## 3 Results
### Optimal Conditions for HPLC
#### 3.1.3 Influence of the Stationary Phase
For the Synchronis C18 column, all the compounds analyzed in the mixture were successfully retained and separated with not broadened peaks. Due to double endcapping of Zorbax Extend-C18 stationary phase with a porosity of 80 A, symmetrical peaks were obtained for all analyzed compounds with good intensity. The use of the HSS T3 column for the analysis of the OATD-02 mixture and impurities allowed for the separation of all compounds, and the obtained signals were symmetrical. The use of the XSelect CSH-C18 column to analyze the OATD-02 mixture and three impurities enabled obtaining the best separation results from all comparable columns. The Waters XSelect CSH-C18 column was designed using BEH, a stationary phase with an ethylene-bridged hybrid inorganic-organic packing, which allows for better separation and longer retention of polar compounds [31]. The CSH (Charged Surface Hybrid) technology consists in additional modification of the column surface with charge, which makes possibility to using this column for analysis of compounds with different polarity and in a wide pH range of the mobile phase (pH 1-11). Table 2 summarizes the obtained most important chromatographic parameters for the determination of OATD-02 in crude samples on different columns. The peak areas of OATD-02 were the largest values on the columns: Luna C18, XSelect HSS T3 and XSelect CSH-C18, but only on the XSelect CSH-C18 column was the tailing factor of OATD-02 peak below 1.5. Moreover, the XSelect CSH-C18 can be used in the widest range of mobile phase pH values (under pH 11), which is very important when using a basic mobile phase with sodium bicarbonate. The comparisons of the selectivity differences of stationary phases were presented in Figure 4A,B as the selectivity charts for reversed phase packings. In Figure 4, the logarithm of selectivity between nearest compounds vs. the retention factor of each compound was plotted. Different stationary phases can be discriminated by the alpha value of two compounds. Moreover, it is useful for finding stationary phases with similar selectivity properties. The retention of basic analytes can be used as a measure for the silanophilic activity of stationary phases [32]. The retention factor of OATD-02 or OAT-9016 on the x-axis is a measure of the hydrophobicity of stationary phases. The relative retention alfa OATD-02 /OAT-9014 or OAT-9016/OATD-02 shown on the y-axis is a selectivity comparison. On selectivity chart Figure 4A for retention of OATD-02 the most hydrophilic stationary phase is phenyl-hexyl phase, because of occurrence of aromatic rings, but the \begin{table}
\begin{tabular}{c c c c c c c c} \hline \hline  & & \multicolumn{6}{c}{**OATD-02**} \\ \hline
**Type of Column** & \(\mathbf{k_{R}}\) & **Area, \(\mathbf{\mu V\times Min}\)** & **Height, \(\mathbf{\mu V}\)** & \(\mathbf{T_{f}}\) & **N/m** & \(\mathbf{W_{h1/2}}\)**, Min** & **S/N** \\ \hline Luna C18 & 10.0 & 5,114,031 & 666,204 & 1.52 & 223,820 & 0.11 & 278 \\ \hline Kinetex Phe-hex & 3.0 & 1,138,000 & 87,869 & 4.01 & 89,284 & 0.15 & 34 \\ \hline Syncronis C18 & 10.4 & 3,997,187 & 548,064 & 1.29 & 260,230 & 0.10 & 344 \\ \hline Kinetex XB-C18 & 6.9 & 4,661,412 & 670,962 & 1.73 & 321,730 & 0.10 & 328 \\ \hline XSelect HSS T3 & 10.1 & 4,941,138 & 779,688 & 1.57 & 303,920 & 0.09 & 440 \\ \hline Zorbax Extend-C18 & 10.4 & 4,555,981 & 644,835 & 1.53 & 175,340 & 0.10 & 239 \\ \hline XSelect CSH-C18 & 6.6 & 4,894,797 & 662,409 & 1.49 & 303,210 & 0.11 & 318 \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Table summarizing chromatographic parameters of determination of OATD-02 in crude samples on different columns. taining factor of OATD-02 on this stationary phase is over 4. Furthermore for OAT-9016 compound there is no retention on this stationary phase, probably there is small broadened peak, which cannot be integrated. On selectivity chart Figure 4B for retention of OAT-9016 the most suitable stationary phase is CSH-C18 phase.