## 3 Results  
The results of this study demonstrated high linearity for both flualprazolam and isotonitazene, as evidenced by the correlation coefficients (r\({}^{2}\)) of 0.997 and 0.999 for flualprazolam and isotonitazene, respectively. These values indicate a highly linear relationship between the concentrations of the analytes and the corresponding responses in the LC-MS/MS system. The calibration equations were established as Y = \(-0.000023\) + 0.0662x for flualprazolam and Y = \(-0.0025\) + 0.2087x for isotonitazene. These calibration curves were derived from multiple replicates across a wide concentration range, ensuring the reliability of the method (Table 1; Figure 2). The method demonstrated high sensitivity, with the limit of detection (LOD) and limit of quantification (LOQ) determined for both analytes (Table 2). The LODs for flualprazol and isotonitazone were calculated as 0.608 ng/mL and 0.192 ng/mL, respectively, indicating the method's ability to detect trace levels of these substances in serum samples. Similarly, the LOQ values were determined as 1.842 ng/mL for flualprazol and 0.584 ng/mL for isotonitazone, representing the lowest concentrations that can be quantified with acceptable precision and accuracy (Table 2). The recovery test results demonstrated high accuracy for both flualprazol and isotonitazone, with all measurements falling within the acceptable range of 70-120%. For flualprazol, the measured concentrations were 9.8 ng/mL and 48.5 ng/mL against theoretical concentrations of 10.0 ng/mL and 50.0 ng/mL, resulting in recovery percentages of 98.0% and 97.0%, respectively. Similarly, isotonitazone showed measured concentrations of 10.5 ng/mL and 49.2 ng/mL against theoretical concentrations of 10.0 ng/mL and 50.0 ng/mL, yielding recovery percentages of 105.0% and 98.4%. These findings confirm the high reliability and accuracy of the analytical method, ensuring its suitability for detecting and quantifying these compounds in biological matrices (Table 3). \begin{table}
\begin{tabular}{c c c} \hline
**Chemical Analyte** & **LOD** & **LOQ** \\ \hline Flualprazol (ng/mL) & 0.608 & 1.842 \\ Isotonitazone (ng/mL) & 0.192 & 0.584 \\ \hline \end{tabular}
\end{table} 
Table 2: LOD and LOQ results. Figure 2: Calibration curves of flualprazol and isotonitazone. The repeatability results (RSDr) for flualprazol and isotonitazene demonstrated reliable performance across all tested fortification levels. For flualprazolam at 10 ng/mL, the mean measured concentrations for two analyses were 11.49 ng/mL and 11.50 ng/mL, with standard deviations (SDs) of 0.44 and 0.42 and relative standard deviations (%RSDs) of 3.80% and 3.61%, respectively. The mean recovery percentages were 114.9% and 115.0%. At 50 ng/mL, the mean concentrations were 54.89 ng/mL and 56.27 ng/mL, with SD values of 3.88 and 1.38, %RSDs of 7.07% and 2.45%, and mean recoveries of 109.8% and 112.5%. For isotonitazene at 10 ng/mL, the mean concentrations were 7.55 ng/mL and 7.56 ng/mL, with SD values of 0.37 and 0.28, %RSDs of 4.94% and 3.64%, and mean recoveries of 75.5% and 75.6%. At 50 ng/mL, the mean concentrations were 35.96 ng/mL and 35.18 ng/mL, with SD values of 2.25 and 1.85, %RSDs of 6.24% and 5.26%, and mean recoveries of 71.9% and 70.4%. These results indicate good repeatability for flualprazolam, with recoveries within the acceptable range for both fortification levels. However, isotonitazene demonstrated lower recoveries, particularly at the higher fortification level, indicating potential matrix effects or challenges in quantification accuracy for this compound (Table 4). The reproducibility results (RSDwR%) for flualprazolam and isotonitazene across fortification levels of 10 ng/mL and 50 ng/mL demonstrated variability in performance. For flualprazolam at 10 ng/mL, the mean concentrations were 11.78 ng/mL and 11.48 ng/mL, with standard deviations (SD) of 0.22 and 0.42 and RSDwR values of 1.88% and 3.65%, respectively. The mean recoveries were 117.8% and 114.8%.