Spectral change over the drying period
The study was based on three relevant body fluids i.e., semen, saliva, and urine except blood as a similar study is already reported on blood6. In this study, the major change is observed in two regions of the spectra i.e., 3300-2800 cm-1 (lipids are the major contributors) and 1700-950 cm-1 (proteins, nucleic acids and carbohydrates are the major contributors)33,34. The list of peaks identified in the spectra of all three fluids is summarized in table 1 with their individual vibrational assignments. Although several peaks were identified in the spectra of each fluid during the study, only a few (age-linked peaks) showed linear changes in their absorption intensity with time. At the initial stage, a similar phenomenon has been observed in the drying of every body fluid. For the first several minutes, only two strong absorption peaks were visible (figure 1.a to 1.f). After a certain amount of time multiple significant peaks corresponding to the biochemical profile of the fluids revealed throughout the fingerprint region of the IR spectra (figure 2.a to 2.f). Following the trend in each fluid, the whole drying time was divided into two phases.
The dilution of the fluids showed an extended (2-4 minutes) phase I due to the excess amount of water in the diluted sample. The longest phase I observed in the Semen samples and shortest in the saliva samples. Phase II was relatively similar in the spectra of both raw and diluted samples. The duration of phase II of three body fluids was relatively the same (10-12 minutes). Table 2 demonstrates the minimum, maximum, and mean values of both phases. The difference in the drying time of all three fluids is potentially a result of the qualitative and quantitative variability in their biochemical components.
Few researchers reported the correlation between the evaporation of distilled water and time.35,36 Although, their experimental parameters were different. Phase I included two strong peaks of amide A (3270-3273 cm-1) and amide I (1636.97 cm-1) is dominated by the water content of these body fluids (figure 1.a to 1.f). Except for similar height, the peak corresponding to amide A was broader than the peak due to amide I. Water is the major component of all the secreted fluids in the human body. The O-H stretching of water masks the absorption of IR radiation by other minor but significant components of the fluid.6 Except for urine, the absorption intensity of these two peaks showed insignificant change throughout phase I in the spectra of semen and saliva (figure 1.a to 1.f and figure 3). In the contrary, Zhang et al.6 found a different result for blood as the peak at 3308 cm-1 showed very weak but linear absorption change during the early stage. Only the spectra of urine (100% and 40%), showed analogous results with the study by Zhang et al.6 as the peak at 3272.77 cm-1 showed a significant decline in the mean absorbance with time during the phase I (figure 2.c, 2.f and 3.c).
Phase II is the fast declination stage where a significant amount of water evaporates rapidly and reveal the other peaks and their intensity changes with time in each body fluid. The peaks at 3270.84 and 1636.97 cm-1 showed no shift during the whole drying (phases I and II) process of semen stain but the former one sharpens with time and rapidly declined during the phase II (figure 1.a, 1.d, 2.a and 2.d). In the phase II drying of semen droplets, one strong (1546.31 cm-1: Amide II), two medium (1446 cm-1: methylene; CH2 and CH3 and 1065.99 cm-1: Glycosylated proteins: probably prostate-specific antigen) and three weak (2967.98 cm-1: CH3 stretching, 1395.85 cm-1: fatty acids and polysaccharides and 1243.46 cm-1: Amide III) significant age-linked peaks were observed (figure 2.a and 2.d). Zha et al.5 investigated the changes in few similar peaks at marginally different positions i.e., 1539 cm-1 (Amide II), 1448 cm-1 (Methylene: CH2 and CH3), 1392 cm-1 (Fatty acids and polysaccharides), 1059 cm-1 (Prostate-specific antigen). Few more researchers reported the IR spectra of semen in several body fluid identification research articles.37-39 Phase II spectra of saliva showed the shift of strong peaks at 3272.77 cm-1 (amide A) to 3286.27 cm-1 (amide A) and 1636.97 cm-1 (amide I) to 1644.69 cm-1 (amide I) that indicated the initiation of this phase (figure 2.b and 2.e). The shifted peak of amide A sharpens following the trend of semen samples. Among others, one strong and sharp (1546.31 cm-1: amide II), four