Metabolite profiling analytical methods

As in the other -omics, analyses may be directed at a specific metabolite, at all metabolites in a given system in a shot-gun approach, or at accessible groups of molecules in profiling experiments. In that also the technology varies. In addition, the chemistry of different metabolites is very heterogeneous since it involves hydrophobic lipids, hydrophilic carbohydrates, ionic inorganic species, and other secondary natural products and already the choice of solvent in metabolite extraction dictates which types of molecules will be present (Fig. 10.8). Therefore, total metabolome profiling is not possible, because no analytical method will be able to accommodate all the different molecule classes at once. 

This paper will review the known metabolic pathways of CW agents, excretion profiles where these have been measured, and methods for the analysis of metabolites in urine or blood. Examples are provided of detection in cases of human exposure. The review focuses mainly on sulfur mustard and nerve agents that represent the greatest global CW threat, and for which most analytical methods have been developed. 

In what many consider to be a landmark publication on metabolomics, Fiehn et al. (2000) state it is crucial to perform unbiased (metabolite) analyses in order to define precisely the biochemical function of plant metabolism. The authors argue that for metabolomics/metabolite profiling to become a robust and sensitive method suited to automation, a mature technology such as gas chromatography-mass spectrometry (GC-MS) is required as an analytical technique. The authors go on to describe a simple sample preparation and analysis regime that allowed for the detection and quantification of more than 300 compounds from a single-leaf sample extract. 

Quantitative analytical methods have been developed to identify metabolites in pathways or classes of compounds. This collective directed approach has been called metabolite profiling or metabolomics. Semiquantitative, NMR-based metabolic fingerprinting has also been applied to high abundance metabolites and has been termed metabonomics [188], Peaks detected in NMR spectra carry information regarding the structure of the metabolites,... 

Whatever the biological test system is, ideally the identification and quantification of the metabolites would be based on a single analytical method. However the identification and the quantitation of metabolites and metabolite profiles remain a daunting task, even with the help of the most modern analytical tools. LC-MS is practical for small-molecular lipid-soluble chemicals (admittedly, a majority of compounds are such), but it does not cover many other types of chemicals. Consequently it is important to consider the scope or the chemical space of the chosen analytical method no single analytical technique is able to identify all potential metabolites [6],... 

Wong, M.C. et al., An approach towards method development for untargeted urinary metabolite profiling in metabonomic research using UPLC/QToF MS, J. Chromatogr. B Analyt. Technol. Biomed. Life Sci., 871(2), 341, 2008. 

ATSDR publi.shes toxicological profiles for hazardous substances. The toxicological profiles for the pesticides chlorpyrifos, chlorfenvinphos, diazinon, dichlorvos, disul-folon, ethion, malathion, and methyl parathion have been published. Each of these profiles contain.s a section on analytical methods for the pesticide and its metabolites. The references for various analytical methods are given. 

Accelerator mass spectrometry (AMS) is an ultrasensitive analytical method for radioactivity analysis. AMS offers 10 -10 -fold increases in sensitivity over LSC or other decay counting methods so that levels as low as 0.0001 DPM can be detected (Brown et al., 2005, 2006). AMS has been applied to mass balance determination, pharmacokinetic studies of total radioactivity, and measurement of chemically modified DNA and proteins in humans after the administration of a low radioisotope dose (approximately lOnCi/person for mass balance and drug metabolism studies) (Buchholz et al., 1999 Garner, 2000 Garner et al., 2002 Liberman et al., 2004 White and Brown, 2004). In addition, off-line HPLC-AMS has been explored for metabolite profiling after... 

The determination of the metabolism profile of medicinal herbs and plant extracts is very difficult due to their inherent complexity. In the present work, a rapid analytical method using LC—MS" techniques for flavonol component screening in crude herbal extract and biosamples was established. Mass spectrometric fragmentation behavior and metabolic pathway complement each other in structural identification and correlating the metabolites and their parent forms. This information provides a basis for research that involves the metabolism profile of complex mixtures. 

In-vivo and real-time monitoring of secondary metabolites, released from living animals and plants, using field-induced direct ionization MS, was also presented (Figure 13.8) [83]. Although real-time monitoring of metabolite profiles was not clearly demonstrated, one may suspect that the method could potentially be adopted for this purpose. However, considering that animals are positioned in front of the mass spectrometer (in the presence of an electric field and hot gas), one can only speculate on the practicality of this analytical approach. 

Gas-Phase Analytical Methods for the Study of Human Metabolites. Metabolic Profiles Obtained by Open Tubular Capillary Chromatography J. Chromatogr. 91 367-378 (1974) ... 

The first two approaches, target metabolomics and metabolite profiling, focus on the detection and quatification of biomarkers. Biomarkers are specific metabolites used to distinguish a normal biological state from a pathogenic process. Analytical methods have been developed to monitor selected biomarkers by well-calibrated methods of analyses. If a patient is already sick, target or profile metabolomics are capable of fast diagnostics since patient symptoms can narrow the analysis to the most affected metabolite(s). 

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