Metabolism studies metabolite identification

Drug metabolism studies including identification of bioactive metabolites and blocking of metabolic inactivation. 

Residues are evaluated to determine the extent of uptake of the veterinary drug, its distribution throughout the body, and its elimination. Normally, contemporary residue depletion studies establish tissue concentrations in a radiolabeled drug study, in which total residues and parent compound are determined at several pre-determined times between zero time and a time beyond the proposed withdrawal time. As well as total residues, which include free and bound components, the study quantifies major metabolites. These are compounds contributing 10% or more of total radioactivity or that are present at a concentration of > 0.10 mg/kg. Metabolism studies enable identification of the marker residue and target tissue. The marker residue must give assurance that, when its concentration is at or below the MRL, total residues satisfy ADI requirements. 

Nnane IP, Damani LA, and Hutt AJ (1995) Dmg metabolism studies Metabolite isolation and identification. In Townsend A, Worsfold PJ, Haswell SJ, Macrae R, Werner HW, and Wilson ID (eds.) Encyclopedia of Analytical Science, vol. 2, pp. 944—951. London Academic Press. 

Most of the information on the metabolism of hexachloroethane has been collected by in vitro techniques using rat liver slices or rat liver microsomes. Figure 2-3 summarizes the results of these studies. The identification of tetrachloroethene and pentachloroethane as the initial metabolites of hexachloroethane metabolism in vitro agrees with in vivo data from sheep that were orally exposed to doses of 500-1,000 mg/kg hexachloroethane (Fowler 1969b). 

Hopfgartner, G. Zell, M. Q trap MS a new tool for metabolite identification, in Using Mass Spectrometry for Drug Metabolism Studies, ed. Korf-macher, W. A., CRC Press, 2004. 

Metabolism studies showed that the major metabolites of the components of ivermectin in cattle, sheep, and rats were 24-hydroxymethyl compounds, whereas major metabolites in swine were 3-0-desmethyl compounds. Identification of the 24-hydroxymethyl metabolites has not been yet achieved in swine, whereas identification of Hie 3-O-desmetlryl metabolites has not been made possible in cattle or sheep (54, 55). Recent metabolism studies (56) in cattle, swine, and rats have indicated, however, diat the metabolism of avermectins was qualitatively similar for all three species. There were quantitative differences both between species and between compounds for a given species, but all three species produced... 

This paper is the only one in the liquid chromatography portion of this symposium which will attempt to deal with chromatography specifically from the viewpoint of the pesticide metabolism chemist. A residue analyst knows what compound he must analyze for, and develops his method with the properties of that substance in mind. On the other hand, the pesticide metabolism chemist has a different problem. At the conclusion of the treatment, exposure, and harvest phases of a radiolabeled metabolism study, he divides his material into appropriate samples, and extracts each sample with selected solvents to obtain the radioactive materials in soluble form. Typically these extracts consist of low levels (ppm) of carbon-14 labeled metabolites in a complicated mixture of normal natural products from the plant, animal, or soil source. The identity of each metabolite is unknown, and each must be isolated from the natural background and from other labeled metabolites in sufficient quantity and in adequate purity for identification studies, usually by mass spectrometry. The situation is rather like looking for the proverbial "needle in the haystack" when one does not know the size, shape,or composition of the needle, or even how many needles there are in the stack. At this point a separation technique must be selected with certain important requirements in mind. 

Until very recently, metabolic stability screening and metabolite identification (metabolite ID) have been sequential processes that is, the metabolic stability assays are typically performed hrst to identify rapidly metabolized compounds and a follow-up metabolite ID study is performed next, typically at a 10-fold higher substrate concentration to ensure generation of sufficiently high-quality MS/MS information to support structure elucidation. 

While metabolite identification studies provide critical information on drug candidates, these studies have typically been reserved for compounds late in the development phase. These studies are not amenable to high throughput as each compound will give a different metabolic profile, and evaluation of the data can be a lengthy and labor-intensive process. Traditional studies required radio labeled compounds,... 

Metabolism studies play an important role in dmg discovery and development. The advent of combinatorial dmg synthesis has increased the need for a fast assessment of drag metabolism [1], LC-MS is an important tool in the identification of drag metabolites [2-4], The soft ionization conditions in electrospray ionization (ESI) or atmospheric-pressure chemical ionization (APCI) facilitate the detection of metabolism in biological samples. Many common biotransformation, e.g., oxidation, hydroxy lation, hydrolysis, and reduction, can be detected from just the knowledge of the molecular mass of the metabolites. Further confirmation and/or stractnre elncidation of metabolites is possible using the variety of MS-MS platforms. 

A variety of MS-MS instruments are available for metabolite identification. So far, results from triple-quadrapole and ion-trap instrument were discussed. The performance of these instraments was evaluated by Gangl et al. [13] in the study on ritonavir metabolism (Ch. 10.3.3). The ion trap shows better performance in full-spectrum MS and MS-MS, which is often necessary in metabolite identification studies. However, a fundamental limitation of the ion trap in full-spectrum MS-MS operation may be its inability to trap fragment ions with m/z less than one-third of the precursor m/z. This means that highly informative fragment peaks at the low-w/z end of spectrum are missed. For this reason, pseudo-MS on a triple quadrupole was preferred over MS on an ion trap for further stractme elucidation. 

TOF and Q-TOF instraments are freqnently applied in metabolism studies. The identity of the epothilone B metabolites fonnd by precnrsor-ion analysis (Figure 10.5, Ch. 10.4.2) was confirmed using accnrate-mass determination on a LC-TOF-MS instmment [29]. Some other examples are the characterization of metabolites of moclobemide and remikiren [32], the identification of ketobemidone Phase-I and Phase-II metabolites [33], and the identification of in vitro metabolites of ethoxidine [34]. The nse of a five-channel multiplexed ESI interface (four chaimels for parallel LC-MS and one channel for lock-mass componnd infusion) on a Q-TOF instrument was recently described to speed np metabolite identification and to enhance the efficient nse of the costly instrument [35]. 

Analysis of In Vitro Metabolism Reactions by LC/MS. An increasingly useful approach in metabolism studies involves a preliminary in vitro metabolism study using liver or kidney microsomal preparations (9,10). When combined with LC/MS, such a preliminary study can rapidly provide information about potential metabolites expected from subsequent in vivo studies. A flow chart of a typical experimental procedure for the microsomal reaction with mass spectral identification is shown in Figure 4. The herbicide substrates are generally labeled with both 13C and 14C, which allows monitoring of the reaction by radioactivity detection, and facilitates metabolite identification based on characteristic doublet ions in the mass spectra. The entire procedure can be completed in several hours on a microgram scale, generating a survey of potential metabolites. 

It is not uncommon in dmg development programs for specific toxicities to be uncovered. In most cases, additional studies are then carried out that will attempt to elucidate additional information with regard to the mechanism of the effect. For example, the identification of a non-specific behavioral effect (e.g. tremors and/or convulsions) may trigger the performance of a neurotoxicity study, which includes an exhaustive evaluation of the potential effects on the central and the peripheral nervous tissues. The identification of an effect on reproduction may warrant the performance of detailed studies to identify the specific mechanism or phase of the reproductive cycle that is affected. In-depth metabolic studies may prove that the effect is related to a metabolite in animals that has no relevance to man, and prevent the abandonment of an otherwise promising dmg. It is rare that a dmg development program does not involve some type of special study. 

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