Structure Elucidation Case Study: in vitro Buspirone Metabolite
Key Concepts
- Refinement of approaches for rapid profiling and detailed elucidation of metabolites from in vitro and in vivo origins
- Investigate new approaches for multi-technique detailed structure elucidation
- Better integrate rapid metabolite profiling using LC/MS/MS with detailed structure work
- Detailed structure elucidation using MSn and capillary NMR
Overview
- Perform metabolic incubation at typical screening levels (low micromolar concentration)
- Analyze samples using Rapid Metabolite Profiling system
- Find all non-blank parent-related metabolites using MetLab Profiler automated data processing software…use results to propose metabolic “hot-spots”
- Obtain MSn information on metabolite of interest to further refine proposed structure…substructure localization
- Scale-up metabolic incubation to levels suitable for Detailed Metabolite Elucidation…typically 10-50x greater than screening levels
- Isolate desired metabolite using automated SepNMR system
- Analyze isolated metabolite using high sensitivity capillary NMR…elucidate site of attachment, etc. on modified substructure found during profiling
Rapid Metabolite Profiling
In recent years, pharmaceutical scientists have increasingly used metabolic stability and metabolite profiling early in the drug discovery and development process. One of the major goals of these initiatives is to translate metabolic tendencies into structural knowledge that help direct upstream chemical decision-making and identify downstream development problems. The bottleneck today is not rapid assay or analysis methods, but rather automated and intelligent data processing tools. This trend is most prevalent when attempting to find and identify metabolites from complex LC/MS/MS mixtures. Most metabolism software packages today focus on using the normal full scan MS data and background subtraction to locate new peaks that appear in an unknown sample. Often, the user must still manually plow through piles of data to validate any candidate metabolites as being parent-related components. Our approach utilizes: 1) An automated system capable of obtaining both metabolic stability and metabolite profiling information on a single sample; and 2) Automated data processing software capable of finding, confirming and identifying non-blank parent-related metabolites using a proprietary algorithm called AutoShift and software package called MetLab. This combination greatly accelerates the process of providing metabolic “hot-spot” information during drug discovery and early development. Figure 1 shows the output from MetLab of the LC/MS data from a buspirone HLM incubation where peaks are labeled with putative modifications. Table 1 shows the MetLab summary results in tabular format.
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 Figure 1. LC/MS of Buspirone HLM Incubation |
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 Table 1. MetLab Report of Putative Metabolites |
Detailed Metabolite Elucidation
Often, more detailed information is required on a potential metabolite, such as, site of attachment and complete structure elucidation. More advanced MS/MS techniques, such as data-dependent MSn analysis can get part of the way towards this goal. However, multi-dimensional NMR analysis is often required to propose complete structures. Traditionally this transition from LC/MS to NMR requires tremendous scale-up of the metabolite to provide milligram levels of material for analysis: prep-scale chromatography and elaborate purification schemes are the norm. With the advent of capillary NMR the scale requirements have been greatly reduced to low microgram levels, but the issue of rapid and efficient isolation still remains. Our approach utilizes: 1) An automated system capable of analytical scale separation, peak isolation, on-line sample enrichment and NMR-friendly sample preparation; and 2) A high-field NMR equipped with a capillary NMR probe for low-volume, low-level sample analysis. This combination greatly reduces the sample requirements for detailed NMR analysis and goes a long way towards approaching the scale typical of front-line LC/MS analysis.
Isolation of Buspirone Metabolite for NMR Structural Studies
| Novatia’s SepNMR system allows for quick and easy isolation of components from complex mixtures. Figure 2 shows an HPLC trace of a buspirone – human liver microsomal incubation. The major peak in the center of the chromatogram is unmodified buspirone while other peaks are buspirone metabolites. The largest peak near buspirone was isolated from a single injection and Figure 3 shows the reinjected buspirone metabolite to be free of other components with good overall recovery. |  Figure 2. LC/UV Analysis of Buspirone Metabolite Incubation |
 Figure 3. LC/UV Analysis of Isolated Metabolite |
MSn Analysis of Metabolite Isolate
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 Figure 4. MS3 Data and Assignments |
NMR Characterization of Isolated Buspirone Metabolite
| Figure 4 shows the additional detailed information that was obtained from MS3 suggesting that the metabolite contains an additional hydroxyl group on the right-hand portion of the molecule. Figure 5 shows the 1D 1H NMR spectrum of buspirone on the top and the isolated metabolite on the bottom. A numbered structure for buspirone is also included on the right-hand side of the figure (all resonances on each spectrum are labeled according the numbered figure). The MS data from the rapid profiling phase indicated that the modification is on the “north” end of the molecule (indicated by the green circles on the buspirone struture). The 1D NMR data show that the degenerate protons in position 13, 20 are split and that a new resonance appears at about 3.6 PPM. These data show that the site of attachment of the OH group in this metabolite is at either the 13 or 20 position. |  Figure 5. 1D 1H NMR Spectrum of Isolated Buspirone Metabolite |