weak (1447.93 and 1403.07 cm-1: Methylene, 1077.56, and 1042.84 cm-1: glycosylated proteins) significant age-linked peaks were found (figure 2.b and 2.e). Including the peak corresponding to amide II, two peaks at 1447.93 cm-1 and 1077.56 cm-1 are similar to the peaks at 1446 and 1065.99 cm-1 in the spectra of semen and placed at marginally different positions. But the intensity of the peak corresponding to glycosylated protein is relatively weaker in the spectra of saliva. The peaks in phase I spectra of urine bifurcated in phase II. The peak at 3272.77 cm-1 divided into 3346.07 and 3205.25 cm-1 and 1636.97 cm-1 divided into 1623.47 and 1658.19 cm-1 (figure 2.c and 2.f). One strong (1658.19 cm-1: amide I), one medium (3346.07 cm-1: H-O-H stretching), and two weak (1156.65 cm-1: urea and 1081.42 cm-1: Glycosylated proteins) peaks were observed in the phase II spectra of urine samples (figure 2.c and 2.f) that significantly changes during the drying process. The peak at 1081.42 cm-1 is similar to the peaks at 1065.99 and 1077.56 cm-1 of semen and saliva, respectively. Due to the presence of a significant quantity of prostate-specific antigen in semen, the peak corresponding to glycosylated protein is stronger in its spectra than saliva and urine.33 In several previous literatures on the IR signature of saliva and urine, the above-mentioned peaks were reported by researchers.37,38,40-42 Amide A, I, II, and glycosylated proteins are the common biochemical components found in all the three body fluids. Elkins, Orphanou, and Takamura et al.35,39,40 previously reported the presence of the common biochemicals in all these body fluids on their article of body fluid identification by ATR-FTIR. In the Phase II, all the age-linked peaks of each fluid showed a linear relationship between the mean absorbance at each time point and TSD (figure 2.a to 2.f). The calculated
Coefficient of determination (R2) measures that whether a regression line can explain most of the variance of the representing data. Hence, higher R2 value for a particular peak indicates it as a good predictor. R2 values were calculated for significantly transforming peaks of semen, saliva and urine and most of them except the peak at 3346.07 cm-1 corresponding to H-O-H stretching (R2 value: 0.8863), have the R2 value above 0.9 (max: 0.9994; min: 0.9016) (Table 3a. to 3.c). The ‘p’ values of all these correlation equations are <0.05, which is also statistically significant. The mean absorbance of the selected peaks at each time point increased with time for both raw and diluted fluid samples. Only the peak at 3346.07 cm-1 in the spectra of urine decreased with time, potentially due to the faster evaporation of water.
Estimation of TSD in Phase II of drying
Body fluids show significant ex-vivo degradation when exposed for a long period. While, early changes are very limited as only a few relevant peaks for aging are visible. Hence, the TSD estimation was performed only on the age-linked peaks of three body fluids. Both Principal Component Regression (PCR) Analysis and Partial Least Square Regression (PLSR) are strong chemometric tool for the estimation of TSD of body fluids as reported in previous studies.1,4,5,10,29 The calculated R2 values for the calibration and prediction of both models are more than 0.9. While the root mean square error of cross validation (RMSECV) and prediction (RMSEP) values in both the models for all the three fluids except diluted urine, showed low values (Table 4.a and 4.b). Although the initial changes in the IR spectra of body fluids are relatively less distinguishable in comparison to the samples exposed for longer period, the High R2 and low RMSE values indicate a good prediction of TSD during this timeframe. In diluted urine samples, the RMSECV (PCRA: 0.77; PLSR: 0.76) and RMSEP (PCRA:0.74; PLSR: 0.73) (Table 4.a and 4.b) in both the models are relatively higher, that potentially interfere in accurate age estimation. While, diluted semen also showed higher RMSECV (0.72) and RMSEP (0.68) in PCRA regression model, while PLSR predicts the age for the same condition with significantly higher accuracy with RMSECV and RMSEP values of 0.19 and 0.15, (Table 4.a and 4.b) respectively. Comparatively, PLSR showed better efficiency of prediction than PCRA as for every fluid it records relatively higher R2 and lower RMSE values than in PCRA. Figure 4 and 5 depicts the PCR and PLS plots of actual vs predicted regression lines for age linked peaks of three body fluids.