Identification of metabolites

The choice of the method of analysis depends on the question to address. Spectrophotometry is sufficient for total curcuminoid content determination in a turmeric extract. Separation techniques coupled to mass spectrometry detection and MALDl-TOF are highly sensitive techniques that are more adapted to the identification of metabolites in biological fluids such as urine or plasma. ... 

Kelley I, JP Freeman, CE Cerniglia (1990) Identification of metabolites from degradation of naphthalene by a Mycobacterium sp. Biodegradation 1 283-290. 

Wetzstein H-G, M Stadler, H-V Tichy, A Dalhoff, W Karl (1999) Degradation of ciprofloxacin by basidiomycetes and identification of metabolites generated by the brown rot fungus Gloeophyllum striatum. Appl Environ Microbiol 65 1556-1563. 

Wetzstein H-G, N Schmeer, W Karl (1997) Degradation of the fluoroquinolone enrofloxacin by the brown-rot fungus Gleophyllum striatum identification of metabolites. Appl Environ Microbiol 63 4272-4281. Wondrack LM, C-A Hsu, MT Abbott (1978) Thymine-7-hydroxylase and pyrimidine deoxyribonucleoside 2 -hydroxylase activities in Rhodotorula glutinis. J Biol Chem 253 6511-6515. 

The fungal transformation of estrogens, by enzymes or cells, seems to produce metabolites with reduced estrogenic activity in short periods, usually days or even few hours. However, only oligomeric products have been identified in enzymatic processes and a lack in the identification of metabolites derived from whole cell fungal systems is still observed. 

Recent work in our laboratories has confirmed the existence of a similar pathway in the oxidation of vindoline in mammals (777). The availability of compounds such as 59 as analytical standards, along with published mass spectral and NMR spectral properties of this compound, served to facilitate identification of metabolites formed in mammalian liver microsome incubations. Two compounds are produced during incubations with mouse liver microsome preparations 17-deacetylvindoline, and the dihydrovindoline ether dimer 59. Both compounds were isolated and completely characterized by spectral comparison to authentic standards. This work emphasizes the prospective value of microbial and enzymatic transformation studies in predicting pathways of metabolism in mammalian systems. This work would also suggest the involvement of cytochrome P-450 enzyme system(s) in the oxidation process. Whether the first steps involve direct introduction of molecular oxygen at position 3 of vindoline or an initial abstraction of electrons, as in Scheme 15, remains unknown. The establishment of a metabolic pathway in mammals, identical to those found in Strep-tomycetes, with copper oxidases and peroxidases again confirms the prospective value of the microbial models of mammalian metabolism concept. 

However, the temptation to link glutathione (GSH) adducts to MBI must be avoided since the identification of metabolite adducts with GSH does not confirm MBI, albeit it may be an indicator of other adverse drug reactions (Walgren et al., 2005). By definition, exogenous nucleophiles such as GSH should offer no protection from MBI [161], and in principle, even when GSH adducts are formed from the same chemical entity responsible for MBI, the exogenous adducts themselves may not be related to the enzyme-inactivating species [174]. 

Roubal et al. (8) applied TLC to the separation and identification of metabolites of l c-iabeled naphthalene administered to coho salmon fingerlings via intraperitoneal injection. 1-Naphthol, a dihydrodiol, mercapturic acid, 1-naphthyl glucuronic acid and a glycoside/sulfate fraction were identified in brain, liver, gall bladder, and muscle 1-naphthol, a dihydrodiol, and 1-naphthyl glucuronic acid were the only metabolites found in heart. 

The prediction of retention times in a given eluent from log P has been proposed for aromatic hydrocarbons.19 The log A values of phenols21 and nitrogen-containing compounds22 were also related to their logP, and the calculated log P was used for the qualitative analysis of urinary aromatic acids, i.e. for the identification of metabolites in urine from the differences of log P in reversed-phase liquid chromatography.23,24... 

S. Suwanrumpha, R. B. Freas, Identification of Metabolites of Ampicillin Using Liquid Chromatography/Thermospray Mass Spectrometry and Fast Atom Bombardment Tandem Mass Spectroscopy , Biomed. Environ. Mass Spectrom. 1989, 18, 983-994. 

T. Nishimaki-Mogami, K. Minegishi, A. Tanaka, M. Sato, Isolation and Identification of Metabolites of 2-Ethylhexyl Diphenyl Phosphate in Rats , Arch. Toxicol. 1988, 61, 259-264. 

