# The Metabolic Stability of Antimicrobial Peptides IK8 in Plasma and Liver S9
## 3 Results
### Liver S9 Metabolic Stability  
2.1 Incubation Chromatograms between Three Configurations IK8 and Liver S9 of Different Species (Using Rat Liver S9 Incubation as an Example)  
By comparing the HPLC chromatogram between 0 and 60 min (Figure 6), it became apparent that night metabolites were generated after incubation of IK8-L, and two metabolites were generated for IK8-2D. In addition, there was no metabolite generated for IK8-D. As shown in Table 2, the metabolites of IK8-L incubated in liver S9 of various species had certain species distinctions. In addition to canine liver S9, M4 was the most abundant metabolite in liver S9 of the other four species.  
\begin{table}
\begin{tabular}{c c c c c} \hline  & **M1** & **M2** & **M3** & **M4** \\ \hline Human & 2.3 & 19.1 & 11.8 & 66.8 \\ Monkey & 6.7 & 15.4 & 9.8 & 68.0 \\ Dog & ND & ND & ND & 100 \\ Rat & 2.4 & 22.2 & 6.7 & 68.7 \\ Mouse & ND & 2.5 & ND & 97.5 \\ \hline \end{tabular}
\end{table}
Table 1: Relative contents of main metabolites of IK8-L in plasma of five species (%).  
Figure 1: HPLC chromatograms of three configurations IK8 and its metabolites in plasma: (a) blank plasm; (b) IK8-L; (c) IK8-D; (d) IK8-2D.  
Figure 3: Relevant structure information of M2: (**a**) the structure of M2 is inferred as KIRIK-NH\({}_{2}\); (**b**) primary mass spectrum of M2; (**c**) secondary mass spectrum of M2 ([M + 2H]2*); (**d**) the assignment of ion fragments in secondary mass spectrum.  
Figure 2: Relevant structure information of M1: (**a**) the structure of M1 is inferred as IRIK-NH\({}_{2}\); (**b**) primary mass spectrum of M1; (**c**) secondary mass spectrum of M1 ([M + 2H]2*); (**d**) the assignment of ion fragments in secondary mass spectrum.  
Figure 4: Relevant structure information of M3: (**a**) the structure of M3 is inferred as IKIRIR-NH\({}_{2}\); (**b**) primary mass spectrum of M3; (**c**) secondary mass spectrum of M3 ([M + 2H]\({}^{2+}\)); (**d**) the assignment of ion fragments in secondary mass spectrum.  
Figure 5: Relevant structure information of M4: (**a**) the structure of M4 is inferred as \(\rm{RIRIRIK\mbox{-}NH_{2}}\); (**b**) primary mass spectrum of M4; (**c**) secondary mass spectrum of M4 ([M + 2H]\({}^{2+}\)); (**d**) the assignment of ion fragments in secondary mass spectrum.  
Figure 6: HPLC chromatograms of three configurations IK8 and its metabolites in liver S9: (**a**) IK8-L; (**b**) IK8-D; (**c**) IK8-2D.  
2.2 Identification of Metabolites after Incubation between Three Configurations IK8 and Liver S9 of Different Species (Using Rat Liver S9 Incubation as an Example)  
The HPLC chromatograms of IK8-L, IK8-D, and IK8-2D incubated with rat liver S9 are shown in Figure 6. Compared with the results in rat plasma (Figure 1), there were five more metabolites of IK8-L in rat liver S9. The results indicated that the metabolites M1, M2, M3, and M4 were identical to that in plasma. Moreover, the structural information of the rest metabolites--M5, M6, and M7--are presented in Figures 7-9. Unfortunately, the structures of M8 and M9 were not identified by the Q-TOF data. For IK8-2D, it can be illustrated, through primary and secondary mass spectrometry, that the structures of M4\({}^{\prime}\) and M7\({}^{\prime\prime}\) were RIKrIK-NH\({}_{2}\) and IRIkIrIK, respectively.