GEN-MKT-18-7897-A
Apr 25, 2024 | Blogs, LC, Pharma | 0 comments
Read time: 3 minutes
The dream state
If we lived in an ideal world, it would be possible to unambiguously identify metabolites using a single analytical experiment. This analytical technique would need to be efficient and easily generate the information needed from a routine assay that is also robust, enabling confident decision-making during drug discovery.
At SCIEX, we believe that metabolite identification using the ZenoTOF 7600 system gets close to this dream state.
The science
Currently, the starting point for metabolite identification is typically a liquid chromatography with high-resolution mass spectrometry (LC-HRMS) experiment using fragmentation to allow structural elucidation of the analyte. The “go to” fragmentation technique is collision-induced dissociation (CID), which provides a wealth of information but doesn’t always allow for explicit identification of the metabolite.
The ZenoTOF 7600 system offers both CID and electron-activated dissociation (EAD) fragmentation, which complement each other. EAD can cause ions in an LC-MS/MS experiment to fragment in locations that are different from where they fragment with CID, providing scientists with additional information in a single experiment.
For metabolite identification, this could mean confidently identifying a single site of metabolism instead of two or three possible sites using CID only and removing the need for additional safety testing.
The evidence
A recent peer-reviewed paper, Electron activated dissociation – a complementary fragmentation technique to collision-induced dissociation for metabolite identification of synthetic cathinone positional isomers – ScienceDirect, reported the following when researching the metabolism of new psychoactive substances (NPS):
“The tentative structural elucidation of metabolites of NPS formed using in vitro models is typically carried out using liquid chromatography combined with high-resolution tandem mass spectrometry (LC-HRMS2) with collision-induced dissociation (CID) as a fragmentation method. However, the thermally excited ions produced with CID may not be sufficient for unambiguous identification of metabolites or their complete characterization. Electron-activated dissociation (EAD), a relatively new fragmentation approach that can be used to fragment singly charged ions, may provide complementary structural information that can be used to further improve the confidence in metabolite identification.”
“The EAD product ion mass spectra showed different fragmentation patterns compared to CID, where unique and abundant product ions were observed in EAD but not in CID. More importantly, certain EAD exclusive product ions play a significant role in structural elucidation of some metabolites. These results highlight the important role that EAD fragmentation can play in metabolite identification workflows, by providing additional fragmentation data compared with CID and, thus, enhancing the confidence in structural elucidation of drug metabolites.”
To learn more about how EAD could be used in your laboratory, this webinar may be of interest: Discover how Bristol-Myers Squibb uses electron-activated dissociation (EAD) to confidently identify drug metabolites and localize site of metabolism (theanalyticalscientist.com). In the webinar, Ming Yao, Principal Scientist, CPPDB, at Bristol-Myers Squibb, discussed how the additional information from EAD can help in confident localization and identification of metabolites compared to CID alone. The study demonstrated that these fragments can be crucial to locating the metabolic modification sites, such as conjugations.
For more information on the options SCIEX offers for metabolite identification, please speak to your account manager or visit our web page on Comprehensive Metabolite Identification | SCIEX.
As an analytical strategy, middle-down mass spectrometry (MS) workflows characterize biotherapeutic proteins by analyzing large, digested protein fragments or defined subunits, rather than fully intact proteins (top-down) or digested peptides (bottom-up). A middle-down strategy combines the strengths of top-down and bottom-up approaches by delivering high sequence coverage and structural specificity while maintaining relatively simple sample preparation. In practice, middle-down analysis enables accurate mass measurement, rapid sequence confirmation, and localization of key post-translational modifications (PTMs) on protein subunits that are directly relevant to product quality.
In biopharmaceutical development, sequence variants (SV) are considered an inherent risk of producing complex proteins in living systems. Sequence variants are unintended changes to the amino acid sequence of a biotherapeutic and can be caused by errors in transcription or translation in the host cell, or cell culture and process conditions. Detailed analysis of SVs is important in process and product development to ensure the drug’s safety and efficacy. Even low‑level sequence variants can have significant implications for product quality, safety, and efficacy, making their accurate detection and characterization a critical requirement across development, process optimization, and regulatory submission.
CE‑SDS remains a cornerstone assay for characterizing fragmentation, aggregation, and product‑related impurities in therapeutic proteins. UV detection has been the long‑standing standard. However, it frequently struggles with baseline noise, limited sensitivity for minor fragments, and subjective integration.
Posted by
You must be logged in to post a comment.
Share this post with your network