Metabolism study procedure

The activities of enforcement laboratories should not be focused on irrelevant problems. Therefore, a clear definition of the relevant residue is needed. In the crops and food sector, procedures are well established to derive the two residue definitions, one for risk assessment and one for monitoring, from metabolism studies. As far as environmental samples are concerned, there is much potential for improvement. There are no clear criteria as to which metabolites should be included in monitoring and control programs. Additionally, the development of criteria for nonpriority pesticides, e.g., naturally occurring compounds or low-risk products, which can be excluded from monitoring exercises would be helpful for laboratories and evaluators. 

The analytical method to determine carfentrazone-ethyl and the major animal metabolites (C-Cl-PAc and C-Pac) in bovine matrices is similar to the method for crop matrices. The hexane-aqueous partition to separate carfentrazone-ethyl from the acid metabolites can be replaced by a Cig SPE cartridge. After the SPE, use 12 mL of water-acetonitrile (7 3, v/v) to elute the metabolites and then use 12 mL of hexane-ethyl acetate (4 1, v/v) to elute carfentrazone-ethyl after drying the cartridge. Follow the rest of the respective analytical procedures for carfentrazone-ethyl and the acid metabolites described in Sections 6.3 and 6.4. However, no reflux under boiling is necessary for the analysis of acid metabolites based on a goat metabolism study, because no conjugated acid metabolites were detected. Also, since HM-C-Cl-Pac is not analyzed for in the bovine matrices, no acylation is needed in the method. Analyze the metabolites by GC/MS, and monitor the ions at m/z 362 for C-Cl-Pac and 303 for C-PAc. 

The extraction efficiencies using a blender and a shaker were compared and both methods gave similar results. A corn sample treated with radiolabeled carfentrazone-ethyl and collected from a metabolism study was used for comparison. Multiple samples can be extracted simultaneously if extraction is performed by shaking. In addition, since the extraction procedures in the residue study closely followed the extraction scheme in the metabolism study, the resulting extraction efficiencies from both studies were almost identical. 

The same procedure, or modifications of it, was used by Zak et al (80), Talseth (42,82,82,83), and Haegele et al (46) for metabolic studies. Zak et al (80) point out that hydrolysis of conjugates of the drug may cause analytical results on biological samples to be variable, depending on the acid concentration during derivatization, and that selective analysis for unchanged hydralazine and acid-labile metabolites can be carried out by suitable adjustment of the acid concentration. 

The first studies with isolated human hepatocytes concentrated on the characterization of these hepatocytes, as well as on the improvement of the isolation procedure, and the possibilities of culturing these cells [16,19-24]. Thereafter studies were performed to investigate the metabolism of drugs [25-28], in which emphasis was often put on the activity and concentration of cytochrome P450 isoforms. Nowadays, hepatocytes are more generally used in metabolic studies of specific compounds, in order to unravel potential species differences and... 

The development of PET as a research tool and diagnostic procedure has had particularly valuable CNS applications. Metabolic studies using [ F]-2-fluoro-2-dexoy glucose ([ F]-FDG), quantitation of regional dopaminergic function with... 

A relatively complex area to address is procedural SOPs for chemical analyses in metabolism studies. One approach is to address the major operations that are common to the studies, for example characterization of metabolites in soil. The SOP can describe the general process, options available in the process and requirements for acceptance or rejection of data. Study-specific procedures that complement the SOPs can be outlined in detail and retained as part of the study records. These study-specific procedures can be prepared in the form of a work sheet and used for entering original documentation, such as the person who performed the procedure and the date it was performed. 

Different reversed phase [195,239,240], mixed mode (ion exchange and reversed phase) SPE cartridges [173,218] and online SPE column [193, 238] have been also reported for samples preparation and extraction. Some of these assays combined both PP and SPE in order to achieve an extensive sample cleanup [193, 195, 238-240], Likewise SPE, LLE provides cleaner plasma extracts than PP. Nevertheless, LLE procedure does not always provide satisfactory results with regard to extraction recovery and selectivity, especially with polar analytes and particularly in the case of multicomponent analysis such as in drug-metabolism studies, where analytes polarity varies widely. This issue was addressed by Zweigenbaum J and Henion J [235] and extraction solvent optimization, using isoamyl alcohol, to achieve acceptable extraction selectivity and recovery for polar analytes has been discussed. 

Need for micromanipulatory procedure. The metabolic studies require the observation of dynamic changes triggered by microinjection of metabolites. The usefulness of both spectral and topographic operation is critically dependent upon the ability to micromanipulate (microinject) living cells, in order to measure changes in injected cells and their neighbors. 

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. 

Analysis of In Vivo Fecal Metabolites by LC/MS. Besides the analysis of urinary metabolites, animal metabolism studies of course require characterization of fecal metabolic products. We have found that rapid analysis of fecal extracts can also be accomplished by LC/MS, although more extensive cleanup is required. A general procedure for the extraction and LC/MS analysis of rat fecal metabolites is given in Figure 14. This procedure can easily be completed in an afternoon to provide a preliminary indication of metabolite structures. Based on this information the appropriate derivatives can be prepared for additional characterization, if necessary. 

NMR spectroscopy is one of the cornerstones of modern natural products research and is used routinely to monitor compound purity through the isolation procedure and to determine the structure of novel bioactives. NMR also has a long history of use in metabolism studies utilizing proton and phosphorus nuclei in both liquid and solid states and in vivo and in vitro. More recently, NMR has been applied in metabolomics (or metabonomics) for metabolite profiling, quantification, and structure elucidation.4... 

Anesthetics. The use of anesthetics or sedatives for restraint in sampling or cannulation procedures can, as in mammals, cause difficulties when used in conjunction with pharmacokinetic and metabolic studies. Anesthesia with MS-222, one of the most common fish anesthetics, has been shown under certain... 

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