Metabolomics, the comprehensive analysis of metabolites in a biological sample, is a huge analytical challenge due to the physico-chemical diversity of analytes, their concentration ranges, and properties of the biological matrices. Separation techniques greatly enhance the analytical capabilities of mass spectrometry (MS) where accurate annotation is vital for data interpretation; however, metabolite identification is still a major bottleneck in untargeted metabolomics.
Along this presentation, I will discuss different analytical challenges in metabolite identification that we have faced at CEMBIO and how we have approached them using CE-TOF-MS, LC-QTOF-MS, and LC-IM-QTOF-MS.
We have demonstrated the robustness of the in-source fragmentation (ISF) in CE-TOF-MS, which makes possible the annotation of compounds by means of their fragmentation pattern . ISF together with the mechanisms of the major fragmentation reactions observed for 57 amino acid standard compounds allowed us to establish a workflow for targeted extraction of modified amino acids  with high biochemical value which led us to the identification of unknown epi-metabolites.
A more recent technique, ion mobility spectrometry (IMS) allows the separation of molecules on the gas phase based on their spatial configuration. Combined with LC-MS, stands out as an excellent tool to study different isomeric compounds. Oxylipins and esterified oxidized lipids are bioactive compounds that play crucial roles in physio-pathological processes, like infections, cancer, and Alzheimer’s. Their analysis, usually done by LC-MS, is hindered by the presence of multiple isomers which in some cases can be disease-specific. Therefore, its identification could reveal reliable diagnostic markers for the diseases they are involved in. We have used LC-IMS-MS to improve the annotation of this type of compounds both in standards and plasma samples of patients suffering different diseases. This allowed the confirmation of elevated oxidized lipids as well as the identification of new and previously unresolved species.
 M. Mamani-Huanca, A. Gilde la Fuente, A. Otero, A. Gradillas, J. Godzien, C. Barbas, Á. López‐Gonzálvez, J. Chromatogr. A, 1635 (2021) 461758.
 M. Mamani-Huanca, A. Gradillas, A. Gilde la Fuente, Á. López‐Gonzálvez, C. Barbas, Anal. Chem. 92 (7), (2020) 4848–4857.