The Role of the Preanalytical Step for Human Saliva Analysis via Vibrational Spectroscopy

Abstract

Saliva is an easily sampled matrix containing a variety of biochemical information, which can be correlated with the individual health status. The fast, straightforward analysis of saliva by vibrational (ATR-FTIR and Raman) spectroscopy is a good premise for large-scale preclinical studies to aid translation into clinics. In this work, the effects of saliva collection (spitting/swab) and processing (two different deproteinization procedures) were explored by principal component analysis (PCA) of ATR-FTIR and Raman data and by investigating the effects on the main saliva metabolites by reversed-phase chromatography (RPC-HPLC-DAD). Our results show that, depending on the bioanalytical information needed, special care must be taken when saliva is collected with swabs because the polymeric material significantly interacts with some saliva components. Moreover, the analysis of saliva before and after deproteinization by FTIR and Raman spectroscopy allows to obtain complementary biological information.

Keywords: ATR-FTIR; Raman; saliva; sample processing.

Figure 1

Saliva sample dried spot (SDS) from 50 μL deposition onto PP sheet and “printing” on ATR-FTIR crystal.

Figure 2

Representative ATR-FTIR spectra of saliva analyzed as is in 4000–2000 cm⁻¹ (a) and 1800–600 cm⁻¹ regions (b).

Figure 3

ATR-FTIR spectra of all representative saliva samples analyzed before and after deproteinization with ethanol (EtOH) and ultrafiltration with 3000 Da cut-off (CO) in 4000–2000 cm⁻¹ (a) and 1800–600 cm⁻¹ regions (b). (c) ATR-FTIR of N = 5 replicates of saliva sample after ultrafiltration with 3000 Da cut-off as example of reproducibility of the spectra. HMWsaliva_CO and HMWsalivette_CO refer to high-molecular-weight (HMW) compounds remaining in the upper part of 3 kDa cut-off filtering units (w = wiping, p = printing) as explained in the experimental part.

Figure 3

ATR-FTIR spectra of all representative saliva samples analyzed before and after deproteinization with ethanol (EtOH) and ultrafiltration with 3000 Da cut-off (CO) in 4000–2000 cm⁻¹ (a) and 1800–600 cm⁻¹ regions (b). (c) ATR-FTIR of N = 5 replicates of saliva sample after ultrafiltration with 3000 Da cut-off as example of reproducibility of the spectra. HMWsaliva_CO and HMWsalivette_CO refer to high-molecular-weight (HMW) compounds remaining in the upper part of 3 kDa cut-off filtering units (w = wiping, p = printing) as explained in the experimental part.

Figure 4

PCA results of SNV-normalized and centered ATR-FTIR spectra of saliva samples. (a) Score plot (87.7% of total variance); (b) loading plot of PC1 (blue line) and PC2 (red line). HMWsaliva_CO and HMWsalivette_CO refer to high-molecular-weight compounds (HMWCs) remaining in the upper part of 3 kDa cut-off filtering units (w = wiping, p = printing) as explained in the experimental part.

Figure 5

Box plots of the main metabolites determined in saliva/salivette samples before and after deproteinization with 3 kDa cut-off filtration. Red cross = mean value; black dots = minimum/maximum value; box = 1st quartile–3rd quartile range; bar = standard deviation.

Figure 6

Comparison of Raman spectra at 785 nm of saliva before (a) and after (b) filtering with 3 kDa filters.