# A Narrative Review of Metabolomic Insights into Olive Oil's Nutritional Value
## 3 Techniques Used for Metabolite Profiling and Evolution  
Metabolite profiling allows for the identification and quantification of metabolites. To prepare samples, the compounds of interest must normally be isolated from the matrix through chromatographic separation, and detection is performed using mass spectrometry (MS) or nuclear magnetic resonance (NMR) spectroscopy [71]. Analysis of the different components of olive oil, particularly VOO, is essential, considering that this field allows for the discernment of potential adulteration with other vegetable oils, characterization of bioactive minor compounds, and detection of metabolites of pesticide residues, thus evaluating the overall safety of olive oil [72]. Metabolite analysis has evolved significantly over the years, from basic techniques such as chromatography to advanced methods such as MS and NMR spectroscopy. Therefore, a timeline for techniques used for phenolic compound analysis can be established. In 1973, the Folin-Ciocalteu method was commonly used to determine the total phenolic content in a sample, but it was modified because a purification step via paper chromatography or thin layer chromatography (TLC) was added (Figure 2) [73]. This method involves the reaction of polyphenols with a redox agent, namely the Folin-Ciocalteu reagent, which leads to the formation of a blue complex that can be accurately measured and quantified using visible light spectroscopy [74]. In the 1980s and the early 1990s, gas-liquid chromatography (GLC) and high-performance liquid chromatography (HPLC) coupled with MS emerged as new approaches for the analysis and separation of these compounds. In general, HPLC methods are more efficient, owing to the utilization of ultraviolet detection and the fact that they do not require chemical or physical modifications of the compounds before quantification. High-resolution gas chromatography (HRGC) was developed in 1987 (Figure 2) [73]. For the unsaponifiable components, different approaches took place in the late 1990s and the early 2000s, depending on whether the compound of interest was a hydrocarbon, caroten, locopherol, linear or triterpene alcohol, 4-methylsterol, sterol, or sterol oxidation product. Nevertheless, a saponification step, followed by TLC, was common to all components and allowed the separation of the different types. GC or HPLC can be used for hydrocarbons, although the latter separates polycyclic aromatic hydrocarbons, which originate from environmental pollution, from other hydrocarbons more efficiently. HPLC is preferred for carotenoids because GC degrades them. Tocopherols were often eluted with other compounds in TLC and were then analyzed as trimethylsilyl derivatives in non-capillary GC columns (cGCs), which have also been used for linear and triterpene alcohols. However, it is difficult to separate them through TLC because of their higher polarity. In fact, cGCs were used for both 4-methylsterols and sterols, although TLC could separate sterols into two bands: one corresponding to 7-sterols and the other corresponding to 5-sterols. Finally, analysis of sterol oxidation products focused on cholesterol, whose separation occurred through cold saponification and enrichment by silica or aminopropyl solid-phase extraction. Among these compounds, cGCs offered better results than HPLC [75]. Currently, metabolomics can be targeted or untargeted. The first involves methods that determine a particular group of metabolites of interest, whereas the second involves measuring the maximum number of metabolites without focusing on any particular group [76]. MS and NMR are the two main types of spectroscopy used for metabolite profiling. Some crucial advantages of MS are its high sensitivity and resolution, which allow the visualization of chemical diversity over a wide dynamic range. Furthermore, it enables the determination of the molecular weight and the structure of unknown compounds.