## 4 Discussion  
In our study, we developed and validated a sensitive reproducible and accurate LC-MS/MS method for the quantification of flualprazolam and isotonitazene in serum samples. The method demonstrated high linearity with correlation coefficients (r\({}^{2}\)) of 0.997 and 0.999 for both analytes. confirming its reliability for quantitative analysis. The LOD and LOQ values were determined to be as low as 0.608 ng/mL and 1.842 ng/mL for flualprazolam and 0.192 ng/mL and 0.584 ng/mL for isotonitazene, respectively, reflecting the method's exceptional sensitivity. The recovery rates across two fortification levels (10 ng/mL and 50 ng/mL) ranged from 70.4% to 115.0%, falling within the acceptable range of 70-120%. Additionally, the %RSD values for RSDr and RSDwR were consistently below 10%, indicating high precision and robustness. These findings underscore the method's suitability for forensic and clinical applications in detecting and quantifying these potent substances. Limited studies in the literature have demonstrated the importance of sensitivity and reproducibility in detecting these substances [2; 7; 17]. Krotulski et al. (2020) analyzed 197 forensic cases and reported flualprazolam concentrations ranging from 2.0 to 620 ng/mL in postmortem samples, with a median of 8.2 ng/mL [2]. Similarly, Kriikku et al. examined 33 postmortem cases, finding flualprazolam concentrations between 3.0 and 68 ng/g, with a median of 18.0 ng/g [17]. Additionally, Vekic et al. identified flualprazolam in 395 death investigations and 108 impaired driving cases, with blood concentrations ranging from <1.3 to 227 ng/mL [7]. These studies highlight the prevalence of flualprazolam in forensic contexts and underscore the necessity for sensitive and reproducible analytical methods. Our method's high linearity (r\({}^{2}\) > 0.99) and recovery rates (97.0-115.0%) align with these findings, demonstrating its efficacy in accurately quantifying flualprazolam in serum samples. Notably, our focus on serum matrices addresses a critical gap in the literature, as previous studies primarily analyzed whole blood or urine samples. The use of serum eliminates potential interference from cellular components in whole blood, leading to improved analytical accuracy and reproducibility. In terms of isotonitazene, Krotulski et al. reported isotonitazene concentrations in postmortem blood samples ranging from 0.4 to 9.5 ng/mL with an average of 2.2 ng/mL [2]. These findings emphasize the need for highly sensitive analytical methods to detect low concentrations, consistent with our method's low LOD (0.192 ng/mL) and LOQ (0.584 ng/mL), which allow for reliable quantification, even at trace levels [2]. Similarly, Vitrano et al. validated an analytical method for isotonitazene in dried blood spots and reported recovery rates ranging from 72% to 89% depending on the fortification level and extraction method. These values are comparable to the recovery rates observed in our study, which ranged \begin{table}
\begin{tabular}{c c c c c c c c c c} \hline \hline \multirow{3}{*}{
\begin{tabular}{c} **Chemical** \\ **Analyte** \\ \end{tabular} } & \multirow{3}{*}{**Mean**} & \multirow{3}{*}{**SD**} & \multirow{3}{*}{**\% RSD\({}_{\text{wR}}\)**} & \multirow{3}{*}{**\% Recovery (Mean)**} & \multirow{3}{*}{**Mean**} & \multirow{3}{*}{**SD**} & \multirow{3}{*}{**\% RSD\({}_{\text{wR}}\)**} & \multirow{3}{*}{**\% Recovery (Mean)**} \\ \cline{4-4} \cline{6-10}  & & & & & & & & & \\ \hline Flualprazolam & 1 & 11.78 & 0.22 & 1.88 & 117.8 & 55.45 & 3.67 & 6.63 & 110.9 \\  & 2 & 11.48 & 0.42 & 3.65 & 114.8 & 56.94 & 2.20 & 3.86 & 113.9 \\ \hline Isotonitazene & 1 & 7.48 & 0.29 & 3.91 & 74.8 & 36.19 & 0.65 & 1.81 & 72.4 \\  & 2 & 7.44 & 0.41 & 5.54 & 74.4 & 36.78 & 1.17 & 3.19 & 73.6 \\ \hline \hline \end{tabular}
\end{table} 
Table 5: Reproducibility and recovery of the proposed method. from 70.4% to 74.8% at fortification levels of 10 ng/mL and 50 ng/mL, respectively [18].