# Infrared Spectroscopy for the Analysis of Bioactive Analytes in Wheat: A Proof-of-Concept Study
## 1 Introduction  
Common or bread wheat (_Tritcium aestivum_ L) is the second-most frequently grown grain crop worldwide (after corn), with over 760 million tonnes harvested in 2020 [1]. It is a vital crop for ensuring global food security, supplying approximately 20% of dietary calorie requirements across the globe [2]. Furthermore, wheat is the largest broadacre crop in Australia, with 14 million tonnes produced in the 2019-2020 season [3]. Although the health benefits of wheat are generally considered to be lower than those of pulse crops [4, 5], it does contain several potentially health-benefitting compound classes, including polyphenols, carotenoids, vitamin E and phytosterols [6, 7]. Although these compounds are most abundant in wholegrain products [6], they are also found in refined wheat products, albeit at lower levels [8]. The major polyphenol compounds previously identified from wheat include gallic acid, 4-hydroxybenzoic acid, vanillic acid protocatech acid, syringic acid, ferulic acid, caffe acid, chlorogenic acid, sinapic acid, \(p\)-cumaric acid, cinnamic acid, rutin, quercetin, kaempferol, vitexin, isovitexin and resveratrol [9, 10]. There is also increasing interest in developing wheat lines with elevated phenolic acid contents [11, 12]. As with other grain crops, infrared (IR) spectroscopy has been widely applied for the quality assessment of wheat and wheat flour, including the prediction of protein [13], fibre [14], starch [15] and other quality parameters important for bread-making [16]. Infrared spectroscopy is an analytical technique that uses the characteristic absorption of infrared light by dipole-active covalent bonds to infer the chemical composition of a sample. In brief, the full spectrum of IR light is shone onto the sample, where certain bonds will absorb the IR light at characteristic wavelengths (depending on the bond's constituent atoms, and to a lesser degree the surrounding atoms). By measuring the IR light that is not absorbed (either from reflectance or transmittance measurements), the chemical bonds found in the sample can be predicted, along with their relative concentrations. More detail on the principles behind IR spectroscopy can be found in several recent reviews [17; 18; 19]. Near-infrared spectroscopy (NIRS) is most commonly used for food analysis, and uses wavelengths in the near-infrared (750-2500 nm) region. It has the benefit of increased IR penetration into the sample, which provides results that are more representative of the true sample composition. However, the absorption is generally less specific, making it more difficult to detect trace constituents. The other type of infrared spectroscopy that is applied to food products is mid-infrared spectroscopy (MIRS), which uses wavelengths between 4000 and 400 cm\({}^{-1}\). It provides increased specificity for the chemical bond type, but decreased sample penetration. Consequently, more MIR instruments require firm contact between the instrument and the sample to be analysed, e.g., using an attenuated total reflectance (ATR) platform. For analytes found in sufficiently high concentrations, IR spectroscopy can provide rapid, non-destructive and environmentally friendly method of compositional analysis. However, its accuracy will almost always be less than that found using traditional analytical techniques. One major benefit of using infrared spectroscopy for compositional analysis is that multiple analytes (e.g., protein, moisture, starch content, etc.) can be simultaneously predicted from a single infrared spectrum. This provides further time savings over 'wet' chemical analysis, which is often a'serial' process using multiple analytical instruments. Recent work has reported the prediction of micro-nutrients (such as phenolics or antioxidant compounds) in several grain crops, including rice [20], gluten-free grains [21] and mungebean.