High-performance LC (HPLC), as the most versatile separation method, allows separation of compounds of a wide range of polarity. The coupling of liquid chromatography (LC) to MS (LC-MS) facilitates metabolite identification and quantitation by reducing sample complexity and allowing metabolite separation prior to detection. Among them, QqQ has been considered as a reference tool for absolute quantitation of small molecules due to its sensitivity and specificity using selected reaction monitoring (SRM), which has been applied for quantitation of trace-level metabolites with detection limit of ng/mL in sample matrices (e.g., plasma, serum, or cellular media). This is achieved through ion fragmentation by collision-induced dissociation (CID) in either quadruple-based tandem in-space instruments, or ion-trap-based tandem in-time instruments. Meanwhile, versatile mass analyzers working in tandem or hybrid configuration can further aid metabolite identification by acquiring highly resolved and accurate MS/MS spectra. A single ionization source containing combinations of ESI and APCI or ESI and APPI currently has become the trend in source configuration.
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These ionization approaches can be complementary to ESI for the analysis of non-polar and thermally-stable compounds (e.g., lipids). Similar to ESI, APCI and APPI typically induce little or no fragmentation and are considered robust and relatively tolerant to high buffer concentrations. Among them, ESI is often preferred for profiling “unknown” metabolites, since this “soft” ionization approach forms intact molecular ions and aids initial identification. For example, the availability of various atmospheric pressure ionization (API) methods in both positive and negative modes enables ionization of diverse classes of metabolites. MS offers quantitative analysis of metabolites with high sensitivity and selectivity and potential to identify metabolites. Among them, mass spectrometry (MS), nuclear magnetic resonance (NMR) spectroscopy, and Fourier transform infrared (FT-IR) spectroscopy are the most commonly employed.
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Due to the complexity and dynamic nature of the metabolome, multiple analytical platforms are needed to cover the full spectrum of metabolites. Metabolomics has been applied in various research areas including environmental and biological-stress studies, biomarker discovery, functional genomics and integrative systems biology. Untargeted approach is typically carried out for hypothesis generation, followed by targeted profiling for more confident quantitation of relevant metabolites. The untargeted approach is a global analysis of metabolic changes in response to disease, environmental or genetic perturbations. The targeted approach focuses on the analysis of specific group of metabolites related to certain metabolic pathway or a class of compounds. It also provides a diagnostic aid to diseases.Ĭurrent metabolomics investigations can be categorized as two complementary approaches: targeted and untargeted. It facilitates understanding of the mechanisms of biological and biochemical processes in complex systems.
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Metabolomics is primarily concerned with identification and quantitation of small-molecule metabolites (<1500 Da) in the metabolome.