Standardized protocol for unstimulated saliva analysis by ATR-FTIR spectroscopy

Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR) spectroscopy has gained relevance as a non-invasive technique for analyzing the biochemical composition of biofluids such as saliva, with increasing interest in clinical diagnostics. However, spectral quality and repeatability are strongly influenced by sample preparation, saliva type, and drying method. This study aimed to standardize a protocol for the collection, processing, and spectroscopic acquisition of unstimulated saliva. Spectra were obtained from liquid, lyophilized, and air-dried samples of both whole and clarified saliva. Liquid samples exhibited strong water absorption, masking characteristic biomolecular bands. Lyophilization produced macroscopically heterogeneous residues, resulting in variable band intensity and poor repeatability. In contrast, air-drying at room temperature yielded uniform and reproducible spectra, with 3 µL identified as the optimal sample volume. Clarified saliva, obtained by centrifugation, produced spectra with sharper and more consistent biomolecular bands, particularly in the Amide II region, by reducing biological noise from cells and particulate matter. Although signal intensity and SNR values were lower, these changes reflected reduced scattering rather than molecular loss. The Wilcoxon test confirmed significant differences between sample types only for the Amide II region (p < 0.05). Overall, air-dried clarified saliva provides a clean, stable, and reproducible spectral matrix, supporting its suitability for reliable biochemical and diagnostic ATR-FTIR applications. • Air-drying produced more uniform and repeatable saliva spectra than lyophilization. • Significant differences between whole and clarified saliva were observed only in the Amide II region. • Clarification improved spectral definition without altering biochemical composition. • Air-dried clarified saliva provides a cleaner and more stable diagnostic matrix.