Metabolites screening procedure

FIGURE 7.6 Targeted metabolite screening procedure, showing a flowchart that could be followed to determine whether to report a potential metabolite observed in a sample assay. (Source Adapted from Wainhaus, S. et al., Am. Drug Dis., 2007, 2, 6. With permission.)... 

A difficulty in finding inborn errors of pyrimidine metabolism by metabolite screening procedures is the absence of a typical end-product, like uric acid is in the purine metabolism. Moreover, pyrimidine metabolism is not easily accessible for simple chromatographic screening techniques. Nevertheless, with more complicated methods we are able to evaluate patterns of urinary pyrimidine bases and nucleosides. With routine gas-liquid chromatography (GLC) as is used for urinary organic acid analysis strongly increased uracil and thymine concentrations can be discovered. 

One of the best tools for metabolite profiling is the hybrid QTRAP MS/MS system (Applied Biosystems).119-121 While the hybrid QTRAP MS/MS was initially considered a premier tool for metabolite identification, it has more recently been seen as a tool for quantitation and metabolite profiling. Li et al.122 described the use of a hybrid QTRAP MS/MS system for discovery PK assays plus metabolite profiling in the same analytical procedure. Because QTRAP MS/MS may be used as a triple quadrupole MS system, it can be used as part of a quantitative HPLC/MS/MS system. Because QTRAP MS/MS also has linear ion trap capabilities, it can be used for metabolite screening and characterization—essentially it combines the capabilities of a triple quadrupole mass spectrometer and a linear ion trap mass spectrometer. 

H. H. Maurer, Identification and differentiation of barbiturates, other sedative-hypnotics and their metabolites in urine integrated in a general screening procedure using computerized gas chroma-tography-mass spectrometry, J. Chromatogr., 530 307 (1990). 

Maurer HH, Bickeboeller-Friedrich J (2000) Screening procedure for detection of antidepressants of the selective serotonin reuptake inhibitor type and their metabolites in urine as part of a modified systematic toxicological analysis procedure using gas chromatography-mass spectrometry. J Anal Toxicol 24 340-347... 

The process for the identification of trapped reactive intermediates can be a slow, painstaking task. Strategies to screen for the occurrence of these reactive metabolites to increase the attrition of potentially toxic drugs can significantly reduce the cost of the development. Much of what follows in this chapter is focused on the development of such screening procedures. 

Another use of in vitro systems is to identify metabolites that will be produced in vivo. Several authors have described procedures for the identification of metabolites produced by in vitro systems [16-22]. Tiller and Romanyshyn [18] describe the use of LC-MS/MS procedures along with metabolite screening in vitro as part of a lead optimization process. Hop et al. [17] discuss the use of fast-gradient procedures specifically for in vitro metabolite identification as part of the drug-discovery process their article suggests that rapid metabolite screening data can be obtained from HPLC gradients that last for as little as 2 minutes. 

Retention times and MRMs for internal standards anabolic agents, beta-blockers, p2-agonists, and metabolites in the LC-MS/MS screening procedure... 

Retention times and MRMs (precursor/product ion transitions) for the internal standards, anabolic agents and metabolites, beta-blockers, (32-agonists, and glucocorticoids are shown in Tables 2 and 3. Depending on the compound, 1-3 product ions are monitored in MRM mode for the screening procedure. 

We describe here our LC-MS/MS general unknown screening procedure, based on a nonspecific solid-phase extraction, reverse-phase HPLC, full-scan detection and full-scan identification, which has been proved to elicit the identification of drugs from many different therapeutic classes and some of their metabolites (11). 

Picard N, Dridi D, Sauvage FL, Boughattas NA, Marquet P (2009) General unknown screening procedure for the characterization of human drug metabolites. Application to loratadine phase I metabolism. J Sep Sci 32 2209-2217... 

Beyer J, Peters F, Mamer HH(2005) Screening procedure for detection of stimulant laxatives and/or their metabolites in human urine using gas chromatography-mass spectrometry after enzymatic cleavage of conjugates and extractive methylation. Ther Drug Monit 27 151-157... 

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