Metabolite Identification and Quantitation

The final screen in the drug discovery process is the identification and quantitative estimation of metabolites a description of the metabolite identification strategies available (Clarke 2001) is still valid although subsequent developments in mass spectrometric technology may have changed the emphasis somewhat. There is now considerable accumulated experience in the nature of metabolites likely to be observed for candidate pharmaceuticals in mammals this information is summarized in Table 9.4. 

Although the very low duty cycle of a triple quadrupole instrument in full spectral scan mode severely limits the sensitivity, this design is in principle ideal for finding 

Results of the rapid oral pharmacokinetic screen in the rat (10 mg/kg n = 2 rats Pooled each hour) 

Samples below the limit of quantitation (100 ng/ml) are reported as zero 

However, metabolite identification has more recently been significantly aided by introduction of the new QqQtrap instrument (Section 6.4.6) that combines the capabilities of a triple quadrupole with those of a linear ion 

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Clinical Trials Human PK and metabolite Identification and quantitation Yes Medium to high... 

If history is any guide, then we foresee great potential for growth in the field of carotenoid analysis in the coming years. Sample preparation methods that quickly and effectively break down and remove sample matrix while preserving carotenoids intact will improve the accuracy of both parent carotenoid and carotenoid metabolite identification and quantitation. 

A recent innovation is the commercial availabihty of linear two-dimensional ion traps [54], The linear ion trap (LIT) is found to be less prone to space-charging effects, enabling a higher number of ions to be accumulated, which results in enhanced sensitivity. In the conunercial instrument, the linear ion trap is the third quadrapole in a triple-quadrapole arrangement, i.e., (J-q o -LlT. In that setup, it can be used to accumulate product ions generated by CID in a LINAC colhsion cell, providing enhanced sensitivity and lack of low-mass cut-off. Further stages of MS-MS can be performed in the linear ion-trap, which then has similar features as the three-dimensional ion-trap. Early reports described the application of the hnear ion trap in metabolite identification and quantitative bioanalysis [55-56],... 

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. 

Hop, C.E., Use of nano-electrospray for metabolite identification and quantitative absorption, distribution, metabolism and excretion studies, Cum Drug Metab., 7(5), 557, 2006. 

This LC-MS experimental approaches used in NEF metabolite identification are also routinely employed for in vitro metabolism comparisons across species, in which liver microsomes or hepatocytes from humans and animal species are used. In addition, liver microsomal incubations followed by metabolite identification and quantitative estimation using liquid chromatography-ultraviolet/ mass spectrometry (LC-UV/MS) is an approach commonly taken to determine metabolic soft spots, where a major metabolic reaction takes place (Table 6.10). Use of UV detection allows for quantitative analysis of major metabolites in the absence of chemical standards, with the assumption that the metabolic reaction did not disturb the molecule s UV chromophore. 

Validation of true extraction efficiency normally requires the identification and quantitation of field-applied radiolabeled analyte(s), including resulting metabolites and all other degradation products. The manufacturer of a new pesticide has to perform such experiments and is able to determine the extraction efficiency of aged residues. Without any identification of residue components the calculation of the ratio between extracted radioactivity and total radioactivity inside the sample before extraction gives a first impression of the extraction efficiency of solvents. At best, this ratio is nearly 1 (i.e., a traceability of about 100%) and no further information is required. Such an efficient extraction solvent may serve as a reference solvent for any comparison with other extraction procedures. 

Hearn W., Pablo J., Hime G., Mash D. Identification and quantitation of ibogaine and an O-demeth-ylated metabolite in brain and biological fluids using gas chromatography mass spectrometry. J. Anal. Toxicol. 19 427, 1995. 

Tweedie DJ, Burke MD. (1987). Metabolism of the beta-carbolines, harmine and harmol, by liver microsomes from phenobarbitone- or 3-methylcholanthrenetreated mice. Identification and quantitation of two novel harmine metabolites. Drug Metab Dispos. 15(1) 74-81. 

In many instances, such as in studies of metabolism or degradation where the sensitivity is not so important, TLC and radioscanning provide a quick and simple technique for the identification and quantitation of radiolabeled metabolic products. An example of a radiochromatogram scan is shown in Fig. 2.13 for the analysis of some metabolites of the insecticide ethyl parathion in a microsomal enzyme preparation from rat liver [SI]. 

Gamache, P., Solomon, M., Acworth, I. N., and Cole, R. (2004a). Rapid on-line electrochemical synthesis of pharmaceutical degradants and metabolites for profibng, identification and quantitation. Poster presented at Pittcon, Chicago, IL. 

