Mass metabolites

Grootveld, M., Henderson, E.B., Farrell, A.J., Blake, D.R, Parkes, H.G. and Haycock, P. (1991). Oxidative damage to hyaluronate and gjucose in synovial fluid during exercise of the inflamed rheumatoid joint detection of abnormal low-molecular-mass metabolites by proton NMR spectroscopy. Biochem. J. 273, 459-467. 

Figure 23.5 Separation with UHPLC [after T. Moritz, Nestle Research Center, Lausanne, with permission see also S.J. Bruce et a .. Anal. Biochem., 372, 237 (2008)]. Conditions sample, extract from human plasma column, 10 cm 2.1mm i.d. stationary phase, C8 UPLC 1.7pm mobile phase, 0.5mlmin water-acetonitrile with 0.1% formic acid, linear gradient pressure, 600 bar detector, TOF-MS with positive ESI. The first peaks are amino acids and low-mass metabolites, the ones between 8 and 11 min are phospholipids (besides background peaks). 

Between 12 and IS billion US per year are spent for analytical purposes worldwide. In this sum the analytical usage of enzymes in clinical chemistry, food and cosmetic industry, and biotechnology for the routine measurement of about 80 substances, mainly low-molecular mass metabolites but also effectors, inhibitors, and the activity of enzymes themselves, is included. A wide range of immunoassays for low-molecular mass haptens, macro-molecules, and microorganisms have been made available in recent years through the enormous progress in immunological research, especially by the preparation of monoclonal antibodies. About 1 billion immunoassays are sold per year. 

In general, the obtained lysates are cleared by centrifugation to remove cellttlar debris, and the supernatant or supemate is considered a whole cell protein extract composed of a complex mixture of proteins, other cell constituents such as lipids, nucleic acids, polysaccharides, low molecular mass metabolites, and all additives. These additives include buffers, chaotropes, detergents, or cocktails of proteinase inhibitors, which are added to aid in protein extraction and preserve the integrity of a proteome. 

Perfused rat liver rapidly converts 4-m thyI-5-/3-chloroethy]thiazole to 2-hydroxy -4-methylthiazol-5-y) acetic acid (40. 41). Finally, tw o new human metabolites of chlormethiazole have been isolated and identified by mass spectra as 2-hydroxy-4-methyl-5-/S-chloroethylthiazole and 2-hydroxy-4-methyl-5-ethylthiazole (42). 

Pfleger, K., Maurer, H.H., and Weber, A., Mass Spectral and GC Data of Drugs, Poisons, Pesticides, Pollutants and Their Metabolites, VCH, Weinheim, Germany, 1992. 

Pyridinium iodide, 4,4 (l,3,4-thiadiazole-2,5-diyl)-bis(l-methyl)-reduction, 6, 564 Pyridinium ion, Af-methyl-as metabolite of pyridine, 1, 234 Pyridinium ions hydrogen bonding to water mass spectrometry, 2, 135 magnetic circular dichroism, 2, 129 NMR, 2, 121... 

The degradation of 2,6-xylenol (2,6-dimethylphenol) by bacteria produces a metabolite with elemental composition C8///0O2 as determined by high-resolution mass spectrometry Which carbon skeleton and which relative configuration are deducible from the NMR experiments 44, all obtained from one 1.5 mg sample ... 

The C NMR spectrum of the metabolite shows 16 signals instead of 8 as expected from the elemental composition determined by high-resolution mass spectrometry. Moreover, aromaticity of the 2,6-xylenol is obviously lost after metabolism because two ketonic carbonyl carbon atoms (5c = 203.1 and 214.4) and four instead of twelve carbon signals are observed in the shift range of trigonal carbon nuclei (5c = 133.1, 135.4, 135.6 and 139.4) in the C NMR spectra. To conclude, metabolism involves oxidation of the benzenoid ring. 

