Gas chromatography-mass spectrometr

Gas chromatography-mass spectrometryGC-MS provides robust analyte separation, reproducible retention times, and peak widths of seconds. GC requires volatile metabolites and gas-phase chemistry; however, most biological metabolites are not volatile by their nature and must be chemically derivatized, which requires dried metabolite extracts, treatment of samples at high temperature and exposure of metabolites to harsh solution conditions. As a result, GC-MS is not the method of choice for chemically-labile, thermally unstable, and easily degradable metabolites (e.g. acyl-carnitines, acyl-CoAs, intact lipid species, phosphorylated intermediates, α-keto-acids). Despite this drawback, GC-MS is used routinely for profiling of organic/amino acids in tissues and bodily fluids32–34 as well as breakdown products of therapeutic and drugs of abuse.35, 36 GC-MS based metabolomics has also been used for non-esterified and esterified fatty acid analyses in plasma samples,37, 38 although GC-MS is not the preferred approach for wide-scale lipidomics.Of GC platforms, the single quadrupole GC-MS is currently the instrument package most often applied to both targeted and nontargeted metabolomics. Nontargeted applications rely on an established in-house retention time/spectral library, since accurate mass determination is not possible.33, 39, 40 For GC-MS with electron impact (EI) ionization, identification of unknown metabolites is aided by a standardized electron energy (i.e. 70eV) for spectral reporting. The standardized electron energy allows easy method transfer between instruments and laboratories, which has resulted in the establishment of large repositories of EI-spectra for unknown metabolite identification, e.g. the NIST database.41Advancements in GC-Q-TOF and GC-Q-Orbitrap technologies have made high-resolution nontargeted metabolomics studies a possibility for GC-MS systems. Coupling the separation capability of GC with accurate mass determination and MS/MS spectral data can enhance discovery-based metabolomics. However, the general pitfalls of GC-MS techniques, including lack of suitability for labile compounds and limited metabolite coverage are still relevant to the more advanced systems.Liquid chromatography-mass spectrometryDue to improvements in high-performance LC (HPLC) and ultra-high performance LC (UHPLC) technology and a variety of column chemistries, LC is now able to separate a wide-range of metabolites, making it very suitable for high-throughput, comprehensive metabolomic analyses of bodily fluids and tissue samples. The commonly used stationary phase chemistries include reverse phase (RP), hydrophilic interaction chromatrography (HILIC), and porous graphitized carbon (PGC). Reverse phase and HILIC are orthogonal chromatographic approaches. Reverse phase chromatography is best applied for nonpolar metabolites analysis, such as glycerolipids, phospholipids, fatty acids, acyl-carnitines and medium/long chain acyl-CoAs. HILIC approaches are more suitable for polar metabolites, including sugar monomers, nucleotides/nucleosides, phosphate compounds, and organic acids. The HILIC technique provides limited separation for very polar molecules, and hence PGC has been applied for separation of such compounds, for example glycans, oligosaccharides and sugar phosphates.42, 43 As the field continues to advance, two-dimensional chromatography approaches may provide a means to interrogate metabolites of wide-ranging polarity in a single method.44, 45 Despite the versatile column chemistries available, an ongoing challenge for LC approaches is their less reproducible chromatography compared to GC methods. Continued improvement of column stationary phases and the flexibility of mobile phase and ion pairing reagent selection will likely narrow this gap in the future.Unlike GC-MS, LC-MS methods do not require volatile analytes and often require very little sample preparation post-extraction. Another benefit of LC-MS techniques is mimimal in-source fragmentation compared to EI-based GC/MS. Thus, the metabolite’s chemical backbone is preserved, which aids in the molecular weight and atomic composition determination of an unknown metabolite. Further fragmentation for MS/MS spectral determination can be performed, if desired. In addition, multiple polarity modes for the LC-MS ion source provides coverage of both positively and negatively charged metabolites.For targeted analyses, LC-MS instruments are typically triple quadrupole due to the high sensitivity and selectivity of monitoring specific ion transitions. For nontargeted and spectral library-assisted applications, TOF and Orbitrap platforms are prevalent due to the instruments’ high-resolution, accurate mass determination and the rich spectral data set that can be collected across a wide m/z range, e.g. the cardiac lipidome.46 Nontargeted TOF and Orbitrap approaches rely on spectral library tools for unknown metabolite identification, but this