# Infrared Spectroscopy for the Analysis of Bioactive Analytes in Wheat: A Proof-of-Concept Study
## 3 Results
### Near-Infrared Spectra  
Figure 1 shows the mean raw and SNV-processed NIR spectra of all 65 wheat flour samples. The NIR spectra showed major peaks at 1467 nm and 1934 nm, corresponding to the second and first overtones of the OH bond (from moisture in the sample or other R-OH bonds), respectively [29]. Other smaller peaks included 1205 nm (CH second overtone from lipids and structural carbohydrates), 2107 nm (amide bond from protein), broad peaks between 1680-1850 nm (CH first overtone stretch) and 2260-2370 nm (CH stretching deformation of CH\({}_{2}\) and CH\({}_{3}\) bonds), and shoulders at 1360 nm (second overtone of CH\({}_{3}\) or ArOH) and 1580 nm (tentatively assigned to CH\({}_{3}\)/ArCH first overtone from starch) [30; 31]. \begin{table}
\begin{tabular}{c c c c c} \hline \hline  & \multicolumn{2}{c}{**Calibration Set (\(n\) = 50)**} & \multicolumn{2}{c}{**Test Set (\(n\) = 15)**} \\ \hline \multirow{2}{*}{**Parameter**} & \multirow{2}{*}{**Range**} & **Mean \(\pm\) SD** & \multirow{2}{*}{**Range**} & **Mean \(\pm\) SD** \\  & & **(Median)** & & **(Median)** \\ \hline \multirow{2}{*}{Moisture (\%)} & \multirow{2}{*}{10.1–14.5} & 11.90 \(\pm\) 1.39 & \multirow{2}{*}{8.7–14.0} & 11.70 \(\pm\) 1.30 \\  & & (11.40) & & (11.30) \\ \multirow{2}{*}{Protein (\%)} & \multirow{2}{*}{10.66–17.46} & 14.16 \(\pm\) 1.98 & \multirow{2}{*}{11.43–16.84} & 13.97 \(\pm\) 2.25 \\  & & (13.84) & & (14.19) \\ \multirow{2}{*}{FRAP (mg TE/100 g)} & \multirow{2}{*}{14.4–64.0} & 32.8 \(\pm\) 16.4 & \multirow{2}{*}{19.4–53.5} & 25.3 \(\pm\) 8.5 \\  & & (24.6) & & (23.4) \\ \multirow{2}{*}{TPC (mg GAE/100 g)} & \multirow{2}{*}{129.7–179.8} & 149.5 \(\pm\) 11.3 & \multirow{2}{*}{139.0–167.6} & 151.9 \(\pm\) 9.7 \\  & & (151.9) & & (146.4) \\ \multirow{2}{*}{TMA (mg} & \multirow{2}{*}{0.0–10.0} & 2.5 \(\pm\) 2.2 & \multirow{2}{*}{0.0–3.9} & 2.0 \(\pm\) 1.1 \\  & & (2.2) & & (2.6) \\ \hline \hline \end{tabular}  
* \(\mathrm{cyd}\)-3-glu = cyanidin-3-glucoside equivalents; FRAP = ferric reducing antioxidant potential; GAE = gallic acid equivalents SD = standard deviation; TE = Trolox equivalents; TMA = total monomeric anthocyanin content; TPC = total phenolic content.  
\end{table} 
Table 1: Descriptive statistics for the parameters measured in this study, for both the calibration and test sets. The PLSR models with the optimised spectral pre-processing methods for each analyte are detailed in Table 2. The best-performing model was that for protein content (Figure 2), which showed an R2 test of 0.991 and RMSEP of 0.22%. Furthermore, the model showed minimal bias and the slope and intercept were extremely close to their ideal values of 1 and 0, respectively (Table 2). Examination of the loadings plot for the protein prediction model (Figure 3) showed predominant contributions in the N-H asymmetric stretch and amide II region (1928-1941 nm), as anticipated [32]. Other minor contributions were at 2226 nm (potentially corresponding to the amide I and III region) and 1732 nm (which may be due to CH first overtones in gluten, the major protein found in wheat) [32]. \begin{table}
\begin{tabular}{c c c c c c c c c c c} \hline \hline \multirow{2}{*}{**Parameter**} & \multicolumn{2}{c}{**Spectral**} & \multirow{2}{*}{**Components**} & \multirow{2}{*}{**R2 cv**} & \multirow{2}{*}{**RMSECV**} & \multirow{2}{*}{**RPD**} & \multirow{2}{*}{**R2 test**} & \multirow{2}{*}{**RMSEP**} & \multirow{2}{*}{**Bias**} & \multirow{2}{*}{**Slope**} & \multirow{2}{*}{**Intercept**} \\  & & & & & & & & & & & \\ \hline Moisture & SNV + 1d15 & 6 & 0.84 & 0.56 & 2.49 & 0.76 & 0.62 & \(-\)0.04 & 0.966 & 0.35 \\ Protein & SNV & 5 & 0.974 & 0.32 & 6.25 & 0.991 & 0.22 & \(-\)0.06 & 0.998 & \(-\)0.03 \\ FRAP & SNV & 6 & 0.88 & 5.6 & 2.95 & 0.917 & 5.4 & \(-\)0.64 & 0.620 & 9.2 \\ TPC & SNV + 1d21 & 5 & 0.61 & 7.0 & 1.62 & 0.83 & 3.9 & \(-\)0.10 & 0.907 & 14.1 \\ TMA & 1d11 & 4 & 0.05 & 2.1 & 1.03 & 0.00 & 1.9 & \(-\)0.63 & 0.02 & 1.98 \\ \hline \hline \end{tabular}
\end{table}