Metabolite identification structure elucidation

Data-dependent MS or MS various metabolites identification structure elucidation... 

LC-MS/MS Approaches to the Identification/Structural Elucidation of Alkaloid Drug Metabolites... 

E. W. Taylor, W. Jia, M. Bush, and G. D. Dollinger, Accelerating the drug optimization process Identification, structure elucidation, and quantification of in vivo metabolites using stable isotopes with LC/MS" and the chemiluminescent nitrogen detector, Anal Chem. 74 (2002), 3232-3238. 

Taylor, E.W. Jia, W. Bush, M. Dollinger, G.D. Accelerating the Drug Optimization Process Identification, Structure Elucidation, and Quantification of In Vivo Metabolites using stable isotopes with LC/MSn and the chemiluminescent nitrogen detector, AnaZ. Chem. 74(13), 3232-3238 (2002). 

It is needless to mention that this is only a tentative identification of those metabolites and further work has to be done in the synthesis of the starting material and the preparative-scale fermentation to produce enough quantities of those metabolites for structural elucidation and other biological studies. 

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. 

Further research should focus on (1) the description of the mechanisms involved in the degradation in situ (2) the identification of the metabolites released during the process to better estimate the toxicity of the residues (3) the relationship between the WRF and the microbiota, which may counteract the degrading capacity and clearly, (4) the optimization of the process and (5) the scale-up necessary to implement a possible real application. Given the challenge of structural elucidation studies with complex environmental matrices such as sludge, the work performed so far respect point (2) in liquid media is reviewed in the next chapters of the book for PhC [151], UV-F [158] and BFR [157]. 

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. 

This chapter has two aims. First, techniques that use mass spectrometry for the identification and structural elucidation of neuroactive chemicals are reviewed. Second, and with greater emphasis, this chapter attempts to describe the routine use and pitfalls encountered when using mass spectrometry— especially GC/MS and LC/MS—for rapid, specific, quantitative analyses of neuroactive compounds and metabolites in a variety of biological matrices. 

As mentioned in the previous section, triple-quadrupole instruments are very good at finding low levels and structurally related compounds in the presence of biological matrices as well as being the gold standard technique for quantitation. Ion trap mass spectrometers, on the other hand, have the capabilities to obtain high-sensitivity full-scan MS and MS/MS spectra therefore, they are widely used for qualitative analysis, such as structural elucidation and unknown identification. For complete metabolite identification, it is important to have both the sensitivity and selectivity of triple-quadrupole instruments and the full-scan data quality of ion traps. 

Conventionally, metabolite identification and more specifically reactive metabolite screening typically uses an array of chromatographic and mass spectrometric methods, and may require multiple injections of the same sample. This is to ensure that enough information has been collected to detect all metabolites and to have sufficient fragmentation information available to elucidate structures. 

Zhu, M., Zhang, D., and Skiles, G. L. (2005). Quantification and structural elucidation of low quantities of radiolabeled metabolites using microplate scintillation counting techniques in conjunction with LC-MS. In Identification and Quantification of Drugs, Metabolites and Metabolizing Enzymes by LC-MS (Chowdhury, S. K., Ed.). Elsevier, Amsterdam, pp. 195-223. 

Until very recently, metabolic stability screening and metabolite identification (metabolite ID) have been sequential processes that is, the metabolic stability assays are typically performed hrst to identify rapidly metabolized compounds and a follow-up metabolite ID study is performed next, typically at a 10-fold higher substrate concentration to ensure generation of sufficiently high-quality MS/MS information to support structure elucidation. 

It is common opinion [5] that the use of in silico methods to predict a hypothetical metabolite structure, combined with the most recent experimental techniques, can speed up the process of metabolite identification by focusing experimental work on specific target structures, thus improving the method of metabolite structure confirmation and elucidation. 

Many of the most important chemical questions in the pharmaceutical industry involve the analysis of complex mixtures. Identification of low-level metabolites and drug substance impurities usually requires high-performance liquid chromatography for the separation of these mixtures or isolation of a compound of interest from a sample matrix. In these analyses, the structural information obtainable for the low-level compounds is limited by the type of detection used. The coupling of HPLC and mass spectrometry has become routine and provides useful molecular weight and fragmentation information, but this is often not enough for complete structure elucidation. 

Mass spectrometry (MS), employing most of the ionization techniques, has been used either independently, or in combination with HPLC, for the determination of the dissociation scheme of paclitaxel or for the identification of taxanes and paclitaxel metabolites. Tandem MS has enhanced identification and structure elucidation capabilities in the analysis of either biological or plant samples. 

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