# NMR in Metabolomics: From Conventional Statistics to Machine Learning and Neural Network Approaches
## Appendix A Technical Aspects
In this case, if nuclear spins are totally relaxed and no polarization transfer sequences are applied, the intensity of each acquired proton signal is correlated with the corresponding concentration levels in the molecules, and the area under each peak is directly proportional to the number of \({}^{1}\)H constituting the corresponding residue, giving a real distribution of the individual metabolites in the sample mixture. This quantification is possible without previous calibration, thanks to the large linear dynamic range and signal response that characterize proton NMR spectroscopy. Another important aspect in the analysis of the \({}^{1}\)H NMR spectra is the solvent suppression, and in this way, several protocols can be used. Commonly, the protonated solvent can be replaced with a deuterated one; this procedure can also require the lyophilization of the sample and the subsequent dispersion in the deuterated solvent. When this is not possible, the solvent peak can be suppressed by using proper pulse sequences [144]. Regarding the identification of metabolites constituting a biological matrix, when they have a unique and high reproducible fingerprint at specific conditions (pH, solvent, temperature), the non-target strategy can be adopted [145]. This consists of the employment of multimodal models, which clarify how the NMR fingerprint of each sample and among the groups correlate with each other, providing a static analysis. This strategy is very important to give a first overview about the sample composition; however, it is not sufficient to analyze very complex samples. In the latter case, it is more common to adopt the target strategy, which consists in the comparison of the acquired data with available metabolite databases, such as the Human Metabolome Database, Biological Magnetic Resonance Data Bank, Birmingham Metabolite Library, Bbiorefcode (Bruker Biospin Ltd., Billerica, MA, USA) and Chenomx library (Chenomx Inc., Edmonton, AB, USA) [145]. Figure 11 reports a \({}^{1}\)H NMR spectrum acquired from human serum at 700 MHz: 55 different metabolites were identified and labeled in the recorded spectrum [3]. In particular, each proton signal can be attributed to the different components of the biofluid, thanks to the high sensitivity of \({}^{1}\)H nuclei, its natural abundance and the remarkably narrow line widths, giving a remarkable spectrum resolution. Note that the high intensity of the lactate peak is due to a conversion of the glucose in lactate during the preparation of the sample. To reach a certain assignment of the detected metabolic peaks 1D \({}^{1}\)H NMR is sometimes not sufficient. This is due to the relevant numbers of resonances with an ambiguous assignment, and to a peak overlap of the matrix's components. Thus, bi-dimensional (2D) NMR techniques, which investigate the spin-spin correlation among \({}^{1}\)H-\({}^{1}\)H nuclei or with heteroatoms, such as \({}^{13}\)C, \({}^{15}\)N, \({}^{31}\)P, are adopted. In metabolomic studies, typical 2D NMR techniques are \({}^{1}\)H-\({}^{1}\)H correlated spectroscopy (COSY) and total correlation spectroscopy (TOCSY), \({}^{1}\)H-\({}^{13}\)C heteronuclear single quantum coherence (HSQC) and heteronuclear multiple bond correlation (HMBC). HSQC is a great experiment for metabolites identification, which gives information on the direct connectivity between protons and heteroatoms. In particular, the large chemical shift scale of \({}^{13}\)C helps to solve the tough issue of the overlapped signals in the proton spectrum, and the variety of HSQC experiments can provide different sets of information on the investigated sample. For instance, the potentiality of the HSQC technique was proved in the identification of methyl groups of betaine and trimethylamine-N-oxide (TMAO). The proton resonances of methyl groups in TMAO and betaine organic compounds are both close to \(\delta\) = 3.26 ppm, and thus, the signals are not distinguishable. Instead, the carbon chemical shift of methyl groups in TMAO is assigned at 62.2 ppm, while that of betaine is at 55.8 ppm.