Saliva has garnered a lot of interest as a non-invasive, easy to collect, and biochemical rich sample for Attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) based disease diagnosis. Although large number of studies have explored its potential, the preparation methods used differ greatly. For large scale clinical studies to aid translation into clinics, collection/processing methodology needs to be standardized. Therefore, in this study, we have explored different saliva collection (spitting-method A/cotton soaking-method B) and processing protocols (unprepared-TS, supernatant from centrifugation-CS, and drying-C) to find which gives the best ATR-FTIR signals. Analysis showed highest proteins, carbohydrates, amino acids, and nucleic acid + proteins/lipids in BTS, BCS, ACS, and BC, respectively. Notably, only BC shows 1377 cm-1 nucleic acid band is also uniquely identified in multivariate analysis. We conclude that collection-processing protocol should be based on biochemical that best gives a differential diagnosis.
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