Definitive Metabolite Identification

[Orbitrap_SciLib]

The detection and identification of metabolites of pharmaceutical agents is an important part of the discovery and development process.  In discovery, metabolite identification can aid in the design of compounds with good drug-like properties.  In development, understanding the metabolic fate of a drug candidate is a requirement of regulatory bodies and helps to assure that a new drug is safe by exposing potential toxicities or drug interactions.  The complex biological matrices studied, where the metabolites of interest represent only a few of the myriad components, make detection difficult even before identification is attempted.  High-resolution, accurate-mass mass spectroscopy powered by Orbitrap™ technology is a powerful tool for metabolite identification.  With multiple fragmentation techniques, acquisition triggering techniques, MSⁿ fragmentation, and structure-based interpretation software, finding and identifying metabolites is significantly easier than by any other method.

Overview

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.

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Sample Preparation

Sample Preparation Workflow for Definitive
Metabolite ID

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.

resources

Re-interrogation of Verapamil Metabolites in Bile Using Time-based Data-dependent Mass Lists and a M...

Lee L, Chen Y, et al.
Application Note 418

Mass Spectrometry

Mass Spectrometry Workflow for Definitive
Metabolite ID

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.

Resources

Multiple Fragmentation Methods for Small Molecule Characterization on a Dual Pressure Linear Ion Tra...

Comstock K, Huang Y.
Application Note 540
 

Metabolite Identification in Complex Matrices by Structurally Intelligent Acquisition and Processing

StrattonT, Huang Y, et al.
MDO/ISSX 2012 Poster

Data Analysis

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.

 

resources

Metabolite Identification of Omeprazole in Complex Matrices by Structurally Intelligent Acquisition ...

Stratton T, Huang Y.
GLDMDG 2012 Poster
 

Thermo Scientific Mass Frontier Software

Product Brochure
 

High-Resolution, Accurate Mass Measurements and Metabolite Identification: An Automated Approach Usi...

Wank J, Wagner-Redeker W, et al.
Application Note 537