## 3 Results and Discussion
### Case Study Review: Analytical Advantages Offered by \({}^{1}\)H NMR Analysis of LOPs in CFOs
#### 3.6.1 Spectrophotometric Conjugated Dienes Assay  
From an analytical food chemistry standpoint, the conjugated dienes (CDs) spectrophotometric assay method employed by researchers, e.g., by [PERSON] et al. (1996) [63], [PERSON] et al. (2014) [64] and [PERSON] et al. (2016) [44], has a very poor selectivity than that afforded by peroxide value (PV) analysis, which is also commonly performed [44]. Likewise, this selectivity is very poor when compared to that of the \({}^{1}\)H NMR spectroscopic technologies presented here. This CDs method is based on the absorption of UV light by conjugated double bonds within CHPD molecules (the conversion of PUFAs to such CHPDs involves a rearrangement of >C=C< double bonds present in these FAs). However, at the wavelength range employed for this relatively simple test (230-235 nm), many interfering compounds also absorb in this spectral region, notably carotenoids, tocopherols and phenolic antioxidants, not to mention possible contributions from \(\alpha\),\(\beta\)-unsaturated aldehydes generated from the fragmentation of CHPDs [65;66], especially the more highly unsaturated ones [67]. Indeed, \(\alpha\),\(\beta\)-unsaturated aldehydes and ketones have electronic absorption spectra with maxima located at ca. 230 (\(\pi\)\(\rightarrow\)\(\pi^{\star}\) absorption, \(\varepsilon\) = ca. 1.3 \(\times\) 10\({}^{4}\) M\({}^{-1}\) cm\({}^{-1}\), where \(\varepsilon\) represents molar extinction coefficient), and 310 nm (n\(\rightarrow\)\(\pi^{\star}\) absorption, \(\varepsilon\) = ca. 1.5 \(\times\) 10\({}^{2}\) M\({}^{-1}\) cm\({}^{-1}\)) [67], and total concentrations of the former aldehyde classifications in repeatedly thermo-oxidised CFOs may attain values as high as 50 mmol kg\({}^{-1}\) (approximately equivalent to 15 mmol aldehyde/mol FA). Hence, if the total \(\alpha\),\(\beta\)-unsaturated aldehyde concentration in such an oxidised CFO product is 20 mmol kg\({}^{-1}\), then prior to any dilution it will have absorbance values of ca. 260 and 3 at ca. 230 and 310 nm, respectively. This recommended CD assay system employs a 1/600 dilution with a hexane solvent [63], and hence on consideration of an \(\varepsilon\) value of 2.90 \(\times\) 10\({}^{4}\) mol\({}^{-1}\) cm\({}^{-1}\) for \(\alpha\),\(\beta\)-unsaturated aldehydes at 234 nm, the absorbance contribution from these aldehyde interferants alone would be as high as ca. 0.4 at this wavelength, undoubtedly a significant interfering value. However, conjugated triene hydroperoxide species arising from lineolopolycerol peroxidation absorb strongly at a presumably more specific wavelength of 268 nm [64].  
However, when applied to unheated or unused CFOs, this CD assay system may be suitable, since interferences from aldehydes will be minimal, although not those arising from mutraceuticals such as alpha-tocopherol (\(\alpha\)-TOH) and any other phenolic antioxidants present. Indeed, [PERSON] et al. [64] have reported a strong correlation between CFO hydroperoxide levels and CDs determined by this method. However, it should be noted that HPMs derived from the peroxidation of UFAs are, of course, unresponsive in the CD assay, but are in the classical peroxide value one, and hence the former method's suitability for application to MUFA-rich oils such as olive oil is limited to hydroperoxides derived from PUFAs only.  
Therefore, overall such CD assay results are of a limited value, most especially when applied to thermally-stressed CFOs, and hence there is a major call for researchers to determine the relative (proportionate) contributions of all interferants, notably those arising from \(\alpha,\beta\)-unsaturated aldehydes, towards presumed CD absorbance values at 234 nm.