This paid-for advertorial by SCIEX appeared in DDW Volume 24 – Issue 2, Spring 2023
Imagine having the ability to consistently identify metabolic soft spots from an experiment that uses liquid chromatography coupled to mass spectrometry (LC-MS), reducing the need for additional safety testing in the drug discovery phase.
Metabolite identification
A key step in the drug discovery process is identifying a drug’s metabolites and confidently identifying the site of metabolism. The correct identification of metabolites is essential to understand the behaviour of drugs in humans. This critical step allows scientists to understand the drug’s efficacy, toxicity, and drug-to-drug interaction.
Analytical requirements
Scientists need an analytical method that provides the following information about their metabolites: detection at low concentrations, reliable metabolite identification, and quantification in complex matrices.
A commonly deployed analytical technique has been liquid chromatography coupled to mass spectrometry (LC-MS). This technique separates the complex sample by chromatography before measuring the mass-to-charge ratio of the analyte and its fragments. Structural elucidation (a chemical jigsaw puzzle based on mass measurements) of the fragment ions is completed to confirm the chemical structure of the analytes of interest.
Typically, these fragments are created by collision-induced dissociation (CID). CID is widely deployed because it is simple to use and well-understood, making it the first choice for metabolite identification work. However, if this approach doesn’t provide enough information (optimal fragmentation) to identify the metabolites at a confidence level that satisfies regulatory bodies, alternative testing must be done. Secondary analysis is both costly and time-consuming.
Figure 1. EAD enables the confident identification of glucuronide conjugation using diagnostic fragments. CID generated two possible sites of glucuronide conjugation. However, the power of EAD provides site-specific characterisation that narrows it down to a single metabolite candidate. Here, the glucuronide conjugation was localised on the imidazole ring.Recent advancements
A recent innovation in mass spectrometry has been the introduction of an alternative fragmentation technique called electron-activated dissociation (EAD), which is commercially available on the ZenoTOF 7600 system from SCIEX. EAD induces fragmentation differently, which causes the analyte to fragment in different places, providing additional information to the analytical scientist. The ability to complete the jigsaw puzzle becomes easier and meeting regulatory requirements in a single analysis can become possible.
EAD in action
A recent technical note published by SCIEX describes the characterisation and identification of glucuronide metabolites from hepatocyte incubations of midazolam using CID and EAD. Additional fragment information was obtained by EAD which aided the identification process of the challenging metabolites, Figure 1.
Data processing software can then be used to support the structural elucidation process. In this example, the software assigned the fragment ions using both CID and EAD to propose one chemical structure for the target analyte. In contrast, when only CID was used, the software predicted two possibilities for hydroxylation. More information about these experiments can be found on https://sciex.li/MetID in the technical note; Confident identification of phase 1 metabolites using electron-activated dissociation (EAD).
