Workflow Overview for Definitive Metabolite ID
The complex matrices commonly analyzed for metabolite identification pose a challenge to acquiring the correct fragmentation data. The need for high resolving power to separate matrix components from real metabolites, combined with the need to have accurate scan-to-scan mass accuracy to assure a good elemental composition, means that Orbitrap™ technology is the perfect platform for this application.
The combination of fast, accurate, acquisition and structurally intelligent processing speeds up the challenging task of metabolite identification and improves our results.
Sample Preparation Workflow for Definitive
The variety of matrices studied for drug metabolism require an equal variety of sample preparation techniques. However, the need to assure minimal loss of analytes of interest limits the sample cleanup options available to analysts and places greater demands on both chromatographic separation and instrument performance. In vitro samples can typically be prepared by precipitation with organic solvent and centrifugation. For bile and urine, desalting by solid phase extraction remains a common and useful tool that offers the ability to concentrate the analytes while centrifuge filtration provides a rapid means to remove particulates.
Lee L, Chen Y, et al.
Application Note 418
Mass Spectrometry Workflow for Definitive
The separation of analytes from matrix components has been improved by the increasing use of UHPLC systems, like the Dionex UltiMate3000 system, which offer increased separation efficiency. A wide range of UHPLC-compatible column chemistries gives the analyst options tailored to their specific separation. When chromatographic separation isn’t enough, Thermo Scientific Orbitrap™ instruments offer significantly higher resolution over similar LC/MS techniques to resolve components of interest from the matrix. In addition, high mass accuracy MSn fragmentation data obtained through multiple techniques is critical in the structural determination of metabolites. Intelligent acquisition, either through mass tags, isotopic triggering, or structure based fragmentation triggering, provides superior data quality.
Below is a typical approach for acquiring metabolite identification data on extremely complex samples by using the fast scanning speed of the Thermo Scientific Orbitrap Elite MS to acquire multiple HR/AM MS2 scans on a sample, which are interpreted with advanced structure-based tools in Thermo Scientific Mass Frontier software to determine related materials. These peaks can then be selectively fragmented for powerful MSn data.
Comstock K, Huang Y.
Application Note 540
StrattonT, Huang Y, et al.
MDO/ISSX 2012 Poster
Data Analysis Workflow for Definitive Metabolite ID
Within complex samples, locating and identifying metabolites in the analytical data is the last and, often, the most difficult problem. Thermo Scientific MetWorks and Mass Frontier software provide the tools to both locate related components and to determine structures. By automating the search for related fragments and neutral losses, with and without metabolic transformation, fragment ion search (FISh) in Mass Frontier™ software quickly detects metabolites based on their relation to the parent while simultaneously providing structure interpretation.
Fragment ion searching works with both accurate and nominal mass data to reduce a complex sample to the fragment-related components. Shown here is the process on a human urine sample where the metabolites of omeprazoleare quickly detected and the color-coded fragmentation interpreted to assign a structure.
Stratton T, Huang Y.
GLDMDG 2012 Poster
Wank J, Wagner-Redeker W, et al.
Application Note 537
Orbitrap of Choice for Definitive Metabolite ID
With a combination of true high resolution (240,000 FWHM at m/z 400), accurate mass, MSn, fast scanning, and multiple fragmentation techniques, the Thermo Scientific Orbitrap Elite MS is the ideal instrument for complex structure identification. The ability to use fragment heritage from MSn aids in the more definitive identification of the site of biotransformation, beyond what may be possible with MS2 alone.
Complex samples can be quickly studied with rapid HCD MS2 fragmentation scanning (TOP5) utilizing dynamic exclusion to dig deep into the unknowns. Subsequent analysis of the fragmentation data by fragment ion search (FISh) localizes the metabolites in the sample, allowing for intelligent, structurally targeted MSn analysis to obtain definitive identification data.
Orbitrap of choice: The Orbitrap Elite™ MS
- The Orbitrap Elite hybrid ion trap-Orbitrap mass spectrometer provides the highest resolving power of all Orbitrap-based instruments (up to 240,000 at m/z 400) to achieve ultimate detection limits and selectivity in full-scan MS for analysis of complex samples.
- Fast, high-quality MS and MSn scans allow unambiguous identification of compounds in a complex matrix.
- Complementary fragmentation techniques, including collision-induced dissociation (CID) and higher-energy collisional dissociation (HCD), provide fragment information for confident identification of structure.