# Defining Blood Plasma and Serum Metabolome by GC-MS
## 5 Workflow of GC-MS Analysis of Blood Metabolome
### Sample Preparation
#### 5.1.3 Extraction  
Extraction releases metabolites from the biological matrix and concentrates them in a smaller volume (Figure 3, stage 3). The breadth of the research problem dictates the choice of extraction methods. Target metabolomics allows to qualitatively and quantitatively characterize a specific, often relatively narrow, group of metabolites [76]. In this case, at the extraction stage, it is required to reduce interference from off-target compounds and increase the completeness of extraction of target groups of metabolites. Panoramic metabolomics is aimed at the broadest possible coverage of a wide variety of chemically diverse metabolites [76]. For such global tasks, an unselective extractant is selected that will effectively extract substances belonging to different chemical classes to ensure adequate depth of metabolite coverage and represent the actual composition of the sample under study. For metabolomic studies of serum and plasma, liquid-phase and solid-phase extraction are used. Liquid extraction of metabolites can be performed by one- or two-phases systems. Single-phase extraction systems are of particular interest since they decrease the complexity of the experimental procedure and allow for the simultaneous deproteinization and extraction of a comprehensive metabolic fraction. In addition, such designs are attractive for studies on a limited amount of available biological material, inadequate for multiple specific protocol protocols suited for different compounds. Biphasic extraction is based on the metabolites transfer from one liquid phase to another immiscible liquid phase in which they are more soluble. The most frequently applied organic extractants include acetonitrile, chloroform, acetone, methanol, ethanol, and mixtures at various ratios. The solvents and their proportions can significantly affect the total number of metabolites extracted. An organized attempt was made to compare various extraction options with each other and select those conditions under which it is possible to extract the largest number of metabolites from the plasma with maximum efficiency: the choice of the extracting agent, its volume, as well as the time and temperature at which the extraction took place, was evaluated [72]. The temperature of the solvent does not affect the peak area at all or shows a slight increase in the case of acetone. A much more critical parameter turned out to be the volume and content of the solvent. Nine parts of methanol:water mixture (8:1, _v/v_[72]), added to one part of a plasma, provide optimal results in terms of completeness, efficiency, and reproducibility of extraction in comparison to other tested solvents (ethanol, acetonitrile, acetone, chloroform). The stability of the procedure for the extraction of low molecular weight compounds from plasma with methanol is also emphasized in a similar scientific work [77]. To increase the range of extractable metabolites (for example, for the reliable recovery of fatty acids), many protocols use complex solvent combinations, such as methanol:chloroform or methanol:chloroform:water [72]. For example, the protocol by [PERSON] uses acetonitrile:isopropanol:water (3:3:2, \(v/v\)) mixture for extraction. The combination of hydrophilic, lipophilic, and medium-polarity solvents demonstrated high analytical precision and comprehensiveness of extracted metabolome [56]. Another solvent mixture of methanol:chloroform (3:1, \(v/v\)) mixture, which, due to nonpolar chloroform, provided better extraction of lipophilic metabolites from the serum sample than methanol alone [78]. The MeOH:MTBE:H\({}_{2}\)O mixture (2:10:3, \(v/v/v\)) was also highly appreciated for its versatility for various chemical classes in comparison with pure methanol and a mixture of methanol:water (3:1, \(v/v\)) with subsequent treatment with a mixture of chloroform:water (3:1, \(v/v\)) [77]. The composition of the extraction mixture and the extraction time are not the only parameters that can be varied to achieve better recovery.