## 3 Results
### Method Validation  
The developed method was validated according to the International Conference of Harmonization (ICH) guideline [2]. Validation parameters were tested and the results were within the acceptable criteria, which proves that the analytical method is a validated method. To evaluate the linearity range of the method, five different concentrations were prepared: 0.8, 0.9, 1.0, 1.1 and 1.2 mg/mL. The peak area under the curve was plotted against concentration. The developed method showed to be a linear relation (R\({}^{2}\) = 0.999); the regression line equation was found to be y = 47,791x + 2569. The specified range was established by confirming that the analytical method provides an acceptable linearity from 80 to 120 percent of the test concentration [2], which was set to 1 mg/mL. This depended on the intended application of the analytical procedure for the assay of a drug substance. The linearity in the lower concentration range was also checked. The method was also evaluated for accuracy, the LOD, the LOQ, precision, recovery validation parameters. The mean percentage recovery (preparing three concentrations of the sample: 0.8, 1.0, 1.2 mg/mL, which were injected in the HPLC in triplicate) was 99.2%. The RSD was calculated for every sample concentration (0.8, 1.0, and 1.2 mg/mL) and was found to be 0.66%, 0.36%, and 0.12%, respectively (Table 4). The relative standard deviation for repeatability were calculated 0.4%. Instrumental precision was assessed by injecting one concentration five times, and the relative standard deviation (RSD) of the area was calculated and found to be 0.6%, which was within the acceptable criteria. The results, mean, standard deviation and relative standard deviation were presented in Table 5.  
For intermediate precision, the two analysts' results for the mean percent recovery and RSD were 99.5% and 2.0%, respectively. According to the results, the sample solution \begin{table}
\begin{tabular}{c c c c} \hline \multirow{2}{*}{**Sample \#**} & \multicolumn{3}{c}{**Recovery**} \\ \cline{2-4}  & **Accuracy III (80\%), \%** & **Accuracy II (100\%), \%** & **Accuracy I (120\%), \%** \\ \hline
1 & 99.6 & 99.4 & 99.0 \\ \hline
2 & 98.5 & 99.5 & 99.2 \\ \hline
3 & 99.6 & 98.8 & 99.2 \\ \hline RSD, \% & 0.66 & 0.36 & 0.12 \\ \hline Mean Accuracy, \% & 99.2 & \\ \hline SD & 0.38 & \\ \hline RSD, \% & 0.39 & \\ \hline \end{tabular}
\end{table} 
Table 4: The accuracy results for OATD-02. \begin{table}
\begin{tabular}{c c} \hline \hline
**Injection \#** & **Peak Area, \(\mu\)V \(\times\) Min** \\ \hline
1 & 4,718,036 \\ \hline
2 & 4,734,661 \\ \hline
3 & 4,767,180 \\ \hline
4 & 4,781,615 \\ \hline
5 & 4,775,214 \\ \hline Mean & 4,755,341 \\ \hline SD & 27,590 \\ \hline RSD, \% & 0.58 \\ \hline \end{tabular}
\end{table} 
Table 5: The injection precision results for OATD-02. was stable under normal laboratory condition for 96 h (the % assay of OATD-02 in the test solution was 99.5%). The method was also found to be robust under the varied parameters; the injected concentration of OATD-02 was 1 mg/mL. Robustness was checked by systematic variations in the organic proportion (+5%), column temperature (+5 \({}^{\circ}\)C), another lot number of column, the flow rate of mobile phase and alizarin solution (\(\pm\)0.2 mL/min), detector wavelength \(\lambda_{\text{ex}}\) and \(\lambda_{\text{em}}\) (\(\pm\)2 nm). The efficiency and tailing factor were then compared to the original method, which was presented in Table 6. The method can be regarded as robust to the above small changes and the acceptance criteria for plate number \(\geq\)20,000 and tailing factor \(\leq\)1.5 were met. The sensitivity of the method was assessed by the LOD and the LOQ. The minimum quantity of OATD-02 which the method can detect is expressed as the LOD and was determined by injection of diluted samples. The signal-to-noise ratio (S/N) of 3:1, which represents the LOD, was found to be 0.02 mg/mL. The signal-to-noise ratio 10:1 was determined as the LOQ and was found to be 0.05 mg/mL.