## 5 Salivary Metabolomics: Techniques and Applications  
Salivary metabolomics differs among individuals; for this reason, the most appropriate way to analyze them is through longitudinal studies, instead of cross-sectional studies. The composition of salivary metabolomics reflects the physiological or pathological condition of the individual and depends on the characteristics of the individual's oral microbiome [49]. Moreover, variation in the microbiome influencing the characteristics of salivary metabolomics may reflect a local or systemic disease [50; 51]. Untargeted metabolomics gives the possibility to examine the whole spectrum of metabolites contained in the saliva. In this way, we can find metabolites that would have been discarded previously, but that can be involved in some disease's biochemical process. Due to the vast diversity of metabolites in the metabolome, each with distinct chemical and physical  
Figure 3: Collection and preparation of salivary samples. Sample collection and preparation are crucial steps in salivary analysis. If some step is incorrectly performed, the whole analysis may be damaged. The patient is invited to spit into a test tube an amount of 1.5–2 mL of saliva. The collected sample undergoes a quenching process, that is, the inhibition of all the biochemical reactions by freezing the sample at \(-80\)\({}^{\circ}\)C or \(-25\)\({}^{\circ}\)C, to preserve the real composition of such sample. When it is ready to be analyzed, the sample is centrifuged to remove all cell debris. Centrifugation is followed by metabolite extraction. At the end, the analysis may be conducted by using MS or NMR. properties, relying on a single analytical method for their detection is impractical. Therefore, adopting an approach that integrates multiple analytical techniques is essential to ensure comprehensive metabolite coverage [52]. The analysis of salivary metabolomics is driven by different methods, including MS in conjunction with either HPLC-MS or 2 DGC-MS, and NMR spectroscopy. FTIR spectroscopy analyzes molecular composition using infrared light. Different molecular bonds absorb specific wavelengths; thus, by examining the resulting spectra, it is possible to determine compounds and their concentrations. PAS is a variation of FTIR, and it is particularly effective for gas analysis. However, both techniques share the same limitation: they are highly sensitive to water, requiring sample drying before measurement to ensure accuracy [12; 16; 17] (Table 1) (Figure 3). In 2015, Dame et al. [64] gave a panoramic view of salivary metabolomics by analyzing salivary metabolomics using NMR. In their research, they identified a total of 76 different metabolites, some of which were previously reported as detected but not quantified. The researchers detected the following salivary compounds: short-chain organic acids (acetic, acetoacetic, butyric, citric, formic, glycolic, lactic, propionic, and pyruvate acids); amino acids (l-alanine, d-glutamic acid, l-valine, l-methionine, l-threonine, l-leucine, l-tyrosine, and l-phenylalanine); alcohols (e.g., ethanol), amines (e.g., ethanolamine, dimethylamine, and methylamine); sugars (e.g., d-glucose); and pharmaceutical adjuvants (e.g., propylene glycol). It was reported that the most representative compounds were short-chain organic acids, with acetic acid being the most abundant. The scientists of the study also reported higher rates of concentrations of acetic acid (6.8 \(\pm\) 4.3 mM) and succinic acid (0.125 \(\pm\) 0.181 mM) than previously reported in the literature [65] (Table 2) (Figure 4).