Feung, C. S., Hanrilton, R.H., and Mumma, R.O. Metabolism of 2,4-dichlorophenoxyacetic acid. V. Identification of metabolites in soybean callus tissue cultures. J. Agric. Food Chem., 21(4) 637-640, 1973. 

Advances in high resolution mass analyzers (TOF, FT-ICR, orbitrap) have greatly improved the detection and identification of metabolites based on accurate mass measurements. In single MS mode accurate mass determination is mainly used to differentiate between isobaric ions. Combined with LC-MS, it allows the detection of predicted metabolites by performing extracted ion current profiles... 

Berdy J, Kadar-Pauncz J, Mehesfalvi-Vajna Z, Horvath Cf Gyimesi J (1977) Metabolites of gentamicin producing Micromonospora species. Part 1. Isolation and identification of metabolites. J Antibiot 30 945-953. 

The decomposition by the fungus, Trametes versicolor, of diatrizoate, iodi-pamide and related triiodinated benzoates was investigated by Rode et al. [121]. All compounds were degraded, albeit not completely, under iodide release. Isolation and identification of metabolites suggested stepwise reductive de-iodina-tions as initial transformation steps. 

Wetzstein H-G., N. Schmeer, and W. Karl (1997). Degradation of the fluoroquinolone etrto-floxacin by the brown rot fungus Gloeophyllum striatum-. Identification of metabolites. Applied and Environmental Microbiology 63 4272-4281. 

Although analysis of urine samples for the presence of these metabolites represents a potential means of assessing recent human exposure to diazinon, these metabolites can originate from exposure to other organophosphorus compounds and, therefore, are not specific for diazinon exposure. Additionally, these studies do not report a quantitative association between metabolite levels and exposure to diazinon in humans. Thus, these biomarkers are only indicative of exposure to diazinon (or other organophosphorus compounds) and are not specifically useful for diazinon exposure nor for dosimetric analysis. Further studies designed to refine the identification of metabolites specific to diazinon and provide dosimetric data will be useful in the search for a more dependable biomarker of diazinon exposure. 

Climie, I.J., Hutson, D.H. Stoydin, G. (1981) Metabolism of the epoxy resin component 2,2-bis[4-(2,3-epoxypropoxy)phenyl]propane, the diglycidyl ether of bisphenol A (DGEBPA) in the mouse. Part ii. Identification of metabolites in urine and faeces following a single oral dose of -DGEBPA. Xenobiotica, 11, 401-424... 

Chee-Sanford, J. C. Tiedje, J.M. (1994). Detection and identification of metabolites produced during anaerobic toluene degradation by Azoarcus sp. (TOL-4). Abstracts of the 94th Annual Meeting, American Society for Microbiology, Q-335. 

Kelley, I., Freeman, J. P., Evans, F. E. Cerniglia, C. E. (1993). Identification of metabolites from the degradation of fluoranthene by Mycobacterium sp. strain PYR-1. Applied and Environmental Microbiology, 59, 800-6. 

The reasons for identification of metabolites are manyfold but all boil down to human safety of drugs under clinical investigation. Initially metabolites of a drug are characterized with in vitro systems (microsomes, hepatocytes, S9 fractions, etc.) and later lead compounds are assessed using mouse, rat, rabbit, dog, and/or monkey. Subsequently, metabolites in humans are identified following dmg administration to assure that the nonclinical species undergoing safety assessment are adequately exposed to human metabolites of the dmg (Smith and Obach, 2006). 

Gallagher, R. T., Wilson, I. D., and Hobby, K. (2008). New approach for identification of metabolites of a model drug Partial isotope-enrichment combined with novel mass spectral modeling software. In Proceedings of the 56th ASMS Conference on Mass Spectrometry and Allied Topics. ASMS, Denver, CO. 

Kantharaj, E., Tuytelaars, A., Proost, P. E., Ongel, Z., Van Assouw, H. P., and Gilissen, R. A. (2003). Simultaneous measurement of drug metabolic stability and identification of metabolites using ion-trap mass spectrometry. Rapid Commun. Mass Spectrom. 17 2661-2668. 

Even with these notable limitations, multi-MRM is a powerful and essential experiment for identification of metabolites present at lowlevel. Figure 3.22 shows an example of detection of a variety of low-level buspirone metabolites by using a set of 27 theoretical MRMs. It is especially important with this type of experiment to have sensitive ion trap product ion scans to confirm and validate the nature of these trace-level metabolites. 

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