Specific metabolites, analysis

Griebler C, M Safinowski, A Vieth, HH Richnow, RU Meckenstock (2004) Combined application of stable carbon isotope analysis and specific metabolites determination for assessing in situ degradation of aromatic hydrocarbons in a tar oil-contaminated aquifer. Environ Sci Technol 38 617-631. 

Some of the motivations to pursue spectral simulation in a clinical MRS setting include providing metabolite prior information for use in parametric spectral analysis procedures, pulse sequence parameter optimization for observation of specific metabolite structures and shortening times for pulse sequence development. This section will describe, in some detail, examples of each with particular regard for the design and level of prior information inclusion of each simulation and the clinical use of the results. 

It has been shown [34,35] that the prevention of intercellular communication can lead to synchronisation of whole cultures, thus facilitating the production of cell-cycle specific metabolites of pharmaceutical importance. Perhaps a similar approach could be used in pharmacological analysis of drugs and their effects on individual cells rather than on whole tissue. 

Although techniques such as NMR and IR spectroscopy have found some utility in metabolite analysis, the most common approach has been to draw upon the versatility, speed, and high degree of specificity and sensitivity inherent in tandem MS. 9 In the case of complex samples, this specificity and sensitivity can be enhanced by interfacing the mass spectrometer to some form of high-resolution chromatography such as GC, nano-LC, or CE. 

Quantitative metabolomics, on the other hand, can be described as a targeted approach focused on the analysis of specific metabolite species. In this method, multivariate statistical analysis follows metabolite identification and quantitation. Because of the reliable peak identification and measurement of metabolite integrals, quantitative metabolomics promises greater insights into the dynamics and fluxes of metabolites, as well as robust statistical models for distinguishing classes with better classification accuracy. A major requirement for quantitative metabolomics is good-quality spectral analysis to provide reliable peak assignments and metabolite identification. 

State of the art PCB exposvure assessment utilizing human serum, milk, and/or tissues should not only include congener specific PCB analysis, but analyze persistent PCB metabolites. Since certain hydroxylated and methylsulfonyl (MeS02) PCB metabolites are present in some cases at levels higher than their respective parent compounds, it is necessary to further investigate the potential biological and/or toxicological activities of these persistent metabolites. 

The methods of analysis for many of the specific metabolites have been reviewed by Aprea et al. (2002). Most of the specific metabolites arc conjugate glucuromides or sulfates thus, enzyme or acid hydrolysis is required to liberate the metabolites. Most of the methods for the analysis of specific metabolites use GC/ECD or GC/MS. The urine sample (after hydrolysis) is extracted with a solvent, such as eiher or toluene, The filtration of urine is performed through a Sep-Pak C18 cartridge followed by extraction of Ihe eluate with solvents. Several derivatizing reagents such a.s BSA lMO-bis(trimethylsilyl)acetamide], I-chloro-3-iodopentane, or MTBSTFA [A -(tertbutyldiniethylsilyl)-Af-methyl trifluo-roacetamide) have been used to convert the metabolites into volatile compounds. 

Tomaszewaska and Hebert (2003) reported a method for the analysis of ( , -dimethyl hydrogen phosphorothioate (0,5-DMPT) in urine. 0,5-DMFT is a specific metabolite of methamidophos, The urine sample was extracted with a CIS column, and the sample was lyophilized at low temperature to prevent loss of highly volatile and thermally unstable metabolite (0,5-DMPT). The lyophilized residue was derivattzed using MTBSTFA and 1% terl-butyl-dimethylchlorosilane in acetonitrile. After filtration, the derivatized product was analyzed with GC/FPD (pulse FPD) in the phosphorus mode. The limit of detection for the method is reported as 0.004 ppm, with a mean recovery of 108%. 

Prior to P450 reaction phenotyping or inhibition experiments, it is important to determine enzyme kinetic parameters such as Km and Umax for the formation of selected metabolites that are subjected to quantitative analysis by LC-MS. For example, -warfarin is catalyzed by CYP2C9 to a specific metabolite, 7-hydroxy-5 -warfarin (Fig. 15.13). Thus, a CYP2C9 inhibition assay is developed based on the reaction. In the assay, Y-warfarin is incubated with HLM in the presence of a test compound, followed by quantification of 7-hydroxy-5 -warfarin by LC—MS (Zhang et ah, 2001). To set up this assay in our lab, enzyme kinetics for the formation of 7-hydroxy-iS-warfarin in HLM was determined. In this experiment, warfarin was incubated at concentrations from 0 to 250 >M with HLM at optimized conditions. Rates of 7-hydroxy-S -warfarin formation at various substrate concentrations were determined as shown in Figure 15.13a, from which Km and Umax values were calculated. The warfarin assay represented an analytical challenge since the turnover of warfarin in the HLM system was extremely low. To be able to quantitatively determine low concentrations of 7-hydroxy-5 -warfarin in the incubations, a very sensitive LC—MS method that used MRM with a 4000 QTRAP has been developed (Fig. 15.13a). 

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