## 2 Materials and Methods
### Preparation of Blood Samples for Calibration  
Drug-free human serum samples used for calibration were obtained from the Red Crescent Blood Center from individuals who were confirmed not to use drugs. For calibration, 1 mL of drug-free human serum was used. To each calibration sample, 20 \(\upmu\)L of internal standard solution was added, followed by 2.5 mL of distilled water. After thorough mixing, different concentrations of flualprazolam and isotonitazene standard solutions (1, 2.5, 5, 10, 25, 50, and 100 ng/mL) were spiked into the samples to construct the calibration curve. After centrifugation at 3500 rpm for 10 min, the supernatant was transferred to Onsis HLB cartridges, which were conditioned with 2 mL of methanol and 2 mL of distilled water. For the elution step, a 70% acetone solution was used, as acetone facilitates the release of analytes from the solid-phase extraction (SPE) cartridges. However, since isotonitazene exhibits higher polarity compared to flualprazolam, its extraction efficiency may be affected by the composition of the elution solvent. The impact of acetone percentage on isotonitazene recovery was noted, and future optimizations may consider alternative solvent compositions to enhance recovery efficiency. The resulting eluent was evaporated under nitrogen, followed by the addition of 1 mL of methanol and vortex mixing. Finally, the methanol mixture was transferred to vials through microfilters for LC-MS/MS analysis.  
To calculate the LOD and LOQ values, 10 injections at the base-level concentration (1 ppb) were introduced to the system, and the mean and standard deviation were calculated. The LOD was determined as 0.03 ppb and 3 ppb for flualprazolam and isotonitazene, respectively. The LOQ value was determined as 0.1 ppb and 9.14 ppb for flualprazolam and isotonitazene, respectively. The peaks resulting from the LOD and LOQ levels were observed.  
A linear range was established to determine the concentration range where the method would provide quantitatively accurate results. Linearity was assessed by preparing calibration samples with known concentrations of flualprazolam and isotonitazene (1, 2.5, 5, 10, 25, 50, and 100 ng/mL) spiked into drug-free human serum. Each sample also contained a fixed amount of internal standard (20 \(\upmu\)L). Following the spiking procedure, the samples underwent extraction and analysis, and the calibration curve was generated based on the peak area ratios of the analytes to the internal standard. Nine independent blood samples were prepared and extracted for each concentration. A linear graph was drawn using the concentrations and the analyte peak area and internal standard peak area relationship corresponding to those concentrations, and the linear regression formula and correlation coefficient of the calibration curve were automatically given by the software. According to the validation guideline, \(r^{2}\geq 0.999\) indicates that the method is linear for the studied linear range [15].  
The analyte recovery was measured using the solid-phase extraction (SPE) method at five different occasions following 10 ppb injections and calculated as 87.85 and 70.02 for flualprazolam and isotonitazene, respectively.  
The recovery of substances from the matrix and the precision of the method were determined by analyzing samples fortified at two different concentration levels (10 ng/mL and 50 ng/mL) in five replicates. The repeatability (RSDr) was evaluated through same-day experiments by two different analysts. Intra-laboratory reproducibility (RSDwR) was performed by two analysts at two different concentrations (10 ng/mL and 50 ng/mL) at five different consecutive times. Precision values are expressed as the relative standard deviation (RSD). By calculating the %RSD values, it was checked whether these values met the \(\leq\)20% condition. Recovery was checked via the compliance of the recovery values obtained from the repeatability and reproducibility studies within the 70-120% range.