## 4 Discussion
Our study shows that NIRs can distinguish different concentrations of aqueous biomarker solutions which can be further developed to distinguish patients with OPMD from patients suffering from oral cancer. [PERSON] et al. [47] found using UV-VIS spectrophotometry that serum L-fucose levels are elevated in patients is suffering from oral cancer 13.85 \(\pm\) 4.34 mg/dL (0.1385 \(\pm\) 0.0434 mg/mL) compared to patients suffering from oral leukoplakia 8.95 \(\pm\) 1.92 mg/dL (0.0895 \(\pm\) 0.0192 mg/mL), and healthy controls 5.29 \(\pm\) 2.18 mg/dL (0.0529 \(\pm\) 0.0218 mg/mL). [PERSON] et al. [33] used NIR Raman spectroscopy to characterize salivary biomarkers to discriminate the oral premalignant and malignant conditions. They observed wavelength regions of molecular bonds of major components in the saliva between 800-1800 cm\({}^{-1}\) (5555-12,500 nm) and did a principal component analysis (PCA) together with linear discriminant analysis (LDA). Their sample set was larger and more divergent than in this study, but they concluded that more studies identifying the key biomolecules are required. Our preliminary study used two key salivary metabolomic biomarkers but further studies with larger sample sets from diverse patient groups are required. [PERSON] et al. [40] conducted a Fourier transformation infrared and photoacoustic (FTIR-PA) spectroscopy study using saliva collected from healthy patients whereas our two samples were from OSCC patients. They found three major spectral bands characterizing proteins 1500-1750 cm\({}^{-1}\) (5714-6666 nm), carbohydrates 1050-2000 cm\({}^{-1}\) (5000-9523 nm), and SCN\({}^{-}\) anions. They compared known SCN\({}^{-}\) concentrations to spectral data of saliva samples obtaining a correlation of r > 0.990 for transmission and r = 0.967 for PA mode. Similarly, further studies comparing L-fucose and L-proline solutions of known concentrations against NIR spectra of larger sets of saliva collected from healthy donors and patients suffering from oral malignancies could produce a statistically significant correlation. One of the advantages of our study was the small sample volume required to measure a NIR absorbance spectrum. In the NMR spectroscopy study by [PERSON] et al. [45], the sample volume size was 0.5 mL. In our study, we found that one 20 \(\upmu\)L drop of aqueous solution and saliva was enough to measure the NIR spectrum. This means that less saliva is required from the patient. Saliva drops dry non-homogenously on the silicon sample surface [41]. One drop of saliva spreads unevenly on the reflective material which is a disadvantage. One, three, and six 15 \(\upmu\)L drops of saliva were dried on a silicon surface in an FTIR-PA spectroscopy study by [PERSON] et al. [40]. Another advantage of our study is the small sample preparation time required before measuring. Pipetting one drop of liquid on a sample holder for measuring without having to dry the drop, as in our study, is less time-consuming than drying several drops onto a sample holder, which could take up to 20 h [40], before measuring. The NIRS set-up used in this study is mobile and clinically applicable; furthermore, a probe specifically designed for liquids can enhance the technique even further. The NIR spectra of two saliva samples differ from water's spectrum in L-fucose and L-proline specific wavelength areas that were identified. A fine kNN validation was carried out, and it showed relatively poorer sensitivity for the solutions that had low concentrations. The results of this proof-of-concept study are qualitative as the sample set was small, and due to the slight uneven spreading of the sample drops. The volume of the drop should be 20 uL to avoid saturation of the signal. To get quantitative results, measurements with larger sets of saliva samples and a liquid measurement-specific probe are required. Saliva samples should be collected from patients suffering from OPMDs, from patients suffering from oral cancer, and from healthy controls.