A9-THC is the major psychoactive constituent of Cannabis. Its detection and quantitation pose a difficult analytical problem because of its low concentration in biological fluids. Much work has been done on the identification and quantitation of A9-THC, its metabolites and cannabinoids by standard methods such as radio-immunoassay (1,2), gas chromatography, either alone (3-6) or coupled with mass spectrometry (7,8) and fluorometry (9-15). All these methods endeavor to satisfy two major criteria specificity and sensitivity. 

Bartczak A, Kline SA, Yu R, et al. 1994. Evaluation of assays for the identification and quantitation of muconic acid, a benzene metabolite in human urine. J Toxicol Environ Health 42 245-258. 

Moller, M. R., Fey, P., and Rimbach, S., Identification and quantitation of cocaine and its metabolites, benzoylecgonine and ecgoiune methyl ester, in hair of Bolivian coca chewers by gas chromatogra-phy/mass spectrometry, J.Anal. Toxicol, 16, 291,1992. 

Chromatographic techniques are the most powerful tools for the identification and quantitation of drugs in hair due to their separation ability and their detection sensitivity. TLC, HPLC, and GC were used in several cases for the detection of various drugs and/or drug metabolites. However, the majority of detection procedures for individual compounds are based on GC/MS. ° ... 

There are a number of reviews of the application of various forms of LC-MS to the detection and identification and quantitation of specific analytes, whether they be natural products, synthetic drug leads, or metabolites. For example, Korfmacher [ 1 ] provides a good general introduction to LC-MS by describing its basic principles and how it facilitates new drug discovery. Prasain et ai. [2] have reviewed mass... 

The mass spectrometer is the most powerful GLC detector used so far. However, the GLC-MS combination is expensive for routine and repetitive analyses but is invaluable for rapid identification of unknown peaks. It has been widely applied in steroid analysis (B16, H23), identification of drugs (L4) and for the identification and quantitation of unusual metabolites in some inborn errors of metabolism (C23). The high sensitivity and specificity of the GLC-MS combination enables all metabolites in a urine to be unequivocally identified. Inexpensive mass spectrometers, with a limited range of m/e values, are becoming available and could become useful for repeated analyses of similar samples for a limited range of constituents. 

Thin layer chromatographic analysis is also highly applicable to the determination of aromatic organic acids.In human organisms, aromatic acids are synthesized as metabolites in intoxication by toluene, xylene, and ethyl benzene. These compounds are easily absorbed through the skin or respiratory system, and are oxidized to aromatic acids. The separation, identification, and quantitative analyses of aromatic acids are also necessary because they appear as semiproducts of the biosynthesis of aromatic amino acids in plants (phenolic acids), and metabolites of numerous toxic substances, drugs, and catecholamines. Polar adsorbents and polar-... 

Several investigators have suggested that the identification and quantitation of ascorbic acid metabolites will aid in our understanding the metabohc role of ascorbic acid (47,53,54). Ascorbic acid probably functions in more ways than as a hydroxylation cofactor and as a redox... 

NMR spectroscopy is in many respects an ideal analytical technology for metabolic profiling because it is equally sensitive to all metabolites, it can provide quantitative results, direct analysis, metabolite identification, and broad metabolite class coverage. However, NMR spectroscopy gives information only for the abundant metabolites (due to the method s relatively poor... 

Identification and quantitative analyses of 25 compounds in steam distillates of burley tobacco stalk were accomplished. Compounds included twelve C,-C. compounds that were probable fatty acid oxidation products and 13 compounds >C that varied in origin. The latter included oxidation products of fatty acids, a C. prenyl pyrophosphate metabolite, and biodegradation products of carotenoids and chlorophyll. About 1/3 of the distillate mass was accounted for. Burley tobacco stalk headspace volatiles were also studied. When compared to the steam distillate, the headspace contained greater concentrations of sesquiterpenoids but lower concentrations of C, and C aldehydes and alcohols. Volatiles in steam distillates of tobacco stalk were not quantitatively different in a fungal resistant and a fungal sensitive variety of tobacco. Yield comparisons were made of headspace volatiles from tobacco and wheat. 

Chu, I. and Nomeir, A.A., In vitro DMPK screening in drug discovery, role of LC-MS/ MS, identification and quantitation of drugs, metabolites and metabolizing enzymes by LC-MS, in Progress in Pharmaceutical and Biomedical Analysis, Vol. 6, Chowdhury, S.K. Ed., Elsevier, the Netherlands, Chapter 5, 2005. 

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