## 4 Discussion
This similarity underscores the reproducibility of recovery rates across different sample types and methods. Additionally, Walton et al. investigated the stability of isotonitazene in blood samples under various storage conditions and found significant degradation at room temperature after 14 days, with concentrations decreasing by approximately 20% [6]. Our study did not specifically evaluate stability, but the lower recovery rates observed at higher fortification levels suggest potential challenges related to matrix effects or compound stability during sample preparation and analysis. These findings collectively demonstrate that while our method achieves comparable sensitivity and precision to existing methods, further optimization is needed to improve the recovery rates and account for matrix effects. The low LOD and LOQ values obtained in our study, however, offer a significant advantage in detecting and quantifying trace levels of isotonitazene in serum samples. The method's high sensitivity with LOD values of 0.608 ng/mL for flualprazolam and 0.192 ng/mL for isotonitazene enables the detection of trace levels of these substances, which is critical for forensic investigations. The low LOQ values similarly ensure quantification with high precision. These characteristics make the method particularly valuable for postmortem toxicology, where low analyte concentrations are common. In the literature, limited studies have been conducted on the determination of isotonitazene in various biological matrices, including bodily fluids, the liver, and brain [14; 15; 19]. Notably, Pardi et al. analyzed isotonitazene in bodily fluids and other biological samples, reporting similar LOD values [14]. Their study primarily focused on urine and other biological fluids, while analyses in serum samples have been relatively limited [14]. Our study contributes to filling this gap by providing a validated method with low LOD and LOQ values for the quantification of isotonitazene in serum samples, thereby enhancing the understanding of its pharmacokinetic properties. Additionally, the SPE method used in our study enables the more accurate extraction of isotonitazene from serum samples. In this context, although both studies achieved comparable LOD values, differences in the biological matrices and extraction methods highlight that the proposed method for serum samples offers a more sensitive and reliable approach for analysis. In our study, the lower recovery rates observed for isotonitazene can be attributed to the interaction between the SPE method and the chemical properties of the compound. The SPE method can yield different recovery rates depending on the hydrophobicity or hydrophilicity of the analytes. Compared to flualprazolam, isotonitazene may have higher polarity, which could affect extraction efficiency and lead to lower recovery rates. Furthermore, the solubility of isotonitazene in the aqueous-organic phase is a crucial factor in the SPE elution step. In this study, a 70% acetone solution was used for elution. The acetone concentration may influence the elution efficiency of isotonitazene, potentially contributing to the lower recovery rates. Literature reports suggest that highly polar compounds may experience losses during the SPE process due to their solubility balance between the aqueous and organic phases. Increasing the acetone percentage or testing alternative elution solvents may enhance the recovery of isotonitazene. Future studies should focus on optimizing the extraction parameters to improve the overall recovery efficiency. Our study's RSDr results align with the existing literature, providing robust data on the detection and recovery of flualprazolam and isotonitazene in serum samples. Krikku et al. reported flualprazolam concentrations in femoral blood between 3.0 and 68 ng/mL [17], while Papsun et al. found levels ranging from 2.0 to 620 ng/mL, with a median of 8.2 ng/mL in postmortem samples [20].