## 4 Salivary Samples: The Importance of the Correct Collection and Preparation  
The pre-analytical processing of biological samples is a crucial aspect of metabolomic and proteomic research; in fact, it directly impacts the accuracy and reliability of metabolite and protein measurements, ultimately influencing the overall quality of the data. This stage involves various procedures, including sample collection, quenching, extraction, and storage.  
Sample preparation protocols are essential for providing an accurate representation of the metabolic and protein profile of the biological system that is studied [35]. In human metabolomics and proteomics research related to oral diseases, participants are typically advised to refrain from eating, drinking, chewing gum, using oral hygiene products, taking medication, or smoking before sample collection to minimize potential factors that could affect the results [36]. Consequently, incorrect sampling timing and inadequate sample collection or preparation can significantly compromise the accuracy and reliability of the results [37] (Figure 3).  
Another critical factor to consider is metabolic flux, which is the movement of metabolites through a reaction system over time. To ensure that samples remain metabolically inactive after collection, it is essential to prevent biochemical reactions that can lead to rapid metabolite turnover within seconds to minutes [38]. Sample quenching plays an important role in arresting or slowing down these metabolic processes, preserving the metabolic profile during sample handling. This step is typically performed at the moment of collection and before extraction [39]. To minimize potential alterations after sample collection, samples are typically flash-frozen in liquid nitrogen and stored at \(-80\)\({}^{\circ}\)C immediately. If \(-80\)\({}^{\circ}\)C freezers are unavailable at the clinical site, an alternative approach involves freezing samples at \(-25\)\({}^{\circ}\)C or collecting them in sterile containers with a preservative like sodium azide, followed by immediate freezing in liquid nitrogen [40; 41; 42]. Before analysis, saliva samples should be thawed in ice and then centrifuged at low temperatures to remove cell debris. There is a two-step centrifugation protocol. An initial centrifugation at 2600-3000\(\times\)\(g\) for 10-15 min at 4 \({}^{\circ}\)C is performed to remove large debris, epithelial cells, and food particles. This is followed by a higher-speed centrifugation at 10,000-15,000\(\times\)\(g\) for 10-15 min to eliminate residual cellular material and microorganisms. For studies focusing on extracellular vesicles or aiming for maximum sample purity, an optional ultracentrifugation step at \(\geq\)100,000\(\times\)\(g\) for60-90 min may be applied [43]. After centrifugation, metabolite extraction is conducted using solvents that provide broad coverage of both polar and non-polar compounds. Commonly used solvents include methanol: water (80:20 or 70:30, _v/v_) for effective protein precipitation and polar metabolite recovery, or methanol:chloroform: water (2:2:1.8, _v/v/v_) for biphasic extraction of both hydrophilic and lipophilic molecules. Acetonitrile: water mixtures (1:1 or 3:1) are also widely used due to their efficiency in protein removal and compatibility with both MS and NMR platforms [44].  
In an untargeted approach, the extraction process aims to recover the majority of metabolites and proteins from the biological sample while ensuring reproducibility and accuracy, particularly when analyzing a large number of samples. In contrast, a targeted approach tailors the extraction process to the specific metabolites or proteins of interest. Additionally, the extraction method should be simple and efficient to prevent alterations in metabolite or protein composition due to prolonged preparation times [41; 45; 46; 47; 48].