1. Investigación

GC-MS for untargeted metabolomics is a well-established technique. Small molecules and molecules made volatile by derivatization can be measured and those compounds are key players in main biological pathways. This tutorial provides ready-to-use protocols for GC-MS-based metabolomics, using either the well-known low-resolution approach (GC-Q-MS) with nominal mass or the more recent high-resolution approach (GC-QTOF-MS) with accurate mass, discussing their corresponding strengths and limitations. Analytical procedures are covered for different types of biofluids (plasma/serum, bronchoalveolar lavage, urine, amniotic fluid) tissue samples (brain/hippocampus, optic nerve, lung, kidney, liver, pancreas) and samples obtained from cell cultures (adipocytes, macrophages, Leishmania promastigotes, mitochondria, culture media). Together with the sample preparation and data acquisition, data processing strategies are described specially focused on Agilent equipments, including deconvolution software and database annotation using spectral libraries. Manual curation strategies and quality control are also deemed. Finally, considerations to obtain a semiquantitative value for the metabolites are also described. As a case study, an illustrative example from one of our experiments at CEMBIO Research Centre, is described and findings discussed.

The metabolome is the complete set of small molecules coming from protein activity (anabolism and catabolism) in living systems. They have a broad range of chemical structures and physico-chemical properties and therefore different analytical methodologies are necessary. Highly polar metabolites, such as sugars and most amino acids are not retained by conventional reversed phase liquid (RP-LC) chromatography columns. Without sufficient retention, coelution may result in identification problems while the detection of compounds by mass spectrometry at low concentrations may also be problematic due to ion suppression. In order to retain compounds based on their hydrophilicity, polar stationary phases and hydrophilic interaction liquid chromatography (HILIC) provide a complementary tool to RP-LC for untargeted comprehensive metabolite fingerprinting. However, robustness of the methods is still limiting their applications. This review focuses on sample pre-treatment, stationary phases, analytical methods and applications for polar compound analysis in biological matrices.