# Infrared Spectroscopy for the Analysis of Bioactive Analytes in Wheat: A Proof-of-Concept Study
## 3 Results
### Moving Window Analysis  
Finally, moving window analysis was performed for the best-performing analytes (protein, FRAP, TPC), using both the NIR and MIR spectra. The optimum pre-processing method was determined using regular PLSR (Tables 2 and 3). Figure 11: Actual vs. MIRS-predicted ferric reducing antioxidant potential (FRAP) values for the calibration (**A**) and test (**B**) samples, using 6 components. Figure 12: Loadings plot for the MIRS prediction of ferric reducing antioxidant potential (FRAP) in wheat flour. Note that “comp” stands for the number of principal components in the model. As shown in Figure 15, most wavelength ranges gave reasonably good PLSR models for the prediction of protein from the NIR spectra. However, it was evident that wavelengths below approximately 1400 nm did not provide any useful information for this analyte. As shown by the darker blue stripe across the top of Figure 15, the inclusion of wavelengths between approximately 2150 and 2500 nm appeared to be quite important for the prediction of protein. The optimum PLSR model was found to be between 1210 and 2340 nm, which included the most important wavelengths previously seen in the loadings plot for this analyte (Figure 3). The model created on the optimised wavelengths showed a moderate improvement in R\({}^{2}\)cv and RMSECV values compared to the full-wavelength model (Table 4). Figure 14: Loadings plot for the MIRS prediction of total phenolic content in wheat flour. Note that “comp” stands for the number of principal components in the model. Figure 13: Actual vs. MIRS-predicted TP values for the calibration (**A**) and test (**B**) samples, using 5 components. The FRAP moving window analysis showed a smaller number of high-performing wavelength ranges (Figure 16). Again, the inclusion of wavelengths between approximately 2300-2450 nm were important for high-accuracy models. The optimal wavelength range for the prediction of FRAP was between 1600-2310 nm (Table 4). Again, this generally agreed with the loadings plot for this analyte (Figure 5). \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Parameter** & **Method** & **Wavelength Range** & \(\mathbf{R^{2}_{cv}}\) & **RMSECV** \\ \hline
**NIRS** & & **(nm)** & & \\ \hline Protein & Full range & 1000–2500 & 0.976 & 0.302 \\ Protein & Optimised & 1210–2340 & 0.987 & 0.225 \\ FRAP & Full range & 1000–2500 & 0.891 & 5.36 \\ FRAP & Optimised & 1600–2310 & 0.918 & 4.66 \\ TPC & Full range & 1000–2500 & 0.521 & 7.78 \\ TPC & Optimised & 1200–2100 & 0.664 & 6.51 \\ \hline
**MIRS** & & **(cm\({}^{-1}\))** & & \\ \hline Protein & Full range & 4000–400 & 0.841 & 0.803 \\ Protein & Optimised & 1515–1315 & 0.916 & 0.584 \\ FRAP & Full range & 4000–400 & 0.750 & 7.56 \\ FRAP & Optimised & 920–800 & 0.846 & 5.92 \\ TPC & Full range & 4000–400 & 0.306 & 9.06 \\ TPC & Optimised & 410–1760 & 0.452 & 8.05 \\ \hline \hline \end{tabular}
\end{table} 
Table 4: Comparison of PLSR models created using the full wavelength range, and the optimised wavelength range found through moving window PLSR optimisation, for selected analytes using both NIRS and MIRS. Note that the RMSECV and \(\mathrm{R^{2}_{cv}}\) values may vary from those reported in Tables 2 and 3, as the optimum number of components was selected using an algorithm for the moving window comparison, rather than manually selected. Figure 15: Results of the moving window optimisation for the prediction of protein using the NIR spectra. Note that the figure shows RMSECV values. The final NIRS moving window analysis was performed for TPC. It showed a much lower number of high-performing PLSR models, with nearly all of the best models beginning at a wavelength of 1000-1400 nm and ending between 2050 and 2450 nm (Figure 17). The optimal model was found to be between 1200 and 2100 nm (Table 4). Interestingly, this excluded several of the prominent peaks seen above 2100 nm in the loadings plot for TPC (Figure 7), suggesting that these peaks may not be specific to TPC.