Metabolites separation methods

The application areas for LC-MS, as will be illustrated later, are diverse, encompassing both qualitative and quantitative determinations of both high-and low-molecular-weight materials, including synthetic polymers, biopolymers, environmental pollutants, pharmaceutical compounds (drugs and their metabolites) and natural products. In essence, it is used for any compounds which are found in complex matrices for which HPLC is the separation method of choice and where the mass spectrometer provides the necessary selectivity and sensitivity to provide quantitative information and/or it provides structural information that cannot be obtained by using other detectors. 

Separation methods, multichromato-graphic la 56 Serine la 246,356 lb 132 Serotonin la 70,76,239,240,262,355, 380 lb 37-39,231,243,348 Serotonin metabolites lb 327 Serum lipids la 89 Serum proteins la 74 Sesquiterpene derivatives lb 239,446 Sesquiterpene esters lb 239 Sesquiterpene glucosides la 327 Sesquiterpene lactones lb 448 Sevin lb 387-389 Si 50 000, specific surface area la 91 Silica gel, caffeine-impregnated la 85 -, surface modified la 3 Silica gel 60, specific surface area la 91... 

Jia, L., Liu, B., Terabe, S., Nishioka, T. (2004). Two-dimensional separation method for analysis of bacillus subtilis metabolites via hyphenation of micro-liquid chromatography and capillary electrophoresis. Anal. Chem. 76, 1419-1428. 

High polarity is one of the reasons why both the ionic and amphoteric surfactants, and especially their metabolites, are difficult to detect. This property, however, is important for the application tasks of surface-active compounds, but is also the reason for their high water solubility. Due to this fact, their extraction and concentration from the water phase, which can be carried out in a number of very different ways, is not always straightforward. Furthermore, they are often not volatile without decomposition, which thus prevents application of gas chromatographic (GC) separation techniques combined with appropriate detection. This very effective separation method in environmental analysis is thus applicable only for short-chain surfactants and their metabolites following derivatisation of the various polar groups in order to improve their volatility. 

For the quantitative description of the metabolic state of a cell, and likewise which is of particular interest within this review as input for metabolic models, experimental information about the level of metabolites is pivotal. Over the last decades, a variety of experimental methods for metabolite quantification have been developed, each with specific scopes and limits. While some methods aim at an exact quantification of single metabolites, other methods aim to capture relative levels of as many metabolites as possible. However, before providing an overview about the different methods for metabolite measurements, it is essential to recall that the time scales of metabolism are very fast Accordingly, for invasive methods samples have to be taken quickly and metabolism has to be stopped, usually by quick-freezing, for example, in liquid nitrogen. Subsequently, all further processing has to be performed in a way that prevents enzymatic reactions to proceed, either by separating enzymes and metabolites or by suspension in a nonpolar solvent. 

We employed various substrates to check for MFO in two bivalve species, a salt water mussel (Mytilus edulis) and a fresh water clam (Anodonta sp). Cytochrome P-450 was also studied. Organisms were exposed to 100 PPM Venezuelan crude in a stagnant system for up to one month. Enzyme assays were carried out with digestive gland 9000 g homogenates (17) and cytochrome P-450 analysis, with microsomes (21). The hydrocarbon substrates investigated included 1I+C-labelled benzo(a)pyrene, fluorene, anthracene, and naphthalene. The method used for separation of BP metabolites by thin layer radiochromatography has been described (7). The metabolite detection method for the other aromatic hydrocarbons was essentially the same except methylene chloride was used as metabolite extractant as well as TLC developer. Besides the hydrocarbon substrates, we also checked for other MFO reactions, N-dealkylase with C-imipramine (22) and 0-dealkylase with ethoxycoumarin (15). 

For isohexide nitrates gas-liquid chromatographic separation methods using OF-1 (Ref. 52), OV-101 (Refs. 53-59), OV-17 (Ref 60), or OV-210 (Ref 61) columns have been reported. Most of the methods described are used to detect traces of nitrates, as well as of their metabolites, in urine and plasma probes.62-69 All known isohexide nitrates can be measured in mixtures by using a DB-5 capillary column.70-71 Electron-capture detection was applied for the g.l.c. determination of isosorbide dinitrate71 and its metabo-lites.7,b... 

Recent research efforts have included the use of NMR coupled with MS, thereby enabling more complete identification of metabolites. The sensitivity of MS methods is a clear advantage over NMR and the ability to separate metabolites by HPLC or GC coupled with MS and/or NMR methods. The GC-based separation methods provide for analysis of volatile compounds or compounds made to be volatile by derivitization, whereas HPLC methods, although biased by solvent systems and columns, can accommodate a larger range of molecules. In both cases, the separation methods afford investigators the opportunity to increase resolution and separation of metabolites, and this enhancement in overall selectivity is particularly useful when novel biomarkers are sought. 

In order to achieve highest sensitivity of VitD metabolites detection, the efforts for method development were focused on three components of the analytical flow path (1) the selective SPE extraction procedure, (2) the ionization and SRM method for MS/MS, and (3) the pLC loading and separation method. 

Tsai reviewed the separation methods used in the determination of choline and acetylcholine [10]. This review surveyed the array of analytical techniques that have been adopted for the measurement of acetylcholine or its main precursor/metabolite (choline), ranging from simple (bioassay, radio enzymatic assay, gas chromatography-flame ionization detection, gas chromatography-mass spectrometry, high... 

Each separation method has benefits and drawbacks. HPLC looks promising for the separation of high molecular weight substances and metabolites, but suffers a lack of resolution. 

The counting efficiencies obtained with in situ methods depend on various concurrent factors. The metabolites separated on the gel column must be fixed in a spaced way to minimize self-absorption. The spaced fixation must not be disturbed during dehydration and the following procedures. However, the counting efficiencies of the in situ method depends on the concentration of the gel. This is even more obvious when compared with those obtained with the in situ method described earlier (Gezelius, 1977) for a mixed gel of 2% polyacrylamide and 0.5% (Table 2 ). The reduction of the counting efficiencies in more concentrated gels is probably due to increased selfabsorption. 

Nowadays, interfacing separation methods such as high-performance hquid chromatography (HPLC) or CE [54] to MS is already a routine technique. MS methods based on so-called soft ionization techniques, which include fast atom bombardment, laser desorption and electrospray ionization (ESI), have allowed the analysis of biological macromolecules that in the past could have been analyzed only by extensive cleavage and derivatization. Of these methods, the two most preferred for biomolecules are ESI [55] and matrix-assisted laser desorption/ionization(MALDI) [56,57], for which time-of-flight (TOE) and ion trap mass analyzers are the most frequentiy used mass analysis methods. However, these methods are equally suitable for small molecules, such as metabolites or the products of organochemical reactions. 

Methods of Metabolite Separation and Mass Spectrometric Analysis... 

F.M. Rubino, Separation methods for methotrexate, its structural analogues and metabolites, J. Chromatogr. B, 2001, 764, 217-254. 

Gas chromatography-based separation methods for the identification of bacterial metabolites are well established. The maximum molecular weight of compoimds... 

IJavidin is incubated with biotin (standard or unknown sample) and the avidin with unoccupied sites is then bound to a plate precoated with biotinylated bovine serum albumin. After washing, the plate wells are counted, giving a measure of the biotin concentration. This method has also been used for postcolumn detection of biotin and its metabolites separated by HPLC (27). The sensitivity is 2 pg per well. 

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