Metabolite identification analytical techniques

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. 

For these reasons, researchers have recently focused on developing faster robotic systems and more sensitive analytical metabolite identification tools [5-9]. However, such techniques are usually resource demanding, consuming a considerable amount of compound, and cannot be used before compound synthesis. Also, because of the increasing number of potential candidates, experimental metabolite identification remains a huge challenge. 

Superior sensitivity, efficiency, and specificity have made high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS), the predominant analytical technique for characterization and quantitative analysis of metabolites (Kostiainen et al., 2003 Ma et al., 2006 Prakash et al., 2007). Ion trap, triple-quadrupole, and quadmpole time-of-flight (Q-TOF) mass spectrometers are routinely used to profile and characterize metabolites in plasma and excreta (Ma et al., 2006). The combination of scan types and features available on mass spectrometers of different design (product ion, MS", neutral loss, precursor ion scans, accurate mass measurements) allows identification and characterization of putative and unexpected metabolites with or without little prior knowledge of biotransformation pathways of a given dmg molecule. 

The analysis of phytochemicals is a tedious process involving several steps in which care must be taken to avoid degradation and contamination. Recent advancements in extraction, concentration, purification and analytical procedures of phytochemicals have been made, but additional developments are needed to assist in the identification and quantification of the diverse array of phytochemicals present in plants and foods, as well as metabolites in biological samples. Specifically there is a need to automate sample extraction, clean-up, and concentration steps to facilitate the screening of phytochemicals develop analytical methods with improved sensitivity, resolution and throughput that utilize less organic solvents and develop concentration and purification methods to produce analytical standards that are not available commercially. Continued advancements in sample preparation and analytical techniques will assist researchers in their quest to identify and quantify the vast array of phytochemicals present in plants... 

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],... 

A prerequisite for detection, identification, and quantification of any species by MS is that all analytes must be converted into gas-phase ions before they enter the mass analyzer. API techniques are most widely used for metabolite identification, mainly due to their ability to couple to liquid chromatography and generate intact gas-phase molecular ions at very high sensitivity (Rossi, 2002 Voyksner, 1997). 

Nanospray ionization is a variation of regular ESI in that the typical flow rate is reduced to between 30 and 200 nL/min. Because of the very low flow rate at which samples are consumed, this technique is rapidly being integrated in many analytical applications including proteomics, metabolite characterization, and pharmaceutical analysis. An approach of combining fraction collection with automated chip-based NSI MS was recently introduced for metabolite identification (Hop, 2006 Staack et al., 2005). LC effluent was collected into a 96-well plate and the fraetions of interest were infused using an automated chip-based nanospray system for structure elucidation. 

The newer MS experiments in a data-dependent acquisition mode provide the MS and MS" data from a single injection. Accurate mass measurements, software-assisted data acquisition, and processing methods have been very useful for metabolite detection and identification. In addition, when MS is combined with other analytical techniques such as derivatization, H/D exchange, and stable isotope labeling have been proven very useful for structural characterization of unusual, uncommon, and difficult metabolites. Further, the flexibility and broad applications of mass spectrometry have allowed for the creation of hybrid instruments and coupling to other powerful analytical techniques, most notably nuclear magnetic resonance (NMR), to further enhance the utility in the field of drug metabolism. 

Liquid chromatography—mass spectroscopy has become one of the most powerful analytical techniques in the drug discovery and development proeess. LC tandem MS is obviously the technique of choice for the identification of metabolites as illustrated in this chapter. Exact mass measurements and elemental composition assignment are essential for the characterization of the metabolites. Accurate mass measurements of the product ions, formed in an MS/MS experiment, greatly facilitate the structure elucidation of the metabolites. With the ever evolving technological advances in mass spectrometry and separation science, LC MS will continue to play an important role in metabolite identification in the future. 

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