phosmet, and tributylphos-phorotrithioate had recoveries of over 125% at both spiked levels. Over 95% of compounds identified at both fortified levels had RSD of less than 15%. Compounds were not identified at the lower fortified level but detected at 100 mg/kg including amitraz, phorate, fenitrothion, validamycin, and prothiofos, with the recovery of 59.3%–125% and RSD of 6.17%–14.7%. Compounds only detected at the spiked level of 500 mg/kg are bromophos ethyl, cyfluthrin, parathion, with recovery of 85.3%–105% and RSD of less than 20%. All the quantification results were obtained with less than 20% RSD. Because of the soft matrix enhancement, there were some compounds with high recoveries at both fortified levels for quantification with the standard matched solvent, especially for chlorpyrifos, phosmet, and tributylphos-phorotrithioate. The details of recovery and RSD are presented in Table 1. It is noticed that some compounds did not meet the recovery criteria at one or both of the fortified levels, which could be attributed to high volatility and easy converting properties.

#### 2.7.3. Calibration and linearity

As the matrix e ffect on the response of the fishery drug sample is quite low, and the recovery results satisfied the semiquantification analysis for most of the compounds in positive samples, the standard solution without matrix matched, and internal standards can be amenable for calibration of positive samples from the perspective of economic costs. In our research, di fferent concentrations of mixed pesticide standards were prepared directly with MeOH–water (1:1, *v*/*v*). Results on mass spectrometer demonstrated that the R-squared of 81 pesticides were no less than 0.990, and 5 other pesticides, including chlorpyrifos, flumethrin, flucythrinate, tau-fluvalinate, and deltamethrin showed R-square between 0.982 and 0.990. The detailed linear profile for 82 compounds is listed in the electronic Supplementary Material (Table S1). The distribution pie chart of the linear range of these compounds is presented in Figure 4.



#### *Molecules* **2019**, *24*, 3375



**Figure 4.** The percentages of different linear range of 82 targeted pesticides.
