## 2 Material and Methods
### Assay Validation Procedures  
The assay validation was performed based on the recommendation described in the recent bioanalytical guideline for method validation [24]. The assay selectivity was determined by comparing the response of 6 different blank plasma samples (obtained from different lots of human plasma) with the response of plasma spiked at lower limit of quantification (LLOQ) and IS concentration. All samples were prepared by following the extraction procedure and were analyzed by developed assay. \begin{table}
\begin{tabular}{c c c c c c c} \hline
**Compound** & **t\({}_{\text{R}}\) (min)** & **Q1 [M + H]\({}^{+}\)** & **CV (V)** & **Q3 [M + H]\({}^{+}\)** & **CE (eV)** & **dt (s)** \\ \hline
5-AIQ & 0.93 & 145.0 & 50 & 91.0 & 22 & 0.160 \\  & & & & 117.4 \({}^{\ast}\) & 20 & 0.160 \\ IS & 0.96 & 384.02 & 20 & 138.05 & 30 & 0.160 \\ \hline \end{tabular}
\end{table} 
Table 1: Optimized UPLC-MS/MS parameters for 5-AIQ and IS. For method acceptance, the interference in the blank plasma at the retention time of analyte and IS should be \(<\)20% and \(<\)5%, respectively. The assay linearity was determined by preparation of the calibration curve by using eight different concentrations of CSs (1-666 ng/mL) in plasma excluding blank samples. A linear regression model of 1/\(\chi^{2}\) was used to express the area ratio between 5-AIQ/IS against the corresponding 5-AIQ concentration and the assay linearity was confirmed by back calculation of the concentration of each CSs using calibration plots. The value of r\({}^{2}\) (representing the goodness-of-fit) needed to not exceed 0.995; the back-calculated concentration values should be within 15% deviation of the nominal concentration (excluding LLOQ; \(\leq\)20%) and a minimum of 75% of the nonzero calibration should be within the range of the acceptance criteria. The intra-day and inter-day precision and bias from nominal value of the developed UPLC-MS/MS method was determined at LLOQ, LQC, MQC, and HQC concentration using five replicates. Precision and bias were expressed by calculating the relative standard deviation (RSD, %) and relative error (RE, %), respectively. The intra-day precision and bias were calculated by analyzing the samples on same day analysis; however, inter-day was calculated by analyzing samples on three different days. For assay acceptance, the precision should not exceed 15% and bias should be within 15%, respectively, for all three QC concentration, except LLOQ which should be within \(\leq\)20% and \(\pm\)20%, respectively, for both intra and inter-day studies. Recovery and matrix effects (ME) of the assay was determined at LQC, MQC and HQC concentration level by spiking them in blank plasma samples. The percentage recoveries were determined by comparing the mean peak response of 5-AIQ between pre- and post-extraction samples. The % ME were determined by comparing the peak response of 5-AIQ between post extracted and aqueous samples. A similar procedure was followed for the determination of ME for IS samples. For assay acceptance, the % ME should be \(\leq\)15%. The stability of 5-AIQ in plasma at different anticipated conditions of storage, handling, preparation and analysis were examined by analyzing the recovery of LQC and HQC samples in five replicates. The evaluation parameters include short-term storage, freeze-thaw cycles, auto-sampler and long-term storage stability of 5-AIQ prior to analysis. The short-term stability was determined by keeping the samples at benchtop at least for 8 h while long-term stability was determined by storing the sample at \(-\)70 \({}^{\circ}\)C for two months before analysis. The freeze-thaw cycles stability was determined by exposing samples for three cycles of freeze (\(-\)70 \({}^{\circ}\)C) and thaw (room temperature) before analysis. The auto-sampler stability was determined by placing the processed sample in an instrument auto-sampler for 24 h at 15 \({}^{\circ}\)C before analysis.