## 2 Main Metabolomics Methodologies
### Detection Techniques  
MS and NMR are the most widely used analytical techniques in metabolomics [45]. MS provides a mix of rapid, sensitive and selective qualitative and quantitative analyses with good skill to identify metabolites [46]. Mass spectrometers act by ion formation, which entails the separation of ions according to their mass-to-charge (m/z) ratio, and the detection of separated ions [9; 47]. MS is rapidly gaining interest in metabolomics, though it is more often associated with other techniques, such as chromatography [48]. In fact, MS, which was widely developed over the last decades, holds a distinguished position in the field of determination, quantification and separation of mixtures of compounds. Recent advances in MS-based metabolomics produced the potential to quantify the levels of hundreds of metabolites that are intermediate or final products of cellular processes [49]. Thanks to its high sensitivity, selectivity and wide range of covered metabolites, MS has become the most widely used technique in metabolomics studies [2]. Another advantage of MS is derived from its reproducible quantitative analysis and its power to analyse samples with extremely high molecular complexity [50; 51]. The objectives of developing MS for metabolomics range from the structural characterization of important metabolites to the detection of metabolite variations [52]. Moreover, MS can be applied to analyse biological samples, either by direct-injection or following chromatographic separations [3; 53; 54]. Recent progress and improvements in mass accuracy and precision have drastically increased the range of metabolites that can be analysed by MS, and have enhanced the accuracy of compound identification [48]. Usually, both of the above mentioned methods are used in metabolomics studies in order to identify and quantify all metabolites of the biological system under analysis [55,56]. Additionally, NMR provides users a vision of intact molecules at the atomic level and enables the viewing not only of 1-H atoms, but also many other kinds of atoms (\({}^{13}\)C, \({}^{15}\)N) or biologically reactive groups, including phosphate atoms (\({}^{31}\)P) [57, 58, 59, 60]. In addition, this technique needs minimal or no sample preparation and it is non-destructive, unlike MS [9,61]. [PERSON] was a pioneer in the use of NMR spectroscopy in the toxicological field [62]. Therefore, it is suitable for studies of cell extracts, and for cell cultures and tissues in vitro or in vivo. Among other advantages, it is highly reproducable and has the ability to identify unknown metabolites [63,64]. Our metabolomics work exclusively used \({}^{1}\)H-NMR for the analyses of marine samples, but other nuclides (i.e., \({}^{13}\)C, \({}^{31}\)P, \({}^{15}\)N, \({}^{19}\)F, and \({}^{2}\)H) may provide more information about this set of metabolites. The main limits of this technique concern the spectral resolution and sensitivity, which can be improved by high intensity magnetic field [57,65]. Furthermore, as compared to LC-MS and GC-MS, \({}^{1}\)H-NMR spectroscopy is almost 100 times less sensitive. This means that a typical \({}^{1}\)H-NMR-based metabolomics study only gives back information on 50-200 identified metabolites with concentrations >1 uM, while a typical LC-MS study can render information on more than 1000 identified metabolites with concentrations of >10 to 100 nM [57].