Metabolite identification

The rapid structure identification of metabolites provides an early perspective on the metabolically labile sites or soft spots of a drug candidate [86], This information is useful during lead optimization and can serve to initiate research efforts that deal with metabolism-guided structural modification and toxicity. 

Metabolite identification using LC-MS techniques is based on the fact that metabolites generally retain most of the core structure of the parent drug [56,87,88], Therefore, the parent drug and the corresponding metabolites would be expected to undergo similar fragmentations and produce mass spectra that indicate major substructures. 

Using Mass Spectrometry for Drug Metabolism Studies 

Typically, the LC-MS full scan mass spectra of a drug molecule contain abundant [M + H]+ ions with little detectable fragmentation. The product ion mass spectrum contains product ions associated with diagnostic substructures of the drug molecule. There is no need, and more importantly, there is no time in a drug discovery setting to identify all fragment ions observed in the product ion mass spectra. Instead, streamlined approaches based on standard methods and structural template motifs are used [89], 

This LC-MS-based methodology can be automated to fit specialized needs within drug discovery based on throughput [62], The recent application of mass defect filtering [90] and high-resolution accurate mass analysis [33] provides further automated protocols for metabolite identification (see Chapters 5 and 6 for more on this topic). 

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The Use of Cone-Voltage Fragmentation in Conjunction with High-Accuracy Mass Measurements and LC-MS for Metabolite Identification... 

When using microbial products for mammalian metabolite identification, it is suggested to compare all the analytical data available. For example, slight differences in MS2 or MS3 spectra may indicate that the microbial products are not the same as the mammalian metabolite. Owing to matrix effects, HPLC retention time often varies from run to run, so it is good practice to spike a comparable amount of purified microbial product into the in vitro, in vivo or purified samples that contain the mammalian metabolite of interest. If the microbial metabolite and the mammalian metabolite are the same compound, then they should co-elute under different HPLC conditions, including different solvent pH, and the MS and/or UV peak area would increase accordingly. 

Owing to rapid development in analytical techniques, metabolite identification and structure elucidation have become possible even with trace levels of metabolites generated with in vitro or in vivo mammalian systems. However, the microbial bioreactor is still a valuable system for metabolite structure determination, especially when the metabolite of interest presents at a low level in in vitro or in vivo mammalian systems and the isolation from these matrices is hindered by the interference of other metabolites, the parent drug or endogenous compounds, or the structure determination requires appreciable amounts of samples due to structure complexity. 

UV Filters Biodegradation by Fungi, Metabolites Identification and Biological Activity Assessment... 

If there is only interest in determining the elimination or degradation percentages, as in the studies mentioned above, experiments can be performed at low contaminant concentrations, similar to those found in the environment. However, if metabolites identification analysis is wanted to be performed, higher concentrations are needed. That is, because degradation products are usually at much lower concentration than the initial parent compound. 

Packard GC/MS instruments. We exploit both custom and commercial libraries for metabolite identifications. By using this approach we have identified a large number (-130 currently) of primary metabolites in M. truncatula (Fig.3.4). This method has also been used to compare the profiles of various M. truncatula tissues (data not shown). 

Multiple mass analyzers exist that can perform tandem mass spectrometry. Some use a tandem-in-space configuration, such as the triple quadrupole mass analyzers illustrated (Fig.3.9). Others use a tandem-in-time configuration and include instruments such as ion-traps (ITMS) and Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS or FTMS). A triple quadrupole mass spectrometer can only perform the tandem process once for an isolated precursor ion (e.g., MS/MS), but trapping or tandem-in-time instruments can perform repetitive tandem mass spectrometry (MS ), thus adding n 1 degrees of structural characterization and elucidation. When an ion-trap is combined with HPLC and photodiode array detection, the net result is a profiling tool that is a powerful tool for both metabolite profiling and metabolite identification. 

Dmg Development Metabolite identification Identification and quantitation No Medium to high... 

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