## 3 Results and Discussion  
### Methodological Evaluation  
The analytical method was validated for linearity, limit of detection (LOD), limit of quantification (LOQ), matrix effect, precision, and accuracy. Thiamethoxam-d\({}_{3}\), clothianidin-d\({}_{3}\), imidalcoprid-d\({}_{4}\), and acetamipid-d\({}_{3}\) were used as IS for THIA, CLO, IMI, and ACE, respectively. In order to improve accuracy and precision, thiamethoxam-d\({}_{3}\) was used as IS for normalizing the response of the analytes lacking their specific IS. Linearity was evaluated using solvent and matrix-matched calibration standards. The matrix effect was evaluated by comparing standards in solvent with matrix-matched standards. Calibration curves of solvents and the milk matrix were obtained by plotting the quotients of peak areas of THIA, CLO, IMI, and ACE and their corresponding d\({}_{3}\)- or d\({}_{4}\)-labeled IS versus the concentrations of the standards. The proposed method exhibited a good linearity of \(>\)0.992 in the range of 0.2-20 \(\mu\)g/kg with linear correlation coefficients R\({}^{2}\) (Table 2). The matrix effect of the seven NEOs was between 69% and 109%, indicating that the matrix effect is negligible for NEOs, except for THIA. Consequently, the matrix-matched standard solutions were selected for calibration. LOD and LOQ were defined as the level with a signal-to-noise ratio of 3 and 10, respectively, and were in the range of 0.004-0.15 and 0.01-0.36 \(\mu\)g/kg, respectively. The precision and accuracy of the method were evaluated using blank milk samples spiked with three levels (8, 12, and 16 \(\mu\)g/kg). Intra- and inter-day precisions were determined using the relative standard deviation (RSD). Intraday precision was analyzed by comparing standard deviation in the recovery percentage of the spiked samples run on the same day. Interday precision was determined by analyzing the spiked samples over three different days. Excellent recoveries were obtained at the three spiked levels, and the average recovery range was 89-119% with intra- and inter-day RSD ranges of 1.3-10.3% and 2.1-23.8%, respectively (Table 3). Thiamethoxam-d\({}_{3}\) was applied for normalizing the response of the three analytes lacking their specific IS, which improve accuracy and precision successfully. Further studies with the isotope labeled standards are essential and encouraged for very accurate quantification in order to compensate matrix effects and mitigate measurement uncertainty [21]. The method provided satisfactory results for detecting trace NEOs in the milk samples because the lowest MRL in milk for these NEOs was 20 \(\mu\)g/kg. \begin{table}
\begin{tabular}{c c c c c c c c c} \hline \hline \multirow{2}{*}{**Analyytes**} & \multicolumn{3}{c}{**Matrix-Matched**} & \multicolumn{3}{c}{**Calibration Curve in**} & \multirow{2}{*}{\begin{tabular}{c} **Matrix** \\ **Solvent** \\ \end{tabular} } & \multirow{2}{*}{\
\begin{tabular}{c} **LOQ** \\ **Effect (\%)** \\ \end{tabular} } & \multirow{2}{*}{
\begin{tabular}{c} **LOQ** \\ **(\%)** \\ \end{tabular} } \\  & & & & & & & \\ \hline Dinotefuran & y = 28910\(\times\) + 258145 & 0.99 & y = 3134x1 \(-\) 17851 & 0.996 & 92 & 0.15 & 0.36 \\ Nitenpyram & y = 4070.3x \(-\) 9237.4 & 0.999 & y = 3733.6x \(-\) 2563.6 & 0.992 & 109 & 0.05 & 0.17 \\ Thiamethoxam & y = 0.1197x \(+\) 0.8938 & 0.999 & y = 0.1733x \(+\) 0.3865 & 0.992 & 69 & 0.01 & 0.03 \\ Cothianid & y = 14532\(-\) 2.296 & 0.999 & y = 1692x \(-\) 32707 & 0.999 & 86 & 0.01 & 0.03 \\ Imidacoprid & y = 0.1361x \(-\) 0.0283 & 0.999 & y = 1.0445x \(+\) 0.2521 & 0.994 & 94 & 0.02 & 0.05 \\ Acetamipid & y = 0.0121x \(-\) 0.0014 & 0.999 & y = 0.012x \(-\) 0.001 & 0.999 & 101 & 0.06 & 0.20 \\ Thiamethoxam & y = 602482x \(+\) 304377 & 0.999 & y = 620973x \(+\) 329412 & 0.999 & 97 & 0.004 & 0.01 \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Matrix effect (ME), limit of detection (LOD), and limit of quantification (LOQ) of seven neonicotinoids.