Also the mirror image of the strueture I, eorreetly denoted as exo-3,10-dihydroxy-3,5,8,10-tetra-methyltrieyelo[6.2.2.0 ]dodeea-5,l 1-diene-4,9-dione, would be possible sinee enantiomers are not differentiated by NMR. A retro-Diels-Alder fragmentation of I to CsH/oO explains why the moleeular ion eorresponding to the moleeular formula C16//20O4 is not deteeted in the mass spee-trum. The metabolite I eould be formed by Diels-Alder dimerisation of 1,5-dimethyleyelohexa-l,3-dien-5-ol-6-one J as the primary metabolite which acts as diene and dienophile as well... 

In gas chromatography/mass spectrometry (GC/MS), the effluent from a gas chromatograph is passed into a mass spectrometer and a mass spectrum is taken every few milliseconds. Thus gas chromatography is used to separate a mixture, and mass spectrometry used to analyze it. GC/MS is a very powerful analytical technique. One of its more visible applications involves the testing of athletes for steroids, stimulants, and other performance-enhancing drugs. These drugs are converted in the body to derivatives called metabolites, which are then excreted in the... 

There are many reports of the use of mass spectroscopy coupled to chromatography outlets for detection and identification of dmgs and metabolites. An example is compound 126 (99MI2, 99MI3). Carboxylic acids have been converted into hydrazides and hence into 3-substituted [l,2,4]triazolo... 

P. O. Edlund, E. Bowers and J. Henion, Detemination of methandrostenolone and its metabolites in equine plasma and urine by coupled-column liquid cliromatography with ulti aviolet detection and confimation by tandem mass spectrometiy , 7. Chromatogr. 487 341-356(1989). 

J. Cai and J. Henion, On-line immunoaffinity exti action-coupled column capillary liquid cliromatography/tandem mass specb omeb y trace analysis of LSD analogs and metabolites in human urine . Anal. Chem. 68 72-78 (1996). 

E. M. Benson, A. J. Tomlinson, A. N. Mayeno, G. J. Gleich, D. Wells and S. Naylor, Membrane preconcentration-capillaiy electi ophoresis-mass specti ometi y (niPC-CE-MS) analysis of 3-phenylamino-l,2-propanediol (PAP) metabolites , 7. High Resolut. Chromatogr. 19 291-293(1996). 

In order to reduce or eliminate off-line sample preparation, multidimensional chromatographic techniques have been employed in these difficult analyses. LC-GC has been employed in numerous applications that involve the analysis of poisonous compounds or metabolites from biological matrices such as fats and tissues, while GC-GC has been employed for complex samples, such as arson propellants and for samples in which special selectivity, such as chiral recognition, is required. Other techniques include on-line sample preparation methods, such as supercritical fluid extraction (SFE)-GC and LC-GC-GC. In many of these applications, the chromatographic method is coupled to mass spectrometry or another spectrometiic detector for final confirmation of the analyte identity, as required by many courts of law. 

E. Podehrad, M. Heil, S. Leih, B. Geier, T. Beck, A. Mosandl, A. C. Sewell and H. Bohles, Analytical approach in diagnosis of inherited metabolic diseases maple syrup urine disease (MSUD)-simultaneous analysis of metabolites in urine by enantioselective multidimensional capillary gas chromatography-mass specti ometiy (enantio-MDGC-MS) , 7. High Resolut. Chromatogr. 20 355-362(1997). 

Gudzinowicz, B. J. and Gudzinowicz, M. J. Analysis of Drugs and Metabolites by Gas Chromatography Mass Spectrometry (Vols. 1-5). New York Marcel Dekker, 1977. 

By carefully examining the fragmentation pattern of the metabolite and comparison with the mass spectra of the precursor molecule, it is often possible to determine not only the nature of the biotransformation, but also its position in the molecule. In the proceeding example, accurate mass measurement was used to determine that a hydroxyl group had been added to the benzene ring containing the fluorine substituent. 

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