## 3 Results and Discussion
### Chromatographic Method Validation  
Methods for quantification of OGNs are widely used during clinical studies, as well as in metabolism investigations. Thus, in our studies, we investigated the method for extraction and RP-UHPLC-DAD method for separation and determination of OGNs: DNA-16, DNA-18, and DNA-20. DNA-20 is a parent compound, while DNA-18 and DNA-16 are potential synthetic metabolites (shorter, with two or four nucleotides at the 3\({}^{\prime}\) end).  
Based on earlier investigations performed in our group [28], separation of the OGNs mixture was performed by using an octadecyl UHPLC column and an aqueous solution of organic salt with MeOH addition as a mobile phase. Both ammonium acetate and ammonium formate were tested, in concentrations of 5, 10, and 25 mM (data not shown). The satisfactory separation was achieved by using a 10 mM solution of ammonium acetate in a gradient program in 15 min. Figure 7 presents an exemplary chromatogram for the separated mixture.  
The extraction procedure that was developed during the present studies (Table 4) was tested for the extraction of parent OGN and its metabolites from standard solution. Recoveries obtained for components of the mixture at 5 \(\upmu\)M concentration were equal to \(89.8\pm 1.9\%\), \(91.5\pm 1.6\%\), and \(92.6\pm 1.9\%\) for DNA-16,  
Figure 7: Chromatograms for the separation of DNA-20 and its synthetic metabolites DNA-18 and DNA-16 from standard solution (black line) and extract, obtained with developed MDSPE (gray line). Experimental conditions: mobile phase, 10 mM NH\({}_{4}\)OAc and MeOH; gradient elution, 5–20% of MeOH in 5 min and next 20–25% of MeOH in 10 min; chromatographic column ACE Excel C18, 1.7 \(\upmu\)m; UV detection at \(\uplambda=260\) nm; column and autosampler temperature, 30 \({}^{\circ}\)C; flow rate, 0.15 mL min\({}^{-1}\); injection volume, 2 \(\upmu\)L.  
tively. These results showed that the method is suitable for usage in the extraction of OGN mixtures.  
Finally, the developed extraction procedure was used as a sample preparation method for the OGNs mixture from fortified serum. Moreover, the samples were analyzed with the validated UHPLC-DAD method. Recovery and validation parameters for an analysis method (linearity, LOQ, LOD, and intraday and interday precision) were determined and are collected in Table 5. Intraday precision values were lower than 4.5% for low (1.25 uM), 3.6% for medium (7.5 uM), and 1.6% for high (15.0 uM) concentration. Repeatability did not exceed 7.5%. The LOD values were in the range of 0.28-0.32 uM (Table 5).  
The recoveries values of OGNs from enriched serum samples were in the range of 82-85% (Table 5), wherein the matrix effect was in the range of 2.8-3.5%. As can be seen, the developed MDSPE procedure can be applied to real samples. It is important to note that the extraction of OGNs with the MNP coated with zwitterionic poly(ionic liquid) can be performed without any preliminary purification. This is the great advantage of the presented method, which confirms our previous studies on the application of poly(ionic liquid)s for OGNs extraction purposes [22]. Application of other commonly used adsorbents required an additional purification step to remove proteins, usually LLE or proteinase K digestion [9]. Moreover, the developed procedure was characterized by good recovery and repeatability.