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Untargeted metabolomics is a useful approach for the simultaneous analysis of a vast array of compounds from a single extract. Metabolomic profiling is the relative multi-parallel quantification of a mixture of low molecular weight compounds, or classes of compounds, and it is most often performed by using ultra performance liquid chromatography (UPLC) coupled with mass spectrometry (MS). Being an extension of the classical targeted methods, this approach allows a broader view of the main biochemical events within a particular sample. This chapter exemplifies and provides experimental details on the basic steps to perform a non-targeted metabolomic analysis on plant leaf tissues: sample collection and homogenization, extraction of metabolites, raw data acquisition, and processing into formats for data mining and informatics. In particular, the approach was applied to two spring wheat varieties with different level of drought tolerance (Kavir, drought-resistant; Bahar, drought-sensitive) developed by the CIMMYT (International Center for the Improvement of Corn and Wheat).

In a systemic viewpoint, relevant biological information on living systems can be grasped from the study of small, albeit pivotal molecules which constitute the fundamental bricks of metabolic pathways. This holds true for liver which plays, among its unique functions, a key role in metabolism. The nonbiased analysis of all this small-molecule complement in its entirety is known as metabolomics. However, no practical approach currently exists to investigate all metabolic species simultaneously without including a technical bias towards acidic or basic compounds, especially when performing mass spectrometry-based investigations. Technical aspects of rapid resolution reversed phase HPLC online with mass spectrometry are hereby described. Such an approach allows to discriminate and quantify a wide array of metabolites with extreme specificity and sensitivity, thus enabling to perform complex investigations even on extremely low quantities of biological material. The advantages also include the possibility to perform targeted investigations on a single (or a handful of) metabolite(s) simoultaneously through single (multiple) reaction monitoring, which further improves the dynamic range of concentrations to be monitored.Such an approach has already proven to represent a valid tool in the direct (on the liver) or indirect (on human red blood cell metabolism which is hereby presented as a representative model, but also on blood plasma or other biological fluids) assessment of metabolic poise modulation and pharmacokinetics for drug development.