## 3 Results
### Optimal Conditions for HPLC
#### 3.1.3 Influence of the Stationary Phase  
Various stationary phases were tested: Luna C18, 3 \(\upmu\)m, 100 mm \(\times\) 2 mm, 100 A; Kinetex Phenyl-Hexyl, 5 \(\upmu\)m, 250 mm \(\times\) 4.6 mm, 100 A; Thermo Scientific Syncronis C18, 3 \(\upmu\)m, 100 mm \(\times\) 2.1 mm, 100 A; Kinetex XB-C18 2.6 \(\upmu\)m, 100 mm \(\times\) 3 mm, 100 A; Waters XSelect HSS T3, 2.5 \(\upmu\)m, 100 mm \(\times\) 2.1 mm, 100 A and Zorbax Extend-C18, 3.5 \(\upmu\)m, 50 mm \(\times\) 2.1 mm, 100 A and the flow rates were adjusted to column diameters. The comparison of the main chromatographic parameters for OATD-02 and related substances on different columns was presented in Figure 3A-C. The narrowest peaks, especially for OAT-9016, was achieved for XSelect CSH-C18. Considering the results displayed in Figure 3B the highest efficiencies of the main compound and the latest impurity were obtained on the XSelect CSH-C18 column. Good peak shape can be defined by tailing factor, which was presented in Figure 3C. The tailing factors below or approximately 1.5 for the main compound and impurities were achieved on the columns: Syncronis C18, XSelect HSS-T3 and XSelect CSH-C18. The application of the Luna C18 column allowed separation of all compounds, but the intensity of the last peak of OAT-9016 was insufficient, which can cause analysis problems for low concentrations. The only two compounds were retained on phenyl-hexyl stationary phase. It happened because the stationary phase with a phenyl group is intended for aromatic compounds. Phenyl-type stationary phases show better selectivity for aromatic compounds than C18 stationary phases because \(\uppi\)-\(\uppi\) interactions occur between the aromatic analyte and the \begin{table}
\begin{tabular}{c c c c c c c c c c} \hline \hline  & & & & **10 mM HCOONH\({}_{4}\)** & & & & & & \\ \hline \hline
**\#Peak** & **Name** & **Retention** & **Area,** & **Height,** & **Failure** & **Tailing** & **Efficiency** & **Rs** & **Width** & **S/N** \\ \hline
1 & OAT-9015 & 1.86 & 351,598 & 29,603 & - & 321 & - & - & 6 \\ \hline
2 & OAT-9014 & 2.01 & 355,836 & 20,317 & - & 12 & 0.1 & - & 4 \\ \hline
3 & OATD-02 & 3.99 & 2,824,963 & 61,963 & - & 196 & 1.1 & 0.58 & 13 \\ \hline
4 & OAT-9016 & 5.67 & 84,399 & 10,901 & 1.47 & 10,689 & 2.5 & 0.12 & 2 \\ \hline \hline
**\#Peak** & **Name** & **Retention** & **Area,** & **Height,** & **Failure** & **Efficiency** & **Rs** & **Width** & **S/N** \\ \hline
1 & OAT-9015 & 4.57 & 111,014 & 17,364 & 1.62 & 11,161 & - & 0.09 & 6 \\ \hline
2 & OAT-9014 & 5.67 & 904,701 & 73,438 & 1.20 & 5206 & 4.5 & 0.18 & 26 \\ \hline
3 & OATD-02 & 8.21 & 5,217,590 & 208,729 & 0.73 & 1965 & 4.8 & 0.41 & 73 \\ \hline
4 & OAT-9016 & 9.28 & 186,428 & 15,203 & 1.40 & 13,737 & 2.0 & 0.19 & 5 \\ \hline \hline
**\#Peak** & **Name** & **Retention** & **Area,** & **Failure** & **Failure** & **Failure** & **Efficiency** & **Rs** & **Width** & **S/N** \\ \hline
1 & OAT-9015 & 4.75 & 96,289 & 18,010 & 1.38 & 15,272 & - & 0.08 & 9 \\ \hline
2 & OAT-9014 & 6.12 & 735,667 & 101,383 & 1.45 & 15,615 & 7.8 & 0.11 & 49 \\ \hline
3 & OATD-02 & 8.57 & 4,894,797 & 662,409 & 1.49 & 30,321 & 12.5 & 0.11 & 318 \\ \hline
4 & OAT-9016 & 12.96 & 167,195 & 21,002 & 1.54 & 55,442 & 21.0 & 0.12 & 10 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Comparison of the analytical parameters for OATD-02 and related substances in different mobile phase compositions. phenyl group of these stationary phases [30]. Therefore, the analysis of the OATD-02 molecule, which is not an aromatic compound, on a phenyl-hexyl column did not yield high-intensity chromatographic signals. In the results obtained for the mixture of OATD-02 and impurities using the Kinetex XB-C18 column, only two of the three impurities can be seen. In addition, the chromatographic signals obtained were broadened. Despite endcapping, i.e., binding of residual silanol groups, there may still be free silanol groups, and this may lead to undesirable interactions with the analytes and, consequently, to peak asymmetry.