# A Narrative Review of Metabolomic Insights into Olive Oil's Nutritional Value
## 3 Techniques Used for Metabolite Profiling and Evolution
However, in NMR, there is no need to preselect the conditions to perform the analysis, which permits the generation of spectra with a high information content. Moreover, this technique allows easy quantification of metabolites, and the NMR chemical shifts are relatively stable, which guarantees that the results are reproducible under consistent experimental conditions (Figure 2) [77]. MS uses a mass spectrometer to measure the masses of different molecules in a sample of interest. This process focuses on specific signals, such as the ion current generated by molecules of a selected and specific mass. The detector in the mass spectrometer senses the relative differences in these signals, manifested as peaks or drops, and calculates them as the response ratios. These ratios are determined by comparing the chromatographic changes with the baseline signal [78]. Several analytical tools can be used for MS-based metabolomics, such as liquid chromatography (LC) coupled with MS (LC-MS), two-dimensional LC coupled with MS (LCxLC-MS), GC coupled with MS (GC-MS), two-dimensional GC coupled with MS (GCxGC-MS), direct infusion with MS (DI-MS), which involves direct introduction of the sample into the mass spectrometer, and capillary electrophoresis coupled with MS (CE-MS) [76]. Figure 2: Schematic representation of the already-used methods for the identification and quantification of olive oil metabolites. Chromatography allows the separation of compounds into two distinct phases: stationary and mobile. Retention of these compounds in the stationary phase results in a lower velocity of movement through the chromatographic system [79]. In LC-MS, the mobile phase is a liquid, as in TLC and capillary electrochromatography (CEC), although TLC uses capillary forces, and CEC uses the electroosmotic flow. The stationary phase in LC-MS can be liquid (liquid-liquid chromatography) or solid (liquid-solid chromatography). In general, liquid chromatography (LC) can be divided into four categories. First, normal-phase LC uses an absorbent material in the stationary phase, which is highly polar and has a relatively nonpolar mobile phase. In reverse-phase LC, the stationary phase is nonpolar, and the mobile phase is polar. Additionally, in ion-exchange LC, the analyte interacts with the stationary phase because of the ionic interactions that result in retention in the column. Finally, size exclusion LC involves separation based on size. Molecules with larger sizes do not penetrate the porous material of the stationary phase and move faster [80]. LC-MS allows the detection and analysis of phenolic compounds and unsaponifiable components and is faster than GC-MS because it does not require derivatization prior to analysis. It is possible to integrate ionization methods that can aid in the simultaneous examination of numerous classes of metabolites. Electrospray ionization (ESI) does not allow additional extraction steps in LC-ESI-MS. Nevertheless, when required, the extraction of metabolites is a fundamental step that constitutes pretreatment and can be of distinct types, including liquid-liquid extraction, liquid-liquid microextraction, ultrasound-assisted extraction, and solid-phase extraction [76]. GC can have a liquid or solid stationary phase that influences the appearance of the system. In a GC with a liquid stationary phase, the system consists of a lengthy capillary covered with a thin layer of a relatively viscous liquid or, alternatively, a polymer with liquid-like properties. A GC with a solid stationary phase uses a capillary featuring a thin porous layer on its walls or columns filled with porous particles [80]. GC-MS allows the separation and analysis of organic compounds, fatty acids, and fatty acid alkyl esters in olive oil, which are either volatile or semi-volatile. To obtain volatile compounds, chemical derivatization can be performed prior to analysis, which increases the thermal stability, sensitivity, volatility, and detector response.