## 2 Results and Discussion
### Application in Biological Samples
#### 2.4.3 Degree of Penetration of Piperacillin and Imipenem to BAL  
In the case of piperacillin, the experimentally determined degree of penetration to BAL was in the range 1.88-2.71% (Table 9). According to the research of [PERSON] et al. (2019), the average degree of penetration of piperacillin into the pulmonary epithelial lining fluid was 43.9%; however, the values determined in their study were in the very wide range of 2.0-515.9%. This demonstrates the problem with the unpredictability of piperacillin penetration into the lungs and shows that the optimal dosing of piperacillin in the critically ill is a true challenge. The low penetration of piperacillin into the epithelial lining fluid may lead to sub-therapeutic concentrations; hence, the current treatment regimens may be ineffective in patients suffering from ventilation-associated pneumonia. Similarly, the distribution of impenem in critically ill patients is much smaller compared with the respective distribution values in healthy individuals. In this regard, [PERSON] et al. (2019) have shown that the maximum concentration of unbound impenem in infected organs in critically ill patients was only 5% of the maximum concentration achieved in plasma, due to its weak perfusion into internal organs. The penetration of unbound impenem into BAL determined in the presented study (Table 9) ranged between 1.49 and 11.15% of its plasma levels. It should also be mentioned that mechanically ventilated patients suffering from VAP were enrolled in this study, in whom lung perfusion could be unstable and variable during hospitalization.  
It should be stated that in the presented study both BAL and blood samples were collected from each patient at the same time (i.e., 1 h after infusion); hence, sampling bias was eliminated and could not deteriorate the comparison of antibiotic concentrations in both matrices. Indeed, good relationship was observed between drug concentration in BAL and plasma (Figure 6). Noticeably, the applied TFME technique only extracts the free fraction of drugs, i.e., those that are unbound to the plasma proteins; hence, the final LC-MS/MS analysis directly determines the biologically active part of antibiotics. This means that the concentrations were not recalculated from the total drug concentration using published protein binding values, which were proved by other researchers to significantly differ in healthy volunteers and critically ill patients ([PERSON] et al., 2019). This fact certainly contributes to the differences in blood/BAL partition observed here and in other studies.  
\begin{table}
\begin{tabular}{c c c c c} \hline \hline \multirow{2}{*}{**Pat. \#**} & **Sample** & \multicolumn{2}{c}{**Concentration after**} & **Concentration** & \multirow{2}{*}{**BAL/Plasma \%**} \\  & **Number** & **Infusion in PLASMA** & & **after Infusion in BAL [mg/L]** & \\ \hline \multicolumn{5}{c}{Patients receiving PIPERACILIN} \\ \hline
1 & PIP-I & 201.687 & 3.794 & 1.88 \\
2 & PIP-II & 263.928 & 6.271 & 2.38 \\
3 & PIP-III & 309.428 & 6.995 & 2.26 \\
4 & PIP-IV & 109.166 & 2.236 & 2.05 \\
5 & PIP-V & 98.457 & 2.673 & 2.71 \\
6 & PIP-VI & 307.027 & 7.711 & 2.51 \\ \hline \multicolumn{5}{c}{Patients receiving IMPENEM} \\ \hline
1 & IMI-I & 25.113 & 2.801 & 11.15 \\
2 & IMI-II & 10.219 & 0.576 & 5.64 \\
2 & IMI-III & 13.399 & 0.298 & 2.22 \\  & IMI-IV & 13.164 & 1.189 & 9.03 \\
3 & IMI-V & 5.852 & 0.087 & 1.49 \\ \hline \hline \end{tabular}
\end{table}
Table 9: The degree of penetration of unbound piperacillin and unbound impenem to BAL.