Confirmation of in vivo metabolites structures (M1 and M3) by LC-SPE-NMR/MS
Microsome metabolites were separated by HPLC followed by MS and UV detection (Figure 5A ) with subsequent post column trapping of analytes on the SPE cartridges. Molecular formulas of the metabolites were obtained from high resolution mass spectrometry during a preparation run. The final isolation of the metabolites was based on UV response in order to reduce the risk of contamination of the ion source because of the large amount of sample injected on the column. M1 and M3 were separated by HPLC and detected by MS as sodium adducts [M+Na]+ ions (i.e. m/z 883 and m/z 913, respectively) (Figure 5B ). The NMR spectra confirmed that the demethylation of M1 occurred at C3″ (labeled in yellow, Figure 6A ) and that the hydroxylation of M3 occurred at C4 (labeled in yellow,Figure 6B ). The evaluation of HSQC and HMBC spectra of IVM, M1, and M3 shows the location of biotransformation (Table 3, Figure 6, NMR supplemental report ). Chemical shift values for proton and carbon resonances are shown in Table 3.
As the sensitivity of the 500MHz instrument was not sufficient to obtain an HMBC spectrum within the period of the nitrogen refill cycle (one week), the isolated metabolites were analyzed using an 800MHz instrument. This provided a complete set of NMR data (within 72 h) for structure elucidation. The HMBC correlations for IVM, M1 and M3 together with the proton spectrum obtained at 800MHz are detailed in the NMR supplement.