# Infrared Spectroscopy for the Analysis of Bioactive Analytes in Wheat: A Proof-of-Concept Study
## 1 Introduction
However, predicting the levels of micro-nutrients (such as phenolics or antioxidant compounds) is considered to be more challenging in wheat, as it generally has much lower phenolic and antioxidant concentrations compared to other grain crops such as pulses (e.g., faba beans, mungebeans, chickpeas, and lentils) [22]. One recent study by [PERSON] et al. [23] investigated the use of NIRS for the prediction of TPC in whole wheat flour, reporting a high level of linearity (R\({}^{2}_{\text{val}}\) = 0.90) and precision (RMSEV = 7.1 mg GAE/100 g) on a dependent test set. However, the authors did not consider the prediction of antioxidant capacity in this study. Additionally, there are few studies investigating the use of MIRS for the quality assessment of wheat. Consequently, this work investigated and evaluated the performance of NIRS and MIRS for the prediction of phenolic content, anthocyanin content and antioxidant capacity in Australian-grown wheat. The samples were impact milled to produce whole seed flour (Falling No. grinder, 0.8 mm screen). Moisture content was determined by the American Association of Cereal Chemists (AACC) International Method 44-15.02; briefly, this involved drying the samples in a laboratory oven at 130 \({}^{\circ}\)C for 1 h. All subsequent results are expressed on a dry weight basis. Protein content was determined by combustion using a LECO TruMac N protein analyser (LECO, Saint Joseph, MI, USA) with a conversion factor of \(\times\)6.25 [24]. Ferric reducing antioxidant potential (FRAP), total phenolic content (TPC) and total monomeric anthocyanin (TMA) content were determined following previously described methods [22; 25]. Antioxidant compounds were first extracted by combining 0.5 g of sample (in duplicate) with 7 mL of 90% methanol, followed by end-over-end shaking at 50 rpm for 60 min. After centrifugation and collecting the supernatant, the pellet was re-extracted using another 7 mL of 90% methanol and end-over-end shaking for 20 min. The combined supernatants were made up to 15 mL volume. The FRAP was measured by combining 100 \(\mu\)L of methanol extract with 3 mL of FRAP reagent (comprising 300 mM acetate buffer at pH 3.56, 20 mM aqueous ferric chloride and 10 mM TTPZ (made in 40 mM HCl) in the ratio 10:1:1). After vortexing (10 s) and incubation at 37 \({}^{\circ}\)C for 4 min, the absorbance was measured at 593 nm. Results are expressed in Trolox equivalents (TE). The TPC was determined by combining 400 \(\mu\)L of methanol extract with 2 mL of Folin-Cicollet reagent (previously diluted 10\(\times\) in Milli-Q water), incubating in darkness at room temperature for 10 min, then adding 2 mL of 7.5% \(w/v\) aqueous sodium carbonate. After a further incubation in a covered waterbath for 30 min at 40 \({}^{\circ}\)C, the absorbance was measured at 760 nm. The results are expressed as gallic acid equivalents (GAE). Finally, the TMA content was measured by combining 400 \(\mu\)L of the methanol extract with 1.6 mL of pH 1 buffer (comprising 0.025 M aqueous KCl and adjusted to pH 1 with HCl). In a separate vessel, 400 \(\mu\)L of the same methanol extract was combined with 1.6 mL of pH 4.5 buffer (comprising 00.4 M aqueous sodium acetate and adjusted to pH 4.5 with HCl). After incubation in darkness at room temperature for 15 min, the absorbance was measured at 510 and 700 nm, with the anthocyanin content calculated using the formula below. Results are expressed in equivalents of cyanidin-3-glucose (cyt-3-glu).