# Defining Blood Plasma and Serum Metabolome by GC-MS
## 5 Workflow of GC-MS Analysis of Blood Metabolome
### Mass Spectrometry
#### 5.3.2 Mass Analyzers
FT-ICR and Orbitraps provide the unbeaten mass resolution and accuracy of any other mass analyzer, even in routine analyses. In Orbitrap mass analyzers, \(m/z\) ratios are estimated through the frequency of harmonic oscillations of the ions along the electric field axis. This type of trap provides high sensitivity and resolution (resolution up to 240,000 at \(m/z\) 400 [160]). The enormous capabilities of Orbitrap made it a solution-of-choice in proteomic research [161]; however, these mass analyzers are also widely used in metabolomics [150]. The already mentioned study of standard NIST 1950 plasma demonstrated the advantages of Orbitrap: it separated four times more peaks than the Q mass analyzer (41,588 vs. 8850) [162]. Although the Orbitrap acquisition speed is not fast enough for GC\(\times\)GC, this mass analyzer performs effectively even in tandem with one-dimensional GC. Thus, in non-human primate serum, the GC\(\times\)GC-TOF-MS option was able to identify 384 metabolites, while the GC-Orbitrap-MS identified 200 compounds [163]. Ion-cyclotron resonance mass analyzers claim to be the most accurate devices. FT-ICR separates ions by their rotational--cyclotron--frequency in the magnetic field, which is inversely proportional to the \(m/z\). Fourier transformation is used to get and transform signals from these bouncing and rotating ions [164]. The high accuracy of this method makes it attractive in metabolomic studies [165], although it is achieved at the cost of expensive and bulky laboratory solutions. In general, in metabolomics, FT-ICR is used without preliminary chromatographic separation. However, a successful attempt to combine GC and FT-ICR has been made to analyze small molecules in gasoline samples [166]. FT-ICR coupled with high-performance LC was used to profile the endogenous metabolites in plasma of rats with pyrexia treated with different medicines [167]. The main disadvantage of FT-ICR is the slow speed of spectrum acquisition, up to minutes. The number of points over the chromatographic peak may be insufficient when FT-ICR is combined with modern fast chromatography systems [165]. Several metabolomic studies were performed by DI-MS, direct infusion of the sample into the FT-ICR mass spectrometer [168]. This method is suitable for a qualitative and quantitative metabolic analysis of human plasma. DI-FTICR-MS has demonstrated its effectiveness in rapid metabolic profiling. In a study of blood serum from 49 experimental animals, more than 400 metabolites were detected in about one day of analysis, which is much faster than mass spectrometric analysis with preliminary chromatographic separation [169]. Among high-resolution analyzers in metabolomics, TOF mass analyzers have a reputation for reliable and robust devices. TOF devices analyze how long it takes for ions with different \(m/z\) ratios but the same initial kinetic energy and constant accelerating voltage to cover a fixed distance. The smaller \(m/z\) is, the shorter time will be required to fly through a vacuum chamber. Time-of-flight analyzers operate with a high scan rate, which allows obtaining sufficient points across the entire peak for better resolution of coeluting peaks. This makes TOF the only mass analyzer fully compatible with GC\(\times\)GC, which requires a high scan rate [123]. The high resolution of two-dimensional chromatography with a time-of-flight mass analyzer is demonstrated in a study where more than 1000 metabolites were found in blood plasma [134]. Mass analyzers can be combined: so, in addition to the usual sequence of three quadrupole mass analyzers, the various hybrids of mass analyzers are used, e.g., Q-TOF [170] and Q-Orbitrap [171]. Table 2 presents a comparison of the essential characteristics of the most popular mass analyzers used for GC-MS analysis of serum and plasma. Indisputably, for estimation of the performance of a particular mass analyzer, one should consider the analyte and its matrix, the method of preliminary separation, ionization technique, and not only intrinsic characteristics of the device.