Structure metabolite elucidation

Microbial Bioreactors Used in Metabolite Structure Elucidation 193... 

Metabolite biosynthesis has demonstrated its utility in drug metabolite preparation and characterization, and contributed to drag discovery and development. Although metabolite biosynthesis is a prerequisite step for metabolite structure elucidation in many cases, it is complementary to chemical synthesis in large-scale metabolite preparations. The merits for using these techniques should be determined on a case-by-case fashion. New techniques, such as recombinant enzyme and microbial glucuronidation systems, would have a great impact on the field. 

Hopfgartner, G., and Vilbois, F. (2000). The impact of accurate mass measurements using quadrupole/time-of-flight mass spectrometry on the characterisation and screening of drug metabolites Structure elucidation by LC-MS. Analusis 28 906-914. 

K. I. Burton, J. R. Everett, M. J. Newman, F. S. Pullen, D. S. Richards, and A. G. Swanson, On-line liqnid chromatography conpled with high field NMR and mass spectrometry (LC-NMR-MS) A new techniqne for drug metabolite structure elucidation, J. Pharma. Biomed. Anal. 15 (1997), 1903-1912. 

In practical application scanning can be manipulated on-the-fly within a chromatographic separation to obtain maximum information. In metabolism studies or as a chemical dosimeter, the structural feature of the parent compound and its unique neutral loss occurring on collisional activation, marks the metabolite. The mass of the metabolite is then obtained from the TSP mass spectrum at Q1 and the product ion spectrum of the metabolite molecule ion is obtained by product ion scanning. Recent publications have discussed additional applications of both tandem mass spectrometry (26. 271 and thermospray tandem mass spectrometry (28) in metabolite structure elucidation. 

In the pharmaceutical industry, the techniques are being used to examine off-target effects particularly for the early identification of toxicity. MOA can be studied through metabolomics and can also be used as a quality control tool for complex mixtures such as foods or herbal medicines. Similarly, the tools and expertise of natural products chemists are essential in metabolomics, particularly in biomarker discovery (see also Volume 9). Biomarker discovery via untargeted metabolomics can lead to metabolite signatures (nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry (MS), etc.) that are not present in current metabolomics databases. This is particularly true for plant secondary metabolism studies and nonmammalian metabolites. Structure elucidation then becomes critical to understanding the metabolomics results and for biomarker development. 

Knecht, A., Cramer, B., and Humpf, H. U. 2006. New Monascus metabolites Structure elucidation and toxicological properties studied with immortalized human kidney epithelial cells. Mol. Nutr. Food Res. 50(3), 314-321. 

The selectivity of capillary HPLC coupled with the sensitivity and improved S/N of microcoil cryoprobes may allow rapid analysis of biotransformation products with minimal purification or preconcentration. The increase in sensitivity and speed offered by these approaches may bring NMR metabolite structure elucidation into a timescale useful for discovery screening. 

Instrument advances in recent years have led to an everincreasing quantity of data that require examination. The automated identification of metabolites and their structures will be crucial to the success of biotransformation studies in drug discovery. Several software packages have been developed that accelerate various stages of this process. One such program is Metabolite ID 1.4 for Analyst from Applied Biosystems/MDS Sciex. Other instrument vendors offer similar software packages to help in metabolite structure elucidation. Metabolite ID 1.4 for Analyst will acquire full-scan chromatographic data of... 

Burton, K.I. Everett, J.R. Newman, M.J. Pullen, F.S. Richards, D.S. Swanson, A.G. On-line Liquid Chromatography Coupled with High Field NMR and Mass Spectrometry (LC-NMR-MS) A New Technique for Drug Metabolite Structure Elucidation, 7. Pharm. Biomed. Anal. 15(12), 1903-1912 (1997). 

Boris, D.J. Cook, S.T. Bowers, G.D. O Mara, M.J. Quinn, K.E. Use of Automated Nanoelectrospray for Drug Metabolite Structure Elucidation, in Proceedings of the 52nd ASMS Conference on Mass Spectrometry and Allied Topics, Nashville, Tennessee, May 23-27, 2004. 

In the end, each of the instruments described here provides unique capabilities that are highly useful in metabolite structure elucidation [30], The most effective and comprehensive metabolite characterization experiments utilize a combination of these instruments at different stages of the drug discovery and development in order to answer a specific question. 

In this chapter we provide a brief introduction to NMR theory and describe the most common experiments used for metabolite structure determination by NMR. Selected examples of metabolite structure elucidation are presented to illustrate relevant hardware, key parameters, and NMR methodology. Detailed explanations of NMR fundamentals have been previously reviewed in a series of books that are listed at the end of this chapter (Berger and Brunn, 2004 Breitmaier, 2002 Freeman, 1997 Friebolin, 2005 Homans, 2005 Keeler, 2005 Martin and Zektzer, 1988). 

The CEC-NMR contour plot indicated that all compounds are base-lined separated resulting in distinct NMR rows in the 2D display. This example shows the high application power of CE and CEC-NMR for providing unambiguous information about substances in complex organic molecules. The first steps towards a high-throughput separation system have already been made. For the successful performance of real-world applications, NMR sensitivity must be improved. NMR probes with 1-ng sensitivity should facilitate the application of capillary separations for metabolite structure elucidation and functional food analysis. 

After detection and acquisition of MS spectra of metabolites, the assignment of product ion spectra is followed to elucidate the structure information. Interpretation of the product ion spectrum for metabolite structural elucidation is illustrated below to differentiate regioisomeric metabolites of compound B, 1 -[2-hydroxy-2-(4-hydroxy-phenyl)-1 -methyl-ethyl]-4-phenyl-piperidin-4-ol) (Fig. 9.7). The full-scan LC-MS of metabolites B1 and B2 both displayed a... 

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