# Defining Blood Plasma and Serum Metabolome by GC-MS
## 4 Approaches of Metabolome Exploration
### Tandem of Chromatography and Mass Spectrometry  
Separation-based MS techniques take advantage of both \"ingredients.\" Due to the characteristic pattern of the parent and daughter ions and advanced MS detectors, the specificity of the analysis is ensured without loss of sensitivity [63]. Before MS detection, the procedure of chromatography is carried out to downgrade the complexity of the sample. The sample components are moving through the stationary phase in the flow of the mobile phase. As a result, the complexity of the analyzed mixture entering the mass spectrometer decreases due to the distribution of substances between the mobile and stationary phases of the chromatographic column by their solubility, polarity, and volatility. As the name suggests, liquid chromatography uses liquids as a mobile phase. An analyte solubility and an affinity for a sorbent in a chromatographic column are decisive physical-chemical separation parameters. Advances in LC-MS in comprehensive coverage of the metabolome are reported in more than 1000 publications (Figure 2). LC-MS is a universal platform suitable for analyzing the majority of plasma and serum metabolites. GC-MS and LC-MS techniques can be successfully used together to provide comprehensive metabolome profiling [64]. In gas chromatography, the mixture of metabolites is carried by gas, and the compounds are divided in the column space by their volatility. GC  
Figure 2: Venn diagram and timelines comprising the number of scientific publications on blood metabolome studies via LC-MS, GC-MS, and NMR up to date. The queries “LC-MS blood metabolome”, “GC-MS blood metabolome” and “NMR blood metabolome” were addressed to the PubMed repository. The leader in the number of publications is the most mature NMR technology, followed by gas and liquid chromatography in tandem with MS. Due to the different applicability of NMR, LC-MS, GC-MS for various classes of metabolites, the combination of two or even three analytical platforms has been used in 200+ metabolomics projects. The synergy is especially noticeable between GC-MS and LC-MS, used together approximately in 7% of mass spectrometry-based experiments. MS has long been used for metabolome profiling due to its separation capacity, sensitivity, and selectivity [12]. Reproducible molecular fragmentation patterns of GC-MS make it one of the most reliable tools for exploring metabolomics. However, the application of GC-MS is limited to volatile compounds, and a large portion of small molecular metabolites are within the range of GC separation. Due to its excellent ability to separate complex chemical mixtures, two-dimensional gas chromatography is gaining popularity in metabolomics [65]. Series connection of two chromatography columns with different polarities allows separating compounds that co-elute from the first column. This technique becoming the new frontier of GC-MS-based metabolomics is used to characterize several classes of chemical compounds for panoramic and targeted studies of various biological samples, including blood plasma and serum (see Section 5.2.5 \"Multidimensional chromatography\" for further discussion). Chemical derivatization before GC significantly improves the volatility and thermal stability of polar and non-volatile metabolites. Therefore, the content of chemical compounds that GC-MS can analyze is expanding [66]. We will build further narratives based on GC-MS technology, which offers a compelling balance of sensitivity (more sensitive than NMR) and reliability (more robust than LC-MS). The monumental research by [PERSON] and co-authors highlighted metabolites routinely found in liquid blood components [44]. The study showed that out of the total pool of 4229 metabolites (including lipids) known at that time, NMR measured 1.2%, GC-MS 2.1%, MS/MS with electrospray ionization (lipids profiling) measured 2.3%, direct flow injection -mass spectrometry (lipids profiling) measured of 79.9%, and tandem MS with direct flow injection was able to access 3.3% of the total serum metabolome.