Target analysis, metabolite measurement

Prior to the genomics era, most metabolite measurements were performed in targeted approaches such as spectrophotometric assays or HPLC with UV detection. Today, these methods are still the best choice for the analysis of small numbers of metabolites. In spectrophotometric assays, metabolites are first... 

When a targeted list is not used to drive the process, analysis may be conducted to assess all metabolites present. The observed m/z is compared with all possible elemental compositions that could produce the observed value, within constraints imposed by the user as described below. Accuracy and precision in the m/z measurement become critical in limiting the possible compositions to the greatest extent possible, greatly simplifying data reduction (Grange and Sovocool, 2008 Ruan et al., 2008). These techniques are discussed in Chapters 4 and 5. 

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. 

A biomarker of exposure is defined as "an exogenous substance or its metabolite(s) or the product of the interaction between a xenobiotic agent and target molecule or cell that is measured within a compartment of an organism" [1]. A marker of external exposure is simply the amount of a xenobiotic substance to which a person is subjected, whereas a marker of internal exposure is the amount of a substance absorbed into the body. Markers of internal exposure are a more accurate means of estimating exposure than are markers of external exposure and require the analysis of biological samples. 

Modern triple quadrupole instruments can rapidly switch between many SRM transitions within a single acquisition cycle (MRM), enabling the measurement of multiple analytes simultaneously. It is critical to choose a unique molecular ion and product ion pair (the SRM transition ) for specific monitoring of the targeted analyte. An example of liquid chromatography-multiple reaction monitoring (LC-MRM) analysis of midazolam and its two major metabolites, T-hydroxymidazolam and 4-hydroxy midazolam, is shown in Figure 6.2 [18]. 

---