# Defining Blood Plasma and Serum Metabolome by GC-MS
## 5 Workflow of GC-MS Analysis of Blood Metabolome
### Mass Spectrometry
#### 5.3.1 Ionization  
Mass spectrometers separate ions according to their mass-to-charge ratios (\(m/z\)). The fundamental laws of nature dictate the need to work with ions: the electromagnetic field providing \(m/z\) separation can interact only with charged particles. Thus, the first step required for the mass spectrometric detection of a substance is the conversion of molecules to ions. Electron impact (EI) is the most common ionization method in GC-MS metabolomics. As the name suggests, ionization occurs due to the collision of electrons with gaseous molecules of a substance. The hot filament emits the electrons, which are accelerated by the high voltage towards the ionization chamber. Typically, the electron energy is 70 eV since, at this energy, it is possible to obtain stable mass spectra with a high degree of ionization and fragmentation of the molecule. When bombarded with such a beam of energetic electrons, the gaseous substance loses its electron and becomes a molecular ion M\({}^{+}\). EI is a hard ionization method, and in most cases, due to significant fluctuations in the electric field around the neutral molecule, the original molecular ion is shattered into fragments. It is important to note that this fragmentation is reproducible between different experiments and instrumental solutions. High stability, reproducibility, and specificity of fragmentation spectra have allowed the creation of extensive repositories (FiehnLib GC-MS Library [129], Golm Metabolome Database [138], Mass Bank [139], National Institute of Standards and Technology (NIST) [140], METLIN [141], and several other sources) and re-use the deposited data for spectral identification. In addition to electron ionization, chemical ionization (CI) can be used in GC-MS metabolomics. In CI, new ionized particles are formed when a gaseous molecule interacts not with electrons, as in EI, but with intermediate reactive ions bombarded by electrons. Chemical ionization can be divided into two subtypes, positive (PCI) and negative (NCI). During PCI, the reagent gas (methane, isobutane, ammonia) has a lower affinity for the proton than the analyzed molecules, and the proton is transferred from the reagent gas ions to the analyte molecules forming positively charged ions. In the classic NCI version, the reagent gas contains water due to ionization of which OH-anions are formed. The GC-CI-MS platform made it possible to create a reproducible and sensitive method (up to 10 pg/mL) for the determination of trace amounts of testosterone and nandrolone esters in blood plasma, showing the linearity of the quantitation in the range of 100-2000 pg/mL [142]. PCI was used for the quantitative analysis of dimethylamine [143]. NCI-MS is also used for analyzing endogenous metabolites in human serum [144], for example, for the study of the PPB-derived eicosanoids in human serum [145]. During electron capture negative chemical ionization (ECNCI), the buffer gas slows down the electrons emitted by the heated filament, lowering their energy to \(\sim\)2 eV. This option is well suited for molecules with high electron affinity (e.g., metabolites modified by pentafluoropropionyl [146]). Decelerated electrons are more easily captured by molecules, resulting in the formation of negatively charged ions. Chemical ionization is considered a mild technique because it leads to less fragmentation and a greater chance of encountering an intact parent ion [147]. However, in metabolomics, CI is used less frequently than EI [148]. The lesser popularity may be explained because the metabolomic profile obtained using chemical ionization usually contains fewer identified compounds [149]. For example, in standard NIST SRM 1950 plasma, GC-EI-MS identified 263 metabolites, versus 93 using PCI and 65 using NCI [150]. In general, EI and CI technologies complement each other: EI provides structural information due to comprehensive fragmentation, and CI provides molecular weight information due to the careful preservation of parent ions.